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Publications | ATLAS-D2K Center

Publications

2023

  1. Functional maturation of kidney organoid tubules: Piezo1-mediated Ca ^\textrm2+ signaling

    Carrisoza-Gaytán, Rolando; Kroll, Katharina T.; Hiratsuka, Ken; Gupta, Navin R.; Morizane, Ryuji; Lewis, Jennifer A.; Satlin, Lisa M.. American Journal of Physiology-Cell Physiology . February 2023.

    Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared to static controls (Homan et al., 2019; Takasato et al., 2015). However, their physiological function has yet to be systematically investigated. Here, we measured mechanoinduced changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in tubules isolated from organoids cultured for 21-64 d, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca 2+ ] i. A rapid \textgreater2.5-fold increase in [Ca 2+ ] i from a baseline of 195.0±22.1 nM (n=9; p≤0.001) was observed when microperfused tubules from organoids \textgreater40 d in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids \textless30 d in culture. Nonperfused tubules (41 d) subjected to a 10-fold increase in bath flow rate also exhibited a 3-fold increase in [Ca 2+ ] i from baseline (p\textless0.001). Mechanosensitive Piezo1 channels contribute to the flow-induced [Ca 2+ ] i response in mouse distal tubule (Carrisoza-Gaytan et al., EB 2019). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 d in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca 2+ ] i (p≤0.001) in segments from organoids cultured for \textgreater30 d, with peak [Ca 2+ ] i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca 2+ channels, including PIEZO1, in tubules of static organoids in culture.

  2. Lineage Tracing and Single-Nucleus Multiomics Reveal Novel Features of Adaptive and Maladaptive Repair after Acute Kidney Injury

    Gerhardt, Louisa M.S.; Koppitch, Kari; van Gestel, Jordi; Guo, Jinjin; Cho, Sam; Wu, Haojia; Kirita, Yuhei; Humphreys, Benjamin D.; McMahon, Andrew P.. Journal of the American Society of Nephrology . Publish Ahead of Print. January 2023.

2022

  1. Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing

    Li, Haikuo; Humphreys, Benjamin D.. STAR Protocols . 3(4):101904. 2022.

    Summary Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).1

  2. Identifying Common Molecular Mechanisms in Experimental and Human Acute Kidney Injury

    Gerhardt, Louisa M.S.; McMahon, Andrew P.. Seminars in Nephrology . 2022.

    Summary Acute kidney injury (AKI) is a highly prevalent, heterogeneous syndrome, associated with increased short- and long-term mortality. A multitude of different factors cause AKI including ischemia, sepsis, nephrotoxic drugs, and urinary tract obstruction. Upon injury, the kidney initiates an intrinsic repair program that can result in adaptive repair with regeneration of damaged nephrons and functional recovery of epithelial activity, or maladaptive repair and persistence of damaged epithelial cells with a characteristic proinflammatory, profibrotic molecular signature. Maladaptive repair is linked to disease progression from AKI to chronic kidney disease. Despite extensive efforts, no therapeutic strategies provide consistent benefit to AKI patients. Since kidney biopsies are rarely performed in the acute injury phase in humans, most of our understanding of AKI pathophysiology is derived from preclinical AKI models. This raises the question of how well experimental models of AKI reflect the molecular and cellular mechanisms underlying human AKI? Here, we provide a brief overview of available AKI models, discuss their strengths and limitations, and consider important aspects of the AKI response in mice and humans, with a particular focus on the role of proximal tubule cells in adaptive and maladaptive repair.

  3. Autophagy Enhances Longevity of Induced Pluripotent Stem Cell-Derived Endothelium via mTOR-Independent ULK1 Kinase

    Hekman, Katherine E; Koss, Kyle M; Ivancic, David Z; He, Congcong; Wertheim, Jason A. Stem Cells Translational Medicine . September 2022.

    Abstract Stem cells are enabling an improved understanding of the peripheral arterial disease, and patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic modality. However, applications of patient-specific induced pluripotent stem cell (iPSC)-derived ECs are limited by rapid loss of mature cellular function in culture. We hypothesized that changes in autophagy impact the phenotype and cellular proliferation of iPSC-ECs. Endothelial cells were differentiated from distinct induced pluripotent stem cell lines in 2D culture and purified for CD144 positive cells. Autophagy, mitochondrial morphology, and proliferation were characterized during differentiation and over serial passages in culture. We found that autophagy activity was stimulated during differentiation but stagnated in mature iPSC-ECs. Mitochondria remodeled through mitophagy during differentiation and demonstrated increasing membrane potential and mass through serial passages; however, these plateaued, coinciding with decreased proliferation. To evaluate for oxidative damage, iPSC-ECs were alternatively grown under hypoxic culture conditions; however, hypoxia only transiently improved the proliferation. Stimulating mTOR-independent ULK1-mediated autophagy with a plant derivative AMP kinase activator Rg2 significantly improved proliferative capacity of iPSC-ECs over multiple passages. Therefore, autophagy, a known mediator of longevity, played an active role in remodeling mitochondria during maturation from pluripotency to a terminally differentiated state. Autophagy failed to compensate for increasing mitochondrial mass over serial passages, which correlated with loss of proliferation in iPSC-ECs. Stimulating ULK1-kinase-driven autophagy conferred improved proliferation and longevity over multiple passages in culture. This represents a novel approach to overcoming a major barrier limiting the use of iPSC-ECs for clinical and research applications.

  4. Mapping the single-cell transcriptomic response of murine diabetic kidney disease to therapies

    Wu, Haojia; Villalobos, Romer Gonzalez; Yao, Xiang; Reilly, Dermot; Chen, Tao; Rankin, Matthew; Myshkin, Eugene; Breyer, Matthew D; Humphreys, Benjamin D. Cell Metabolism . 34(7):1064–1078.e6. July 2022.

    Diabetic kidney disease (DKD) occurs in ∼40% of patients with diabetes and causes kidney failure, cardiovascular disease, and premature death. We analyzed the response of a murine DKD model to five treatment regimens using single-cell RNA sequencing (scRNA-seq). Our atlas of ∼1 million cells revealed a heterogeneous response of all kidney cell types both to DKD and its treatment. Both monotherapy and combination therapies targeted differing cell types and induced distinct and non-overlapping transcriptional changes. The early effects of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on the S1 segment of the proximal tubule suggest that this drug class induces fasting mimicry and hypoxia responses. Diabetes downregulated the spliceosome regulator serine/arginine-rich splicing factor 7 (Srsf7) in proximal tubule that was specifically rescued by SGLT2i. In vitro proximal tubule knockdown of Srsf7 induced a pro-inflammatory phenotype, implicating alternative splicing as a driver of DKD and suggesting SGLT2i regulation of proximal tubule alternative splicing as a potential mechanism of action for this drug class.

  5. Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary

    Liu, Yulong; Kossack, Michelle E; McFaul, Matthew E; Christensen, Lana N; Siebert, Stefan; Wyatt, Sydney R; Kamei, Caramai N; Horst, Samuel; Arroyo, Nayeli; Drummond, Iain A; Juliano, Celina E; Draper, Bruce W. eLife . 11:e76014. May 2022.

    Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 days post-fertilization). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA-sequencing studies that identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in the development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells, and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function, and the transcriptome profiles will provide a valuable resource for future studies.

  6. Biomanufacturing human tissues via organ building blocks

    Wolf, Kayla J.; Weiss, Jonathan D.; Uzel, Sebastien G.M.; Skylar-Scott, Mark A.; Lewis, Jennifer A. Cell Stem Cell . 29(5):667–677. May 2022.

    The construction of human organs on demand remains a tantalizing vision to solve the organ donor shortage. Yet, engineering tissues that recapitulate the cellular and architectural complexity of native organs is a grand challenge. The use of organ building blocks (OBBs) composed of multicellular spheroids, organoids, and assembloids offers an important pathway for creating organ-specific tissues with the desired cellular-to-tissue-level organization. Here, we review the differentiation, maturation, and 3D assembly of OBBs into functional human tissues and, ultimately, organs for therapeutic repair and replacement. We also highlight future challenges and areas of opportunity for this nascent field.

  7. Modelling ciliopathy phenotypes in human tissues derived from pluripotent stem cells with genetically ablated cilia

    Cruz, Nelly M.; Reddy, Raghava; McFaline-Figueroa, José L.; Tran, Christine; Fu, Hongxia; Freedman, Benjamin S. Nature Biomedical Engineering . 6(4):463–475. April 2022.

    The functions of cilia—antenna-like organelles associated with a spectrum of disease states—are poorly understood, particularly in human cells. Here we show that human pluripotent stem cells (hPSCs) edited via CRISPR to knock out the kinesin-2 subunits KIF3A or KIF3B can be used to model ciliopathy phenotypes and to reveal ciliary functions at the tissue scale. KIF3A–/– and KIF3B–/– hPSCs lacked cilia, yet remained robustly self-renewing and pluripotent. Tissues and organoids derived from these hPSCs displayed phenotypes that recapitulated defective neurogenesis and nephrogenesis, polycystic kidney disease (PKD) and other features of the ciliopathy spectrum. We also show that human cilia mediate a critical switch in hedgehog signaling during organoid differentiation, and that they constitutively release extracellular vesicles containing signaling molecules associated with ciliopathy phenotypes. The capacity of KIF3A–/– and KIF3B–/– hPSCs to reveal endogenous mechanisms underlying complex ciliary phenotypes may facilitate the discovery of candidate therapeutics.

  8. Chapter Nine - Growth control of the kidney

    Oxburgh, Leif. Current Topics in Developmental Biology. 148:237–263. 2022.

    The functional mass of kidney tissue in an adult is an important determinant of human health. Kidney formation during development is an essential determinant of the final nephron endowment of the adult organ, and no evidence has been reported that mice or humans are able to generate new nephrons after the developmental period. Mechanisms controlling organ growth after development are essential to establish the final adult organ size. The potential for organ growth is maintained in adult life and the size of one kidney may be significantly increased by loss of the contralateral kidney. The mouse has provided a model system for investigators to critically explore genetic, cell biological, and hormonal control of developmental and juvenile kidney growth. This article reviews three basic aspects of kidney size regulation: (1) Mechanisms that control nephron formation and how these are altered by the cessation of nephrogenesis at the end of the developmental period. (2) Applicability of the general model for growth hormone-insulin like growth factor control to kidney growth both pre- and postnatally. (3) Commonalities between mechanisms of juvenile kidney growth and the compensatory growth that is stimulated in adult life by reduction of kidney mass. Understanding the mechanisms that determine set-points for cell numbers and size in the kidney may inform ongoing efforts to generate kidney tissue from stem cells.

  9. Chapter Seven - Plumbing our organs: Lessons from vascular development to instruct lab generated tissues

    Ryan, Anne R.; Cleaver, Ondine. Current Topics in Developmental Biology. 148:165–194. 2022.

    The formation, growth and maintenance of our organs, such as our kidneys or pancreas, requires their coordinated growth alongside the intricate vasculature that pervades them. Blood vessels course through nearly every tissue in our bodies, facilitating the essential exchange of gases, nutrition and wastes, as well as the rapid circulation of hormones and other signaling molecules. Endothelial cells (ECs) that line all of our blood vessels are therefore the gatekeepers for communication between the circulation and organ-specific cell types. We and many others have sought to understand: (1) how endothelial cell progenitors initially assemble to form blood vessels in the embryo, and (2) how the embryonic vascular tree expands to perfuse growing organs. Here, we review what we have learned from embryonic blood vessels and how this knowledge instructs our approaches to vascularize laboratory generated tissues, such as organoids. We will assess our general understanding of blood vessel formation, and discuss recent studies of the growing kidney vasculature. Furthermore, we will assess the challenges and limitations faced by organoid technologies, including the difficulties in achieving the patterned vascular network that is essential to organ function. Lastly, we will then review recent studies of kidney organoid blood vessels and propose approaches that improve vascularization. Understanding the ontogeny of organ-specific vasculatures will help propel regenerative therapeutic approaches.

  10. Physiology Assays in Human Kidney Organoids

    Freedman, Benjamin. American Journal of Physiology-Renal Physiology . April 2022.

    Kidney organoids derived from human pluripotent stem cells constitute a novel model of disease, development, and regenerative therapy. Organoids are human, experimentally accessible, high throughput, and enable reconstitution of tissue-scale biology in a petri dish. While gene expression patterns in organoid cells have been analyzed extensively, less is known about the functionality of these structures. Here we review assays of physiological function in human kidney organoids, best practices for quality control, and future applications. Tubular structures in organoids accumulate specific molecules through active transport, including dextran and organic anions, and swell with fluid in response to cyclic adenosine monophosphate stimulation. When engrafted into animal models in vivo, organoids form vascularized glomerulus-like structures capable of size-selective filtration. Organoids exhibit metabolic, endocrine, injury, and infection phenotypes, although their specificity is not yet fully clear. To properly interpret organoid physiology assays, it is important to incorporate appropriate negative and positive controls, statistical methods, data presentation, molecular mechanisms, and clinical datasets. Improvements in organoid perfusion, patterning, and maturation are needed to enable branching morphogenesis, urine production, and renal replacement. Reconstituting renal physiology with kidney organoids is a new field with potential to provide fresh insights into classical phenomena.

  11. Smad4 controls proliferation of interstitial cells in the neonatal kidney

    McCarthy, Sarah S.; Karolak, Michele; Oxburgh, Leif. Development . 149(1):dev199984. January 2022.

    ABSTRACT Expansion of interstitial cells in the adult kidney is a hallmark of chronic disease, whereas their proliferation during fetal development is necessary for organ formation. An intriguing difference between adult and neonatal kidneys is that the neonatal kidney has the capacity to control interstitial cell proliferation when the target number has been reached. In this study, we define the consequences of inactivating the TGFβ/Smad response in the mouse interstitial cell lineage. We find that pathway inactivation through loss of Smad4 leads to overproliferation of interstitial cells regionally in the kidney medulla. Analysis of markers for BMP and TGFβ pathway activation reveals that loss of Smad4 primarily reduces TGFβ signaling in the interstitium. Whereas TGFβ signaling is reduced in these cells, marker analysis shows that Wnt/β-catenin signaling is increased. Our analysis supports a model in which Wnt/β-catenin-mediated proliferation is attenuated by TGFβ/Smad to ensure that proliferation ceases when the target number of interstitial cells has been reached in the neonatal medulla.

  12. Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium

    Naved, Bilal A.; Bonventre, Joseph V.; Hubbell, Jeffrey A.; Hukriede, Neil A.; Humphreys, Benjamin D.; Kesselman, Carl; Valerius, M. Todd; McMahon, Andrew P.; Shankland, Stuart J.; Wertheim, Jason A.; White, Michael J.V.; de Caestecker, Mark P.; Drummond, Iain A. Kidney International . March 2022.

  13. Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair

    Gupta, Navin; Matsumoto, Takuya; Hiratsuka, Ken; Garcia Saiz, Edgar; Galichon, Pierre; Miyoshi, Tomoya; Susa, Koichiro; Tatsumoto, Narihito; Yamashita, Michifumi; Morizane, Ryuji. Science Translational Medicine . 14(634):eabj4772. March 2022.

    Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 ( FANCD2 ) and RAD51 recombinase ( RAD51 ) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies. , Disease modeling in kidney organoids identifies a conserved RAD51/FANCD2-mediated process that promotes effective repair. , Fingering FANCD2 in tubular repair The transition from acute kidney injury, characterized by intrinsic repair, to incomplete repair and chronic damage has been difficult to study. Here, Gupta and colleagues modeled the transition from intrinsic to incomplete repair using human kidney organoids. A single exposure to cisplatin resulted in intrinsic repair, with preserved tubular architecture and up-regulation of genes associated with homology-directed repair, including Fanconi anemia complementation group D2 ( FANCD2 ). However, with repeated cisplatin exposure, FANCD2 and RAD51 recombinase ( RAD51 ) were down-regulated, leading to incomplete repair. The DNA ligase IV inhibitor SCR7 increased FANCD2-mediated repair and ameliorated progression to chronic injury in the organoids, suggesting that targeting the FANCD2/RAD51 pathway may have potential to treat kidney disease.

  14. Validation of HDAC8 Inhibitors as Drug Discovery Starting Points to Treat Acute Kidney Injury

    Long, Keith; Vaughn, Zoe; McDaniels, Michael David; Joyasawal, Sipak; Przepiorski, Aneta; Parasky, Emily; Sander, Veronika; Close, David; Johnston, Paul A.; Davidson, Alan J.; de Caestecker, Mark; Hukriede, Neil A.; Huryn, Donna M.. ACS Pharmacology & Translational Science . March 2022.

    Acute kidney injury (AKI), a sudden loss of kidney function, is a common and serious condition for which there are no approved specific therapies. While there are multiple approaches to treat the underlying causes of AKI, no targets have been clinically validated. Here, we assessed a series of potent, selective competitive inhibitors of histone deacetylase 8 (HDAC8), a promising therapeutic target in an AKI setting. Using biochemical assays, zebrafish AKI phenotypic assays, and human kidney organoid assays, we show that selective HDAC8 inhibitors can lead to efficacy in increasingly stringent models. One of these, PCI-34051, was efficacious in a rodent model of AKI, further supporting the potential for HDAC8 inhibitors and, in particular, this scaffold as a therapeutic approach to AKI.

  15. Experimental models of acute kidney injury for translational research

    Hukriede, Neil A.; Soranno, Danielle E.; Sander, Veronika; Perreau, Tayla; Starr, Michelle C.; Yuen, Peter S. T.; Siskind, Leah J.; Hutchens, Michael P.; Davidson, Alan J.; Burmeister, David M.; Faubel, Sarah; de Caestecker, Mark P.. Nature Reviews Nephrology . February 2022.

    Preclinical models of human disease provide powerful tools for therapeutic discovery but have limitations. This problem is especially apparent in the field of acute kidney injury (AKI), in which clinical trial failures have been attributed to inaccurate modelling performed largely in rodents. Multidisciplinary efforts such as the Kidney Precision Medicine Project are now starting to identify molecular subtypes of human AKI. In addition, over the past decade, there have been developments in human pluripotent stem cell-derived kidney organoids as well as zebrafish, rodent and large animal models of AKI. These organoid and AKI models are being deployed at different stages of preclinical therapeutic development. However, the traditionally siloed, preclinical investigator-driven approaches that have been used to evaluate AKI therapeutics to date rarely account for the limitations of the model systems used and have given rise to false expectations of clinical efficacy in patients with different AKI pathophysiologies. To address this problem, there is a need to develop more flexible and integrated approaches, involving teams of investigators with expertise in a range of different model systems, working closely with clinical investigators, to develop robust preclinical evidence to support more focused interventions in patients with AKI.

  16. Modeling oxidative injury response in human kidney organoids

    Przepiorski, Aneta; Vanichapol, Thitinee; Espiritu, Eugenel B.; Crunk, Amanda E.; Parasky, Emily; McDaniels, Michael D.; Emlet, Dave R.; Salisbury, Ryan; Happ, Cassandra L.; Vernetti, Lawrence A.; MacDonald, Matthew L.; Kellum, John A.; Kleyman, Thomas R.; Baty, Catherine J.; Davidson, Alan J.; Hukriede, Neil A.. Stem Cell Research & Therapy . 13(1):76. February 2022.

    Background Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. Methods To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. Results We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. Conclusion This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

  17. Regrow or Repair: An Update on Potential Regenerative Therapies for the Kidney

    Little, Melissa H.; Humphreys, Benjamin D. Journal of the American Society of Nephrology: JASN . 33(1):15–32. January 2022.

    Fifteen years ago, this journal published a review outlining future options for regenerating the kidney. At that time, stem cell populations were being identified in multiple tissues, the concept of stem cell recruitment to a site of injury was of great interest, and the possibility of postnatal renal stem cells was growing in momentum. Since that time, we have seen the advent of human induced pluripotent stem cells, substantial advances in our capacity to both sequence and edit the genome, global and spatial transcriptional analysis down to the single-cell level, and a pandemic that has challenged our delivery of health care to all. This article will look back over this period of time to see how our view of kidney development, disease, repair, and regeneration has changed and envision a future for kidney regeneration and repair over the next 15 years.

  18. Engineered collagen-targeting therapeutics reverse lung and kidney fibrosis in mice (Preprint)

    White, Michael JV; Raczy, Michal M; Budina, Erica; Yuba, Eiji; Solanki, Ani; Shim, Ha-Na; Zhang, Zheng Jenny; Gray, Laura T; Cao, Shijie; Alpar, Aaron T.; Hubbell, Jeffrey A. bioRxiv . 2022.

    Fibrotic diseases are involved in 45% of deaths in the United States. In particular, fibrosis of the kidney and lung are major public health concerns due to their high prevalence and lack of existing treatment options. Here, we harness the pathophysiological features of fibrotic diseases, namely leaky vasculature and aberrant extracellular matrix (ECM) protein deposition (i.e. collagen), to target an anti-fibrotic biologic and a small molecule drug to disease sites of fibrosis, thus improving their therapeutic potential in mouse models of lung and kidney fibrosis. First, we identify and validate collagen-targeting drug delivery systems that preferentially accumulate in the diseased organs: von Willebrand Factor’s A3 domain (VWF-A3) and decorin-derived collagen-binding peptide-conjugated micelles (CBP-micelles). We then engineer and recombinantly express novel candidate biologic therapies based on the anti-inflammatory cytokine IL-10: A3-IL-10 and A3-Serum Albumin-IL-10 (A3-SA-IL-10). Simultaneously, we stably encapsulate the potential anti-fibrotic water-insoluble drug, rapamycin, in CBP-micelles. We show that these novel formulations of therapeutics bind to collagen in vitro and that their efficacy in mouse models of lung and kidney fibrosis is improved, compared to free, untargeted drugs. Our results demonstrate that collagen-targeted anti-fibrotic drugs may be next generation therapies of high clinical potential.Competing Interest StatementThe authors have declared no competing interest.

  19. Chapter 32: Small molecules in regeneration

    Crunk, Amanda E.; Przepiorski, Aneta; Hukriede, Neil A.. Regenerative Nephrology. 2022.

    Acute kidney injury (AKI) is a major clinical and economic problem worldwide and currently there are no effective treatments. Due to the numerous renal insults that lead to AKI and the complexity of injury, many pathways are dysregulated leading to maladaptive repair, which can lead to chronic kidney disease. The focus of this chapter is to illustrate the pathways that play a role in AKI and how small molecules for therapeutic intervention can be utilized to modulate their activity and enhance productive repair. Given the large number of pathways that could be therapeutic targets, we will focus on only a limited number that have shown promising results in the treatment of AKI.

  20. Chapter 18: Stress-induced senescence of tubular cells

    Baird, David P.; Ferenbach, David A.; Bonventre, Joseph V.. Regenerative Nephrology. 2022.

    Senescent cells, characterized typically by irreversible cell cycle arrest coupled with marked transcriptional changes and a pro-inflammatory secretome, are proposed as key drivers of kidney fibrosis with the epithelial cells of the renal tubule playing a central role. In this chapter, we discuss the accumulating evidence that senescence is associated with aging and a number of renal diseases in humans and that this is associated with worsened kidney function and outcome. We also review the insights gained from studies using a variety of animal models and discuss how these have informed our knowledge of the processes underlying cellular senescence and fibrosis in the kidney.

2021

  1. Single cell transcriptomic analysis of external genitalia reveals complex and sexually dimorphic cell populations in the early genital tubercle

    Armfield, Brooke A.; Cohn, Martin J.. Developmental Biology . 477:145-154. 2021.

    External genital organs are among the most recognizable sexually dimorphic characters. The penis and clitoris develop from the embryonic genital tubercle, an outgrowth at the anterior margin of the cloaca that undergoes an extensive period of development in male and female embryos prior to the onset of sexual differentiation. In mice, differentiation into the penis and clitoris begins around embryonic day (E)15.5. Current knowledge of cell types that comprise the genital tubercle is limited to a few studies that have fate mapped derivatives of endoderm, mesoderm, and ectoderm. Here we use single cell transcriptomics to characterize the cell populations in the genital tubercles of male and female mouse embryos at E14.5, approximately 24 ​h before the onset of sexual differentiation, and we present the first comprehensive atlas of single-cell gene expression during external genital development. Clustering analyses and annotation using marker genes shows 19 distinct cell populations in E14.5 genital tubercles. Mapping of cell clusters to anatomical locations using in situ gene expression patterns revealed granularity of cellular specializations and positional identities. Although E14.5 precedes sexually dimorphic morphogenesis of the genital tubercle, comparative analysis of males and females identified sexual dimorphisms at the single cell level, including male-specific cell clusters with transcriptional signatures of smooth muscle and bone progenitors, both of which are known to be sexually dimorphic in adult genitalia, as well as immune cells. These results provide a new resource for classification of external genital cell types based on gene expression profiles and reveal sex-specific cellular specializations in the early genital tubercle.

  2. A uropathogenic E. coli UTI89 model of prostatic inflammation and collagen accumulation for use in studying aberrant collagen production in the prostate

    Ruetten, Hannah; Sandhu, Jaskiran; Mueller, Brett; Wang, Peiqing; Zhang, Helen L.; Wegner, Kyle A.; Cadena, Mark; Sandhu, Simran; L. Abler, Lisa; Zhu, Jonathan; O’Driscoll, Chelsea A.; Chelgren, Britta; Wang, Zunyi; Shen, Tian; Barasch, Jonathan; Bjorling, Dale E.; Vezina, Chad M.. American Journal of Physiology-Renal Physiology . 320(1):F31-F46. 2021.

    Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation using transurethral instillations of Escherichia coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 µL) distributed exclusively to the bladder, 100- and 200-µL volumes distributed to the bladder and prostate, and a 500-µL volume distributed to the bladder, prostate, and ureter. A threshold optical density of 0.4 E. coli UTI89 in the instillation fluid was necessary for significant (P < 0.05) prostate colonization. E. coli UTI89 infection resulted in a low frequency, high volume spontaneous voiding pattern. This phenotype was due to exposure to E. coli UTI89, not catheterization alone, and was minimally altered by a 50-µL increase in instillation volume and doubling of E. coli concentration. Prostate inflammation was isolated to the dorsal prostate and was accompanied by increased collagen density. This was partnered with increased density of protein tyrosine phosphatase receptor type C+, procollagen type I-α1+ copositive cells and decreased density of α2-smooth muscle actin+, procollagen type I-α1+ copositive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.

  3. Altered sacral neural crest development in Pax3 spina bifida mutants underlies deficits of bladder innervation and function

    Deal, KK; Chandrashekar, AS; Beaman, MM; Branch, MC; Buehler, DP; Conway, SJ; Southard-Smith, EM. Developmental Biology . 2021.

    Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.

  4. Sox10-cre BAC transgenes reveal temporal restriction of mesenchymal cranial neural crest and identify glandular Sox10 expression

    Deal, KK; Rosebrock, JC; Eeds, AM; DeKeyser, JML; Musser, MA; Ireland, SJ; May-Zhang, AA; Buehler, DP; Southard-Smith, EM. Developmental Biology . 471:119–137. 2021.

    Abstract Diversity of neural crest derivatives has been studied with a variety of approaches during embryonic development. In mammals Cre-LoxP lineage tracing is a robust means to fate map neural crest relying on cre driven from regulatory elements of early neural crest genes. Sox10 is an essential transcription factor for normal neural crest development. A variety of efforts have been made to label neural crest derivatives using partial Sox10 regulatory elements to drive cre expression. To date published Sox10-cre lines have focused primarily on lineage tracing in specific tissues or during early fetal development. We describe two new Sox10-cre BAC transgenes, constitutive (cre) and inducible (cre/ERT2), that contain the complete repertoire of Sox10 regulatory elements. We present a thorough expression profile of each, identifying a few novel sites of Sox10 expression not captured by other neural crest cre drivers. Comparative mapping of expression patterns between the Sox10-cre and Sox10-cre/ERT2 transgenes identified a narrow temporal window in which Sox10 expression is present in mesenchymal derivatives prior to becoming restricted to neural elements during embryogenesis. In more caudal structures, such as the intestine and lower urinary tract, our Sox10-cre BAC transgene appears to be more efficient in labeling neural crest-derived cell types than Wnt1-cre. The analysis reveals consistent expression of Sox10 in non-neural crest derived glandular epithelium, including salivary, mammary, and urethral glands of adult mice. These Sox10-cre and Sox10-cre/ERT2 transgenic lines are verified tools that will enable refined temporal and cell-type specific lineage analysis of neural crest derivatives as well as glandular tissues that rely on Sox10 for proper development and function.

  5. Spatiotemporal mapping of sensory and motor innervation of the embryonic and postnatal mouse urinary bladder.

    Smith-Anttila, Casey JA; Morrison, Victoria; Keast, Janet R. Developmental Biology . 2021.

    The primary function of the urinary bladder is to store urine (continence) until a suitable time for voiding (micturition). These distinct processes are determined by the coordinated activation of sensory and motor components of the nervous system, which matures to enable voluntary control at the time of weaning. Our aim was to define the development and maturation of the nerve-organ interface of the mouse urinary bladder by mapping the organ and tissue distribution of major classes of autonomic (motor) and sensory axons. Innervation of the bladder was evident from E13 and progressed dorsoventrally. Increasing defasciculation of axon bundles to single axons within the muscle occurred through the prenatal period, and in several classes of axons underwent further maturation until P7. Urothelial innervation occurred more slowly than muscle innervation and showed a clear regional difference, from E18 the bladder neck having the highest density of urothelial nerves. These features of innervation were similar in males and females but varied in timing and tissue density between different axon classes. We also analysed the pelvic ganglion, the major source of motor axons that innervate the lower urinary tract and other pelvic organs. Cholinergic, nitrergic (subset of cholinergic) and noradrenergic neuronal cell bodies were present prior to visualization of these axon classes within the bladder. Examination of cholinergic structures within the pelvic ganglion indicated that connections from spinal preganglionic neurons to pelvic ganglion neurons were already present by E12, a time at which these autonomic ganglion neurons had not yet innervated the bladder. These putative preganglionic inputs increased in density prior to birth as axon terminal fields continued to expand within the bladder tissues. Our studies also revealed in numerous pelvic ganglion neurons an unexpected transient expression of calcitonin gene-related peptide, a peptide commonly used to visualise the peptidergic class of visceral sensory axons. Together, our outcomes enhance our understanding of neural regulatory elements in the lower urinary tract during development and provide a foundation for studies of plasticity and regenerative capacity in the adult system.

  6. Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

    Helms, Louisa; Marchiano, Silvia; Stanaway, Ian B.; Hsiang, Tien-Ying; Juliar, Benjamin A.; Saini, Shally; Zhao, Yan Ting; Khanna, Akshita; Menon, Rajasree; Alakwaa, Fadhl; Mikacenic, Carmen; Morrell, Eric D.; Wurfel, Mark M.; Kretzler, Matthias; Harder, Jennifer L.; Murry, Charles E.; Himmelfarb, Jonathan; Ruohola-Baker, Hannele; Bhatraju, Pavan K.; Gale, Michael Jr; Freedman, Benjamin S. JCI insight . 6(24). December 2021.

    Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

  7. Spatially Resolved Transcriptomic Analysis of Acute Kidney Injury in a Female Murine Model

    Dixon, Eryn; Wu, Haojia; Muto, Yoshiharu; Wilson, Parker; Humphreys, Benjamin. Journal of the American Society of Nephrology . December 2021.

    Background Single cell sequencing technologies have advanced our understanding of kidney biology and disease but the loss of spatial information in these datasets hinders our interpretation of intercellular communication networks and regional gene expression patterns. New spatial transcriptomic sequencing platforms make it possible to measure the topography of gene expression at genome depth. Methods We optimized and validated a female bilateral ischemia reperfusion injury model. Using the 10X Genomics Visium Spatial Gene Expression solution, we generated spatial maps of gene expression across the injury and repair time course, and applied two open-source computational tools, Giotto and SPOTlight, to increase resolution and measure cell-cell interaction dynamics. Results An ischemia time of 34 minutes in a female murine model resulted in comparable injury to 22 minutes for males. We report a total of 16,856 unique genes mapped across injury and repair time course. Giotto, a computational toolbox for spatial data analysis, enabled increased resolution mapping of genes and cell types. Using a seeded non-negative matrix regression (SPOTlight) to deconvolute the dynamic landscape of cell-cell interactions, we find that injured proximal tubule cells are characterized by increasing macrophage and lymphocyte interactions even at 6 weeks after injury, potentially reflecting the AKI to CKD transition. Conclusions In this transcriptomic atlas, we defined region-specific and injury-induced loss of differentiation markers and their re-expression during repair, as well as region-specific injury and repair transcriptional responses. Lastly, we created a data visualization web application for the scientific community to explore these results (http://humphreyslab.com/SingleCell/; login: humphreyslab_visium password: irivisium).

  8. Multi-omics integration in the age of million single-cell data

    Miao, Zhen; Humphreys, Benjamin D.; McMahon, Andrew P.; Kim, Junhyong. Nature Reviews Nephrology . 17(11):710–724. November 2021.

    An explosion in single-cell technologies has revealed a previously underappreciated heterogeneity of cell types and novel cell-state associations with sex, disease, development and other processes. Starting with transcriptome analyses, single-cell techniques have extended to multi-omics approaches and now enable the simultaneous measurement of data modalities and spatial cellular context. Data are now available for millions of cells, for whole-genome measurements and for multiple modalities. Although analyses of such multimodal datasets have the potential to provide new insights into biological processes that cannot be inferred with a single mode of assay, the integration of very large, complex, multimodal data into biological models and mechanisms represents a considerable challenge. An understanding of the principles of data integration and visualization methods is required to determine what methods are best applied to a particular single-cell dataset. Each class of method has advantages and pitfalls in terms of its ability to achieve various biological goals, including cell-type classification, regulatory network modelling and biological process inference. In choosing a data integration strategy, consideration must be given to whether the multi-omics data are matched (that is, measured on the same cell) or unmatched (that is, measured on different cells) and, more importantly, the overall modelling and visualization goals of the integrated analysis.

  9. Vascular deficiencies in renal organoids and ex vivo kidney organogenesis.

    Ryan, Anne R.; England, Alicia R.; Chaney, Christopher P.; Cowdin, Mitzy A.; Hiltabidle, Max; Daniel, Edward; Gupta, Ashwani Kumar; Oxburgh, Leif; Carroll, Thomas J.; Cleaver, Ondine. Developmental biology . 477:98–116. September 2021.

    Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. Here, we comprehensively assess the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, we show that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress over time in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo.

  10. Podocyte Aging: Why and How Getting Old Matters

    Shankland, Stuart; Wang, Yuliang; Shaw, Andrey; Vaughan, Joshua; Pippin, Jeffrey; Wessely, Oliver. Journal of the American Society of Nephrology . September 2021.

    The effects of healthy aging on the kidney, and how these effects intersect with superimposed diseases, are highly relevant in the context of the population’s increasing longevity. Age-associated changes to podocytes, which are terminally differentiated glomerular epithelial cells, adversely affect kidney health. This review discusses the molecular and cellular mechanisms underlying podocyte aging, how these mechanisms might be augmented by disease in the aged kidney, and approaches to mitigate progressive damage to podocytes. Furthermore, we address how biologic pathways such as those associated with cellular growth confound aging in humans and rodents.

  11. Autonomous Calcium Signaling in Human and Zebrafish Podocytes Controls Kidney Filtration Barrier Morphogenesis

    Djenoune, Lydia; Tomar, Ritu; Dorison, Aude; Ghobrial, Irene; Schenk, Heiko; Hegermann, Jan; Beverly-Staggs, Lynne; Hidalgo-Gonzalez, Alejandro; Little, Melissa H.; Drummond, Iain A.. Journal of the American Society of Nephrology . 32(7):1697–1712. July 2021.

    Podocytes are critical to maintaining the kidney glomerular filtration barrier. Mutations in genes associated with development of nephrotic syndrome lead to elevated cytoplasmic calcium in podocytes and cause disruption of filtration barrier function. Whether calcium signaling plays a role in the initial formation of the filtration barrier is not known. Using live calcium imaging in two models, larval zebrafish and human kidney organoids, the authors demonstrate that podocyte calcium signaling is active during podocyte differentiation, is podocyte-cell autonomous, occurs independently of neighboring cell types, and is required for foot process and slit diaphragm formation. Their findings also show that developmental calcium signaling occurs by a different mechanism than disease-associated calcium perturbations, and represents a critical regulatory signal for podocyte morphogenesis and filtration barrier formation.Background Podocytes are critical to maintaining the glomerular filtration barrier, and mutations in nephrotic syndrome genes are known to affect podocyte calcium signaling. However, the role of calcium signaling during podocyte development remains unknown.Methods We undertook live imaging of calcium signaling in developing podocytes, using zebrafish larvae and human kidney organoids. To evaluate calcium signaling during development and in response to channel blockers and genetic defects, the calcium biosensor GCaMP6s was expressed in zebrafish podocytes. We used electron microscopy to evaluate filtration barrier formation in zebrafish, and Fluo-4 to detect calcium signals in differentiating podocytes in human kidney organoids.Results Immature zebrafish podocytes (2.5 days postfertilization) generated calcium transients that correlated with interactions with forming glomerular capillaries. Calcium transients persisted until 4 days postfertilization, and were absent after glomerular barrier formation was complete. We detected similar calcium transients in maturing human organoid glomeruli, suggesting a conserved mechanism. In both models, inhibitors of SERCA or IP3 receptor calcium-release channels blocked calcium transients in podocytes, whereas lanthanum was ineffective, indicating the calcium source is from intracellular podocyte endoplasmic-reticulum stores. Calcium transients were not affected by blocking heartbeat or by blocking development of endothelium or endoderm, and they persisted in isolated glomeruli, suggesting podocyte-autonomous calcium release. Inhibition of expression of phospholipase C-γ1, but not nephrin or phospholipase C-ε1, led to significantly decreased calcium activity. Finally, blocking calcium release affected glomerular shape and podocyte foot process formation, supporting the critical role of calcium signaling in glomerular morphogenesis.Conclusions These findings establish podocyte cell–autonomous calcium signaling as a prominent and evolutionarily conserved feature of podocyte differentiation and demonstrate its requirement for podocyte foot process formation.

  12. Nephrotoxicity Assessment with Human Kidney Tubuloids using Spherical Nucleic Acid-Based mRNA Nanoflares.

    Wiraja, Christian; Mori, Yutaro; Ichimura, Takaharu; Hwang, Jangsun; Xu, Chenjie; Bonventre, Joseph V.. Nano letters . 21(13):5850–5858. July 2021.

    Drug-induced nephrotoxicity represents an important cause of acute kidney injury with associated patient morbidity and mortality and is often responsible for termination of drug development, after extensive resource allocation. We have developed a human kidney tubuloid system that phenocopies, in 3D culture, kidney proximal tubules, a primary injury site of most nephrotoxicants. Traditional end point assays are often performed on 2D cultures of cells that have lost their differentiated phenotype. Herein, we pair a tubuloid system with Nanoflare (NF) mRNA nanosensors to achieve a facile, real-time assessment of drug nephrotoxicity. Using kidney injury molecule-1 (KIM-1) mRNA as a model injury biomarker, we verify NF specificity in engineered and adenovirus-transfected cells and confirm their efficacy to report tubular cell injury by aristolochic acid and cisplatin. The system also facilitates nephrotoxicity screening as demonstrated with 10 representative anticancer moieties. 5-Fluorouracil and paclitaxel induce acute tubular injury, as reflected by an NF signal increase.

  13. Single-nuclear transcriptomics reveals diversity of proximal tubule cell states in a dynamic response to acute kidney injury

    Gerhardt, Louisa M. S.; Liu, Jing; Koppitch, Kari; Cippà, Pietro E.; McMahon, Andrew P.. Proceedings of the National Academy of Sciences . 118(27). July 2021.

    A single acute kidney injury event increases the risk of progression to chronic kidney disease (CKD). Combining single-nucleus RNA sequencing with genetic tracing of injured proximal tubule cells identified a spatially dynamic, evolving injury response following ischemia–reperfusion injury. Failed proximal tubule repair leads to the persistence of a profibrotic, proinflammatory Vcam1+/Ccl2+ cell type exhibiting a senescence-associated secretory phenotype and a marked transcriptional activation of NF-κB and AP-1 pathway signatures, but no signs of G2/M cell cycle arrest. Insights from this study can inform strategies to improve renal repair and prevent CKD progression.Acute kidney injury (AKI), commonly caused by ischemia, sepsis, or nephrotoxic insult, is associated with increased mortality and a heightened risk of chronic kidney disease (CKD). AKI results in the dysfunction or death of proximal tubule cells (PTCs), triggering a poorly understood autologous cellular repair program. Defective repair associates with a long-term transition to CKD. We performed a mild-to-moderate ischemia–reperfusion injury (IRI) to model injury responses reflective of kidney injury in a variety of clinical settings, including kidney transplant surgery. Single-nucleus RNA sequencing of genetically labeled injured PTCs at 7-d (“early”) and 28-d (“late”) time points post-IRI identified specific gene and pathway activity in the injury–repair transition. In particular, we identified Vcam1+/Ccl2+ PTCs at a late injury stage distinguished by marked activation of NF-κB–, TNF-, and AP-1–signaling pathways. This population of PTCs showed features of a senescence-associated secretory phenotype but did not exhibit G2/M cell cycle arrest, distinct from other reports of maladaptive PTCs following kidney injury. Fate-mapping experiments identified spatially and temporally distinct origins for these cells. At the cortico-medullary boundary (CMB), where injury initiates, the majority of Vcam1+/Ccl2+ PTCs arose from early replicating PTCs. In contrast, in cortical regions, only a subset of Vcam1+/Ccl2+ PTCs could be traced to early repairing cells, suggesting late-arising sites of secondary PTC injury. Together, these data indicate even moderate IRI is associated with a lasting injury, which spreads from the CMB to cortical regions. Remaining failed-repair PTCs are likely triggers for chronic disease progression.The single-nuclei RNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (accession no. GSE171417). All study data are included in the article and/or supporting information. Previously published data were used for this work (https://doi.org/10.1172/jci.insight.123151; https://doi.org/10.1038/s42255-020-0238-1).

  14. Single Cell Technologies: Beyond Microfluidics

    Li, Haikuo; Humphreys, Benjamin D. Kidney360 . July 2021.

    Single cell RNA-sequencing (scRNA-seq) has been widely adopted in recent years due to standardized protocols and automation, reliability and standardized bioinformatic pipelines. The most widely adopted platform is the 10X Genomics solution. While powerful, this system is also limited by its high cost, moderate throughput and the inability to customize due to fixed kit components. This review will cover new approaches that do not rely on microfluidics and thus have low entry costs, are highly customizable and are within the reach of any lab possessing molecular biology expertise.

  15. An efficient method to generate kidney organoids at the air-liquid interface.

    Gupta, Ashwani Kumar; Ivancic, David Z.; Naved, Bilal A.; Wertheim, Jason A.; Oxburgh, Leif. Journal of biological methods . 8(2):e150. June 2021.

    The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

  16. Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

    Li, Li; Galichon, Pierre; Xiao, Xiaoyan; Figueroa-Ramirez, Ana C.; Tamayo, Diana; Lee, Jake J.-K.; Kalocsay, Marian; Gonzalez-Sanchez, David; Chancay, Maria S.; McCracken, Kyle W.; Lee, Nathan N.; Ichimura, Takaharu; Mori, Yutaro; Valerius, M. Todd; Wilflingseder, Julia; Lemos, Dario R.; Edelman, Elazer R.; Bonventre, Joseph V.. EMBO reports . 22(6):e51169. June 2021.

    Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

  17. Bioengineered Kidney Models: Methods and Functional Assessments

    Rizki-Safitri, Astia; Traitteur, Tamara; Morizane, Ryuji. Function . 2(4). May 2021.

    Investigations into bioengineering kidneys have been extensively conducted owing to their potential for preclinical assays and regenerative medicine. Various approaches and methods have been developed to improve the structure and function of bioengineered kidneys. Assessments of functional properties confirm the adequacy of bioengineered kidneys for multipurpose translational applications. This review is to summarize the studies performed in kidney bioengineering in the past decade. We identified 84 original articles from PubMed and Mendeley with keywords of kidney organoid or kidney tissue engineering. Those were categorized into 5 groups based on their approach: de-/recellularization of kidney, reaggregation of kidney cells, kidney organoids, kidney in scaffolds, and kidney-on-a-chip. These models were physiologically assessed by filtration, tubular reabsorption/secretion, hormone production, and nephrotoxicity. We found that bioengineered kidney models have been developed from simple cell cultures to multicellular systems to recapitulate kidney function and diseases. Meanwhile, only about 50\% of these studies conducted functional assessments on their kidney models. Factors including cell composition and organization are likely to alter the applicability of physiological assessments in bioengineered kidneys. Combined with recent technologies, physiological assessments importantly contribute to the improvement of the bioengineered kidney model toward repairing and refunctioning the damaged kidney.

  18. KIM-1 mediates fatty acid uptake by renal tubular cells to promote progressive diabetic kidney disease.

    Mori, Yutaro; Ajay, Amrendra K.; Chang, Jae-Hyung; Mou, Shan; Zhao, Huiping; Kishi, Seiji; Li, Jiahua; Brooks, Craig R.; Xiao, Sheng; Woo, Heung-Myong; Sabbisetti, Venkata S.; Palmer, Suetonia C.; Galichon, Pierre; Li, Li; Henderson, Joel M.; Kuchroo, Vijay K.; Hawkins, Julie; Ichimura, Takaharu; Bonventre, Joseph V.. Cell metabolism . 33(5):1042–1061.e7. May 2021.

    Tubulointerstitial abnormalities are predictive of the progression of diabetic kidney disease (DKD), and their targeting may be an effective means for prevention. Proximal tubular (PT) expression of kidney injury molecule (KIM)-1, as well as blood and urinary levels, are increased early in human diabetes and can predict the rate of disease progression. Here, we report that KIM-1 mediates PT uptake of palmitic acid (PA)-bound albumin, leading to enhanced tubule injury with DNA damage, PT cell-cycle arrest, interstitial inflammation and fibrosis, and secondary glomerulosclerosis. Such injury can be ameliorated by genetic ablation of the KIM-1 mucin domain in a high-fat-fed streptozotocin mouse model of DKD. We also identified TW-37 as a small molecule inhibitor of KIM-1-mediated PA-albumin uptake and showed in vivo in a kidney injury model in mice that it ameliorates renal inflammation and fibrosis. Together, our findings support KIM-1 as a new therapeutic target for DKD.

  19. 3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration

    Wang, Hongbing; Brown, Paul C.; Chow, Edwin C.Y.; Ewart, Lorna; Ferguson, Stephen S.; Fitzpatrick, Suzanne; Freedman, Benjamin S.; Guo, Grace L.; Hedrich, William; Heyward, Scott; Hickman, James; Isoherranen, Nina; Li, Albert P.; Liu, Qi; Mumenthaler, Shannon M.; Polli, James; Proctor, William R.; Ribeiro, Alexandre; Wang, Jian-Ying; Wange, Ronald L.; Huang, Shiew-Mei. Clinical and Translational Science . 14(5):1659-1680. May 2021.

    Abstract Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

  20. 3D kidney organoids for bench-to-bedside translation

    Gupta✉, Navin; Dilmen, Emre; Morizane, Ryuji. Journal of Molecular Medicine . 99(4):477–487. April 2021.

    The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.

  21. A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

    AU - Przepiorski, Aneta; AU - Crunk, Amanda E.; AU - Holm, Teresa M.; AU - Sander, Veronika; AU - Davidson, Alan J.; AU - Hukriede, Neil A.. JoVE . April 2021.

    Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

  22. Myofibroblast differentiation is governed by adhesion mechanics, and inhibition of the stress sensor Talin2 reverses lung fibrosis (Preprint)

    White, Michael JV; Ozkan, Melis; Gomez Medellin, Jorge Emiliano; Zent, Roy; Critchley, David; Hubbell, Jeffrey A. bioRxiv . 2021.

    Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse progression of the disease. In order to find novel targets for fibrosis therapeutics, we developed a model for the differentiation of monocytes to myofibroblasts that allowed us to screen for proteins involved in myofibroblast differentiation. We assessed these screening results for proteins to target for novel fibrosis therapeutics. Here we test whether inhibition of a novel protein target generated by our model, talin2, can prevent and even reverse myofibroblast differentiation. We find that knockdown of talin2 de-differentiates myofibroblasts, altering myofibroblast morphology, α-smooth muscle actin and collagen content, and the secretome. Talin2 inhibition reverses bleomycin-induced lung fibrosis in mice. Talin2 inhibition could be a novel treatment for reversing lung fibrosis.One Sentence Summary Silencing the spring protein Talin2 reverses myofibroblast differentiation and reverses existing fibrosis.Competing Interest StatementMJVW and JH are authors on a patent regarding this work(α-SMA)alpha-smooth muscle actin(ALT)Alanine Transferase(AST)Aspartate Transferase(BAL)broncheo-alveolar lavaged(BUN)blood urea nitrogen(FCS)fetal calf serum(FAs)focal adhesions(FBs)Fibrillar adhesions(IHC)Immunohistochemistry(MRC-5)Human fibroblasts(kilopascals)kPa(MCP1)macrophage-chemotactic protein-1(NIH-3T3)mouse fibroblasts(PBMC)peripheral blood mononuclear cells(PBS)phosphate buffered saline(SFM)serum-free media(siRNA)Silencing RNA(TGFβ)Transforming growth factor β(Tln2)Talin2(TNFα)Tumor necrosis factor α(UUO)Unilateral Ureteral Obstruction

  23. Blocking antibodies against integrin-α3, integrin-αM, and integrin-αMβ2 de-differentiate myofibroblasts and reverse lung and kidney fibroses in a mouse model (Preprint)

    White, Michael JV; Ozkan, Melis; Rączy, Michal M; Gomez-Medellin, Jorge Emiliano; Koss, Kyle M; Alpar, Aaron T.; Naved, Bilal; Wertheim, Jason; Hubbell, Jeffrey A. bioRxiv . 2021.

    Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of the disease. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins α3, αM, and αMβ2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and reverse fibrosis in vivo. Blocking key integrins may be an effective therapeutic for the treatment and reversal of fibrosis. Teaser: Blocking integrin-α3, integrin-αM, and integrin-αMβ2 reverses myofibroblast differentiation and reverses existing fibrosis. Competing Interest Statement: MJVW and JH are authors on a patent regarding this submission Abbreviations: (α-SMA) alpha-smooth muscle actin (CBP-α-αMβ2) CBP-functionalized neutralizing antibodies against αMβ2 (CBP-α-αM) CBP-functionalized neutralizing antibodies against αM (CBP-α-α3) CBP-functionalized neutralizing antibodies against α3 (CBP)(LRELHLNNNC) collagen-binding peptide (FAs) focal adhesions (FCS) fetal calf serum (FBs) Fibrillar adhesions (CY7-α-α3) Fluorescently labeled Cy7-neutralizing antibodies against α3 (CY7-α-αMβ2) Fluorescently labeled Cy7-neutralizing antibodies against αMβ2 (CY7-α-αM) Fluorescently labeled Cy7-neutralizing antibodies against αM (antibody which stabilizes the association of integrin α2β1) Gi14 (MRC-5) Human fibroblasts (called MAC1) heterodimer αMβ2 (IL12p40) IL-12 subunit p40 (α3) Integrin-α3 (αM) Integrin-αM (β2)(CD18) Integrin-β2 (MCP1) macrophage-chemotactic protein-1 (NIH-3T3) mouse fibroblasts (α-αMβ2) neutralizing antibodies against αMβ2 (α-αM) neutralizing antibodies against αM (α-α3) neutralizing antibodies against α3 (PBMC) peripheral blood mononuclear cells (PBS) phosphate buffered saline (SFM) serum-free media (TGFβ) Transforming growth factor β (TNFα) Tumor necrosis factor α (UUO) Unilateral ureteral obstruction

2020

  1. Urethral luminal epithelia are castration-insensitive cells of the proximal prostate.

    Joseph, DB; Henry, GH; Malewska, A; Iqbal, NS; Ruetten, HM; Turco, AE; Abler, LL; Sandhu, SK; Cadena, MT; Malladi, VS; Reese, JC; Mauck, RJ; Gahan, JC; Hutchinson, RC; Roehrborn, CG; Baker, LA; Vezina, CM; Strand, DW. The Prostate . 80(11):872–884. August 2020.

    BACKGROUND: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.

  2. Identification of a Sacral, Visceral Sensory Transcriptome in Embryonic and Adult Mice

    Smith-Anttila, CJA; Mason, EA; Wells, CA; Aronow, BJ; Osborne, PB; Keast, JR. eNeuro . 7(1). 2020.

    Visceral sensory neurons encode distinct sensations from healthy organs and initiate pain states that are resistant to common analgesics. Transcriptome analysis is transforming our understanding of sensory neuron subtypes but has generally focused on somatic sensory neurons or the total population of neurons in which visceral neurons form the minority. Our aim was to define transcripts specifically expressed by sacral visceral sensory neurons, as a step towards understanding the unique biology of these neurons and potentially leading to identification of new analgesic targets for pelvic visceral pain. Our strategy was to identify genes differentially expressed between sacral dorsal root ganglia (DRG) that include somatic neurons and sacral visceral neurons, and adjacent lumbar DRG that comprise exclusively of somatic sensory neurons. This was performed in adult and E18.5 male and female mice. By developing a method to restrict analyses to nociceptive Trpv1 neurons, a larger group of genes were detected as differentially expressed between spinal levels. We identified many novel genes that had not previously been associated with pelvic visceral sensation or nociception. Limited sex differences were detected across the transcriptome of sensory ganglia, but more were revealed in sacral levels and especially in Trpv1 nociceptive neurons. These data will facilitate development of new tools to modify mature and developing sensory neurons and nociceptive pathways.

  3. Protocol for Large-Scale Production of Kidney Organoids from Human Pluripotent Stem Cells.

    Sander, Veronika; Przepiorski, Aneta; Crunk, Amanda E.; Hukriede, Neil A.; Holm, Teresa M.; Davidson, Alan J.. STAR protocols . 1(3):100150. December 2020.

    Kidney organoids represent a physiologically advanced model for studying the mechanisms of kidney development and disease. Here, we describe a simple two-step protocol for the differentiation of human pluripotent stem cells into kidney organoids. Our approach involves suspension culture that allows for rapid and cost-effective bulk production of organoids, which is well suited for large-scale assays such as drug screening. The organoids correspond to fetal human kidney tissue and may be of limited use for modeling adult kidney function. For complete details on the use and execution of this protocol, please refer to Przepiorski et al. (2018).

  4. Returning to kidney development to deliver synthetic kidneys

    Little, Melissa H. Developmental Biology . 2020.

    There is no doubt that the development of transplantable synthetic kidneys could improve the outcome for the many millions of people worldwide suffering from chronic kidney disease. Substantial progress has been made in the last 6 years in the generation of kidney tissue from stem cells. However, the limited scale, incomplete cellular complexity and functional immaturity of such structures suggests we are some way from this goal. While developmental biology has successfully guided advances to date, these human kidney models are limited in their capacity for ongoing nephrogenesis and lack corticomedullary definition, a unified vasculature and a coordinated exit path for urinary filtrate. This review will reassess our developmental understanding of how the mammalian embryo manages to create kidneys, how this has informed our progress to date and how both engineering and developmental biology can continue to guide us towards a synthetic kidney.

  5. Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk

    Howden, Sara E.; Wilson, Sean B.; Groenewegen, Ella; Starks, Lakshi; Forbes, Thomas A.; Tan, Ker Sin; Vanslambrouck, Jessica M.; Holloway, Emily M.; Chen, Yi-Hsien; Jain, Sanjay; Spence, Jason R.; Little, Melissa H. Cell Stem Cell . 2020.

    Summary During development, distinct progenitors contribute to the nephrons versus the ureteric epithelium of the kidney. Indeed, previous human pluripotent stem-cell-derived models of kidney tissue either contain nephrons or pattern specifically to the ureteric epithelium. By re-analyzing the transcriptional distinction between distal nephron and ureteric epithelium in human fetal kidney, we show here that, while existing nephron-containing kidney organoids contain distal nephron epithelium and no ureteric epithelium, this distal nephron segment alone displays significant in vitro plasticity and can adopt a ureteric epithelial tip identity when isolated and cultured in defined conditions. “Induced” ureteric epithelium cultures can be cryopreserved, serially passaged without loss of identity, and transitioned toward a collecting duct fate. Cultures harboring loss-of-function mutations in PKHD1 also recapitulate the cystic phenotype associated with autosomal recessive polycystic kidney disease.

  6. Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation

    Lawlor, Kynan T.; Vanslambrouck, Jessica M.; Higgins, J. William; Chambon, Alison; Bishard, Kristina; Arndt, Derek; Er, Pei Xuan; Wilson, Sean B.; Howden, Sara E.; Tan, Ker Sin; Li, Fanyi; Hale, Lorna J.; Shepherd, Benjamin; Pentoney, Stephen; Presnell, Sharon C.; Chen, Alice E.; Little, Melissa H. Nature Materials . November 2020.

    Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here, we apply extrusion-based three-dimensional cellular bioprinting to deliver rapid and high-throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate the relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, three-dimensional bioprinting enables precise manipulation of biophysical properties, including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitates the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production delivers improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

  7. Identification and characterization of cellular heterogeneity within the developing renal interstitium

    AR, England; CP, Chaney; A, Das; M, Patel; A, Malewska; D, Armendariz; GC, Hon; DW, Strand; KA, Drake; TJ, Carroll. Development . 147(15). August 2020.

    Kidney formation requires the coordinated growth of multiple cell types including the collecting ducts, nephrons, vasculature and interstitium. There is a long-held belief that interactions between progenitors of the collecting ducts and nephrons are primarily responsible for kidney development. However, over the last several years, it has become increasingly clear that multiple aspects of kidney development require signaling from the interstitium. How the interstitium orchestrates these various roles is poorly understood. Here, we show that during development the interstitium is a highly heterogeneous patterned population of cells that occupies distinct positions correlated to the adjacent parenchyma. Our analysis indicates that the heterogeneity is not a mere reflection of different stages in a linear developmental trajectory but instead represents several novel differentiated cell states. Further, we find that β-catenin has a cell autonomous role in the development of a medullary subset of the interstitium and that this non-autonomously affects the development of the adjacent epithelia. These findings suggest the intriguing possibility that the different interstitial subtypes may create microenvironments that play unique roles in development of the adjacent epithelia and endothelia.

  8. Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury.

    Ettou, Sandrine; Jung, Youngsook L.; Miyoshi, Tomoya; Jain, Dhawal; Hiratsuka, Ken; Schumacher, Valerie; Taglienti, Mary E.; Morizane, Ryuji; Park, Peter J.; Kreidberg, Jordan A.. Science advances . 6(30):eabb5460. July 2020.

    In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in reparative responses to injury are not well understood. We have studied the mechanisms of transcriptional reprogramming in disease using murine kidney podocytes as a model for tissue injury. Podocytes are a crucial component of glomeruli, the filtration units of each nephron. Podocyte injury is the initial event in many processes that lead to end-stage kidney disease. Wilms tumor-1 (WT1) is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Using murine models and human kidney organoids, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Reprogramming the transcriptome involved highly dynamic changes in the binding of WT1 to target genes during a reparative injury response, affecting chromatin state and expression levels of target genes.

  9. Effect of luminal flow on doming of mpkCCD cells in a 3D perfusable kidney cortical collecting duct model

    JL, Rein; S, Heja; D, Flores; R, Carrisoza-Gaytán; NYC, Lin; KA, Homan; JA, Lewis; LM, Satlin. American Journal of Physiology-Cell Physiology . 319(1):C136-C147. 2020.

    The cortical collecting duct (CCD) of the mammalian kidney plays a major role in the maintenance of total body electrolyte, acid/base, and fluid homeostasis by tubular reabsorption and excretion. The mammalian CCD is heterogeneous, composed of Na+-absorbing principal cells (PCs) and acid-base-transporting intercalated cells (ICs). Perturbations in luminal flow rate alter hydrodynamic forces to which these cells in the cylindrical tubules are exposed. However, most studies of tubular ion transport have been performed in cell monolayers grown on or epithelial sheets affixed to a flat support, since analysis of transepithelial transport in native tubules by in vitro microperfusion requires considerable expertise. Here, we report on the generation and characterization of an in vitro, perfusable three-dimensional kidney CCD model (3D CCD), in which immortalized mouse PC-like mpkCCD cells are seeded within a cylindrical channel embedded within an engineered extracellular matrix and subjected to luminal fluid flow. We find that a tight epithelial barrier composed of differentiated and polarized PCs forms within 1 wk. Immunofluorescence microscopy reveals the apical epithelial Na+ channel ENaC and basolateral Na+/K+-ATPase. On cessation of luminal flow, benzamil-inhibitable cell doming is observed within these 3D CCDs consistent with the presence of ENaC-mediated Na+ absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.

  10. Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids.

    A, Kumar Gupta; P, Sarkar; JA, Wertheim; X, Pan; TJ, Carroll; L., Oxburgh. Communications biology . 3(1):231. May 2020.

    A fundamental challenge in emulating kidney tissue formation through directed differentiation of human pluripotent stem cells is that kidney development is iterative, and to reproduce the asynchronous mix of differentiation states found in the fetal kidney we combined cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and heterochronic organoids were well vascularized when engrafted under the kidney capsule. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed, but despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study. We propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop similar stromal overgrowth when engrafted under the kidney capsule.

2019

  1. Insight and Resources from a Study of the “Impact of Sex, Androgens, and Prostate Size on C57BL/6J Mouse Urinary Physiology”

    Ruetten, H; Wegner, KA; Zhang, HL; Wang, P; Sandhu, J; Sandhu, S; Morkrid, J; Mueller, B; Wang, Z; Macoska, J; Peterson, RE; Bjorling, DE; Ricke, WA; Marker, PC; Vezina, CM. Toxicol Pathol . 47:1038–1042. December 2019.

    The purpose of this symposium report is to summarize information from a session 3 oral presentation at the Society of Toxicologic Pathology Annual Symposium in Raleigh, North Carolina. Mice are genetically tractable and are likely to play an important role in elucidating environmental, genetic, and aging-related mechanisms of urinary dysfunction in men. We and others have made significant strides in developing quantitative methods for assessing mouse urinary function and our collaborators recently showed that aging male mice, like men, develop urinary dysfunction. Yet, it remains unclear how mouse prostate anatomy and histology relate to urinary function. The purpose of this report is to share foundational resources for evaluating mouse prostate histology and urinary physiology from our recent publication "Impact of Sex, Androgens, and Prostate Size on C57BL/6J Mouse Urinary Physiology\:Functional Assessment." We will begin with a review of prostatic embryology in men and mice, then move to comparative histology resources, and conclude with quantitative measures of rodent urinary physiology.

  2. Pparg promotes differentiation and regulates mitochondrial gene expression in bladder epithelial cells

    Liu, Chang; Tate, Tiffany; Batourina, Ekatherina; Truschel, Steven T.; Potter, Steven; Adam, Mike; Xiang, Tina; Picard, Martin; Reiley, Maia; Schneider, Kerry; Tamargo, Manuel; Lu, Chao; Chen, Xiao; He, Jing; Kim, Hyunwoo; Mendelsohn, Cathy Lee. Nature Communications . 10:4589. October 2019.

    The urothelium is an epithelial barrier lining the bladder that protects against infection, fluid exchange and damage from toxins. The nuclear receptor Pparg promotes urothelial differentiation in vitro, and Pparg mutations are associated with bladder cancer. However, the function of Pparg in the healthy urothelium is unknown. Here we show that Pparg is critical in urothelial cells for mitochondrial biogenesis, cellular differentiation and regulation of inflammation in response to urinary tract infection (UTI). Superficial cells, which are critical for maintaining the urothelial barrier, fail to mature in Pparg mutants and basal cells undergo squamous-like differentiation. Pparg mutants display persistent inflammation after UTI, and Nf-KB, which is transiently activated in response to infection in the wild type urothelium, persists for months. Our observations suggest that in addition to its known roles in adipogegnesis and macrophage differentiation, that Pparg-dependent transcription plays a role in the urothelium controlling mitochondrial function development and regeneration.

  3. A temporal and spatial map of axons in developing mouse prostate.

    Turco, Anne E.; Cadena, Mark T.; Zhang, Helen L.; Sandhu, Jaskiran K.; Oakes, Steven R.; Chathurvedula, Thrishna; Peterson, Richard E.; Keast, Janet R.; Vezina, Chad M.. Histochem Cell Biol . 152(1):35–45. July 2019.

    Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.

  4. Edar is a downstream target of beta-catenin and drives collagen accumulation in the mouse prostate

    Wegner, Kyle A.; Mehta, Vatsal; Johansson, Jeanette A.; Mueller, Brett R.; Keil, Kimberly P.; Abler, Lisa L.; Marker, Paul C.; Taketo, M. Mark; Headon, Denis J.; Vezina, Chad M.. Biology Open . 8(3):bio037945. March 2019.

    Beta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.

  5. Differentiation of human kidney organoids from pluripotent stem cells

    Cruz, Nelly M.; Freedman, Benjamin S.. Methods in Cell Biology . 153:133–150. 2019.

    It is now possible to direct the differentiation of human pluripotent stem cells into three-dimensional nephron-like structures called kidney organoids. Organoids contain proximal and distal tubules as well as podocytes, in addition to a variety of other lineages such as endothelial cells. Organoid technology has great potential for kidney regeneration and has already been proven to be suitable for modeling kidney disease. However, the methodologies that are used for the generation of kidney organoids require expertise and can be daunting for the inexperienced. Here, we describe in detail a well-established and relatively simple method for the generation of human kidney organoids. We include notes on technical and design considerations for these experiments, and highlight key advantages and limitations of the system.

  6. Kidney organoids: accurate models or fortunate accidents.

    Little, Melissa H.; Combes, Alexander N.. Genes & development . 33(19-20):1319–1345. October 2019.

    There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.

  7. A Toolbox to Characterize Human Induced Pluripotent Stem Cell–Derived Kidney Cell Types and Organoids

    Vanslambrouck, Jessica M.; Wilson, Sean B.; Tan, Ker Sin; Soo, Joanne Y.-C.; Scurr, Michelle; Spijker, H. Siebe; Starks, Lakshi T.; Neilson, Amber; Cui, Xiaoxia; Jain, Sanjay; Little, Melissa Helen; Howden, Sara E.. Journal of the American Society of Nephrology . 30(10):1811–1823. 2019.

    Kidney organoids generated from human induced pluripotent stem cells (iPSCs) show great potential for modeling kidney diseases and studying disease pathogenesis. However, the relative accuracy with which kidney organoids model normal morphogenesis, as well as the maturity and identity of the renal cell types they comprise, remain to be fully investigated. The authors describe the generation and validation of ten fluorescent CRISPR/Cas9 gene-edited iPSC reporter lines specifically designed for the visualization, isolation, and characterization of cell types and states within kidney organoids, and demonstrate the use of these lines for cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing applications. These tools offer promise for better understanding this model system and its congruence with human kidney morphogenesis.Background The generation of reporter lines for cell identity, lineage, and physiologic state has provided a powerful tool in advancing the dissection of mouse kidney morphogenesis at a molecular level. Although use of this approach is not an option for studying human development in vivo, its application in human induced pluripotent stem cells (iPSCs) is now feasible.Methods We used CRISPR/Cas9 gene editing to generate ten fluorescence reporter iPSC lines designed to identify nephron progenitors, podocytes, proximal and distal nephron, and ureteric epithelium. Directed differentiation to kidney organoids was performed according to published protocols. Using immunofluorescence and live confocal microscopy, flow cytometry, and cell sorting techniques, we investigated organoid patterning and reporter expression characteristics.Results Each iPSC reporter line formed well patterned kidney organoids. All reporter lines showed congruence of endogenous gene and protein expression, enabling isolation and characterization of kidney cell types of interest. We also demonstrated successful application of reporter lines for time-lapse imaging and mouse transplantation experiments.Conclusions We generated, validated, and applied a suite of fluorescence iPSC reporter lines for the study of morphogenesis within human kidney organoids. This fluorescent iPSC reporter toolbox enables the visualization and isolation of key populations in forming kidney organoids, facilitating a range of applications, including cellular isolation, time-lapse imaging, protocol optimization, and lineage-tracing approaches. These tools offer promise for enhancing our understanding of this model system and its correspondence with human kidney morphogenesis.

  8. Proximal Tubule Translational Profiling during Kidney Fibrosis Reveals Proinflammatory and Long Noncoding RNA Expression Patterns with Sexual Dimorphism

    Wu, Haojia; Lai, Chun-Fu; Chang-Panesso, Monica; Humphreys, Benjamin D. Journal of the American Society of Nephrology . September 2019.

    Having a comprehensive transcriptional profile of the proximal tubule in health and fibrosis would likely enhance understanding of fibrosis and perhaps help explain why CKD progresses more quickly in males versus females. To obtain a more complete picture of gene expression in the proximal tubule, the authors performed deep translational profiling of this segment in a mouse model of kidney fibrosis. Their findings demonstrate substantial sex differences in transcripts expressed in proximal tubule cells of males versus females, and indicate that the proximal tubule drives fibrosis through inflammatory and profibrotic paracrine signaling. The study also identified 439 long noncoding RNAs expressed in the proximal tubule, 143 of which undergo differential regulation in fibrosis, suggesting that this type of RNA has unanticipated regulatory roles kidney fibrosis.Background Proximal tubule injury can initiate CKD, with progression rates that are approximately 50% faster in males versus females. The precise transcriptional changes in this nephron segment during fibrosis and potential differences between sexes remain undefined.Methods We generated mice with proximal tubule–specific expression of an L10a ribosomal subunit protein fused with enhanced green fluorescent protein. We performed unilateral ureteral obstruction surgery on four male and three female mice to induce inflammation and fibrosis, collected proximal tubule–specific and bulk cortex mRNA at day 5 or 10, and sequenced samples to a depth of 30 million reads. We applied computational methods to identify sex-biased and shared molecular responses to fibrotic injury, including up- and downregulated long noncoding RNAs (lncRNAs) and transcriptional regulators, and used in situ hybridization to validate critical genes and pathways.Results We identified >17,000 genes in each proximal tubule group, including 145 G-protein–coupled receptors. More than 700 transcripts were differentially expressed in the proximal tubule of males versus females. The >4000 genes displaying altered expression during fibrosis were enriched for proinflammatory and profibrotic pathways. Our identification of nearly 150 differentially expressed proximal tubule lncRNAs during fibrosis suggests they may have unanticipated regulatory roles. Network analysis prioritized proinflammatory and profibrotic transcription factors such as Irf1, Nfkb1, and Stat3 as drivers of fibrosis progression.Conclusions This comprehensive transcriptomic map of the proximal tubule revealed sexually dimorphic gene expression that may reflect sex-related disparities in CKD, proinflammatory gene modules, and previously unappreciated proximal tubule–specific bidirectional lncRNA regulation.

  9. In Vivo Developmental Trajectories of Human Podocyte Inform In Vitro Differentiation of Pluripotent Stem Cell-Derived Podocytes.

    Tran, Tracy; Lindstrom, Nils O.; Ransick, Andrew; De Sena Brandine, Guilherme; Guo, Qiuyu; Kim, Albert D.; Der, Balint; Peti-Peterdi, Janos; Smith, Andrew D.; Thornton, Matthew; Grubbs, Brendan; McMahon, Jill A.; McMahon, Andrew P. Dev Cell . 50(1):102–116.e6. July 2019.

    The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.

  10. Kidney-in-a-lymph node: A novel organogenesis assay to model human renal development and test nephron progenitor cell fates.

    Francipane, Maria Giovanna; Han, Bing; Oxburgh, Leif; Sims-Lucas, Sunder; Li, Zhongwei; Lagasse, Eric. J Tissue Eng Regen Med . July 2019.

    Stem cell-derived organoids are emerging as sophisticated models for studying development and disease and as potential sources for developing organ substitutes. Unfortunately, although organoids containing renal structures have been generated from mouse and human pluripotent stem cells, there are still critical unanswered questions that are difficult to attain via in vitro systems, including whether these nonvascularized organoids have a stable and physiologically relevant phenotype or whether a suitable transplantation site for long-term in vivo studies can be identified. Even orthotopic engraftment of organoid cultures in the adult does not provide an environment conducive to vascularization and functional differentiation. Previously, we showed that the lymph node offers an alternative transplantation site where mouse metanephroi can differentiate into mature renal structures with excretory, homeostatic, and endocrine functions. Here, we show that the lymph node lends itself well as a niche to also grow human primary kidney rudiments and can additionally be viewed as a platform to interrogate emerging renal organoid cultures. Our study has a wide-ranging impact for tissue engineering approaches to rebuild functional tissues in vivo including-but not limited to-the kidney.

  11. EGFR is required for Wnt9a–Fzd9b signalling specificity in haematopoietic stem cells

    Grainger, Stephanie; Nguyen, Nicole; Richter, Jenna; Setayesh, Jordan; Lonquich, Brianna; Oon, Chet Huan; Wozniak, Jacob M.; Barahona, Rocio; Kamei, Caramai N.; Houston, Jack; Carrillo-Terrazas, Marvic; Drummond, Iain A.; Gonzalez, David; Willert, Karl; Traver, David. Nature Cell Biology . 21(6):721–730. June 2019.

    Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand–receptor (Wnt–Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit β-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a–Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a–Fzd9b–LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt–Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.

  12. Fibroblast growth factor signaling mediates progenitor cell aggregation and nephron regeneration in the adult zebrafish kidney.

    Gallegos, Thomas F.; Kamei, Caramai N.; Rohly, Michael; Drummond, Iain A. Dev Biol . June 2019.

    The zebrafish kidney regenerates after injury by development of new nephrons from resident adult kidney stem cells. Although adult kidney progenitor cells have been characterized by transplantation and single cell RNA seq, signals that stimulate new nephron formation are not known. Here we demonstrate that fibroblast growth factors and FGF signaling is rapidly induced after kidney injury and that FGF signaling is required for recruitment of progenitor cells to sites of new nephron formation. Chemical or dominant negative blockade of Fgfr1 prevented formation of nephron progenitor cell aggregates after injury and during kidney development. Implantation of FGF soaked beads induced local aggregation of lhx1a:EGFP + kidney progenitor cells. Our results reveal a previously unexplored role for FGF signaling in recruitment of renal progenitors to sites of new nephron formation and suggest a role for FGF signaling in maintaining cell adhesion and cell polarity in newly forming kidney epithelia.

  13. Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry

    Singh, Nikhil; Avigan, Zachary M; Kliegel, Judith A; Shuch, Brian M; Montgomery, Ruth R; Moeckel, Gilbert W; Cantley, Lloyd G. JCI Insight . 4(12). June 2019.

    An incomplete understanding of the biology of the human kidney, including the relative abundances of and interactions between intrinsic and immune cells, has long constrained the development of therapies for kidney disease. The small amount of tissue obtained by renal biopsy has previously limited the ability to use patient samples for discovery purposes. Imaging mass cytometry (IMC) is an ideal technology for quantitative interrogation of scarce samples, permitting concurrent analysis of more than 40 markers on a single tissue section. Using a validated panel of metal-conjugated antibodies designed to confer unique signatures on the structural and infiltrating cells comprising the human kidney, we performed simultaneous multiplexed imaging with IMC in 23 channels on 16 histopathologically normal human samples. We devised a machine-learning pipeline (Kidney-MAPPS) to perform single-cell segmentation, phenotyping, and quantification, thus creating a spatially preserved quantitative atlas of the normal human kidney. These data define selected baseline renal cell types, respective numbers, organization, and variability. We demonstrate the utility of IMC coupled to Kidney-MAPPS to qualitatively and quantitatively distinguish individual cell types and reveal expected as well as potentially novel abnormalities in diseased versus normal tissue. Our studies define a critical baseline data set for future quantitative analysis of human kidney disease.

  14. Polycystin 2 regulates mitochondrial Ca(2+) signaling, bioenergetics, and dynamics through mitofusin 2.

    Kuo, Ivana Y.; Brill, Allison L.; Lemos, Fernanda O.; Jiang, Jason Y.; Falcone, Jeffrey L.; Kimmerling, Erica P.; Cai, Yiqiang; Dong, Ke; Kaplan, David L.; Wallace, Darren P.; Hofer, Aldebaran M.; Ehrlich, Barbara E. Sci Signal . 12(580). May 2019.

    Mitochondria and the endoplasmic reticulum (ER) have an intimate functional relationship due to tethering proteins that bring their membranes in close (~30 nm) apposition. One function of this interorganellar junction is to increase the efficiency of Ca(2+) transfer into mitochondria, thus stimulating mitochondrial respiration. Here, we showed that the ER cation-permeant channel polycystin 2 (PC2) functions to reduce mitochondria-ER contacts. In cell culture models, PC2 knockdown led to a 50% increase in mitofusin 2 (MFN2) expression, an outer mitochondrial membrane GTPase. Live-cell super-resolution and electron microscopy analyses revealed enhanced MFN2-dependent tethering between the ER and mitochondria in PC2 knockdown cells. PC2 knockdown also led to increased

  15. Scaffolding kidney organoids on silk.

    Gupta, Ashwani Kumar; Coburn, Jeannine M.; Davis-Knowlton, Jessica; Kimmerling, Erica; Kaplan, David L.; Oxburgh, Leif. J Tissue Eng Regen Med . 13(5):812–822. May 2019.

    End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air-liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue.

  16. Recent Insights into Kidney Injury and Repair from Transcriptomic Analyses.

    Kirita, Yuhei; Chang-Panesso, Monica; Humphreys, Benjamin D. Nephron . 21:1–4. May 2019.

    Injured tubular epithelium exhibits cellular plasticity in that it can dedifferentiate, reenter the cell cycle, and subsequently either redifferentiate or adopt a chronically injured phenotype. Although some nephrogenic genes are reexpressed during injury and repair, developmental pathways are only partially recapitulated and the process is more accurately viewed as an entirely new program intrinsic to the regenerative response to injury. Recent advances in our understanding of the molecular circuitry underpinning epithelial plasticity have come from bulk, cell-specific, and single-cell transcriptomic analyses. These results have begun to define the signaling pathways and gene regulatory networks governing the epithelial injury response. In this review, we highlight recent transcriptomic analyses in kidney injury, repair and fibrosis, and outline the ways that these studies are improving our understanding of kidney regeneration.

  17. Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish.

    Brilli Skvarca, Lauren; Han, Hwa In; Espiritu, Eugenel B.; Missinato, Maria A.; Rochon, Elizabeth R.; McDaniels, Michael D.; Bais, Abha S.; Roman, Beth L.; Waxman, Joshua S.; Watkins, Simon C.; Davidson, Alan J.; Tsang, Michael; Hukriede, Neil A. Dis Model Mech . 12(4). April 2019.

    Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified

  18. Dual lineage tracing shows that glomerular parietal epithelial cells can transdifferentiate toward the adult podocyte fate

    Kaverina, Natalya V.; Eng, Diana G.; Freedman, Benjamin S.; Kutz, J. Nathan; Chozinski, Tyler J.; Vaughan, Joshua C.; Miner, Jeffrey H.; Pippin, Jeffrey W.; Shankland, Stuart J. Kidney International . April 2019.

    Podocytes are differentiated post-mitotic cells that cannot replace themselves after injury. Glomerular parietal epithelial cells are proposed to be podocyte progenitors. To test whether a subset of parietal epithelial cells transdifferentiate to a podocyte fate, dual reporter PEC-rtTA\textbarLC1\textbartdTomato\textbarNphs1-FLPo\textbarFRT-EGFP mice, named PEC-PODO, were generated. Doxycycline administration permanently labeled parietal epithelial cells with tdTomato reporter (red), and upon doxycycline removal, the parietal epithelial cells (PECs) cannot label further. Despite the presence or absence of doxycycline, podocytes cannot label with tdTomato, but are constitutively labeled with an enhanced green fluorescent protein (EGFP) reporter (green). Only activation of the Nphs1-FLPo transgene by labeled parietal epithelial cells can generate a yellow color. At day 28 of experimental focal segmental glomerulosclerosis, podocyte density was 20% lower in 20% of glomeruli. At day 56 of experimental focal segmental glomerulosclerosis, podocyte density was 18% lower in 17% of glomeruli. TdTomato+ parietal epithelial cells were restricted to Bowman?s capsule in healthy mice. However, by days 28 and 56 of experimental disease, two-thirds of tdTomato+ parietal epithelial cells within glomerular tufts were yellow in color. These cells co-expressed the podocyte markers podocin, nephrin, p57 and VEGF164, but not markers of endothelial (ERG) or mesangial (Perlecan) cells. Expansion microscopy showed primary, secondary and minor processes in tdTomato+EGFP+ cells in glomerular tufts. Thus, our studies provide strong evidence that parietal epithelial cells serve as a source of new podocytes in adult mice.

  19. Wnt signaling mediates new nephron formation during zebrafish kidney regeneration

    Kamei, Caramai N.; Gallegos, Thomas F.; Liu, Yan; Hukriede, Neil; Drummond, Iain A. Development . 146(8). April 2019.

    Zebrafish kidneys use resident kidney stem cells to replace damaged tubules with new nephrons: the filtration units of the kidney. What stimulates kidney progenitor cells to form new nephrons is not known. Here, we show that wnt9a and wnt9b are induced in the injured kidney at sites where frizzled9b- and lef1-expressing progenitor cells form new nephrons. New nephron aggregates are patterned by Wnt signaling, with high canonical Wnt-signaling cells forming a single cell thick rosette that demarcates: domains of cell proliferation in the elongating nephron; and tubule fusion where the new nephron plumbs into the distal tubule and establishes blood filtrate drainage. Pharmacological blockade of canonical Wnt signaling inhibited new nephron formation after injury by inhibiting cell proliferation, and resulted in loss of polarized rosette structures in the aggregates. Mutation in frizzled9b reduced total kidney nephron number, caused defects in tubule morphology and reduced regeneration of new nephrons after injury. Our results demonstrate an essential role for Wnt/frizzled signaling in adult zebrafish kidney development and regeneration, highlighting conserved mechanisms underlying both mammalian kidney development and kidney stem cell-directed neonephrogenesis in zebrafish.

  20. Single Cell Transcriptomics and Solid Organ Transplantation.

    Malone, Andrew F.; Humphreys, Benjamin D. Transplantation . April 2019.

    Single cell RNA-sequencing (scRNA-seq) allows the measurement of transcriptomes from individual cells providing new insights into complex biological systems. scRNA-seq has enabled the identification of rare cell types, new cell states and intercellular communication networks that may be masked by traditional bulk transcriptional profiling. Researchers are increasingly using scRNA-seq to comprehensively characterize complex organs in health and disease. The diversity of immune cell types, some present at low frequency, in a transplanted organ undergoing rejection makes scRNA-seq ideally suited to characterize transplant pathologies because it can quantify subtle transcriptional differences between rare cell types. In this review we discuss single cell sequencing methods and their application in transplantation to date, current challenges and future directions. We believe that the remarkably rapid pace of technological development in this field makes it likely that single cell technologies such as scRNA-seq will have an impact in clinical transplantation within a decade.

  21. Renal reabsorption in 3D vascularized proximal tubule models

    Lin, Neil Y. C.; Homan, Kimberly A.; Robinson, Sanlin S.; Kolesky, David B.; Duarte, Nathan; Moisan, Annie; Lewis, Jennifer A. Proceedings of the National Academy of Sciences . 116(12):5399–5404. 2019.

    Current kidney-on-chip models lack the 3D geometry, complexity, and functionality vital for recapitulating in vivo renal tissue. We report the fabrication and perfusion of 3D vascularized proximal tubules embedded within an engineered ECM that exhibit active reabsorption of solutes via tubular–vascular exchange. Using this model, we quantified albumin and glucose reabsorption over time. We also studied hyperglycemic effects in the absence and presence of a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular–vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium–endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

  22. Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells

    Kumar, Santhosh V.; Er, Pei X.; Lawlor, Kynan T.; Motazedian, Ali; Scurr, Michelle; Ghobrial, Irene; Combes, Alexander N.; Zappia, Luke; Oshlack, Alicia; Stanley, Edouard G.; Little, Melissa H. Development . 146(5):dev172361. March 2019.

    Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro. We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.

  23. Reporter‐based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation

    Howden, Sara E; Vanslambrouck, Jessica M; Wilson, Sean B; Tan, Ker Sin; Little, Melissa H. EMBO Rep . March 2019.

    Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self‐renewing Six2‐expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell‐derived kidney organoids, we performed a similar fate‐mapping analysis of the SIX2‐expressing lineage in induced pluripotent stem cell (iPSC)‐derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro. Using CRISPR/Cas9 gene‐edited lineage reporter lines, we show that SIX2‐expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2+ cells to contribute to nephron formation over time, but retention of nephron‐forming capacity if provided an exogenous WNT signal. Hence, while human iPSC‐derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self‐renewal and ongoing nephrogenesis.EMBO Reports (2019) e47483

  24. Single-cell genomics and gene editing: implications for nephrology

    Wilson, Parker C.; Humphreys, Benjamin D. Nature Reviews Nephrology . 15(2):63–64. February 2019.

    Discoveries in 2018 using single-cell sequencing and gene-editing technologies have revealed their transformative potential for the investigation of kidney physiology and disease. Their promise is matched by the speed of their evolution.

  25. Flow-enhanced vascularization and maturation of kidney organoids in vitro

    Homan, Kimberly A.; Gupta, Navin; Kroll, Katharina T.; Kolesky, David B.; Skylar-Scott, Mark; Miyoshi, Tomoya; Mau, Donald; Valerius, M. Todd; Ferrante, Thomas; Bonventre, Joseph V.; Lewis, Jennifer A.; Morizane, Ryuji. Nature Methods . February 2019.

    Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

  26. Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis.

    Wu, Haojia; Kirita, Yuhei; Donnelly, Erinn L.; Humphreys, Benjamin D. J Am Soc Nephrol . 30(1):23–32. January 2019.

    BACKGROUND: A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses. METHODS: We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. RESULTS: A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation-induced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. Our snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways. CONCLUSIONS: snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney.

  27. Evaluation of variability in human kidney organoids

    Phipson, Belinda; Er, Pei X.; Combes, Alexander N.; Forbes, Thomas A.; Howden, Sara E.; Zappia, Luke; Yen, Hsan-Jan; Lawlor, Kynan T.; Hale, Lorna J.; Sun, Jane; Wolvetang, Ernst; Takasato, Minoru; Oshlack, Alicia; Little, Melissa H. Nature Methods . 16(1):79–87. January 2019.

    The utility of human pluripotent stem cell–derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.

  28. Kidney and organoid single-cell transcriptomics: the end of the beginning.

    Wilson, Parker C.; Humphreys, Benjamin D. Pediatr Nephrol . January 2019.

    Single-cell RNA sequencing (scRNA-seq) technologies are increasingly being applied to reveal cellular heterogeneity in kidney development and disease. In just the last year, multiple scRNA-seq datasets have been generated from kidney organoids, developing mouse and human kidney, adult kidney, and kidney cancer. The data generated enables a much deeper understanding of biological processes within and between cells. It has also elucidated unforeseen cell lineage relationships, defined the presence of off-target cell types in kidney organoids, and revealed a diverse inflammatory response in a human kidney allograft undergoing rejection. This review summarizes the recent rapid progress in scRNA-seq of the kidney and outlines future directions for single-cell technologies as applied to the kidney.

  29. Single-cell analysis reveals congruence between kidney organoids and human fetal kidney

    Combes, Alexander N.; Zappia, Luke; Er, Pei Xuan; Oshlack, Alicia; Little, Melissa H. Genome Medicine . 11(1):3. January 2019.

    Human kidney organoids hold promise for studying development, disease modelling and drug screening. However, the utility of stem cell-derived kidney tissues will depend on how faithfully these replicate normal fetal development at the level of cellular identity and complexity.

  30. The Single Cell Transcriptomic Landscape of Early Human Diabetic Nephropathy

    Wilson, Parker C.; Wu, Haojia; Kirita, Yuhei; Uchimura, Kohei; Rennke, Helmut G.; Welling, Paul A.; Waikar, Sushrut S.; Humphreys, Benjamin D. bioRxiv . 2019.

    Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single nucleus RNA sequencing (snRNAseq) on cryopreserved human diabetic kidney samples to generate 23,980 single nucleus transcriptomes from three control and three early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side by side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic loop of Henle, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na-K+-ATPase, WNK1, mineralocorticoid receptor and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.Significance Statement Single nucleus RNA sequencing revealed gene expression changes in early diabetic nephropathy that promote urinary potassium secretion and decreased calcium and magnesium reabsorption. Multiple cell types exhibited angiogenic signatures, which may represent early signs of aberrant angiogenesis. These alterations may help to identify biomarkers for disease progression or signaling pathways amenable to early intervention.

  31. Long-Term Culture of Nephron Progenitor Cells Ex Vivo.

    Brown, Aaron C.; Gupta, Ashwani K.; Oxburgh, Leif. Methods Mol Biol . 1926:63–75. 2019.

    Nephrons differentiate from the cap mesenchyme of the fetal kidney. Nephron progenitor cells that populate the cap mesenchyme efficiently balance self-renewal and epithelial differentiation to enable repeated rounds of nephron formation during development. Here we describe a method to isolate and propagate these cells from the embryonic mouse kidney. Using this method, nephron progenitor cells from a single litter of mice can be propagated to hundreds of millions of cells that express appropriate markers of the undifferentiated state and retain epithelial differentiation capacity in vitro.

2018

  1. A Cellular Anatomy of the Normal Adult Human Prostate and Prostatic Urethra

    Henry, GH; Malewska, A; Joseph, DB; Malladi, VS; Lee, J; Torrealba, J; Mauck, RJ; Gahan, JC; Raj, GV; Roehrborn, CG; Hon, GC; Macconmara, MP; Reese, JC; Hutchinson, RC; Vezina, CM; Strand, DW. Cell Rep . 25(12):3530-3542. December 2018.

    A cellular anatomy of normal human organs is essential for solving the cellular origins of disease. We report the first comprehensive cellular atlas of the young adult human prostate and prostatic urethra using an iterative process of single cell RNA sequencing and flow cytometry on ~98,000 cells taken from different anatomical regions. Two previously unrecognized epithelial cell types were identified by KRT13 and SCGB1A1 expression and found to be highly similar to hillock and club cells of the proximal lung. It was demonstrated by immunohistochemistry that prostate club and hillock cells are similarly concentrated in the proximal prostate. We also optimized a new flow cytometry antibody panel to improve cell type-specific purification based on newly established cellular markers. The molecular classification, anatomical distribution, and purification methods for each cell type in the human prostate create a powerful new resource for experimental design in human prostate disease.

  2. Polyploid Superficial Cells that Maintain the Urothelial Barrier Are Produced via Incomplete Cytokinesis and Endoreplication

    Wang, Jia; Batourina, Ekatherina; Schneider, Kerry; Souza, Spenser; Swayne, Theresa; Liu, Chang; George, Christopher D.; Tate, Tiffany; Dan, Hanbin; Wiessner, Gregory; Zhuravlev, Yelena; Canman, Julie C.; Mysorekar, Indira U.; Mendelsohn, Cathy Lee. Cell Reports . 25(2):464–477.e4. October 2018.

  3. In vivo replacement of damaged bladder urothelium by Wolffian duct epithelial cells.

    Joseph, DB; Chandrashekar, AS; Abler, LL; Chu, LF; Thomson, JA; Mendelsohn, C; Vezina, CM. Proc Natl Acad Sci U S A . 115(33):8394–8399. August 2018.

    The bladder’s remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.

  4. Void spot assay procedural optimization and software for rapid and objective 2 quantification of rodent voiding function, including overlapping urine spots

    Wegner, KA; Abler, LL; Oakes, SR; Mehta, GS; Ritter, KE; Hill, WG; Zwaans, BMM; Lamb, LE; Wang, Z; Bjorling, DE; Ricke, WA; Macoska, J; Marker, PC; Southard-Smith, EM; Eliceiri, KW; Vezina, CM.. Am J Physiol Renal Physiol . July 2018.

    Mouse urinary behavior is quantifiable and used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying non-concentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.

  5. Conserved and Divergent Features of Human and Mouse Kidney Organogenesis.

    Lindström, NO; McMahon, JA; Guo, J; Tran, T; Guo, Q; Rutledge, E; Parvez, RK; Saribekyan, G; Schuler, RE; Liao, C; Kim, AD; Abdelhalim, A; Ruffins, SW; Thornton, ME; Basking, L; Grubbs, B; Kesselman, C; McMahon, AP. J Am Soc Nephrol . February 2018.

    Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.

  6. Conserved and Divergent Features of Mesenchymal Progenitor Cell Types within the Cortical Nephrogenic Niche of the Human and Mouse Kidney

    Lindström, NO; Guo, J; Kim, AD; Tran, T; Guo, Q; De Sena Brandine, G; Ransick, A; Parvez, RK; Thornton, ME; Basking, L; Grubbs, B; McMahon, JA; Smith, AD; McMahon, AP. J Am Soc Nephrol . February 2018.

    Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2+ nephron progenitor cells (NPCs) and Foxd1+ interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2+ NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2+ NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.

  7. Conserved and Divergent Molecular and Anatomic Features of Human and Mouse Nephron Patterning

    Lindström, NO; Tran, T; Guo, J; Rutledge, E; Parvez, RK; Thornton, ME; Grubbs, B; McMahon, JA; McMahon, AP. J Am Soc Nephrol . February 2018.

    The nephron is the functional unit of the kidney, but the mechanism of nephron formation during human development is unclear. We conducted a detailed analysis of nephron development in humans and mice by immunolabeling, and we compared human and mouse nephron patterning to describe conserved and divergent features. We created protein localization maps that highlight the emerging patterns along the proximal–distal axis of the developing nephron and benchmark expectations for localization of functionally important transcription factors, which revealed unanticipated cellular diversity. Moreover, we identified a novel nephron subdomain marked by Wnt4 expression that we fate-mapped to the proximal mature nephron. Significant conservation was observed between human and mouse patterning. We also determined the time at which markers for mature nephron cell types first emerge—critical data for the renal organoid field. These findings have conceptual implications for the evolutionary processes driving the diversity of mammalian organ systems. Furthermore, these findings provide practical insights beyond those gained with mouse and rat models that will guide in vitro efforts to harness the developmental programs necessary to build human kidney structures.

  8. Development of the Human Fetal Kidney from Mid to Late Gestation in Male and Female Infants

    Ryan, D; Sutherland, MR; Flores, TJ; Kent, AL; Dahlstrom, JE; Puelles, VG; Bertram, JF; McMahon, AP; Little, MH; Moore, L; Black, MJ. EBioMedicine . 27:275–283. January 2018.

    BACKGROUND: During normal human kidney development, nephrogenesis (the formation of nephrons) is complete by term birth, with the majority of nephrons formed late in gestation. The aim of this study was to morphologically examine nephrogenesis in fetal human kidneys from 20 to 41weeks of gestation. METHODS: Kidney samples were obtained at autopsy from 71 infants that died acutely in utero or within 24h after birth. Using image analysis, nephrogenic zone width, the number of glomerular generations, renal corpuscle cross-sectional area and the cellular composition of glomeruli were examined. Kidneys from female and male infants were analysed separately. FINDINGS: The number of glomerular generations formed within the fetal kidneys was directly proportional to gestational age, body weight and kidney weight, with variability between individuals in the ultimate number of generations (8 to 12) and in the timing of the cessation of nephrogenesis (still ongoing at 37weeks gestation in one infant). There was a slight but significant (r2=0.30, P=0.001) increase in renal corpuscle cross-sectional area from mid gestation to term in females, but this was not evident in males. The proportions of podocytes, endothelial and non-epithelial cells within mature glomeruli were stable throughout gestation. INTERPRETATION: These findings highlight spatial and temporal variability in nephrogenesis in the developing human kidney, whereas the relative cellular composition of glomeruli does not appear to be influenced by gestational age.

  9. 3D organoid-derived human glomeruli for personalised podocyte disease modelling and drug screening

    Hale, Lorna J.; Howden, Sara E.; Phipson, Belinda; Lonsdale, Andrew; Er, Pei X.; Ghobrial, Irene; Hosawi, Salman; Wilson, Sean; Lawlor, Kynan T.; Khan, Shahnaz; Oshlack, Alicia; Quinlan, Catherine; Lennon, Rachel; Little, Melissa H. Nature Communications . 9(1):5167. December 2018.

    The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.

  10. Reconstructing the Human Renal Vascular-Tubular Unit In Vitro.

    Rayner, Samuel G.; Phong, Kiet T.; Xue, Jun; Lih, Daniel; Shankland, Stuart J.; Kelly, Edward J.; Himmelfarb, Jonathan; Zheng, Ying. Adv Healthc Mater . 7(23):e1801120. December 2018.

    Engineered human kidney-on-a-chip platforms show tremendous promise for disease modeling and drug screening. Outstanding challenges exist, however, in reconstructing the complex architecture, cellular make-up, and matrix composition necessary for the proper modeling of kidney function. Herein, the first fully tunable human kidney-on-a-chip platform is reported that allows the reconstruction of the native architecture of the renal endothelial-epithelial exchange interface using entirely cell-remodelable matrix and patient-derived kidney cells. This platform consists of a double-layer human renal vascular-tubular unit (hRVTU) enabled by a thin collagen membrane that replicates the kidney exchange interface. It is shown that endothelial and epithelial cells lining their respective lumens remodel the membrane in culture into a approximately 1 microm thick exchange interface composed of native basement membrane proteins. This interface displays sufficient mechanical integrity for media flow and blood perfusion. As a proof of principle, it is demonstrated that the hRVTU performs kidney-specific functions including reabsorption of albumin and glucose from the epithelial channel. By incorporating multiple cell populations from single donors, it is demonstrated that the hRVTU may have utility for future precision medicine applications. The success of the system provides new opportunities for the next generation of organ-on-a-chip models.

  11. Cells of NG2 lineage increase in glomeruli of mice following podocyte depletion.

    Suzuki, Taihei; Eng, Diana G.; McClelland, Aaron D.; Pippin, Jeffrey W.; Shankland, Stuart J. Am J Physiol Renal Physiol . 315(5):F1449–F1464. November 2018.

    Under certain circumstances, podocytes can be partially replaced following their loss in disease. The inability of podocytes to proliferate suggests that replacement derives from other cell types. Because neural/glial antigen 2 (NG2)-expressing cells can serve as progenitors in other organs and because herein we showed increased NG2 staining in podocytes following their loss in experimental focal segmental glomerulosclerosis, we used lineage tracing in

  12. Comparative Analysis and Refinement of Human PSC-Derived Kidney Organoid Differentiation with Single-Cell Transcriptomics

    Wu, H; Uchimura, K; Donnelly, E.L.; Kirita, Y; Morris, S.A.; Humphreys, B.D. Cell Stem Cell . November 2018.

    Kidney organoids derived from human pluripotent stem cells have great utility for investigating organogenesis and disease mechanisms and, potentially, as a replacement tissue source, but how closely organoids derived from current protocols replicate adult human kidney is undefined. We compared two directed differentiation protocols by single-cell transcriptomics of 83,130 cells from 65 organoids with single-cell transcriptomes of fetal and adult kidney cells. Both protocols generate a diverse range of kidney cells with differing ratios, but organoid-derived cell types are immature, and 10%?20% of cells are non-renal. Reconstructing lineage relationships by pseudotemporal ordering identified ligands, receptors, and transcription factor networks associated with fate decisions. Brain-derived neurotrophic factor (BDNF) and its cognate receptor NTRK2 were expressed in the neuronal lineage during organoid differentiation. Inhibiting this pathway improved organoid formation by reducing neurons by 90% without affecting kidney differentiation, highlighting the power of single-cell technologies to characterize and improve organoid differentiation.

  13. A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition

    Ó hAinmhire, E; Wu, H; Muto, Y; Donnelly, EL; Machado, FG; Fan, LX; Chang-Panesso, M; Humphreys, BD. Am. J. Physiol. Renal Physiol. . October 2018.

    Gli1-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KG1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation and display robust myofibroblast differentiation upon treatment with TGFb. Co-culture of KG1 cells with endothelium stabilizes capillary formation. Single cell RNA-sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 which potently inhibited TGFb-induced pericyte to myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF) which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial to tubule paracrine signaling axis. Thus KG1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

  14. FoxM1 drives proximal tubule proliferation during repair from acute kidney injury

    Chang-Panesso, Monica; Kadyrov, Farid F.; Lalli, Matthew; Wu, Haojia; Ikeda, Shiyo; Kobayashi, Akio; Humphreys, Benjamin D. bioRxiv . 2018.

    The proximal tubule has a remarkable capacity for repair after acute injury but the cellular lineage and molecular mechanisms underlying this repair response have been poorly characterized. Here, we developed a Kim-1-GFPCreERt2 knockin mouse line (Kim-1-GCE), performed genetic lineage analysis after injury and measured the cellular transcriptome of proximal tubule during repair. Acutely injured genetically labeled clones co-expressed Kim-1, Vimentin, Sox9 and Ki67, indicating a dedifferentiated and proliferative state. Clonal analysis revealed clonal expansion of Kim-1+ cells, indicating that acutely injured, dedifferentiated proximal tubule cells account for repair rather than a fixed tubular progenitor. Translational profiling during injury and repair revealed signatures of both successful and unsuccessful maladaptive repair. The transcription factor FoxM1 was induced early in injury, was required for epithelial proliferation, and was dependent on epidermal growth factor receptor (EGFR) stimulation. In conclusion, dedifferentiated proximal tubule cells effect proximal tubule repair and we reveal a novel EGFR-FoxM1-dependent signaling pathway that drives proliferative repair after injury.

  15. Single-cell analysis of progenitor cell dynamics and lineage specification of the human fetal kidney

    Menon, R; Otto, EA; Kokoruda, A; Zhou, J; Zhang, Z; Yoon, E; Chen, Y; Troyanscaya, O; Spence, J; Kretzler, M; Cebrian, C. Development . 145. August 2018.

    The mammalian kidney develops through reciprocal interactions between the ureteric bud and the metanephric mesenchyme to give rise to the entire collecting system and the nephrons. Most of our knowledge of the developmental regulators driving this process arises from the study of gene expression and functional genetics in mice and other animal models. In order to shed light on human kidney development, we have used single-cell transcriptomics to characterize gene expression in different cell populations, and to study individual cell dynamics and lineage trajectories during development. Single-cell transcriptome analyses of 6414 cells from five individual specimens identified 11 initial clusters of specific renal cell types as defined by their gene expression profile. Further subclustering identifies progenitors, and mature and intermediate stages of differentiation for several renal lineages. Other lineages identified include mesangium, stroma, endothelial and immune cells. Novel markers for these cell types were revealed in the analysis, as were components of key signaling pathways driving renal development in animal models. Altogether, we provide a comprehensive and dynamic gene expression profile of the developing human kidney at the single-cell level.

  16. Volumetric, Nanoscale Optical Imaging of Mouse and Human Kidney via Expansion Microscopy.

    Chozinski, Tyler J.; Mao, Chenyi; Halpern, Aaron R.; Pippin, Jeffrey W.; Shankland, Stuart J.; Alpers, Charles E.; Najafian, Behzad; Vaughan, Joshua C. Sci Rep . 8(1):10396. July 2018.

    Although light microscopy is a powerful tool for the assessment of kidney physiology and pathology, it has traditionally been unable to resolve structures separated by less than the ~250 nm diffraction limit of visible light. Here, we report on the optimization, validation, and application of a recently developed super-resolution fluorescence microscopy method, called expansion microscopy (ExM), for volumetric interrogation of mouse and human kidney tissue with 70-75 nm lateral and ~250 nm axial spatial resolution. Using ExM with a standard confocal microscope, we resolve fine details of structures that have traditionally required visualization by electron microscopy, including podocyte foot processes, the glomerular basement membrane, and the cytoskeleton. This inexpensive and accessible approach to volumetric, nanoscale imaging enables visualization of fine structural details of kidney tissues that were previously difficult or impossible to measure by conventional methodologies.

  17. Human Organ-specific Endothelial Cell Heterogeneity

    Marcu, R; Choi, YJ; Xue, J; Fortin, CL; Wang, Y; Nagao, RJ; Xu, J; MacDonald, JW; Bammler, TK; Murry, CE; Muczynski, K; Stevens, KR; Himmelfarb, J; Schwartz, SM; Zheng, Y. iScience . 4:20–35. June 2018.

    The endothelium first forms in the blood islands in the extra-embryonic yolk sac and then throughout the embryo to establish circulatory networks that further acquire organ-specific properties during development to support diverse organ functions. Here, we investigated the properties of endothelial cells (ECs), isolated from four human major organs—the heart, lung, liver, and kidneys—in individual fetal tissues at three months’ gestation, at gene expression, and at cellular function levels. We showed that organ-specific ECs have distinct expression patterns of gene clusters, which support their specific organ development and functions. These ECs displayed distinct barrier properties, angiogenic potential, and metabolic rate and support specific organ functions. Our findings showed the link between human EC heterogeneity and organ development and can be exploited therapeutically to contribute in organ regeneration, disease modeling, as well as guiding differentiation of tissue-specific ECs from human pluripotent stem cells.

  18. Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response

    Wu, H; Malone, AF; Donnelly, EL; Kirita, Y; Uchimura, K; Ramakrishnan, SM; Gaut, JP; Humphreys, BD. JASN . 29(8):2069–2080. May 2018.

    Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen. Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection. Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16− group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database. Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

  19. Sex differences in transcriptomic profiles in aged kidney cells of renin lineage.

    Wang, Yuliang; Eng, Diana G.; Pippin, Jeffrey W.; Gharib, Sina A.; McClelland, Aaron; Gross, Kenneth W.; Shankland, Stuart J. Aging (Albany NY) . 10(4):606–621. April 2018.

    Renin expressing cells in the kidney’s juxta-glomeruluar compartment likely also serve as progenitors for adult glomerular cells in disease. Although these cells of renin lineage (CoRL) decrease in number with advancing kidney age, accompanied by less responsiveness to typical stimuli such as ACE-inhibition, mechanisms and the impact of sex as a biological variable with age are not known. Accordingly, labeled CoRL were sorted from individual young (2m) and aged (27m) male and female Ren1cCre\textbarZsGreen reporter mice, and their transcriptomic profiles analyzed by RNA seq. When both aged female and male mice were combined, there were 48 differentially expressed genes (DEG) compared to young mice. However, when compared to their young sex-matched mice, aged female and male mice had 159 and 503 DEGs respectively. In addition to marked differences in individual genes between aged female and male mice, gene ontology analysis showed major pathway differences by sex. The majority of DEGs in one sex did not significantly change or changed in the opposite direction in the other sex. These results show that in CoRL of advanced age, individual genes and gene ontologies change, but differ between female and male mice, highlighting sex related differences the aging process.

  20. Simultaneous reprogramming and gene editing of human fibroblasts

    Howden, SE; Thomson, JA; Little, MH. Nature Protocols . 13(5):875–898. April 2018.

    The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6–8 weeks), and cost-effective.

  21. Spatiotemporal heterogeneity and patterning of developing renal blood vessels

    Daniel, E; Azizoglu, DB; Ryan, AR; Walji, TA; Chaney, CP; Sutton, GI; Carroll, TJ; Marciano, DK; Cleaver, O. Angiogenesis . April 2018.

    The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of speci cation by E13.5 when the rst arteries can be identi ed. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immuno uorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.

  22. Detection of renin lineage cell transdifferentiation to podocytes in the kidney glomerulus with dual lineage tracing

    Eng, DG; Kaverina, NV; Schneider, RRS; Freedman, BS; Gross, KW; Miner, JH; Pippin, JW; Shankland, SJ. Kidney International . March 2018.

    Understanding of cellular transdifferentiation is limited by the technical inability to track multiple lineages in vivo. To overcome this we developed a new tool to simultaneously fate map two distinct cell types in the kidney, and genetically test whether cells of renin lineage (CoRL) can transdifferentiate to a podocyte fate. Ren1cCreER/ tdTomato/Nphs1-FLPo/FRT-EGFP mice (CoRL-PODO mice) were generated by crossing Ren1c-CreER/tdTomato CoRL reporter mice with Nphs1-FLPo/FRT-EGFP podocyte reporter mice. Following tamoxifen administration in these animals, CoRL were labeled with red fluorescence (tdTomato) and co-localized with renin. Podocytes were labeled green (enhanced green fluorescent protein) and co-localized with nephrin. Following podocyte loss by nephrotoxic antibody and subsequent enalapril-enhanced partial replacement, tdTomato-EGFP-labeled CoRL were detected as yellow- colored cells in a subset of glomerular tufts, without the use of antibodies. Co-localization with podocin indicated that these cells are podocytes, derived from CoRL origin. Thus, our novel study shows that two distinct cell types can be simultaneously labeled in the mouse kidney and provide strong genetic evidence in vivo that lost podocytes can be replaced in part by CoRL.

  23. Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.

    van den Berg, CW; Ritsma, L; Avramut, MC; Wiersma, LE; van den Berg, BM; Leuning, DG; Lievers, E; Koning, M; Vanslambrouck, JM; Koster, AJ; Howden, SE; Takasato, M; Little, MH; Rabelink, TJ. Stem Cell Reports. . 10(3):751–765. March 2018.

    Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.

  24. Conserved and Divergent Features of Human and Mouse Kidney Organogenesis.

    Lindström, NO; McMahon, JA; Guo, J; Tran, T; Guo, Q; Rutledge, E; Parvez, RK; Saribekyan, G; Schuler, RE; Liao, C; Kim, AD; Abdelhalim, A; Ruffins, SW; Thornton, ME; Basking, L; Grubbs, B; Kesselman, C; McMahon, AP. J Am Soc Nephrol . February 2018.

    Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.

  25. Bringing Renal Biopsy Interpretation Into the Molecular Age With Single-Cell RNA Sequencing

    Malone, AF; Wu, H; Humphreys, BD. Semin Nephrol . January 2018.

    The renal biopsy provides critical diagnostic and prognostic information to clinicians including cases of acute kidney injury, chronic kidney disease, and allograft dysfunction. Today, biopsy specimens are read using a combination of light microscopy, electron microscopy, and indirect immunofluorescence, with a limited number of antibodies. These techniques all were perfected decades ago with only incremental changes since then. By contrast, recent advances in single-cell genomics are transforming scientists’ ability to characterize cells. Rather than measure the expression of several genes at a time by immunofluorescence, it now is possible to measure the expression of thousands of genes in thousands of single cells simultaneously. Here, we argue that the development of single-cell RNA sequencing offers an opportunity to describe human kidney disease comprehensively at a cellular level. It is particularly well suited for the analysis of immune cells, which are characterized by multiple subtypes and changing functions depending on their environment. In this review, we summarize the development of single-cell RNA sequencing methodologies. We discuss how these approaches are being applied in other organs, and the potential for this powerful technology to transform our understanding of kidney disease once applied to the renal biopsy.

2017

  1. An immunohistochemical identification key for cell types in adult mouse prostatic and urethral tissue sections

    Wegner, KA; Cadena, MT; Trevena, R; Turco, AE; Gottschalk, A; Halberg, RB; Guo, J; McMahon, JA; McMahon, AP; Vezina, CM. PLoS ONE . 12(11). November 2017.

  2. Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development

    Adam, M; Potter, AS; Potter, SS. Development . 144(19):3625–3632. October 2017.

    Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively ‘freezing in’ the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.

  3. Migration pathways of sacral neural crest during development of lower urogenital tract innervation

    Wiese, CB; Deal, KK; Ireland, SJ; Cantrell, VA; Southard-Smith, EM. Dev Biol . 429(1):356–369. September 2017.

  4. Dynamic Expression of Serotonin Receptor 5-HT3A in Developing Sensory Innervation of the Lower Urinary Tract

    Ritter, KE; Southard-Smith, EM. Front Neurosci . 10(592). June 2017.

  5. Charting the transcriptional landscape of cells of renin lineage following podocyte depletion

    McClelland, AD; Lichtnekert, J; Eng, DG; Pippin, JW; Gross, KW; Gharib, SA; Shankland, SJ. PLOS ONE . 12(12). December 2017.

    Renin producing cells of the juxtaglomerulus, herein called cells of renin lineage (CoRL), have garnered recent interest for their propensity to act as a progenitor source for various kidney cell types including podocytes. Despite recent advances, the process of transdifferentiation of CoRL to podocytes is poorly understood. In this study, we employed a transgenic reporter mouse line which permanently labels CoRL with ZsGreen fluorescent protein, allowing for isolation by fluorescence-activated cell sorting. At 5 days following induction of abrupt podocyte ablation via anti-podocyte sheep IgG, mice were sacrificed and CoRL were isolated by FACS. RNA was subsequently analyzed by microarray. Gene set enrichment analysis (GSEA) was performed and revealed that CoRL display a distinct phenotype following podocyte ablation, primarily consisting of downregulation of metabolic processes and upregulation of immuno-modulatory processes. Additionally, RNA-biology and cell cycle-related processes were also upregulated. Changes in gene expression or activity of a core set of transcription factors including HNF1 and E2F were identified through changes in enrichment of their respective target genes. However, integration of results from transcription factor and canonical pathway analysis indicated that ERR1 and PU-box family members may be the major contributors to the post-podocyte ablation phenotype of CoRL. Finally, top ranking genes were selected from the microarray-based analysis and confirmed by qPCR. Collectively, our results provide valuable insights into the transcriptional regulation of CoRL following abrupt podocyte ablation.

  6. Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development

    Kim, YK; Refaeli, I; Brooks, CR; Jing, P; Gulieva, RE; Hughes, MR; Cruz, NM; Liu, Y; Churchill, AJ; Wang, Y; Fu, H; Pippin, JW; Lin, LY; Shankland, SJ; Vogl, AW; McNagny, KM; Freedman, BS. Stem Cells . 35(12):2366–2378. December 2017.

    A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378

  7. High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney

    Combes, AN; Phipson, B; Zappia, L; Lawlor, KE; Er, PX; Oshlack, A; Little, MA. bioRxiv . December 2017.

    Recent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of off target populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

  8. The promise of single-cell RNA sequencing for kidney disease investigation

    Wu, H; Humphreys, Benjamin D. Kidney International . 92(6):1334–1342. December 2017.

    Recent techniques for single-cell RNA sequencing (scRNA-seq) at high throughput are leading to profound new discoveries in biology. The ability to generate vast amounts of transcriptomic data at cellular resolution represents a transformative advance, allowing the identification of novel cell types, states, and dynamics. In this review, we summarize the development of scRNA-seq methodologies and highlight their advantages and drawbacks. We discuss available software tools for analyzing scRNA-Seq data and summarize current computational challenges. Finally, we outline ways in which this powerful technology might be applied to discovery research in kidney development and disease.

  9. Transcriptional evaluation of the developmental accuracy, reproducibility and robustness of kidney organoids derived from human pluripotent stem cells

    Phipson, B; Er, PX; Hale, L; Yen, DH; Lawlor, KE; Takasato, M; Sun, J; Wolvetang, E; Oshlack, A; Little, MH. bioRxiv . December 2017.

    We have previously reported a protocol for the directed differentiation of human induced pluripotent stem cells to kidney organoids comprised of nephrons, proximal and distal epithelium, vasculature and surrounding interstitial elements. The utility of this protocol for applications such as disease modelling will rely implicitly on the developmental accuracy of the model, technical robustness of the protocol and transferability between iPSC lines. Here we report extensive transcriptional analyses of the sources of variation across the timecourse of differentiation from pluripotency to complete kidney organoid, focussing on repeated differentiations to day 18 organoid. Individual organoids generated within the same differentiation experiment show Spearmans correlation coefficients of \textgreater0.99. The greatest source of variation was seen between experimental batch, with the enrichment for genes that also varied temporally between day 10 and day 25 organoids implicating nephron maturation as contributing to transcriptional variance between individual differentiation experiments. A morphological analysis revealed a transition from renal vesicle to capillary loop stage nephrons across the same time period. Distinct iPSC clones were also shown to display congruent transcriptional programs with inter-experimental and inter-clonal variation most strongly associated with nephron patterning. Even epithelial cells isolated from organoids showed transcriptional alignment with total organoids of the same day of differentiation. This data provides a framework for managing experimental variation, thereby increasing the utility of this approach for personalised medicine and functional genomics.

  10. Tissue engineering toward organ-specific regeneration and disease modeling

    Mandrycky, C; Phong, K; Zheng, Y. MRS Communications . 7(3):332–347. September 2017.

    Tissue engineering has been recognized as a translational approach to replace damaged tissue or whole organs. Engineering tissue, however, faces an outstanding knowledge gap in the challenge to fully recapitulate complex organ-specific features. Major components, such as cells, matrix, and architecture, must each be carefully controlled to engineer tissue-specific structure and function that mimics what is found in vivo. Here we review different methods to engineer tissue, and discuss critical challenges in recapitulating the unique features and functional units in four major organs-the kidney, liver, heart, and lung, which are also the top four candidates for organ transplantation in the USA. We highlight advances in tissue engineering approaches to enable the regeneration of complex tissue and organ substitutes, and provide tissue-specific models for drug testing and disease modeling. We discuss the current challenges and future perspectives toward engineering human tissue models.

  11. WT1 Is Necessary for the Proliferation and Migration of Cells of Renin Lineage Following Kidney Podocyte Depletion.

    Kaverina, NV; Eng, DG; Largent, AD; Daehn, I; Chang, A; Gross, KW; Pippin, JW; Hohenstein, P; Shankland, SJ. Stem Cell Reports. . pii: S2213-6711(17):30377–6. September 2017.

    Wilms’ tumor suppressor 1 (WT1) plays an important role in cell proliferation and mesenchymal-epithelial balance in normal development and disease. Here, we show that following podocyte depletion in three experimental models, and in patients with focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, WT1 increased significantly in cells of renin lineage (CoRL). In an animal model of FSGS in RenWt1fl/fl reporter mice with inducible deletion of WT1 in CoRL, CoRL proliferation and migration to the glomerulus was reduced, and glomerular disease was worse compared with wild-type mice. To become podocytes, CoRL undergo mesenchymal-to-epithelial transformation (MET), typified by reduced staining for mesenchymal markers (MYH11, SM22, αSMA) and de novo expression of epithelial markers (E-cadherin and cytokeratin18). Evidence for changes in MET markers was barely detected in RenWt1fl/fl mice. Our results show that following podocyte depletion, WT1 plays essential roles in CoRL proliferation and migration toward an adult podocyte fate.

  12. Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds.

    Uzarski, JS; DiVito, MD; Wertheim, JA; Miller, WM. Biomaterials . June 2017.

    Precise measurement of cellularity within bioartificial tissues and extracellular matrix (ECM) scaffolds is necessary to augment rigorous characterization of cellular behavior, as accurate benchmarking of tissue function to cell number allows for comparison of data across experiments and between laboratories. Resazurin, a soluble dye that is reduced to highly fluorescent resorufin in proportion to the metabolic activity of a cell population, is a valuable, noninvasive tool to measure cell number. We investigated experimental conditions in which resazurin reduction is a reliable indicator of cellularity within three-dimensional (3D) ECM scaffolds. Using three renal cell populations, we demonstrate that correlation of viable cell numbers with the rate of resorufin generation may deviate from linearity at higher cell densities, lower resazurin working volumes, or longer incubation times that all contribute to depleting the pool of resazurin. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout of metrics enumerating cellularity and growth within ECM scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and methodological recommendations presented herein may be used as a guide for application-specific optimization of this assay to obtain rigorous and accurate measurement of cellular content in bioengineered tissues.

  13. (Re)Building a Kidney.

    Oxburgh, L; Carroll, TJ; Cleaver, O; Gossett, DR; Hoshizaki, DK; Hubbell, JA; Humphreys, BD; Jain, S; Jensen, J; Kaplan, DL; Kesselman, C; Ketchum, CJ; Little, MH; McMahon, AP; Shankland, SJ; Spence, JR; Valerius, MT; Wertheim, JA; Wessely, O; Zheng, Y; Drummond, IA. J Am Soc Nephrol . 28(5):1370–1378. May 2017.

    (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

  14. Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury.

    Kramann, Rafael; Wongboonsin, Janewit; Chang-Panesso, Monica; Machado, Flavia G; Humphreys, Benjamin D. J Am Soc Nephrol . 2017.

    Peritubular capillary rarefaction is hypothesized to contribute to the increased risk of future CKD after AKI. Here, we directly tested the role of Gli1+ kidney pericytes in the maintenance of peritubular capillary health, and the consequences of pericyte loss during injury. Using bigenic Gli1-CreERt2; R26tdTomato reporter mice, we observed increased distance between Gli1+ pericytes and endothelial cells after AKI (mean6 SEM: 3.360.1 mm before injury versus 12.560.2 mm after injury; P,0.001). Using a genetic ablation model, we asked whether pericyte loss alone is sufficient for capillary destabilization. Ten days after pericyte ablation, we observed endothelial cell damage by electron microscopy. Furthermore, pericyte loss led to significantly reduced capillary number at later time points (mean6SEM capillaries/high-power field: 67.664.7 in control versus 44.164.8 at 56 days; P,0.05) and increased cross-sectional area (mean6 SEM: 21.960.4 mm2 in control versus 24.160.6 mm2 at 10 days; P,0.01 and 24.66 0.6 mm2 at 56 days; P,0.001). Pericyte ablation also led to hypoxic focal and subclinical tubular injury, reflected by transient expression of Kim1 and vimentin in scattered proximal tubule segments. This analysis provides direct evidence that AKI causes pericyte detachment from capillaries, and that pericyte loss is sufficient to trigger transient tubular injury and permanent peritubular capillary rarefaction.

  15. Comparative analysis of kidney organoid and adult human kidney single cell and single nucleus transcriptomes

    Wu, Haojia; Uchimura, Kohei; Donnelly, Erinn; Kirita, Yuhei; Morris, Samantha A; Humphreys, Benjamin D. bioRxiv . January 2017.

    Kidney organoids differentiated from human pluripotent stem cells hold great promise for understanding organogenesis, modeling disease and ultimately as a source of replacement tissue. Realizing the full potential of this technology will require better differentiation strategies based upon knowledge of the cellular diversity and differentiation state of all cells within these organoids. Here we analyze single cell gene expression in 45,227 cells isolated from 23 organoids differentiated using two different protocols. Both generate kidney organoids that contain a diverse range of kidney cells at differing ratios as well as non-renal cell types. We quantified the differentiation state of major organoid kidney cell types by comparing them against a 4,259 single nucleus RNA-seq dataset generated from adult human kidney, revealing immaturity of all kidney organoid cell types. We reconstructed lineage relationships during organoid differentiation through pseudotemporal ordering, and identified transcription factor networks associated with fate decisions. These results define impressive kidney organoid cell diversity, identify incomplete differentiation as a major roadblock for current directed differentiation protocols and provide a human adult kidney snRNA-seq dataset against which to benchmark future progress.

2016

  1. Sex-dependent expression of TRPV1 in bladder arterioles

    Phan, TX; Ton, HT; Chen, Y; Basha, ME; Ahern, GP. Am J Physiol Renal Physiol . 311(5):F1063–F1073. November 2016.

  2. Molecular characterization of the genital organizer: Gene expression profile of the mouse urethral plate epithelium.

    Armfield, BA; Seifert, AW; Zheng, Z; Merton, EM; Rock, JR; Lopez, MC; Baker, HV; Cohn, MJ. J Urol . 196(4):1295–302. October 2016.

  3. Uroplakin 1b is critical in urinary tract development and urothelial differentiation and homeostasis

    Carpenter, AR; Becknell, MB; Ching, CB; Cuaresma, EJ; Chen, X; Hains, DS; McHugh, K. Kidney Int . 89(3):612–24. March 2016.

  4. Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.

    O’Brien, LL; Guo, Q; Lee, Y; Tran, T; Benazet, JD; Whitney, PH; Valouev, A; McMahon, AP. Development . 143(4):595–608. February 2016.

    Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.

  5. Development of the Mammalian Kidney

    McMahon, AP. Curr Top Dev Biol . 117:31–64. January 2016.

    The basic unit of kidney function is the nephron. In the mouse, around 14,000 nephrons form in a 10-day period extending into early neonatal life, while the human fetus forms the adult complement of nephrons in a 32-week period completed prior to birth. This review discusses our current understanding of mammalian nephrogenesis: the contributing cell types and the regulatory processes at play. A conceptual developmental framework has emerged for the mouse kidney. This framework is now guiding studies of human kidney development enabled in part by in vitro systems of pluripotent stem cell-seeded nephrogenesis. A near future goal will be to translate our developmental knowledge-base to the productive engineering of new kidney structures for regenerative medicine.

  6. A strategy for generating kidney organoids: Recapitulating the development in human pluripotent stem cells.

    Takasato, M; Little, MH. Dev Biol . 420(2):210–220. December 2016.

    Directed differentiation of human pluripotent stem cells (hPSCs) can provide us any required tissue/cell types by recapitulating the development in vitro. The kidney is one of the most challenging organs to generate from hPSCs as the kidney progenitors are composed of at least 4 different cell types, including nephron, collecting duct, endothelial and interstitium progenitors, that are developmentally distinguished populations. Although the actual developmental process of the kidney during human embryogenesis has not been clarified yet, studies using model animals accumulated knowledge about the origins of kidney progenitors. The implications of these findings for the directed differentiation of hPSCs into the kidney include the mechanism of the intermediate mesoderm specification and its patterning along with anteroposterior axis. Using this knowledge, we previously reported successful generation of hPSCs-derived kidney organoids that contained all renal components and modelled human kidney development in vitro. In this review, we explain the developmental basis of the strategy behind this differentiation protocol and compare strategies of studies that also recently reported the induction of kidney cells from hPSCs. We also discuss the characterization of such kidney organoids and limitations and future applications of this technology.

  7. Generation of kidney organoids from human pluripotent stem cells.

    Takasat, M; Er, PX; Chiu, HS; Little, MH. Nat Protoc . 11(9):1681–92. September 2016.

    The human kidney develops from four progenitor populations-nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors-resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.

  8. A Plumbing Solution for Stem Cell-Derived Kidneys

    Ó\hAinmhire, Eoghainín; Humphreys, Benjamin D. Transplantation . 100(1):3–4. January 2016.

2015

  1. Developing a functional urinary bladder: a neuronal context

    Keast, JR; Smith-Anttila, CJ; Osborne, PB. Front Cell Dev Biol . 3:53. September 2015.

    The development of organs occurs in parallel with the formation of their nerve supply. The innervation of pelvic organs (lower urinary tract, hindgut, and sexual organs) is complex and we know remarkably little about the mechanisms that form these neural pathways. The goal of this short review is to use the urinary bladder as an example to stimulate interest in this question. The bladder requires a healthy mature nervous system to store urine and release it at behaviorally appropriate times. Understanding the mechanisms underlying the construction of these neural circuits is not only relevant to defining the basis of developmental problems but may also suggest strategies to restore connectivity and function following injury or disease in adults. The bladder nerve supply comprises multiple classes of sensory, and parasympathetic or sympathetic autonomic effector (motor) neurons. First, we define the developmental endpoint by describing this circuitry in adult rodents. Next we discuss the innervation of the developing bladder, identifying challenges posed by this area of research. Last we provide examples of genetically modified mice with bladder dysfunction and suggest potential neural contributors to this state.

  2. Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors

    Ton, HT; Smart, AE; Aguilar, BL; Olson, TT; Kellar, KJ; Ahern, GP. Mol Pharmacol . 88(2):256–64. August 2015.

    The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine’s actions in the brain. We examined menthol’s effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.

  3. An illustrated anatomical ontology of the developing mouse lower urogenital tract

    Georgas, KM; Armstrong, J; Keast, JR; Larkins, CE; McHugh, KM; Southard-Smith, EM; Cohn, MJ; Batourina, E; Dan, H; Schneider, K; Buehler, DP; Wiese, CB; Brennan, J; Davies, JA; Harding, SD; Baldock, RA; Little, MH; Vezina, CM; Mendelsohn, C. Development . 142(10):1893–908. May 2015.

    Malformation of the urogenital tract represents a considerable paediatric burden, with many defects affecting the lower urinary tract (LUT), genital tubercle and associated structures. Understanding the molecular basis of such defects frequently draws on murine models. However, human anatomical terms do not always superimpose on the mouse, and the lack of accurate and standardised nomenclature is hampering the utility of such animal models. We previously developed an anatomical ontology for the murine urogenital system. Here, we present a comprehensive update of this ontology pertaining to mouse LUT, genital tubercle and associated reproductive structures (E10.5 to adult). Ontology changes were based on recently published insights into the cellular and gross anatomy of these structures, and on new analyses of epithelial cell types present in the pelvic urethra and regions of the bladder. Ontology changes include new structures, tissue layers and cell types within the LUT, external genitalia and lower reproductive structures. Representative illustrations, detailed text descriptions and molecular markers that selectively label muscle, nerves/ganglia and epithelia of the lower urogenital system are also presented. The revised ontology will be an important tool for researchers studying urogenital development/malformation in mouse models and will improve our capacity to appropriately interpret these with respect to the human situation.

2014

  1. Translational profiles of medullary myofibroblasts during kidney fibrosis.

    Grgic, Ivica; Krautzberger, A. Michaela; Hofmeister, Andreas; Lalli, Matthew; DiRocco, Derek P.; Fleig, Susanne V.; Liu, Jing; Duffield, Jeremy S.; McMahon, Andrew P.; Aronow, Bruce; Humphreys, Benjamin D.. J Am Soc Nephrol . 25(9):1979–1990. September 2014.

    Myofibroblasts secrete matrix during chronic injury, and their ablation ameliorates fibrosis. Development of new biomarkers and therapies for CKD will be aided by a detailed analysis of myofibroblast gene expression during the early stages of fibrosis. However, dissociating myofibroblasts from fibrotic kidney is challenging. We therefore adapted translational ribosome affinity purification (TRAP) to isolate and profile mRNA from myofibroblasts and their precursors during kidney fibrosis. We generated and characterized a transgenic mouse expressing an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit protein under control of the collagen1alpha1 promoter. We developed a one-step procedure for isolation of polysomal RNA from collagen1alpha1-eGFPL10a mice subject to unilateral ureteral obstruction and analyzed and validated the resulting transcriptional profiles. Pathway analysis revealed strong gene signatures for cell proliferation, migration, and shape change. Numerous novel genes and candidate biomarkers were upregulated during fibrosis, specifically in myofibroblasts, and we validated these results by quantitative PCR, in situ, and Western blot analysis. This study provides a comprehensive analysis of early myofibroblast gene expression during kidney fibrosis and introduces a new technique for cell-specific polysomal mRNA isolation in kidney injury models that is suited for RNA-sequencing technologies.

  2. Single cell dissection of early kidney development: multilineage priming.

    Brunskill, Eric W.; Park, Joo-Seop; Chung, Eunah; Chen, Feng; Magella, Bliss; Potter, S. Steven. Development . 141(15):3093–3101. August 2014.

    We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction.

  3. Cell-specific translational profiling in acute kidney injury.

    Liu, Jing; Krautzberger, A. Michaela; Sui, Shannan H.; Hofmann, Oliver M.; Chen, Ying; Baetscher, Manfred; Grgic, Ivica; Kumar, Sanjeev; Humphreys, Benjamin D.; Hide, Winston A.; McMahon, Andrew P.. J Clin Invest . 124(3):1242–1254. March 2014.

    Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specific cell types. This strategy relies on CRE recombinase-dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with cell type-specific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific injury signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.

  4. Induction and patterning of the metanephric nephron

    O’Brien, LL; McMahon, AP. Semin Cell Dev Biol . 36:31–8. December 2014.

    The functional unit of the mammalian metanephric kidney is the nephron: a complex tubular structure dedicated to blood filtration and maintenance of several important physiological functions. Nephrons are assembled from a nephron-restricted pool of mesenchymal progenitors over an extensive developmental period that is completed prior to (human), or shortly after (mouse), birth. An appropriate balance in the expansion and commitment of nephron progenitors to nephron formation is essential for normal kidney function. Too few nephrons increase risk of kidney disease later in life while the failure of normal progenitor differentiation in Wilm’s tumor patients leads to massive growth of a nephroblast population often necessitating surgical removal of the kidney. An inductive process within the metanephric mesenchyme leads to the formation of a pretubular aggregate which transitions into an epithelial renal vesicle: the precursor for nephron assembly. Growth, morphogenesis and patterning transform this simple cyst-like structure into a highly elongated mature nephron with distinct cell types positioned along a proximal (glomerular) to distal (connecting segment) axis of functional organization. This review discusses our current understanding of the specification, maintenance and commitment of nephron progenitors, and the regulatory processes that transform the renal vesicle into a nephron.

  5. Embryonic origin and compartmental organization of the external genitalia

    Herrera, AM; Cohn, MJ. Sci Rep . 4:6896. November 2014.

    Genital malformations occur at a high frequency in humans, affecting ~1:250 live births. The molecular mechanisms of external genital development are beginning to be identified; however, the origin of cells that give rise to external genitalia is unknown. Here we use cell lineage analysis to show that the genital tubercle, the precursor of the penis and clitoris, arises from two populations of progenitor cells that originate at the lateral edges of the embryo, at the level of the posterior hindlimb buds and anterior tail. During body wall closure, the left and right external genital progenitor pools are brought together at the ventral midline, where they form the paired genital swellings that give rise to the genital tubercle. Unexpectedly, the left and right external genital progenitor pools form two lineage-restricted compartments in the phallus. Together with previous lineage studies of limb buds, our results indicate that, at the pelvic level, the early lateral mesoderm is regionalized from medial to lateral into dorsal limb, ventral limb, and external genital progenitor fields. These findings have implications for the evolutionary diversification of external genitalia and for the association between external genital defects and disruption of body wall closure, as seen in the epispadias-extrophy complex.

  6. Defining the acute kidney injury and repair transcriptome

    Kumar, S; Liu, J; McMahon, AP. Semin Nephrol . 34(4):404–17. July 2014.

    The mammalian kidney has an intrinsic ability to repair after significant injury. However, this process is inefficient: patients are at high risk for the loss of kidney function in later life. No therapy exists to treat established acute kidney injury (AKI) per se: strategies to promote endogenous repair processes and retard associated fibrosis are a high priority. Whole-organ gene expression profiling has been used to identify repair responses initiated with AKI, and factors that may promote the transition from AKI to chronic kidney disease. Transcriptional profiling has shown molecular markers and potential regulatory pathways of renal repair. Activation of a few key developmental pathways has been reported during repair. Whether these are comparable networks with similar target genes with those in earlier nephrogenesis remains unclear. Altered microRNA profiles, persistent tubular injury responses, and distinct late inflammatory responses highlight continuing kidney pathology. Additional insights into injury and repair processes will be gained by study of the repair transcriptome and cell-specific translatome using high-resolution technologies such as RNA sequencing and translational profiling tailored to specific cellular compartments within the kidney. An enhanced understanding holds promise for both the identification of novel therapeutic targets and biomarker-based evaluation of the damage-repair process.

  7. Formation and regeneration of the urothelium

    Yamany, T; Van Batavia, J; Mendelsohn, C. Curr Opin Organ Transplant . 19(3):323–30. June 2014.

    PURPOSE OF REVIEW: This review addresses significant changes in our understanding of urothelial development and regeneration. Understanding urothelial differentiation will be important in the push to find new methods of bladder reconstruction and augmentation, as well as identification of bladder cancer stem cells. RECENT FINDINGS: This review will cover recent findings including the identification of novel progenitor cells in the embryo and adult urothelium, function of the urothelium, and regeneration of the urothelium. Using Cre-lox recombination with cell-type-specific Cre lines, lineage studies from our laboratory have revealed novel urothelial cell types and progenitors that are critical for formation and regeneration of the urothelium. Interestingly, our studies indicate that Keratin-5-expressing basal cells, which have previously been proposed to be urothelial stem cells, are a self-renewing unipotent population, whereas P-cells, a novel urothelial cell type, are progenitors in the embryo, and intermediate cells serve as a progenitor pool in the adult. SUMMARY: These findings could have important implications for our understanding of cancer tumorigenesis and could move the fields of regeneration and reconstruction forward.

  8. Defining kidney biology to understand renal disease

    Little, MH; Brown, D.; Humphreys, BD; McMahon, AP; Miner, JH; Sands, JM; Weisz, OA; Mullins, C; Hoshizaki, D; Kidney Research National Dialogue, (KRND). Clin J Am Soc Nephrol . 9(4):809–11. April 2014.

    The Kidney Research National Dialogue represents a novel effort by the National Institute of Diabetes and Digestive and Kidney Diseases to solicit and prioritize research objectives from the renal research and clinical communities. The present commentary highlights selected scientific opportunities specific to the study of renal development, physiology, and cell biology. Describing such fundamental kidney biology serves as a necessary foundation for translational and clinical studies that will advance disease care and prevention. It is intended that these objectives foster and focus scientific efforts in these areas in the coming decade and beyond.

2013

  1. Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract

    Keil, KP; Altmann, HM; Mehta, V; Abler, LL; Elton, EA; Vezina, CM. Gene Expr Patterns . 13(8):413–24. December 2013.

    The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltransferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.

  2. Retinoid signaling in progenitors controls specification and regeneration of the urothelium

    Gandhi, D; Molotkov, A; Batourina, E; Schneider, K; Dan, H; Reiley, M; Laufer, E; Metzger, D; Liang, F; Liao, Y; Sun, TT; Aronow, B; Rosen, R; Mauney, J; Adam, R; Rosselot, C; Van Batavia, J; McMahon, AP; McMahon, J; Guo, JJ; Mendelsohn, C. Dev Cell . 26(5):469–482. September 2013.

    The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

2012

  1. Six2 and Wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks.

    Park, Joo-Seop; Ma, Wenxiu; O’Brien, Lori L.; Chung, Eunah; Guo, Jin-Jin; Cheng, Jr-Gang; Valerius, M. Todd; McMahon, Jill A.; Wong, Wing Hung; McMahon, Andrew P.. Dev Cell . 23(3):637–651. September 2012.

    A balance between Six2-dependent self-renewal and canonical Wnt signaling-directed commitment regulates mammalian nephrogenesis. Intersectional studies using chromatin immunoprecipitation and transcriptional profiling identified direct target genes shared by each pathway within nephron progenitors. Wnt4 and Fgf8 are essential for progenitor commitment; cis-regulatory modules flanking each gene are cobound by Six2 and beta-catenin and are dependent on conserved Lef/Tcf binding sites for activity. In vitro and in vivo analyses suggest that Six2 and Lef/Tcf factors form a regulatory complex that promotes progenitor maintenance while entry of beta-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and beta-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.

  2. Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice.

    Hoshi, Masato; Batourina, Ekatherina; Mendelsohn, Cathy; Jain, Sanjay. Development . 139(13):2405–2415. July 2012.

    Mutations in the receptor tyrosine kinase RET are associated with congenital anomalies of kidneys or urinary tract (CAKUT). RET tyrosine Y1015 is the docking site for PLCgamma, a major regulator of RET signaling. Abrogating signaling via Y1015 causes CAKUT that are markedly different than renal agenesis in Ret-null or RetY1062F mutant mice. We performed analysis of Y1015F mutant upper and lower urinary tracts in mice to delineate its molecular and developmental roles during early urinary tract formation. We found that the degeneration of the common nephric ducts (CND), the caudal-most Wolffian duct (WD) segment, depends on Y1015 signals. The CNDs in Y1015F mutants persist owing to increased proliferation and reduced apoptosis, and showed abundance of phospho-ERK-positive cells. In the upper urinary tract, the Y1015 signals are required for proper patterning of the mesonephros and metanephros. Timely regression of mesonephric mesenchyme and proper demarcation of mesonephric and metanephric mesenchyme from the WD depends on RetY1015 signaling. We show that the mechanism of de novo ectopic budding is via increased ERK activity due to abnormal mesenchymal GDNF expression. Although reduction in GDNF dosage improved CAKUT it did not affect delayed mesenchyme regression. Experiments using whole-mount immunofluorescence confocal microscopy and explants cultures of early embryos with ERK-specific inhibitors suggest an imbalance between increased proliferation, decreased apoptosis and increased ERK activity as a mechanism for WD defects in RetY1015F mice. Our work demonstrates novel inhibitory roles of RetY1015 and provides a possible mechanistic explanation for some of the confounding broad range phenotypes in individuals with CAKUT.

  3. Wnt inhibitory factor 1 (Wif1) is regulated by androgens and enhances androgen-dependent prostate development

    Keil, KP; Mehta, V; Branam, AM; Abler, LL; Bresh-Stiemke, RA; Joshi, PS; Schmitz, CT; Marker, PC; Vezina, CM. Endocrinology . 153(12):6091–103. December 2012.

    Fetal prostate development from urogenital sinus (UGS) epithelium requires androgen receptor (AR) activation in UGS mesenchyme (UGM). Despite growing awareness of sexually dimorphic gene expression in the UGS, we are still limited in our knowledge of androgen-responsive genes in UGM that initiate prostate ductal development. We found that WNT inhibitory factor 1 (Wif1) mRNA is more abundant in male vs. female mouse UGM in which its expression temporally and spatially overlaps androgen-responsive steroid 5α-reductase 2 (Srd5a2). Wif1 mRNA is also present in prostatic buds during their elongation and branching morphogenesis. Androgens are necessary and sufficient for Wif1 expression in mouse UGS explant mesenchyme, and testicular androgens remain necessary for normal Wif1 expression in adult mouse prostate stroma. WIF1 contributes functionally to prostatic bud formation. In the presence of androgens, exogenous WIF1 protein increases prostatic bud number and UGS basal epithelial cell proliferation without noticeably altering the pattern of WNT/β-catenin-responsive Axin2 or lymphoid enhancer binding factor 1 (Lef1) mRNA. Wif1 mutant male UGSs exhibit increased (Sfrp)2 and (Sfrp)3 expression and form the same number of prostatic buds as the wild-type control males. Collectively our results reveal Wif1 as one of the few known androgen-responsive genes in the fetal mouse UGM and support the hypothesis that androgen-dependent Wif1 expression is linked to the mechanism of androgen-induced prostatic bud formation.

  4. Visualization and quantification of mouse prostate development by in situ hybridization

    Keil, KP; Mehta, V; Abler, LL; Joshi, PS; Schmitz, CT; Vezina, CM. Differentiation . 84(3):232–9. October 2012.

    The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three different prostatic bud marking riboprobes: a previously identified prostatic bud marker, NK-3 transcription factor, locus 1 (Nkx3-1), and two newly identified prostatic bud markers, wingless-related MMTV integration site 10b (Wnt10b) and ectodysplasin-A receptor (Edar). We calculated total buds formed per UGS and the proportion marked by each mRNA after male UGS development in vivo and male and female UGS development in vitro. Nkx3-1 was first to mark the prostate field during UGS development in vivo but all three mRNAs marked prostatic buds during later developmental stages. The mRNAs localized to different domains: Nkx3-1 was present along about half the prostatic bud length while Edar and Wnt10b were restricted to distal bud tips. None of the mRNAs marked all buds formed in vitro and the proportion marked was developmental stage- and gender-dependent. Nkx3-1 marked the highest proportion of prostatic buds during in vitro UGS development. Together, our results reveal that ISH staining of mouse UGS can be used to quantify prostatic bud number, Nkx3-1 is currently the best suited riboprobe for this method, and female UGSs cannot be used interchangeably with male UGSs when conducting prostate development studies in vitro. We also found that Nkx3-1, Edar, and Wnt10b mark different prostatic bud regions and are likely to be useful in future studies of regional differences in prostatic bud gene expression.

  5. A genome-wide screen to identify transcription factors expressed in pelvic Ganglia of the lower urinary tract

    Wiese, CB; Ireland, S; Fleming, NL; Yu, J; Valerius, MT; Georgas, K; Chiu, HS; Brennan, J; Armstrong, J; Little, MH; McMahon, AP; Southard-Smith, EM. Front Neurosci . 6:130. September 2012.

    Relative positions of neurons within mature murine pelvic ganglia based on expression of neurotransmitters have been described. However the spatial organization of developing innervation in the murine urogenital tract (UGT) and the gene networks that regulate specification and maturation of neurons within the pelvic ganglia of the lower urinary tract (LUT) are unknown. We used whole-mount immunohistochemistry and histochemical stains to localize neural elements in 15.5 days post coitus (dpc) fetal mice. To identify potential regulatory factors expressed in pelvic ganglia, we surveyed expression patterns for known or probable transcription factors (TF) annotated in the mouse genome by screening a whole-mount in situ hybridization library of fetal UGTs. Of the 155 genes detected in pelvic ganglia, 88 encode TFs based on the presence of predicted DNA-binding domains. Neural crest (NC)-derived progenitors within the LUT were labeled by Sox10, a well-known regulator of NC development. Genes identified were categorized based on patterns of restricted expression in pelvic ganglia, pelvic ganglia and urethral epithelium, or pelvic ganglia and urethral mesenchyme. Gene expression patterns and the distribution of Sox10+, Phox2b+, Hu+, and PGP9.5+ cells within developing ganglia suggest previously unrecognized regional segregation of Sox10+ progenitors and differentiating neurons in early development of pelvic ganglia. Reverse transcription-PCR of pelvic ganglia RNA from fetal and post-natal stages demonstrated that multiple TFs maintain post-natal expression, although Pax3 is extinguished before weaning. Our analysis identifies multiple potential regulatory genes including TFs that may participate in segregation of discrete lineages within pelvic ganglia. The genes identified here are attractive candidate disease genes that may now be further investigated for their roles in malformation syndromes or in LUT dysfunction.

  6. An optimized procedure for fluorescence-activated cell sorting (FACS) isolation of autonomic neural progenitors from visceral organs of fetal mice

    Buehler, DP; Wiese, CB; Skelton, SB; Southard-Smith, EM. J Vis Exp . 66. August 2012.

    During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine and TH is also present in a subset of adult pelvic ganglia neurons . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (\textgreater70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

  7. Identification of molecular compartments and genetic circuitry in the developing mammalian kidney

    Yu, J; Valerius, MT; Duah, M; Staser, K; Hansard, JK; Guo, JJ; McMahon, J; Vaughan, J; Faria, D; Georgas, K; Rumballe, B; Ren, Q; Krautzberger, AM; Junker, JP; Thiagarajan, RD; Machanick, P; Gray, PA; van Oudenaarden, A; Rowitch, DH; Stiles, CD; Ma, Q; Grimmond, SM; Bailey, TL; Little, MH; McMahon, AP. Development . 139(10):1863–73. May 2012.

    Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.

  8. Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways

    Thiagarajan, RD; Georgas, KM; Rumballe, BA; Lesieur, E; Chiu, HS; Taylor, D; Tang, DT; Grimmond, SM; Little, MH. PLoS ONE . 6(2). February 2012.

    The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as ’anchor’ genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.

  9. Temporal transcriptional profiling of somatic and germ cells reveals biased lineage priming of sexual fate in the fetal mouse gonad

    Jameson, SA; Natarajan, A; Cool, J; DeFalco, T; Maatouk, DM; Mork, L; Munger, SC; Capel, B. PLoS Genet . 8(3). 2012.

    The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.

  10. Identification of novel markers of mouse fetal ovary development

    Chen, H; Palmer, JS; Thiagarajan, RD; Dinger, ME; Lesieur, E; Chiu, H; Schulz, A; Spiller, C; Grimmond, SM; Little, MH; Koopman, P; Wilhelm, D. PLoS ONE . 7(7). 2012.

    In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.

  11. Use of in situ hybridization to examine gene expression in the embryonic, neonatal, and adult urogenital system

    Rumballe, BA; Chiu, HS; Georgas, KM; Little, MH. Methods Mol Biol . 886:223–39. 2012.

    Studies into the molecular basis of morphogenesis frequently begin with investigations into gene expression across time and cell type in that organ. One of the most anatomically informative approaches to such studies is the use of in situ hybridization, either of intact or histologically sectioned tissues. Here, we describe the optimization of this approach for use in the temporal and spatial analysis of gene expression in the urogenital system, from embryonic development to the postnatal period. The methods described are applicable for high throughput analysis of large gene sets. As such, ISH has become a powerful technique for gene expression profiling and is valuable for the validation of profiling analyses performed using other approaches such as microarrays.

  12. Access and use of the GUDMAP database of genitourinary development

    Davies, JA; Little, MH; Aronow, B; Armstrong, J; Brennan, J; Lloyd-MacGilp, S; Armit, C; Harding, S; Piu, X; Roochun, Y; Haggarty, B; Houghton, D; Davidson, D; Baldock, R. Methods Mol Biol . 886:185–201. 2012.

    The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene expression profiling (from laser-dissected and FACS-sorted samples) and in situ hybridization at both low (whole-mount) and high (section) resolutions. Data are annotated to a published, high-resolution anatomical ontology and can be accessed using a variety of search interfaces. Here, we explain how to run typical queries on the database, by gene or anatomical location, how to view data, how to perform complex queries, and how to submit data.

2011

  1. Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways.

    Thiagarajan, Rathi D.; Georgas, Kylie M.; Rumballe, Bree A.; Lesieur, Emmanuelle; Chiu, Han Sheng; Taylor, Darrin; Tang, Dave T. P.; Grimmond, Sean M.; Little, Melissa H.. PLoS One . 6(2):e17286. February 2011.

    The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as ’anchor’ genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARgamma:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.

  2. Nephron formation adopts a novel spatial topology at cessation of nephrogenesis

    Rumballe, BA; Georgas, KM; Combes, AN; Ju, AL; Gilbert, T; Little, MH. Dev Biol . 360(1):110–22. December 2011.

    Nephron number in the mammalian kidney is known to vary dramatically, with postnatal renal function directly influenced by nephron complement. What determines final nephron number is poorly understood but nephron formation in the mouse kidney ceases within the first few days after birth, presumably due to the loss of all remaining nephron progenitors via epithelial differentiation. What initiates this event is not known. Indeed, whether nephron formation occurs in the same way at this time as during embryonic development has also not been examined. In this study, we investigate the key cellular compartments involved in nephron formation; the ureteric tip, cap mesenchyme and early nephrons; from postnatal day (P) 0 to 6 in the mouse. High resolution analyses of gene and protein expression indicate that loss of nephron progenitors precedes loss of ureteric tip identity, but show spatial shifts in the expression of cap mesenchyme genes during this time. In addition, cap mesenchymal volume and rate of proliferation decline prior to birth. Section-based 3D modeling and Optical Projection Tomography revealed a burst of ectopic nephron induction, with the formation of multiple (up to 5) nephrons per ureteric tip evident from P2. While the distal-proximal patterning of these nephrons occurred normally, their spatial relationship with the ureteric compartment was altered. We propose that this phase of nephron formation represents an acceleration of differentiation within the cap mesenchyme due to a displacement of signals within the nephrogenic niche.

  3. Atlas of Wnt and R-spondin gene expression in the developing male mouse lower urogenital tract

    Mehta, V; Abler, LL; Keil, KP; Schmitz, CT; Joshi, PS; Vezina, CM. Dev Dyn . 240(11):2548–60. November 2011.

    Prostate development is influenced by β-catenin signaling, but it is unclear which β-catenin activators are involved, where they are synthesized, and whether their mRNA abundance is influenced by androgens. We identified WNT/β-catenin-responsive β-galactosidase activity in the lower urogenital tract (LUT) of transgenic reporter mice, but β-galactosidase activity differed among the four mouse strains we examined. We used in situ hybridization to compare patterns of Wnts, r-spondins (Rspos, co-activators of β-catenin signaling), β-catenin-responsive mRNAs, and an androgen receptor-responsive mRNA in wild type fetal male, fetal female, and neonatal male LUT. Most Wnt and Rspo mRNAs were present in LUT during prostate development. Sexually dimorphic expression patterns were observed for WNT/β-catenin-responsive genes, and for Wnt2b, Wnt4, Wnt7a, Wnt9b, Wnt10b, Wnt11, Wnt16, and Rspo3 mRNAs. These results reveal sexual differences in WNT/β-catenin signaling in fetal LUT, supporting the idea that this pathway may be directly or indirectly responsive to androgens during prostate ductal development.

  4. A high-resolution molecular atlas of the fetal mouse lower urogenital tract

    Abler, LL; Keil, KP; Mehta, V; Joshi, PS; Schmitz, CT; Vezina, CM. Dev Dyn . 240(10):2364–77. October 2011.

    Epithelial-stromal interactions in the lower urogenital tract (LUT) are integral to prostatic and seminal vesicle development in males, vaginal and uterine development in females, and urethral development in both sexes. Gene expression profiling of isolated LUT stroma and epithelium has unraveled mechanisms of LUT development, but such studies are confounded by heterogeneous and ill-defined cell sub-populations contained within each tissue compartment. We used in situ hybridization to synthesize a high-resolution molecular atlas of 17-day post-coitus fetal mouse LUT. We identified mRNAs that mark selective cell populations of the seminal vesicle, ejaculatory duct, prostate, urethra, and vagina, subdividing these tissues into 16 stromal and 8 epithelial sub-compartments. These results provide a powerful tool for mapping LUT gene expression patterns and also reveal previously uncharacterized sub-compartments that may play mechanistic roles in LUT development of which we were previously unaware.

  5. Defining and redefining the nephron progenitor population

    Hendry, C; Rumballe, B; Moritz, K; Little, MH. Pediatr Nephrol . 26(9):1395–406. September 2011.

    It has long been appreciated that the mammalian kidney arises via reciprocal interactions between an epithelial ureteric epithelium and the surrounding metanephric mesenchyme. More recently, lineage tracing has confirmed that the portion of the metanephric mesenchyme closest to the advancing ureteric tips, the cap mesenchyme, represents the progenitor population for the nephron epithelia. This Six2(+)Cited1(+) population undergoes self-renewal throughout nephrogenesis while retaining the potential to epithelialize. In contrast, the Foxd1(+) portion of the metanephric mesenchyme shows no epithelial potential, developing instead into the interstitial, perivascular, and possibly endothelial elements of the kidney. The cap mesenchyme rests within a nephrogenic niche, surrounded by the stroma and the ureteric tip. While the role of Wnt signaling in nephron induction is known, there remains a lack of clarity over the intrinsic and extrinsic regulation of cap mesenchyme specification, self-renewal, and nephron potential. It is also not known what regulates cessation of nephrogenesis, but there is no nephron generation in response to injury during the postnatal period. In this review, we will examine what is and is not known about this nephron progenitor population and discuss how an increased understanding of the regulation of this population may better explain the observed variation in final nephron number and potentially facilitate the reinitiation or prolongation of nephron formation.

  6. Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling

    Thiagarajan, RD; Cloonan, N; Gardiner, BB; Mercer, TR; Kolle, G; Nourbakhsh, E; Wani, S; Tang, D; Krishnan, K; Georgas, KM; Rumballe, BA; Chiu, HS; Steen, JA; Mattick, JS; Little, MH; Grimmond, SM. BMC Genomics . 12:441. September 2011.

    BACKGROUND: The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic in situ screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models. RESULTS: To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3’ UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section in situ hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs. CONCLUSION: The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.

  7. A high throughput in situ hybridization method to characterize mRNA expression patterns in the fetal mouse lower urogenital tract

    Abler, LL; Mehta, V; Keil, KP; Joshi, PS; Flucus, CL; Hardin, HA; Schmitz, CT; Vezina, CM. J Vis Exp . 54. August 2011.

    Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions(1,2). Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer. The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma(3). This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals(4). The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators. Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

  8. Defining the molecular character of the developing and adult kidney podocyte

    Brunskill, EW; Georgas, K; Rumballe, B; Little, MH; Potter, SS. PLoS ONE . 6(9). 2011.

    BACKGROUND: The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease. METHODOLOGY/PRINCIPAL FINDINGS: In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined. CONCLUSIONS/SIGNIFICANCE: The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells.

  9. The GUDMAP database–an online resource for genitourinary research

    Harding, SD; Armit, C; Armstrong, J; Brennan, J; Cheng, Y; Haggarty, B; Houghton, D; Lloyd-MacGilp, S; Pi, X; Roochun, Y; Sharghi, M; Tindal, C; McMahon, AP; Gottesman, B; Little, MH; Georgas, K; Aronow, B; Potter, SS; Brunskill, EW; Southard-Smith, EM; Mendelsohn, C; Baldock, RA; Davies, JA; Davidson, D. Development . 138(13):2845–53. July 2011.

    The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.

  10. Expression of metanephric nephron-patterning genes in differentiating mesonephric tubules

    Georgas, KM; Chiu, HS; Lesieur, E; Rumballe, BA; Little, MH. Dev Dyn . 240(6):1600–12. June 2011.

    The metanephros is the functional organ in adult amniotes while the mesonephros degenerates. However, parallel tubulogenetic events are thought to exist between mesonephros and metanephros. Mesonephric tubules are retained in males and differentiate into efferent ducts of the male reproductive tract. By examining the murine mesonephric expression of markers of distinct stages and regions of metanephric nephrons during tubule formation and patterning, we provide further evidence to support this common morphogenetic mechanism. Renal vesicle, early proximal and distal tubule, loop of Henle, and renal corpuscle genes were expressed by mesonephric tubules. Vip, Slc6a20b, and Slc18a1 were male-specific. In contrast, mining of the GUDMAP database identified candidate late mesonephros-specific genes, 10 of which were restricted to the male. Among the male-specific genes are candidates for regulating ion/fluid balance within the efferent ducts, thereby regulating sperm maturation and genes marking tubule-associated neurons potentially critical for normal male reproductive tract function.

2010

  1. RET signaling is required for survival and normal function of nonpeptidergic nociceptors.

    Golden, Judith P.; Hoshi, Masato; Nassar, Mohammed A.; Enomoto, Hideki; Wood, John N.; Milbrandt, Jeffrey; Gereau, Robert W. 4th; Johnson, Eugene M. Jr; Jain, Sanjay. J Neurosci . 30(11):3983–3994. March 2010.

    Small unmyelinated sensory neurons classified as nociceptors are divided into two subpopulations based on phenotypic differences, including expression of neurotrophic factor receptors. Approximately half of unmyelinated nociceptors express the NGF receptor TrkA, and half express the GDNF family ligand (GFL) receptor Ret. The function of NGF/TrkA signaling in the TrkA population of nociceptors has been extensively studied, and NGF/TrkA signaling is a well established mediator of pain. The GFLs are analgesic in models of neuropathic pain emphasizing the importance of understanding the physiological function of GFL/Ret signaling in nociceptors. However, perinatal lethality of Ret-null mice has precluded the study of the physiological role of GFL/Ret signaling in the survival, maintenance, and function of nociceptors in viable mice. We deleted Ret exclusively in nociceptors by crossing nociceptor-specific Na(v)1.8 Cre and Ret conditional mice to produce Ret-Na(v)1.8 conditional knock-out (CKO) mice. Loss of Ret exclusively in nociceptors results in a reduction in nociceptor number and size, indicating that Ret signaling is important for the survival and trophic support of these cells. Ret-Na(v)1.8 CKO mice exhibit reduced epidermal innervation but normal central projections. In addition, Ret-Na(v)1.8 CKO mice have increased sensitivity to cold and increased formalin-induced pain, demonstrating that Ret signaling modulates the function of nociceptors in vivo. Enhanced inflammation-induced pain may be mediated by decreased prostatic acid phosphatase (PAP), as PAP levels are markedly reduced in Ret-Na(v)1.8 CKO mice. The results of this study identify the physiological role of endogenous Ret signaling in the survival and function of nociceptors.

  2. Comparative gene expression analysis of genital tubercle development reveals a putative appendicular Wnt7 network for the epidermal differentiation

    Chiu, HS; Szucsik, JC; Georgas, KM; Jones, JL; Rumballe, BA; Tang, D; Grimmond, SM; Lewis, AG; Aronow, B; Lessard, JL; Little, MH. Developmental Biology . 344(2):1071–87. August 2010.

    Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.

  3. Molecular anatomy of the kidney: what have we learned from gene expression and functional genomics?

    Rumballe, B; Georgas, K; Wilkinson, L; Little, MH. Pediatr Nephrol . 25(6):1005–16. June 2010.

    The discipline of paediatric nephrology encompasses the congenital nephritic syndromes, renal dysplasias, neonatal renal tumours, early onset cystic disease, tubulopathies and vesicoureteric reflux, all of which arise due to defects in normal kidney development. Indeed, congenital anomalies of the kidney and urinary tract (CAKUT) represent 20-30% of prenatal anomalies, occurring in 1 in 500 births. Developmental biologists have studied the anatomical and morphogenetic processes involved in kidney development for the last five decades. However, with the advent of transgenic mice, the sequencing of the genome, improvements in mutation detection and the advent of functional genomics, our understanding of the molecular basis of kidney development has grown significantly. Here we discuss how the advent of new genetic and genomics approaches has added to our understanding of kidney development and paediatric renal disease, as well as identifying areas in which we are still lacking knowledge.

2009

  1. The many faces of RET dysfunction in kidney.

    Jain, Sanjay. Organogenesis . 5(4):177–190. October 2009.

    Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret’s role in early kidney and urinary system development. Here we present a brief overview of the "many faces" of Ret dysfunction in kidney with particular emphasis on Ret’s signaling specificity and intergenic interactions that confer normal urinary system development.

  2. High-resolution gene expression analysis of the developing mouse kidney defines novel cellular compartments within the nephron progenitor population

    Mugford, JW; Yu, J; Kobayashi, A; McMahon, AP. Dev Biol . 333(2):312–23. September 2009.

    The functional unit of the kidney is the nephron. During its organogenesis, the mammalian metanephric kidney generates thousands of nephrons over a protracted period of fetal life. All nephrons are derived from a population of self-renewing multi-potent progenitor cells, termed the cap mesenchyme. However, our understanding of the molecular and cellular mechanisms underlying nephron development is at an early stage. In order to identify factors involved in nephrogenesis, we performed a high-resolution, spatial profiling of a number of transcriptional regulators expressed within the cap mesenchyme and early developing nephron. Our results demonstrate novel, stereotypic, spatially defined cellular sub-domains within the cap mesenchyme, which may, in part, reflect induction of nephron precursors. These results suggest a hitherto unappreciated complexity of cell states that accompany the assembly of the metanephric kidney, likely reflecting diverse regulatory actions such as the maintenance and induction of nephron progenitors.

  3. Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment.

    Georgas, K; Rumballe, B; Valerius, MT; Chiu, HS; Thiagarajan, RD; Lesieur, E; Aronow, B; Brunskill, EW; Combes, AN; Tang, D; Taylor, D; Grimmond, SM; Potter, SS; McMahon, AP; Little, MH. Dev Biol . 332(2):273–86. August 2009.

    While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.

  4. Three-dimensional visualization of testis cord morphogenesis, a novel tubulogenic mechanism in development

    Combes, AN; Lesieur, E; Harley, VR; Sinclair, AH; Little, MH; Wilhelm, D; Koopman, P. Dev Dyn . 238(5):1033–41. May 2009.

    Testis cords are specialized tubes essential for generation and export of sperm, yet the mechanisms directing their formation, and the regulation of their position, size, shape, and number remain unclear. Here, we use a novel fluorescence-based three-dimensional modeling approach to show that cords initially form as a network of irregular cell clusters that are subsequently remodeled to form regular parallel loops, joined by a flattened plexus at the mesonephric side. Variation in cord number and structure demonstrates that cord specification is not stereotypic, although cord alignment and diameter becomes relatively consistent, implicating compensatory growth mechanisms. Branched, fused, and internalized cords were commonly observed. We conclude that the tubule-like structure of testis cords arise through a novel form of morphogenesis consisting of coalescence, partitioning, and remodeling. The methods we describe are applicable to investigating defects in testis cord development in mouse models, and more broadly, studying morphogenesis of other tissues.

2008

  1. Atlas of gene expression in the developing kidney at microanatomic resolution

    Brunskill, EW; Aronow, B; Georgas, K; Rumballe, B; Valerius, MT; Aronow, B; Kaimal, V; Jegga, AG; Yu, J; Grimmond, SM; McMahon, AP; Patterson, LT; Little, MH; Potter, SS. Dev Cell . 15(5):781–91. November 2008.

    Kidney development is based on differential cell-type-specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genome-wide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis, and segmentation. We describe here a comprehensive gene expression atlas of the developing mouse kidney based on the isolation of each major compartment by either laser capture microdissection or fluorescence-activated cell sorting, followed by microarray profiling. The resulting data agree with known expression patterns and additional in situ hybridizations. This kidney atlas allows a comprehensive analysis of the progression of gene expression states during nephrogenesis, as well as discovery of potential growth factor-receptor interactions. In addition, the results provide deeper insight into the genetic regulatory mechanisms of kidney development.

  2. Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

    Georgas, K; Rumballe, B; Wilkinson, L; Chiu, HS; Lesieur, E; Gilbert, T; Little, MH. Histochem Cell Biol . 130(5):927–42. November 2008.

    The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

  3. High-throughput paraffin section in situ hybridization and dual immunohistochemistry on mouse tissues

    Rumballe, B; Georgas, K; Little, MH. CSH Protoc . 2008. July 2008.

    Section in situ hybridization (SISH) is a high-resolution tool used to analyze gene expression patterns. This protocol utilizes the Tecan Freedom EVO150 platform to perform high-throughput SISH on paraffin sections to detect mRNA with a digoxigenin (DIG)-labeled probe. The slide is mounted and imaged before performing immunohistochemistry (IHC) on the same section. The dual reaction enables a marker of protein expression to be localized on the same section as the mRNA and facilitates more accurate annotation of the gene expression.

  4. GUDMAP: the genitourinary developmental molecular anatomy project

    McMahon, AP; Aronow, B; Davidson, DR; Davies, JA; Gaido, KW; Grimmond, SM; Lessard, JL; Little, MH; Potter, SS; Wilder, EL; Zhang, P; GUDMAP, Project. J Am Soc Nephrol . 19(4):667–71. April 2008.

    In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.

2007

  1. A high-resolution anatomical ontology of the developing murine genitourinary tract

    Little, MH; Brennan, J; Georgas, K; Davies, JA; Davidson, DR; Baldock, RA; Beverdam, A; Bertram, JF; Capel, B; Chiu, HS; Clements, D; Cullen-McEwen, L; Fleming, J; Gilbert, T; Herzlinger, D; Houghton, D; Kaufman, MH; Kleymenova, E; Koopman, PA; Lewis, AG; McMahon, AP; Mendelsohn, C; Mitchell, EK; Rumballe, BA; Sweeney, DE; Valerius, MT; Yamada, G; Yang, Y; Yu, J. Gene Expr Patterns . 7(6):680–99. June 2007.

    Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.