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The Human Reference Atlas (HRA) aims to map all of the cells of the human body to advance biomedical research and clinical practice. This Perspective presents collaborative work by members of 16 international consortia on two essential and interlinked parts of the HRA: (1) three-dimensional representations of anatomy that are linked to (2) tables that name and interlink major anatomical structures, cell types, plus biomarkers (ASCT+B). We discuss four examples that demonstrate the practical utility of the HRA.

Kidney fibrosis constitutes the shared final pathway of nearly all chronic nephropathies, but biomarkers for the non-invasive assessment of kidney fibrosis are currently not available. To address this, we characterize five candidate biomarkers of kidney fibrosis: Cadherin-11 (CDH11), Sparc-related modular calcium binding protein-2 (SMOC2), Pigment epithelium-derived factor (PEDF), Matrix-Gla protein, and Thrombospondin-2. Gene expression profiles in single-cell and single-nucleus RNA-sequencing (sc/snRNA-seq) datasets from rodent models of fibrosis and human chronic kidney disease (CKD) were explored, and Luminex-based assays for each biomarker were developed. Plasma and urine biomarker levels were measured using independent prospective cohorts of CKD: the Boston Kidney Biopsy Cohort, a cohort of individuals with biopsy-confirmed semiquantitative assessment of kidney fibrosis, and the Seattle Kidney Study, a cohort of patients with common forms of CKD. Ordinal logistic regression and Cox proportional hazards regression models were used to test associations of biomarkers with interstitial fibrosis and tubular atrophy and progression to end-stage kidney disease and death, respectively. Sc/snRNA-seq data confirmed cell-specific expression of biomarker genes in fibroblasts. After multivariable adjustment, higher levels of plasma CDH11, SMOC2, and PEDF and urinary CDH11 and PEDF were significantly associated with increasing severity of interstitial fibrosis and tubular atrophy in the Boston Kidney Biopsy Cohort. In both cohorts, higher levels of plasma and urinary SMOC2 and urinary CDH11 were independently associated with progression to end-stage kidney disease. Higher levels of urinary PEDF associated with end-stage kidney disease in the Seattle Kidney Study, with a similar signal in the Boston Kidney Biopsy Cohort, although the latter narrowly missed statistical significance. Thus, we identified CDH11, SMOC2, and PEDF as promising non-invasive biomarkers of kidney fibrosis.

Organoids serve as a novel tool for disease modeling in three-dimensional multicellular contexts. Static organoids, however, lack the requisite biophysical microenvironment such as fluid flow, limiting their ability to faithfully recapitulate disease pathology. Here, we unite organoids with organ-on-a-chip technology to unravel disease pathology and develop therapies for autosomal recessive polycystic kidney disease. PKHD1 -mutant organoids-on-a-chip are subjected to flow that induces clinically relevant phenotypes of distal nephron dilatation. Transcriptomics discover 229 signal pathways that are not identified by static models. Mechanosensing molecules, RAC1 and FOS, are identified as potential therapeutic targets and validated by patient kidney samples. On the basis of this insight, we tested two U.S. Food and Drug Administration–approved and one investigational new drugs that target RAC1 and FOS in our organoid-on-a-chip model, which suppressed cyst formation. Our observations highlight the vast potential of organoid-on-a-chip models to elucidate complex disease mechanisms for therapeutic testing and discovery.

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Acute kidney injury impacts ∼13.3 million individuals and causes ∼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.

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.

Directed differentiation of human pluripotent stem cells (hPSCs) into functional ureteric and collecting duct (CD) epithelia is essential to kidney regenerative medicine. Here we describe highly efficient, serum-free differentiation of hPSCs into ureteric bud (UB) organoids and functional CD cells. The hPSCs are first induced into pronephric progenitor cells at 90% efficiency and then aggregated into spheres with a molecular signature similar to the nephric duct. In a three-dimensional matrix, the spheres form UB organoids that exhibit branching morphogenesis similar to the fetal UB and correct distal tip localization of RET expression. Organoid-derived cells incorporate into the UB tips of the progenitor niche in chimeric fetal kidney explant culture. At later stages, the UB organoids differentiate into CD organoids, which contain95% CD cell types as estimated by single-cell RNA sequencing. The CD epithelia demonstrate renal electrophysiologic functions, with ENaC-mediated vectorial sodium transport by principal cells and V-type ATPase proton pump activity by FOXI1-induced intercalated cells.

Kidney Precision Medicine Project (KPMP) is building a spatially-specified human tissue atlas at the single-cell resolution with molecular details of the kidney in health and disease. Here, we describe the construction of an integrated reference tissue map of cells, pathways and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 55 subjects. We use single-cell and -nucleus transcriptomics, subsegmental laser microdissection bulk transcriptomics and proteomics, near-single-cell proteomics, 3-D nondestructive and CODEX imaging, and spatial metabolomics data to hierarchically identify genes, pathways and cells. Integrated data from these different technologies coherently describe cell types/subtypes within different nephron segments and interstitium. These spatial profiles identify cell-level functional organization of the kidney tissue as indicative of their physiological functions and map different cell subtypes to genes, proteins, metabolites and pathways. Comparison of transcellular sodium reabsorption along the nephron to levels of mRNAs encoding the different sodium transporter genes indicate that mRNA levels are largely congruent with physiological activity.This reference atlas provides an initial framework for molecular classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

An important need exists to better understand and stratify kidney disease according to its underlying pathophysiology in order to develop more precise and effective therapeutic agents. National collaborative efforts such as the Kidney Precision Medicine Project are working towards this goal through the collection and integration of large, disparate clinical, biological and imaging data from patients with kidney disease. Ontologies are powerful tools that facilitate these efforts by enabling researchers to organize and make sense of different data elements and the relationships between them. Ontologies are critical to support the types of big data analysis necessary for kidney precision medicine, where heterogeneous clinical, imaging and biopsy data from diverse sources must be combined to define a patient's phenotype. The development of two new ontologies - the Kidney Tissue Atlas Ontology and the Ontology of Precision Medicine and Investigation - will support the creation of the Kidney Tissue Atlas, which aims to provide a comprehensive molecular, cellular and anatomical map of the kidney. These ontologies will improve the annotation of kidney-relevant data, and eventually lead to new definitions of kidney disease in support of precision medicine.

Recent studies demonstrated 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 kidneys of patients with chronic kidney disease, fibrotic lungs of patients with idiopathic pulmonary fibrosis, fibrotic human kidney organoids, and fibrotic 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 was sufficient to enhance glycolysis and increase alpha smooth muscle actin (αSMA) and collagen1 levels. Knockout of COUP-TFII decreased glycolysis and collagen1 levels in fibroblasts. Chip-qPCR assays revealed the binding of COUP-TFII on the promoter of PGC1α, a critical regulator of mitochondrial genesis and oxidative metabolism. Overexpression of COUP-TFII reduced the cellular level of PGC1α. In conclusion, COUP-TFII mediates fibrosis by serving as a key regulator of the shift in cellular metabolism of interstitial pericytes/fibroblasts from oxidative respiration to aerobic glycolysis. The fibrogenic response may share a common pathway in different organ injury and failure. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potential other organ fibrosis.

Background and Aims The endogenous repair process of the mammalian kidney allows rapid recovery after acute kidney injury (AKI) through robust proliferation of tubular epithelial cells. There is currently limited understanding of which transcriptional regulators activate these repair programs and how transcriptional dysregulation leads to maladaptive repair. Here we investigate the existence of enhancer dynamics in the regenerating mouse kidney. Method RNA-seq and ChIP-seq (H3K27ac, H3K4m3, BRD4, POL2 and selected transcription factors) were performed on samples from repairing kidney cortex 2 days after ischemia/reperfusion injury (IRI) to identify activated genes, transcription factors, enhancer and super-enhancers associated with kidney repair. Further we investigated the role of super-enhancer activation in kidney repair through pharmacological BET inhibition using the small molecule JQ1 in vitro and in acute kidney injury models in vivo. Results Response to kidney injury leads to genome-wide alteration in enhancer repertoire in-vivo. We identified 16,781 enhancer sites (H3K27ac and BRD4 positive, H3K4me3 negative binding) active in SHAM and IRI samples; 6,512 lost and 9,774 gained after IRI. The lost and gained enhancer sites can be annotated to 62% and 63% of down- and up-regulated transcripts at day 2 after kidney injury, respectively. Super-enhancer analysis revealed 164 lost and 216 gained super-enhancer sites at IRI day 2. 385 super-enhancers maintain activity before and after injury. ChIP-seq profiles of selected transcription factors based on motif analysis show specific binding at corresponding enhancer sites. We observed lost enhancer binding of HNF4A and GR mainly at kidney related enhancer elements. In contrast, STAT3 showed increased binding at injury induces enhancer elements. No dynamic was observed for STAT5. Both transcription factor groups show corresponding mRNA changes after injury. Pharmacological inhibition of enhancer and super-enhancer activity by BRD4 inhibition (JQ1: 50mg/kg/day) before IRI leads to suppression of 40% of injury-induced transcripts associated with cell cycle regulation and significantly increased mortality between days 2 and 3 after AKI. Conclusion This is the first demonstration of enhancer and super-enhancer function in the repairing kidney. In addition, our data call attention to potential caveats for use of small molecule inhibitors of BET proteins that are currently being tested in clinical trials in cancer patients who are at risk for AKI. Our analyses of enhancer dynamics after kidney injury in vivo have the potential to identify new targets for therapeutic intervention.

Formation of a functional kidney depends on the balance between renewal and differentiation of nephron progenitors. Failure to sustain this balance can lead to kidney failure or stem cell tumors. For nearly 60 years, we have known that signals from an epithelial structure known as the ureteric bud were essential for maintaining this balance. More recently it was discovered that one molecule, Wnt9b, was necessary for both renewal and differentiation of the nephron progenitor cells. How one ligand signaling through one transcription factor promoted two seemingly contradictory cellular processes was unclear. In this study, we show that Wnt9b/beta-catenin signaling alone is sufficient to promote both renewal and differentiation. Moreover, we show that discrete levels of beta-catenin can promote these two disparate fates, with low levels fostering progenitor renewal and high levels driving differentiation. These results provide insight into how Wnt9b regulates distinct target genes that balance nephron progenitor renewal and differentiation.

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.

Human pluripotent stem cells (hPSCs) represent a formidable tool for disease modeling, drug discovery, and regenerative medicine using human cells and tissues in vitro. Evolving techniques of targeted genome editing, specifically the CRISPR/Cas9 system, allow for the generation of cell lines bearing gene-specific knock-outs, knock-in reporters, and precise mutations. However, there are increasing concerns related to the transfection efficiency, cell viability, and maintenance of pluripotency provided by genome-editing techniques. The procedure presented here employs transient antibiotic selection that overcomes reduced transfection efficiency, avoids cytotoxic flow sorting for increased viability, and generates multiple genome-edited pluripotent hPSC lines expanded from a single parent cell. Avoidance of xenogeneic contamination from feeder cells and reduced operator workload, owing to single-cell passaging rather than clump passaging, are additional benefits. The outlined methods may enable researchers with limited means and technical experience to create human stem cell lines containing desired gene-specific mutations. © 2018 by John Wiley & Sons, Inc.

Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) may enable organ regeneration, disease modeling and drug screening. We report an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+ SALL1+ WT1+ PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+ LHX1+ renal vesicles that self-organize into nephron structures. In both two- and three-dimensional culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity.

The neonatal mouse kidney retains nephron progenitor cells in a nephrogenic zone for 3 days after birth. We evaluated whether de novo nephrogenesis can be induced postnatally beyond 3 days. Given the long-term implications of nephron number for kidney health, it would be useful to enhance nephrogenesis in the neonate. We induced nephron reduction by cryoinjury with or without contralateral nephrectomy during the neonatal period or after 1 week of age. There was no detectable compensatory de novo nephrogenesis, as determined by glomerular counting and lineage tracing. Contralateral nephrectomy resulted in additional adaptive healing, with little or no fibrosis, but did not also stimulate de novo nephrogenesis. In contrast, injury initiated at 1 week of age led to healing with fibrosis. Thus, despite the presence of progenitor cells and ongoing nephron maturation in the newborn mouse kidney, de novo nephrogenesis is not inducible by acute nephron reduction. This indicates that additional nephron progenitors cannot be recruited after birth despite partial renal ablation providing a reparative stimulus and suggests that nephron number in the mouse is predetermined at birth.

(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.

Nur77 and its family members Nurr1 and Nor-1 are inducible orphan nuclear receptors that orchestrate cellular responses to diverse extracellular signals. In epithelia, Nur77 can act as a potent proapoptotic molecule in response to cellular stress, suggesting a possible role for this nuclear receptor in the tissue response to injury. Here, we found that Nur77 promotes epithelial cell apoptosis after AKI. Injury of proximal tubular epithelial cells rapidly and strongly induced Nur77, Nor-1, and Nurr1 both in vitro and in vivo. After renal ischemia-reperfusion, Nurr77-deficient mice exhibited less apoptosis of tubular epithelial cells and better renal function than wild-type mice. Nur77-mediated renal injury involved a conformational change of Bcl2 and an increase in the protein levels of proapoptotic Bcl-xS. Ligand-activated retinoic acid receptors repressed Nur77 induction and function. Pretreatment of wild-type mice with retinoic acid before renal ischemia-reperfusion blunted the induction of Nur77, conferred protection of renal function, attenuated renal histologic injury, and reduced the expression of epithelial-derived proinflammatory cytokines. Retinoic acid also inhibited hypoxia-mediated induction of proinflammatory cytokines in cultured renal epithelial cells. Results obtained from proximal tubule cultures derived from Nur77-deficient mice suggested that the inhibition of Nur77 expression mediated the renoprotective effects of retinoic acid. In summary, Nur77 promotes epithelial apoptosis after ischemia-reperfusion injury, and retinoic acid-mediated inhibition of Nur77 expression is a promising therapeutic strategy for the prevention of AKI.

The primary excretory organ in vertebrates is the kidney, which is responsible for blood filtration, solute homeostasis and pH balance. These functions are carried out by specialized epithelial cells organized into tubules called nephrons. Each of these cell types arise during embryonic development from a mesenchymal stem cell pool through a process of mesenchymal-to-epithelial transition (MET) that requires sequential action of specific Wnt signals. Induction by Wnt9b directs cells to exit the stem cell niche and express Wnt4, which is both necessary and sufficient for the formation of epithelia. Without either factor, MET fails, nephrons do not form and newborn mice die owing to kidney failure. Ectopic Notch activation in stem cells induces mass differentiation and exhaustion of the stem cell pool. To investigate whether this reflected an interaction between Notch and Wnt, we employed a novel gene manipulation strategy in cultured embryonic kidneys. We show that Notch activation is capable of inducing MET in the absence of both Wnt4 and Wnt9b. Following MET, the presence of Notch directs cells primarily to the proximal tubule fate. Only nephron stem cells have the ability to undergo MET in response to Wnt or Notch, as activation in the closely related stromal mesenchyme has no inductive effect. These data demonstrate that stem cells for renal epithelia are uniquely poised to undergo MET, and that Notch activation can replace key inductive Wnt signals in this process. After MET, Notch provides an instructive signal directing cells towards the proximal tubule lineage at the expense of other renal epithelial fates.

Tissue regeneration and organ development from patient-induced pluripotent stem cells (iPSCs), created through cellular reprogramming,[1][1],[2][2] are new approaches for resolving the inadequate number of organs available for transplantation, including the kidney. Because iPSCs are derived from the

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×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.

The mature nephron forms from a simple epithelial vesicle into an elaborate structure with distinct regions of specialized physiological function. The molecular components driving the process of nephron development are not well understood. To identify genes that may be informative in this process we conducted a transcriptional profiling screen using Wnt4 mutant kidneys. In Wnt4 −/− homozygous mice, condensates and pretubular aggregates are induced, however, epithelial renal vesicles fail to form and subsequent tubulogenesis is blocked. A transcriptional profile comparison between wildtype and Wnt4−/− mutant kidneys at E14.5 was performed using Affymetrix oligonucleotide microarrays to identify nephron-expressed genes. This approach identified 236 genes with expression levels >1.8 fold higher in wildtype versus mutant kidneys, amongst these were a number of known nephron component markers confirming the validity of the screen. These results were further detailed by wholemount in situ hybridization (WISH) of E15.5 urogenital systems (UGS). We annotated the spatial expression pattern of these genes into eight categories of expression. Genes expressed in renal vesicle and their derivatives, structures absent in the mutant, accounted for the largest number of the observed expression patterns. A number of additional genes in areas not directly overlapping the Wnt4 expression domain were also identified including the cap mesenchyme, the collecting duct, and the cortical interstitium. This study provides a useful compendium of molecular markers for the study of nephrogenesis.

SummaryUnderstanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%–95% of tubular epithelial cells, but no interstitial cells, were labeled with either β-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney.

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications., Generating organized kidney tissues from human pluripotent stem cell is a major challenge. Here, Freedman et al. describe a differentiation system forming spheroids and tubular structures, characteristic of these kidney structures, and using CRISPR/Cas9, delete PKD1/2, to model polycystic kidney disease.

Clonal cell lines representing different developmental stages of the metanephric mesenchyme were made from transgenic mice with the Simian Virus 40 T-antigen (SV40 Tag) gene driven by the Hoxa 11 promoter. The resulting mK3 cell line represented early metanephric mesenchyme, prior to induction by the ureteric bud. These cells showed a spindle-shaped, fibroblast morphology. They expressed genes characteristic of early mesenchyme, including Hoxa 11, Hoxd 11, collagen I, and vimentin. Moreover, the mK3 cells displayed early metanephric mesenchyme biological function. In organ co-culture experiments they were able to induce growth and branching of the ureteric bud. Another cell line, mK4, represented later, induced metanephric mesenchyme undergoing epithelial conversion. These cells were more polygonal, or epithelial in shape, and expressed genes diagnostic of late mesenchyme, including Pax-2, Pax-8, Wnt-4, Cadherin-6, Collagen IV, and LFB3. To better define the gene expression patterns of kidney metanephric mesenchyme cells at these two stages of development, RNAs from the mK3 and mK4 cells were hybridized to Affymetrix GeneChip probe arrays. Over 4000 expressed genes were identified and thereby implicated in kidney formation. Comparison of the mK3 and mK4 gene expression profiles revealed 121 genes showing greater than a ten-fold difference in expression level. Several are known to be expressed during metanephric mesenchyme differentiation, but most had not been previously associated with this process. In situ hybridizations were used to confirm that selected novel genes were expressed in the developing kidney.

The kidney contains the functional units, the nephrons, surrounded by the renal interstitium. Previously, we discovered that, once Six2-expressing nephron progenitor cells and Foxd1-expressing renal interstitial progenitor cells form at the onset of kidney development, descendant cells from these populations contribute exclusively to the main body of nephrons and renal interstitial tissues, respectively, indicating a lineage boundary between the nephron and renal interstitial compartments. Currently, it is unclear how lineages are regulated during kidney organogenesis. We demonstrate that nephron progenitor cells lacking Pax2 fail to differentiate into nephron cells, but can switch fates into renal interstitium-like cell types. These data suggest that Pax2 function maintains nephron progenitor cells by repressing a renal interstitial cell program. Thus, the lineage boundary between the nephron and renal interstitial compartments is maintained by the Pax2 activity in nephron progenitor cells during kidney organogenesis.

This report presents a novel mechanism for remodelling a branched epithelial tree. The mouse renal collecting duct develops by growth and repeated branching of an initially unbranched ureteric bud: this mechanism initially produces an almost fractal form with young branches connected to the centre of the kidney via a sequence of nodes (branch points) distributed widely throughout the developing organ. The collecting ducts of a mature kidney have a different form: from the nephrons in the renal cortex, long, straight lengths of collecting duct run almost parallel to one another through the renal medulla, and open together to the renal pelvis. Here we present time-lapse studies of E11.5 kidneys growing in culture: after about 5 days, the collecting duct trees show evidence of ‘node retraction’, in which the node of a ‘Y’-shaped branch moves downwards, shortening the stalk of the ‘Y’, lengthening its arms and narrowing their divergence angle so that the ‘Y’ becomes a ‘V’. Computer simulation suggests that node retraction can transform a spread tree, like that of an early kidney, into one with long, almost-parallel medullary rays similar to those seen in a mature real kidney.

Human pluripotent stem cells (hPSCs) can generate a diversity of cell types, but few methods have been developed to derive cells of the kidney lineage. Here, we report a highly efficient system for differentiating human embryonic stem cells and induced pluripotent stem cells (referred to collectively as hPSCs) into cells expressing markers of the intermediate mesoderm (IM) that subsequently form tubule-like structures. Treatment of hPSCs with the glycogen synthase kinase-3β inhibitor CHIR99021 induced BRACHYURY(+)MIXL1(+) mesendoderm differentiation with nearly 100% efficiency. In the absence of additional exogenous factors, CHIR99021-induced mesendodermal cells preferentially differentiated into cells expressing markers of lateral plate mesoderm with minimal IM differentiation. However, the sequential treatment of hPSCs with CHIR99021 followed by fibroblast growth factor-2 and retinoic acid generated PAX2(+)LHX1(+) cells with 70%-80% efficiency after 3 days of differentiation. Upon growth factor withdrawal, these PAX2(+)LHX1(+) cells gave rise to apically ciliated tubular structures that coexpressed the proximal tubule markers Lotus tetragonolobus lectin, N-cadherin, and kidney-specific protein and partially integrated into embryonic kidney explant cultures. With the addition of FGF9 and activin, PAX2(+)LHX1(+) cells specifically differentiated into cells expressing SIX2, SALL1, and WT1, markers of cap mesenchyme nephron progenitor cells. Our findings demonstrate the effective role of fibroblast growth factor signaling in inducing IM differentiation in hPSCs and establish the most rapid and efficient system whereby hPSCs can be differentiated into cells with features characteristic of kidney lineage cells.

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.

SummaryA 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 β-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 β-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and β-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.

MicroRNAs (miRNAs) are a large and growing class of small, non-coding, regulatory RNAs that control gene expression predominantly at the post-transcriptional level. The production of most functional miRNAs depends on the enzymatic activity of Dicer, an RNase III class enzyme. To address the potential action of Dicer-dependent miRNAs in mammalian kidney development, we conditionally ablated Dicer function within cells of nephron lineage and the ureteric bud-derived collecting duct system. Six2Cre-mediated removal of Dicer activity from the progenitors of the nephron epithelium led to elevated apoptosis and premature termination of nephrogenesis. Thus, Dicer action is important for maintaining the viability of this critical self-renewing progenitor pool and, consequently, development of a normal nephron complement. HoxB7Cre-mediated removal of Dicer function from the ureteric bud epithelium led to the development of renal cysts. This was preceded by excessive cell proliferation and apoptosis, and accompanied by disrupted ciliogenesis within the ureteric bud epithelium. Dicer removal also disrupted branching morphogenesis with the phenotype correlating with downregulation of Wnt11 and c-Ret expression at ureteric tips. Thus Dicer, and by inference Dicer-dependent miRNA activity, have distinct regulatory roles within different components of the developing mouse kidney. Furthermore, an understanding of miRNA action may provide new insights into the etiology and pathogenesis of renal cyst-based kidney disease.

Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor beta(1) treatment. However, using either red fluorescent protein or beta-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive((+)) mesenchymal cells give rise to adult CD73(+), platelet derived growth factor receptor beta(+), smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin(+) myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.

Mammalian nephrons form as a result of a complex morphogenesis and patterning of a simple epithelial precursor, the renal vesicle. Renal vesicles are established from a mesenchymal progenitor population in response to inductive signals. Several lines of evidence support the sequential roles of two Wnt family members, Wnt9b and Wnt4, in renal vesicle induction. Using genetic approaches to specifically manipulate the activity of β-catenin within the mesenchymal progenitor pool in mice, we investigated the potential role of the canonical Wnt pathway in these inductive events. Progenitor-cell-specific removal of β-catenin activity completely blocked both the formation of renal vesicles and the expected molecular signature of an earlier inductive response. By contrast, activation of stabilizedβ-catenin in the same cell population causes ectopic expression of mesenchymal induction markers in vitro and functionally replaces the requirement for Wnt9b and Wnt4 in their inductive roles in vivo. Thus, canonical Wnt signaling is both necessary and sufficient for initiating and maintaining inductive pathways mediated by Wnt9b and Wnt4. However, the failure of induced mesenchyme with high levels of β-catenin activity to form epithelial structures suggests that modulating canonical signaling may be crucial for the cellular transition to the renal vesicle.

The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules)despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a γ-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by aγ-secretase inhibitor. These results establish distinct (non-redundant),instructive roles for Notch receptors in nephron segmentation.

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 to 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.

In the developing brain, transcription factors (TFs) direct the formation of a diverse array of neurons and glia. We identifed 1445 putative TFs in the mouse genome. We used in situ hybridization to map the expression of over 1000 of these TFs and TF-coregulator genes in the brains of developing mice. We found that 349 of these genes showed restricted expression patterns that were adequate to describe the anatomical organization of the brain. We provide a comprehensive inventory of murine TFs and their expression patterns in a searchable brain atlas database.

Recent functional studies in mouse further illustrate the importance of the epithelial-mesenchymal interaction between the ureteric bud epithelium and the metanephric mesenchyme in kidney formation. Genetic ablation of Gdf11, Six1, Slit2/Robo2 reveal a role of these genes in regulating the outgrowth of a single ureteric bud from the Wolffian duct. Studies of Wnt11 and Fras1/Grip1, all expressed in the ureteric bud, show a role for these genes in regulating events in the adjacent metanephric mesenchyme. Furthermore, various approaches were used to address the function of Pod1, Pbx1, the Notch pathway and Brn1 in nephron formation.

Wnt/ β -catenin signaling plays a significant role during kidney development and maturation.[1][1][][2]–[3][3] β -Catenin, the canonical Wnt signal transducer, is involved in nephron induction[4][4],[5][5] and branching morphogenesis.[1][1],[6][6] Importantly, β -catenin modulation is necessary

Mutation of the functionally redundantHoxa 11/Hoxd 11genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms ofHoxa 11/Hoxd 11function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen forHoxa 11-responsive genes in two kidney cell lines.HEK293cells, which usually do not expressHoxa 11, were modified to allow inducibleHoxa 11expression. ThemK10cells, derived specifically for this study fromHoxa 11/Hoxd 11double-mutant mice, were also modified to give cell populations with and withoutHoxa 11expression. Differential display, Gene Discovery Arrays, and Affymetrixgenechipprobe arrays were used to screen for genes up- or down-regulated byHoxa 11. Nine genes,PDGF A,Cathepsin L,annexin A1,Mm.112139,Est2 repressor factor,NrCAM,ZNF192,integrin-associated protein, andGCM1, showed reproducible 3-fold or smaller changes in gene expression in response toHoxa 11. One gene, theIntegrinα8, was up-regulated approximately 20-fold afterHoxa 11expression. TheIntegrinα8gene is expressed together withHoxa 11in metanephric mesenchyme cells, and mutation ofIntegrinα8gives a bud-branching morphogenesis defect very similar to that observed inHoxa 11/Hoxd 11mutant mice.In situhybridizations showed a dramatic regional reduction inIntegrinα8expression in the developing kidneys ofHoxa 11/Hoxd 11mutant mice. This work suggests that theIntegrinα8gene may be a major effector ofHoxa 11/Hoxd 11function in the developing kidney.

Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein‐ protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.

We report the characterization of Gsh-1, a novel murine homeobox gene. Northern blot analysis revealed a transcript of approximately 2 kb in size present at embryonic days 10.5, 11.5, and 12.5 of development. The cDNA sequence encoded a proline rich motif, a polyalanine tract, and a homeodomain with strong homology to those encoded by the clustered Hox genes. The Gsh-1 expression pattern was determined for days E8.5 to E13.5 by whole mount and serial section in situ hybridizations. Gsh-1 transcription was restricted to the central nervous system. Expression is present in the neural tube and hindbrain as two continuous, bilaterally symmetrical stripes within neural epithelial tissue. In the mesencephalon, expression is seen as a band across the most anterior portion. There is also diencephalon expression in the anlagen of the thalamus and the hypothalamus as well as in the optic stalk, optic recess, and the ganglionic eminence. Moreover, through the use of fusion proteins containing the Gsh-1 homeodomain, we have determined the consensus DNA ninding site of the Gsh-1 homeoprotein to be GCT/CA/CATTAG/A. ©1995 Wiley-Liss, Inc.

We have identified a novel homeobox gene, designated K-2, using a reverse transcription PCR cloning strategy. Sequence analysis reveals that the homeobox of K-2 is 77.6% homologous at the nucleotide level and 97% identical at the amino acid sequence level to another murine gene, S8. Homeodomain sequence comparisons indicate that K-2 and S8 represent a distinct subclass of paired type homeobox genes. Northern blot analysis of RNA from murine embryos and adult tissues identified multiple transcripts that are expressed in a developmentally specific and tissue restricted manner. Alternate splicing of K-2 at the 3-coding region leads to the inclusion of a chain terminating sequence. In addition, the developmental expression pattern of this gene at day 12 of gestation was determined by in situ hybridization. Expression was observed in diverse mesenchymal cells in craniofacial, pericardial, primitive dermal, prevertebral, and genital structures.