Nephron Progenitor Cells Research Articles

Rapamycin and dexamethasone during pregnancy prevent tuberous sclerosis complex-associated cystic kidney disease.

Chronic kidney disease is the main cause of mortality in patients with tuberous sclerosis complex (TSC) disease. The mechanisms underlying TSC cystic kidney disease remain unclear, with no available interventions to prevent cyst formation. Using targeted deletion of TSC1 in nephron progenitor cells, we showed that cysts in TSC1-null embryonic kidneys originate from injured proximal tubular cells with high mTOR complex 1 activity. Injection of rapamycin to pregnant mice inhibited the mTOR pathway and tubular cell proliferation in kidneys of TSC1-null offspring. Rapamycin also prevented renal cystogenesis and prolonged the life span of TSC newborns. Gene expression analysis of proximal tubule cells identified sets of genes and pathways that were modified secondary to TSC1 deletion and rescued by rapamycin administration during nephrogenesis. Inflammation with mononuclear infiltration was observed in the cystic areas of TSC1-null kidneys. Dexamethasone administration during pregnancy decreased cyst formation by not only inhibiting the inflammatory response, but also interfering with the mTORC1 pathway. These results reveal mechanisms of cystogenesis in TSC disease and suggest interventions before birth to ameliorate cystic disease in offspring.

The core SWI/SNF catalytic subunit Brg1 regulates nephron progenitor cell proliferation and differentiation

Chromatin-remodeling complexes play critical roles in establishing gene expression patterns in response to developmental signals. How these epigenetic regulators determine the fate of progenitor cells during development of specific organs is not well understood. We found that genetic deletion of Brg1 (Smarca4), the core enzymatic protein in SWI/SNF, in nephron progenitor cells leads to severe renal hypoplasia. Nephron progenitor cells were depleted in Six2-Cre, Brg1flx/flx mice due to reduced cell proliferation. This defect in self-renewal, together with impaired differentiation resulted in a profound nephron deficit in Brg1 mutant kidneys. Sall1, a transcription factor that is required for expansion and maintenance of nephron progenitors, associates with SWI/SNF. Brg1 and Sall1 bind promoters of many progenitor cell genes and regulate expression of key targets that promote their proliferation.

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors.

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here, we show in mice that the signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/β-catenin signalling and their genetic deletion leads to a rapid decline of nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation.

Linking Energy Metabolism to Cell Fate Decision During Kidney Development

ObjectiveMetabolic flux switching accompanies cell fate changes in several organisms and cell types. We recently showed that glycolysis inhibition drives differentiation of nephron progenitor cell (NPC) in embryonic (E) kidney in organ culture experiments. We utilized a transcriptomics approach to uncover renewal vs differentiation genes in pathways targeted by glycolysis vs Wnt‐mediated differentiation, as canonical Wnt signaling is an established inducer of nephrogenesis.MethodsBulk RNASeq was performed on cultured NPCs isolated from E13.5 kidneys after 24h treatment with either the glycolysis inhibitor YN1 (20uM) or GSK3b inhibitor CHIR (3uM) to activate Wnt signaling. NPCs were isolated from 3 separate litters and pooled per litter. Differential expression (DE) thresholds: p‐value<0.05, fold‐change 0.6.We have also carried out organ culture experiments on E12.5 kidneys; paired kidneys were fixed after 24h Pravastatin (10uM) or vehicle treatment for immunostaining to identify NPCs (Six2+) or nascent nephrons (Lhx1+).ResultsCompared to renewing NPCs, approximately 550/15,300 genes were differentially expressed after CHIR or YN1 treatment, respectively. Pathway analysis revealed the common top over‐represented pathways as Cholesterol biosynthesis, Wnt and Calcium signaling. Fatty acid metabolism related pathways dominated in YN1‐treated NPCs and amino acid biosynthesis pathways in CHIR‐treated NPCs. Furthermore, to determine whether the inhibition of cholesterol biosynthesis is a requisite for nephrogenesis or a result of the differentiated state, E12.5 kidneys were treated with HMG‐CoA reductase inhibitor, Pravastatin, resulting in a significant increase in nephrogenesis marker staining (Lhx1+) and decrease in the self‐renewing progenitor pool (Six2+).ConclusionsOur data suggest glycolysis is required to maintain the cholesterol biosynthesis pathway and NPC self‐renewal and that the inhibition of either pathway directs the NPC toward differentiation.Support or Funding InformationThis work was supported by the National Institutes of Health: NIH/NIDDK R01 DK118231

Epigenetic regulation of kidney progenitor cells.

The reciprocal interactions among the different embryonic kidney progenitor populations lay the basis for proper kidney organogenesis. During kidney development, three types of progenitor cells, including nephron progenitor cells, ureteric bud progenitor cells, and interstitial progenitor cells, generate the three major kidney structures—the nephrons, the collecting duct network, and the stroma, respectively. Epigenetic mechanisms are well recognized for playing important roles in organism development, in fine‐tuned control of physiological activities, and in responses to environment stimuli. Recently, evidence supporting the importance of epigenetic mechanisms underlying kidney organogenesis has emerged. In this perspective, we summarize the research progress and discuss the potential contribution of novel stem cell, organoid, and next‐generation sequencing tools in advancing this field in the future.

Dnmt3a and Dnmt3b-Decommissioned Fetal Enhancers are Linked to Kidney Disease.

Cytosine methylation is an epigenetic mark that dictates cell fate and response to stimuli. The timing and establishment of methylation logic during kidney development remains unknown. DNA methyltransferase 3a and 3b are the enzymes capable of establishing de novo methylation. We generated mice with genetic deletion of Dnmt3a and Dnmt3b in nephron progenitor cells (Six2CreDnmt3a/3b) and kidney tubule cells (KspCreDnmt3a/3b). We characterized KspCreDnmt3a/3b mice at baseline and after injury. Unbiased omics profiling, such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing and RNA sequencing were performed on whole-kidney samples and isolated renal tubule cells. KspCreDnmt3a/3b mice showed no obvious morphologic and functional alterations at baseline. Knockout animals exhibited increased resistance to cisplatin-induced kidney injury, but not to folic acid-induced fibrosis. Whole-genome bisulfite sequencing indicated that Dnmt3a and Dnmt3b play an important role in methylation of gene regulatory regions that act as fetal-specific enhancers in the developing kidney but are decommissioned in the mature kidney. Loss of Dnmt3a and Dnmt3b resulted in failure to silence developmental genes. We also found that fetal-enhancer regions methylated by Dnmt3a and Dnmt3b were enriched for kidney disease genetic risk loci. Methylation patterns of kidneys from patients with CKD showed defects similar to those in mice with Dnmt3a and Dnmt3b deletion. Our results indicate a potential locus-specific convergence of genetic, epigenetic, and developmental elements in kidney disease development.

Small molecule TCS21311 can replace BMP7 and facilitate cell proliferation in in vitro expansion culture of nephron progenitor cells

Several groups have developed in vitro expansion cultures for mouse metanephric nephron progenitor cells (NPCs) using cocktails of small molecules and growth factors including BMP7. However, the detailed mechanisms by which BMP7 acts in the NPC expansion remain to be elucidated. Here, by performing chemical screening for BMP substitutes, we identified a small molecule, TCS21311, that can replace BMP7 and revealed a novel inhibitory role of BMP7 in JAK3-STAT3 signaling in NPC expansion culture. Further, we found that TCS21311 facilitates the proliferation of mouse embryonic NPCs and human induced pluripotent stem cell-derived NPCs when added to the expansion culture. These results will contribute to understanding the mechanisms of action of BMP7 in NPC proliferation in vitro and in vivo and to the stable supply of NPCs for regenerative therapy, disease modeling and drug discovery for kidney diseases.

Mutational analysis of genes with ureteric progenitor cell-specific expression in branching morphogenesis of the mouse kidney.

Ureteric progenitor cells (UPCs) within the branch tips of the arborizing ureteric epithelium of the kidney's developing collecting system establish the shape and cellular organization of the collecting network, and drive the nephrogenic program through their interactions with nephron progenitor cells. In a previous study, expression screening identified a cohort of genes showing UPC-enriched expression including D17H6S56E-5, Hs3st3a1, Hs3st3b1, and Tmem59l. Each of these is also enriched in branch tips of assembling airways of the developing lungs. Here, we used Crispr-CAS9 directed gene editing to mutate each of these targets to address their potential role(s) in UPC programs. Single (D17H6S56E-5 and Tmem59l) and double (Hs3st3a1 and Hs3st3b1) mutants were viable, fertile, and displayed varying frequencies of ureter duplications and no overt lung phenotype. Ureter duplications arise spontaneously through multiple outgrowths of the ureteric bud at the onset of kidney development. Tmem59l mutants and Hs3st3a1/Hs3st3b1 compound mutants showed a weakly penetrant, but statistically significant increase in duplicated ureters compared to C57BL6/J and SW wild-type mouse strains. Tmem59l and Hs3st3a1/Hs3st3b1 activities contribute to the regulatory programs restricting ureteric outgrowth in the developing mouse kidney. However, the low penetrance of the observed phenotype precludes a detailed analysis of their specific actions.

Stromal β-catenin activation impacts nephron progenitor differentiation in the developing kidney and may contribute to Wilms tumor.

Wilms' tumor (WT) morphologically resembles the embryonic kidney, consisting of blastema, epithelial and stromal components, suggesting tumors arise from the dysregulation of normal development. β-Catenin activation is observed in a significant proportion of WTs; however, much remains to be understood about how it contributes to tumorigenesis. Although activating β-catenin mutations are observed in both blastema and stromal components of WT, current models assume that activation in the blastemal lineage is causal. Paradoxically, studies performed in mice suggest that activation of β-catenin in the nephrogenic lineage results in loss of nephron progenitor cell (NPC) renewal, a phenotype opposite to WT. Here, we show that activation of β-catenin in the stromal lineage non-autonomously prevents the differentiation of NPCs. Comparisons of the transcriptomes of kidneys expressing an activated allele of β-catenin in the stromal or nephron progenitor cells reveals that human WT more closely resembles the stromal-lineage mutants. These findings suggest that stromal β-catenin activation results in histological and molecular features of human WT, providing insights into how alterations in the stromal microenvironment may play an active role in tumorigenesis.

Knockdown of Nuclear lncRNAs by Locked Nucleic Acid (LNA) Gapmers in Nephron Progenitor Cells.

Despite recent advance in our understanding on the role of long noncoding RNAs (lncRNAs), the function of the vast majority of lncRNAs remains poorly understood. To characterize the function of lncRNAs, knockdown studies are essential. However, the conventional silencing methods for mRNA, such as RNA interference (RNAi), may not be as efficient against lncRNAs, partly due to the mismatch of the localization of lncRNAs and RNAi machinery. To circumvent such limitation, a new technique has recently been developed, i.e., locked nucleic acid (LNA) gapmers. This system utilizes RNase H that distributes evenly in both nucleus and cytoplasm and is expected to knock down lncRNAs of interest more consistently regardless of their localization in the cell. In this chapter, we describe the procedure with tips to silence lncRNAs by LNA gapmers, by using mouse nephron progenitor cells as an example.

\u03b2-catenin regulates the formation of multiple nephron segments in the mouse kidney

The nephron is composed of distinct segments that perform unique physiological functions. Little is known about how multipotent nephron progenitor cells differentiate into different nephron segments. It is well known that β-catenin signaling regulates the maintenance and commitment of mesenchymal nephron progenitors during kidney development. However, it is not fully understood how it regulates nephron segmentation after nephron progenitors undergo mesenchymal-to-epithelial transition. To address this, we performed β-catenin loss-of-function and gain-of-function studies in epithelial nephron progenitors in the mouse kidney. Consistent with a previous report, the formation of the renal corpuscle was defective in the absence of β-catenin. Interestingly, we found that epithelial nephron progenitors lacking β-catenin were able to form presumptive proximal tubules but that they failed to further develop into differentiated proximal tubules, suggesting that β-catenin signaling plays a critical role in proximal tubule development. We also found that epithelial nephron progenitors lacking β-catenin failed to form the distal tubules. Expression of a stable form of β-catenin in epithelial nephron progenitors blocked the proper formation of all nephron segments, suggesting tight regulation of β-catenin signaling during nephron segmentation. This work shows that β-catenin regulates the formation of multiple nephron segments along the proximo-distal axis of the mammalian nephron.

Mesangial cell regeneration from exogenous stromal progenitor by utilizing embryonic kidney

Kidney regenerative medicine is expected to be the solution to the shortage of organs for transplantation. In a previous report, we transplanted exogenous renal progenitor cells (RPCs) including nephron progenitor cells (NPCs), stromal progenitor cells (SPCs), and the ureteric bud (UB) into the nephrogenic zone of animal embryos and succeeded in regenerating new nephrons from exogenous NPCs through a fetal developmental program. However, it was unknown whether the renal stromal lineage cells were regenerated from SPCs. The present study aimed to verify the differentiation of SPCs into mesangial cells and renal stromal lineage cells. Here, we found that simply transplanting RPCs, including SPCs, into the nephrogenic zone of wild-type fetal mice was insufficient for differentiation of SPCs. Therefore, to enrich the purity of SPCs, we sorted cells from RPCs by targeting platelet-derived growth factor receptor alpha (PDGFRa) which is a cell surface marker for immature stromal cells and transplanted the PDGFRa-positive sorted cells. As a result, we succeeded in regenerating a large number of mesangial cells and other renal stromal lineage cells including interstitial fibroblasts, vascular pericytes, and juxtaglomerular cells. We have established the method for regeneration of stromal cells from exogenous SPCs that may contribute to various fields, such as regenerative medicine and kidney embryology, and the creation of disease models for renal stromal disorders.

Kidney Regeneration in Later-Stage Mouse Embryos via Transplanted Renal Progenitor Cells.

The limited availability of donor kidneys for transplantation has spurred interest in investigating alternative strategies, such as regenerating organs from stem cells transplanted into animal embryos. However, there is no known method for transplanting cells into later-stage embryos, which may be the most suitable host stages for organogenesis, particularly into regions useful for kidney regeneration. We demonstrated accurate transplantation of renal progenitor cells expressing green fluorescent protein to the fetal kidney development area by incising the opaque uterine muscle layer but not the transparent amniotic membrane. We allowed renal progenitor cell-transplanted fetuses to develop for 6 days postoperatively before removal for analysis. We also transplanted renal progenitor cells into conditional kidney-deficient mouse embryos. We determined growth and differentiation of transplanted cells in all cases. Renal progenitor cell transplantation into the retroperitoneal cavity of fetuses at E13-E14 produced transplant-derived, vascularized glomeruli with filtration function and did not affect fetal growth or survival. Cells transplanted to the nephrogenic zone produced a chimera in the cap mesenchyme of donor and host nephron progenitor cells. Renal progenitor cells transplanted to conditional kidney-deficient fetuses induced the formation of a new nephron in the fetus that is connected to the host ureteric bud. We developed a cell transplantation method for midstage to late-stage fetuses. In vivo kidney regeneration from renal progenitor cells using the renal developmental environment of the fetus shows promise. Our findings suggest that fetal transplantation methods may contribute to organ regeneration and developmental research.

Human kidney organoids: progress and remaining challenges

Kidney organoids are regarded as important tools with which to study the development of the normal and diseased human kidney. Since the first reports of human pluripotent stem cell-derived kidney organoids 5 years ago, kidney organoids have been successfully used to model glomerular and tubular diseases. In parallel, advances in single-cell RNA sequencing have led to identification of a variety of cell types in the organoids, and have shown these to be similar to, but more immature than, human kidney cells in vivo. Protocols for the in vitro expansion of stem cell-derived nephron progenitor cells (NPCs), as well as those for the selective induction ofspecific lineages, especially glomerular podocytes, have also been reported. Although most current organoids are based on the induction of NPCs, an induction protocol for ureteric buds (collecting duct precursors) has also been developed, and approaches to generate more complex kidney structures may soon be possible. Maturation of organoids is a major challenge, and more detailed analysis of the developing kidney at a single cell level is needed. Eventually, organotypic kidney structures equipped with nephrons, collecting ducts, ureters, stroma and vascular flow are required to generate transplantable kidneys; such attempts are in progress.

Activin Is Superior to BMP7 for Efficient Maintenance of Human iPSC-Derived Nephron Progenitors.

SummaryKidney formation is regulated by the balance between maintenance and differentiation of nephron progenitor cells (NPCs). Now that directed differentiation of NPCs from human induced pluripotent stem cells (iPSCs) can be achieved, maintenance and propagation of NPCs in vitro should be beneficial for regenerative medicine. Although WNT and FGF signals were previously shown to be essential for NPC propagation, the requirement for BMP/TGFβ signaling remains controversial. Here we reveal that activin has superior effects to BMP7 on maintenance efficiency of human iPSC-derived NPCs. Activin expanded ITGA8+/PDGFRA−/SIX2-GFP+ NPCs by 5-fold per week at 80%–90% efficiency, and the propagated cells possessed robust capacity for nephron formation both in vitro and in vivo. The expanded cells also maintained their nephron-forming potential after freezing. Furthermore, the protocol was applicable to multiple non-GFP-tagged iPSC lines. Thus, our activin-based protocol will be applicable to a variety of research fields including disease modeling and drug screening.

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

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.

Generation of Human PSC-Derived Kidney Organoids with Patterned Nephron Segments and a De Novo Vascular Network.

Human pluripotent stem cell-derived kidney organoids recapitulate developmental processes and tissue architecture, but intrinsic limitations, such as lack of vasculature and functionality, have greatly hampered their application. Here we establish a versatile protocol for generating vascularized three-dimensional (3D) kidney organoids. We employ dynamic modulation of WNT signaling to control the relativeproportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. Single-cell RNA sequencing identifies a subset of nephron progenitor cells as a potential source of renal vasculature. These kidney organoids undergo further structural and functional maturation upon implantation. Using this kidney organoid platform,we establish an invitro model of autosomalrecessive polycystic kidney disease (ARPKD), the cystic phenotype of which can be effectively prevented by gene correction or drug treatment. Ourstudies provide new avenues for studying human kidney development, modeling disease pathogenesis,and performing patient-specific drug validation.

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

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.

Single cell analysis of the developing mouse kidney provides deeper insight into marker gene expression and ligand-receptor crosstalk.

Recent advances in the generation of kidney organoids and the culture of primary nephron progenitors from mouse and human have been based on knowledge of the molecular basis of kidney development in mice. Although gene expression during kidney development has been intensely investigated, single cell profiling provides new opportunities to further subsect component cell types and the signalling networks at play. Here, we describe the generation and analysis of 6732 single cell transcriptomes from the fetal mouse kidney [embryonic day (E)18.5] and 7853 sorted nephron progenitor cells (E14.5). These datasets provide improved resolution of cell types and specific markers, including subdivision of the renal stroma and heterogeneity within the nephron progenitor population. Ligand-receptor interaction and pathway analysis reveals novel crosstalk between cellular compartments and associates new pathways with differentiation of nephron and ureteric epithelium cell types. We identify transcriptional congruence between the distal nephron and ureteric epithelium, showing that most markers previously used to identify ureteric epithelium are not specific. Together, this work improves our understanding of metanephric kidney development and provides a template to guide the regeneration of renal tissue.

Von Hippel-Lindau Acts as a Metabolic Switch Controlling Nephron Progenitor Differentiation.

Nephron progenitors, the cell population that give rise to the functional unit of the kidney, are metabolically active and self-renew under glycolytic conditions. A switch from glycolysis to mitochondrial respiration drives these cells toward differentiation, but the mechanisms that control this switch are poorly defined. Studies have demonstrated that kidney formation is highly dependent on oxygen concentration, which is largely regulated by von Hippel-Lindau (VHL; a protein component of a ubiquitin ligase complex) and hypoxia-inducible factors (a family of transcription factors activated by hypoxia). To explore VHL as a regulator defining nephron progenitor self-renewal versus differentiation, we bred Six2-TGCtg mice with VHLlox/lox mice to generate mice with a conditional deletion of VHL from Six2+ nephron progenitors. We used histologic, immunofluorescence, RNA sequencing, and metabolic assays to characterize kidneys from these mice and controls during development and up to postnatal day 21. By embryonic day 15.5, kidneys of nephron progenitor cell-specific VHL knockout mice begin to exhibit reduced maturation of nephron progenitors. Compared with controls, VHL knockout kidneys are smaller and developmentally delayed by postnatal day 1, and have about half the number of glomeruli at postnatal day 21. VHL knockout nephron progenitors also exhibit persistent Six2 and Wt1 expression, as well as decreased mitochondrial respiration and prolonged reliance on glycolysis. Our findings identify a novel role for VHL in mediating nephron progenitor differentiation through metabolic regulation, and suggest that VHL is required for normal kidney development.