Nephron Progenitor Research Articles

Generation of patterned kidney organoids that recapitulate the adult kidney collecting duct system from expandable ureteric bud progenitors

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney’s collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney’s collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.

Clearly imaging and quantifying the kidney in 3D

For decades, measurements of kidney microanatomy using 2-dimensional sections has provided us with a detailed knowledge of kidney morphology under physiological and pathological conditions. However, the rapid development of tissue clearing methods in recent years, in combination with the development of novel 3-dimensional imaging modalities have provided new insights into kidney structure and function. This review article describes a range of novel insights into kidney development and disease obtained recently using these new methodological approaches. For example, in the developing kidney these approaches have provided new understandings of ureteric branching morphogenesis, nephron progenitor cell proliferation and commitment, interactions between ureteric tip cells and nephron progenitor cells, and the establishment of nephron segmentation. In whole adult mouse kidneys, tissue clearing combined with light sheet microscopy can image and quantify the total number of glomeruli, a major breakthrough in the field. Similar approaches have provided new insights into the structure of the renal vasculature and innervation, tubulointerstitial remodeling, podocyte loss and hypertrophy, cyst formation, the evolution of cellular crescents, and the structure of the glomerular filtration barrier. Many more advances in the understanding of kidney biology and pathology can be expected as additional clearing and imaging techniques are developed and adopted by more investigators.

Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF.

ABSTRACTNephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.

A coordinated progression of progenitor cell states initiates urinary tract development

The kidney and upper urinary tract develop through reciprocal interactions between the ureteric bud and the surrounding mesenchyme. Ureteric bud branching forms the arborized collecting duct system of the kidney, while ureteric tips promote nephron formation from dedicated progenitor cells. While nephron progenitor cells are relatively well characterized, the origin of ureteric bud progenitors has received little attention so far. It is well established that the ureteric bud is induced from the nephric duct, an epithelial duct derived from the intermediate mesoderm of the embryo. However, the cell state transitions underlying the progression from intermediate mesoderm to nephric duct and ureteric bud remain unknown. Here we show that nephric duct morphogenesis results from the coordinated organization of four major progenitor cell populations. Using single cell RNA-seq and Cluster RNA-seq, we show that these progenitors emerge in time and space according to a stereotypical pattern. We identify the transcription factors Tfap2a/b and Gata3 as critical coordinators of this progenitor cell progression. This study provides a better understanding of the cellular origin of the renal collecting duct system and associated urinary tract developmental diseases, which may inform guided differentiation of functional kidney tissue.

Importance of Extra-Renal CYP24A1 Expression for Maintaining Mineral Homeostasis

Calcium homeostasis involves a complex interplay between kidneys, parathyroid glands, intestine and bone. Specifically, 1,25(OH)2D3 is a key calciotropic hormone which stimulates intestinal calcium absorption. A growing body of evidence suggests that circulating levels of 1,25(OH)2D3 depend not only on its synthesis under the action of PTH in the kidneys, but also its catabolism by 24-hydroxylase, herein referred to as CYP24A1. The clinical importance of CYP24A1 has been demonstrated by human loss-of-function mutations, which lead to severe hypercalcemia due to exaggerated levels of 1,25(OH)2D3. Despite its growing importance, little is known about its tissue-specific contributions to normal vitamin D metabolism. To explore the physiology of CYP24A1 and delineate renal-specific effects of CYP24A1 in calcium metabolism, we generated a mouse with constitutive kidney-specific deletion of Cyp24a1 (Six2Cre-Cyp24flox). Six2 marks the nephron progenitor population throughout nephrogenesis. We hypothesized that hypercalcemia as seen in CYP24A1 inactivating mutations is related to lack of both renal and extrarenal expression, and that renal deletion does not lead to severe hypercalcemia. To confirm Cyp24a1 deletion, we measured mRNA expression in the kidney using qPCR and RNA in situ hybridization. All mice were fed a standard commercial rodent diet and followed longitudinally for five months with interval calcium measurements. At time of termination, serum PTH levels were measured along with vitamin D-dependent calcium transporters as a functional measure of 1,25(OH)2D3 action. Cyp24a1 expression was significantly knocked down in total kidneys from Six2Cre-Cyp24flox mice as compared to intestinal expression suggesting successful gene deletion. Compared to age-matched wildtype controls, Six2Cre-Cyp24flox mice were mildly but persistently hypercalcemic (diff between means= 0.46 mg/dL, p-value: 0.03, n=8 per group). As expected, 1,25D-dependent calcium transporters in the kidney (Calb1, Trpv5, Slc8a1, Atp2b1) and intestine (Trpv6, s100g) were all increased, consistent with increased systemic 1,25(OH)2D3 activity. PTH levels were appropriately suppressed in the Six2Cre-Cyp24flox mice (diff between means=83 pg/mL, p-value 0.2, n=9 control, n=3 exp) as were renal cyp27b1 mRNA expression. These data suggest that renal CYP24A1 is important for systemic 1,25(OH)2D3 regulation, but the lack of severe hypercalcemia supports critical contributions of extra-renal CYP24A1.

Nephron progenitor cell organization and nephrogenesis is dependent on Shroom3

The functional unit of the kidney, termed the nephron, develops from specialized mesenchyme cells known as the nephron progenitors. Nephron progenitors must undergo significant cell shape changes to regulate cell migration, cell movements, and cell projection formation during nephrogenesis. These dynamic cellular changes are essential for orientating and organizing the cells for progression through various stages of nephrogenesis to form a proper functioning nephron. The actin cytoskeleton regulates these dynamic morphological changes by interacting with intracellular proteins and their extracellular environment. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are strongly associated with poor nephron function and chronic kidney disease. Our previous work demonstrated unique Shroom3 expression patterns in nephron progenitor cells, yet the function of Shroom3 during nephrogenesis is not known. Here, we investigated functional roles for Shroom3 in nephron progenitor organization during nephrogenesis. Analysis of E13.5 and E18.5 kidney sections from Wildtype and Shroom3-/- null mutants demonstrated clustering abnormalities of nephron progenitor cells characterized by large abnormal gaps between cells in Shroom3 mutants. Analysis of Wildtype and Shroom3-/- nephron progenitor cell orientation by GM130 and Integrin-α8 immunofluorescence, markers of cell polarization, displayed altered cell orientation in some but not all cells. Transmission electron microscopy (TEM) confirmed that nephron progenitors of Shroom3-/- were irregularly shaped, abnormally clustered, and found at large distances from the neighboring epithelium. TEM analysis also revealed several filopodia-like long and thin actin-based membrane protrusions from both Wildtype and Shroom3-/- nephron progenitors. However, the broad, thick lamellipodia-like protrusions, found at the leading edge of the cells, were virtually absent from Shroom3-/- nephron progenitors. As nephrogenesis proceeds, Wildtype nephron progenitors develop into renal vesicles that consist of a central lumen surrounded by polarized wedge-shaped epithelial cells, which was not observed in Shroom3-/- renal vesicles. These alterations in the early stages of nephrogenesis in Shroom3-/- kidneys were also associated with a disruption in the epithelial polarization of mature nephrons demonstrated by irregular Sglt2 and Slc12a3 expression, key ion channels localized to the apical aspect in Wildtype epithelia. Our results demonstrate that Shroom3 is an important regulator of nephron progenitor organization and cell morphology in the earliest stages of nephron formation. It also suggests these abnormalities translate to a poorly polarized nephron epithelium and likely poor nephron function. These studies could explain why mutations in Shroom3 are so highly associated with poor nephron function and chronic kidney disease.

The Rhesus Macaque Serves As a Model for Human Lateral Branch Nephrogenesis.

Most nephrons are added in late gestation. Truncated extrauterine nephrogenesis in premature infants results in fewer nephrons and significantly increased risk for CKD in adulthood. To overcome the ethical and technical difficulties associated with studies of late-gestation human fetal kidney development, third-trimester rhesus macaques served as a model to understand lateral branch nephrogenesis (LBN) at the molecular level. Immunostaining and 3D rendering assessed morphology. Single-cell (sc) and single-nucleus (sn) RNA-Seq were performed on four cortically enriched fetal rhesus kidneys of 129-131 days gestational age (GA). An integrative bioinformatics strategy was applied across single-cell modalities, species, and time. RNAScope validation studies were performed on human archival tissue. Third-trimester rhesus kidney undergoes human-like LBN. scRNA-Seq of 23,608 cells revealed 37 transcriptionally distinct cell populations, including naïve nephron progenitor cells (NPCs), with the prior noted marker genes CITED1, MEOX1, and EYA1 (c25). These same populations and markers were reflected in snRNA-Seq of 5972 nuclei. Late-gestation rhesus NPC markers resembled late-gestation murine NPC, whereas early second-trimester human NPC markers aligned to midgestation murine NPCs. New, age-specific rhesus NPCs (SHISA8) and ureteric buds (POU3F4 and TWIST) predicted markers were verified in late-gestation human archival samples. Rhesus macaque is the first model of bona fide LBN, enabling molecular studies of late gestation, human-like nephrogenesis. These molecular findings support the hypothesis that aging nephron progenitors have a distinct molecular signature and align to their earlier human counterparts, with unique markers highlighting LBN-specific progenitor maturation.

Identification of candidate PAX2-regulated genes implicated in human kidney development

PAX2 is a transcription factor essential for kidney development and the main causative gene for renal coloboma syndrome (RCS). The mechanisms of PAX2 action during kidney development have been evaluated in mice but not in humans. This is a critical gap in knowledge since important differences have been reported in kidney development in the two species. In the present study, we hypothesized that key human PAX2-dependent kidney development genes are differentially expressed in nephron progenitor cells from induced pluripotent stem cells (iPSCs) in patients with RCS relative to healthy individuals. Cap analysis of gene expression revealed 189 candidate promoters and 71 candidate enhancers that were differentially activated by PAX2 in this system in three patients with RCS with PAX2 mutations. By comparing this list with the list of candidate Pax2-regulated mouse kidney development genes obtained from the Functional Annotation of the Mouse/Mammalian (FANTOM) database, we prioritized 17 genes. Furthermore, we ranked three genes—PBX1, POSTN, and ITGA9—as the top candidates based on closely aligned expression kinetics with PAX2 in the iPSC culture system and susceptibility to suppression by a Pax2 inhibitor in cultured mouse embryonic kidney explants. Identification of these genes may provide important information to clarify the pathogenesis of RCS, human kidney development, and kidney regeneration.

Single cell regulatory landscape of the mouse kidney highlights cellular differentiation programs and disease targets

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profile open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We reveal key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrates that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells follow a more complex differentiation, where Hfn4a is associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease implicates critical cell types, developmental stages, genes, and regulatory mechanisms. The single cell multi-omics atlas reveals key chromatin remodeling events and gene expression dynamics associated with kidney development.

POS-389 PODOCYTE MATURATION IN HUMAN KIDNEY ORGANOIDS IS ACCELERATED WITH RENIN-ANGIOTENSIN SYSTEM ACTIVATION

The global prevalence of chronic kidney disease (CKD) is approximately 10% and rising. Current renal replacement modalities (dialysis and transplant) carry significant socioeconomic burden and may struggle to meet rising demands. PSC-derived kidney organoids have emerged as a promising alternative for providing functional nephrons to CKD patients. However, this nascent technology faces numerous challenges most pressing of which is maturation. Organoid differentiation protocols rely on application of morphogens in a temporal restricted fashion mimicking ureteric bud and metanephric mesenchyme development. However, organoids are poorly vascularized and resemble first and second trimester fetal kidneys. The renin-angiotensin system (RAS) is critical for normal kidney morphogenesis guiding glomerular, vascular and ureteric formation. Indeed, prenatal exposure to RAS inhibitors is associated with kidney dysgenesis and mortality. Can RAS modulators like angiotensin 2 (ANG2) and RAS inhibitors alter kidney organoid maturation? We hypothesize that ANG2 and losartan perturb developmental programs within nephron progenitor populations, alter cell fate specification and maturation of glomerular epithelial cells (i.e. podocytes). We performed single-cell RNA sequence analyses on human iPSC derived kidney organoids treated acutely (24hrs) with vehicle, ANG2 alone, losartan alone, or ANG2 and losartan on the 28th day of the Takasato protocol. Kidney organoids expressed all major RAS genes. AGT, ACE1 and ACE2 were restricted to proximal tubule clusters while REN (renin), AGTR1 and AGTR2 were preferentially expressed in stromal cells. ACE1 and REN were also found in endothelial cells. ATP6AP2 (pro-renin receptor) was ubiquitously expressed. REN expression was downregulated by ANG2 and normalized with losartan pre-treatment suggesting a functioning RAS network. Unbiased clustering identified 3 late podocyte (LP) clusters (NPHS2hiPODXLhi), 1 early podocyte (EP) cluster (OLFM3+NPHS2loPDOXLlo), 1 parietal epithelial (PEC) cluster (CLDN1+CAV2+) and 2 nephron progenitor (NPC) clusters (SIX2+CITED1+). RNA velocity analysis revealed 2 distinct differentiation trajectories: NPC – EP – LP and NPC – EP – PEC – LP. The EP and NPC transcriptional profiles were disproportionately sensitive to ANG2 as we noted significant upregulation of podocyte differentiation genes such as BMP7, FOXC2 PODXL and NPHS2. Moreover, ANG2 depleted the immature populations (NPC and EP) and expanded mature cell cell states (PEC and LP). On the other hand, losartan downregulated the podocyte differentiation programs and increased the proportion of immature cells (EP and NPC) to mature (PEC and LP) cells. The proportion of glomerular epithelial cells relative to all cells was unchanged across treatments. Human kidney organoids developed a functioning intraorganoid RAS resembling in vivo intrarenal RAS. The lineage plasticity observed between NPCs, EPs, PECs and LPs was sensitive to acute RAS perturbation. ANG2 accelerated podocyte maturity while RAS inhibition held podocytes in an immature state. Next steps include testing different ANG2 treatment durations, validate findings using immunocytochemistry, investigate the podocyte differentiation programs closely and their relationship to RAS, and reproduce this analysis on endothelial and tubule cells.

A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells.

The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

Impact of gestational low-protein intake on embryonic kidney microRNA expression and in nephron progenitor cells of the male fetus.

Here, we have demonstrated that gestational low-protein (LP) intake offspring present lower birth weight, reduced nephron numbers, renal salt excretion, arterial hypertension, and renal failure development compared to regular protein (NP) intake rats in adulthood. We evaluated the expression of various miRNAs and predicted target genes in the kidney in gestational 17-days LP (DG-17) fetal metanephros to identify molecular pathways involved in the proliferation and differentiation of renal embryonic or fetal cells. Pregnant Wistar rats were classified into two groups based on protein supply during pregnancy: NP (regular protein diet, 17%) or LP diet (6%). Renal miRNA sequencing (miRNA-Seq) performed on the MiSeq platform, RT-qPCR of predicted target genes, immunohistochemistry, and morphological analysis of 17-DG NP and LP offspring were performed using previously described methods. A total of 44 miRNAs, of which 19 were up and 25 downregulated, were identified in 17-DG LP fetuses compared to age-matched NP offspring. We selected 7 miRNAs involved in proliferation, differentiation, and cellular apoptosis. Our findings revealed reduced cell number and Six-2 and c-Myc immunoreactivity in metanephros cap (CM) and ureter bud (UB) in 17-DG LP fetuses. Ki-67 immunoreactivity in CM was 48% lesser in LP compared to age-matched NP fetuses. Conversely, in LP CM and UB, β-catenin was 154%, and 85% increased, respectively. Furthermore, mTOR immunoreactivity was higher in LP CM (139%) and UB (104%) compared to that in NP offspring. TGFβ-1 positive cells in the UB increased by approximately 30% in the LP offspring. Moreover, ZEB1 metanephros-stained cells increased by 30% in the LP offspring. ZEB2 immunofluorescence, although present in the entire metanephros, was similar in both experimental groups. Maternal protein restriction changes the expression of miRNAs, mRNAs, and proteins involved in proliferation, differentiation, and apoptosis during renal development. Renal ontogenic dysfunction, caused by maternal protein restriction, promotes reduced reciprocal interaction between CM and UB; consequently, a programmed and expressive decrease in nephron number occurs in the fetus.

Single cell RNA sequencing uncovers cellular developmental sequences and novel potential intercellular communications in embryonic kidney

Kidney development requires the coordinated growth and differentiation of multiple cells. Despite recent single cell profiles in nephrogenesis research, tools for data analysis are rapidly developing, and offer an opportunity to gain additional insight into kidney development. In this study, single-cell RNA sequencing data obtained from embryonic mouse kidney were re-analyzed. Manifold learning based on partition-based graph-abstraction coordinated cells, reflecting their expected lineage relationships. Consequently, the coordination in combination with ForceAtlas2 enabled the inference of parietal epithelial cells of Bowman’s capsule and the inference of cells involved in the developmental process from the S-shaped body to each nephron segment. RNA velocity suggested developmental sequences of proximal tubules and podocytes. In combination with a Markov chain algorithm, RNA velocity suggested the self-renewal processes of nephron progenitors. NicheNet analyses suggested that not only cells belonging to ureteric bud and stroma, but also endothelial cells, macrophages, and pericytes may contribute to the differentiation of cells from nephron progenitors. Organ culture of embryonic mouse kidney demonstrated that nerve growth factor, one of the nephrogenesis-related factors inferred by NicheNet, contributed to mitochondrial biogenesis in developing distal tubules. These approaches suggested previously unrecognized aspects of the underlying mechanisms for kidney development.

Development of the metanephric kidney.

The kidney plays an integral role in filtering the blood-removing metabolic by-products from the body and regulating blood pressure. This requires the establishment of large numbers of efficient and specialized blood filtering units (nephrons) that incorporate a system for vascular exchange and nutrient reabsorption as well as a collecting duct system to remove waste (urine) from the body. Kidney development is a dynamic process which generates these structures through a delicately balanced program of self-renewal and commitment of nephron progenitor cells that inhabit a constantly evolving cellular niche at the tips of a branching ureteric "tree." The former cells build the nephrons and the latter the collecting duct system. Maintaining these processes across fetal development is critical for establishing the normal "endowment" of nephrons in the kidney and perturbations to this process are associated both with mutations in integral genes and with alterations to the fetal environment.

Targeted disruption of the histone lysine 79 methyltransferase Dot1L in nephron progenitors causes congenital renal dysplasia

ABSTRACT The epigenetic regulator Dot1, the only known histone H3K79 methyltransferase, has a conserved role in organismal development and homoeostasis. In yeast, Dot1 is required for telomeric silencing and genomic integrity. In Drosophila, Dot1 (Grappa) regulates homoeotic gene expression. Dysregulation of DOT1L (human homologue of Dot1) causes leukaemia and is implicated in dilated cardiomyopathy. In mice, germline disruption of Dot1L and loss of H3K79me2 disrupt vascular and haematopoietic development. Targeted inactivation of Dot1L in principal cells of the mature collecting duct affects terminal differentiation and cell type patterning. However, the role of H3K79 methylation in mammalian tissue development has been questioned, as it is dispensable in the intestinal epithelium, a rapidly proliferating tissue. Here, we used lineage-specific Cre recombinase to delineate the role of Dot1L methyltransferase activity in the mouse metanephric kidney, an organ that develops via interactions between ureteric epithelial (Hoxb7) and mesenchymal (Six2) cell lineages. The results demonstrate that Dot1LHoxb7 is dispensable for ureteric bud branching morphogenesis. In contrast, Dot1LSix2 is critical for the maintenance and differentiation of Six2+ progenitors into epithelial nephrons. Dot1LSix2 mutant kidneys exhibit congenital nephron deficit and cystic dysplastic kidney disease. Molecular analysis implicates defects in key renal developmental regulators, such as Lhx1, Pax2 and Notch. We conclude that the developmental functions of Dot1L-H3K79 methylation in the kidney are lineage-restricted. The link between H3K79me and renal developmental pathways reaffirms the importance of chromatin-based mechanisms in organogenesis.

Metabolic programming of nephron progenitor cell fate.

Metabolic pathways are one of the first responses at the cellular level to maternal/fetal interface stressors. Studies have revealed the previously unrecognized contributions of intermediary metabolism to developmental programs. Here, we provide an overview of cellular metabolic pathways and the cues that modulate metabolic states. We discuss the developmental and physiological implications of metabolic reprogramming and the key role of metabolites in epigenetic and epiproteomic modifications during embryonic development and with respect to kidney development and nephrogenesis.

Transcription Factor 21 Regulates Nephron Progenitor Cells Self-Renewal/Induction and Podocyte Development

Transcription Factor 21 Regulates Nephron Progenitor Cells Self-Renewal/Induction and Podocyte Development

ADAM10 mediates ectopic proximal tubule development and renal fibrosis through Notch signalling.

Disturbed intrauterine development increases the risk of renal disease. Various studies have reported that Notch signalling plays a significant role in kidney development and kidney diseases. A disintegrin and metalloproteinase domain 10 (ADAM10), an upstream protease of the Notch pathway, is also reportedly involved in renal fibrosis. However, how ADAM10 interacts with the Notch pathway and causes renal fibrosis is not fully understood. In this study, using a prenatal chlorpyrifos (CPF) exposure mouse model, we investigated the role of the ADAM10/Notch axis in kidney development and fibrosis. We found that prenatal CPF‐exposure mice presented overexpression of Adam10, Notch1 and Notch2, and led to premature depletion of Six2+ nephron progenitors and ectopic formation of proximal tubules (PTs) in the embryonic kidney. These abnormal phenotypic changes persisted in mature kidneys due to the continuous activation of ADAM10/Notch and showed aggravated renal fibrosis in adults. Finally, both ADAM10 and NOTCH2 expression were positively correlated with the degree of renal interstitial fibrosis in IgA nephropathy patients, and increased ADAM10 expression was negatively correlated with decreased kidney function evaluated by serum creatinine, cystatin C, and estimated glomerular filtration rate. Regression analysis also indicated that ADAM10 expression was an independent risk factor for fibrosis in IgAN. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Generation of Human Renal Vesicles in Mouse Organ Niche Using Nephron Progenitor Cell Replacement System

Animal fetuses may be used for the regeneration of human organs. We have previously generated a transgenic mouse model that allows diphtheria toxin (DT)-induced ablation of Six2-positive nephron progenitor cells (NPCs). Elimination of existing native host NPCs enables their replacement with donor NPCs, which can generate neo-nephrons. However, this system cannot be applied to human NPCs, because DT induces apoptosis in human cells. Therefore, the present study presents a transgenic mouse model for the ablation of NPCs using tamoxifen, which does not affect human cells. Using this system, we successfully regenerate interspecies neo-nephrons, which exhibit urine-producing abilities, from transplanted rat NPCs in a mouse host. Transplantation of human induced pluripotent stem cell (iPSC)-derived NPCs results in differentiation into renal vesicles, which connect to the ureteric bud of the host. Thus, we demonstrate thepossibility of the regeneration of human kidneys derived from human iPSC-derived NPCs via NPC replacement.

Abstract 9: Histone Deacetylases 1 And 2 Regulate Six2 Function To Maintain Nephron Progenitor Cells During Nephrogenesis

Low nephron endowment is strongly associated with cardiovascular disease, especially hypertension. Sine oculis homeobox 2 (Six2) is the master transcriptional regulator in balancing self-renewal and differentiation of nephron progenitor cells (NPCs) for appropriate nephron endowment. Loss of Six2 in mice causes early-onset loss of self-renewal and premature differentiation of NPCs. However, it is unclear how Six2 is functionally regulated during nephrogenesis. In vivo interaction of histone deacetylase1 and 2 (Hdac1/2) to Six2 was detected in developing kidney by co-immunoprecipitation and proximity ligation assay. Chromatin immunoprecipitation and DNA sequencing experiments in isolated E16.5 NPCs revealed 1,180 (84.58%) of the Six2 peaks overlapped with Hdac2 peaks, implying the involvement of Hdac1/2 in Six2 DNA binding and its function in NPCs. To test whether Hdac1/2 are required for Six2 function to regulate nephron formation, we employed Six2 GC mouse line, in which the eGFP and Cre fusion gene (GC) replaces and fully recapitulates the endogenous Six2 gene expression pattern. Analysis of kidneys at embryonic day (E) 19.5 and newborn (P0) showed that Six2 heterozygous (Six2 GC ) together with three alleles knockout of Hdac1/2 resulted in severely hypoplastic kidneys, while three alleles knockout of Hdac1/2 by transgenic Six2-Cre only led to very subtle phenotypes. Immunostaining at E 19.5 and P0 revealed about 50% reduction of Six2 level in the kidney of Six2 GC only and Six2 GC together with three alleles knockout of Hdac1/2 mice. In the kidneys of Six2 heterozygous mice, no change was observed for most of the NPC identity makers for self-renewal, including the three Six2 targets, Pax2, Sall1 and WT1. However, sequential removal of three alleles of Hdac1/2 of Six2 GC mice did not change Six2 protein level but significantly decreased the expression of Pax2, Sall1 and WT1, suggesting the requirement of Hdac1/2 for Six2’s function to transcriptionally activate the expression of its target genes. We also observed the premature differentiation and decreased nephron formation in mutant kidneys. Therefore, we conclude that Hdac1/2 are required for Six2’s function to promote NPC self-renewal and repress premature differentiation during nephrogenesis.