Single-cell multiomics reveals ENL mutation perturbs kidney developmental trajectory by rewiring gene regulatory landscape.

How disruptions to normal cell differentiation link to tumorigenesis remains incompletely understood. Wilms tumor, an embryonal tumor associated with disrupted organogenesis, often harbors mutations in epigenetic regulators, but their role in kidney development remains unexplored. Here, we show at single-cell resolution that a Wilms tumor-associated mutation in the histone acetylation reader ENL disrupts kidney differentiation in mice by rewiring the gene regulatory landscape. Mutant ENL promotes nephron progenitor commitment while restricting their differentiation by dysregulating transcription factors such as Hox clusters. It also induces abnormal progenitors that lose kidney-associated chromatin identity. Furthermore, mutant ENL alters the transcriptome and chromatin accessibility of stromal progenitors, resulting in hyperactivation of Wnt signaling. The impacts of mutant ENL on both nephron and stroma lineages lead to profound kidney developmental defects and postnatal mortality in mice. Notably, a small molecule inhibiting mutant ENL's histone acetylation binding activity largely reverses these defects. This study provides insights into how mutations in epigenetic regulators disrupt kidney development and suggests a potential therapeutic approach.

Engineering physiological environments to advance kidney organoid models from human pluripotent stem cells.

During embryogenesis, the mammalian kidney arises because of reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM), driving UB branching and nephron induction. These morphogenetic processes involve a series of cellular rearrangements that are tightly controlled by gene regulatory networks and signaling cascades. Here, we discuss how kidney developmental studies have informed the definition of procedures to obtain kidney organoids from human pluripotent stem cells (hPSCs). Moreover, bioengineering techniques have emerged as potential solutions to externally impose controlled microenvironments for organoid generation from hPSCs. Next, we summarize some of these advances with major focus On recent works merging hPSC-derived kidney organoids (hPSC-kidney organoids) with organ-on-chip to develop robust models for drug discovery and disease modeling applications. We foresee that, in the near future, coupling of different organoid models through bioengineering approaches will help advancing to recreate organ-to-organ crosstalk to increase our understanding on kidney disease progression in the human context and search for new therapeutics.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Isthmin-1: A critical regulator of branching morphogenesis and metanephric mesenchyme condensation during early kidney development.

Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 in Xenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8ß1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.

A comparative study of cellular diversity between the Xenopus pronephric and mouse metanephric nephron.

The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the kidney, comprise a blood filter, the glomerulus or glomus, and an epithelial tubule that processes the filtrate from the blood or coelom and selectively reabsorbs solutes, such as sugars, proteins, ions, and water, leaving waste products to be eliminated in the urine. Genes coding for transporters are segmentally expressed, enabling the nephron to sequentially process the filtrate. The Xenopus embryonic kidney, the pronephros, which consists of a single large nephron, has served as a valuable model to identify genes involved in nephron formation and patterning. Therefore, the developmental patterning program that generates these segments is of great interest. Prior work has defined the gene expression profiles of Xenopus nephron segments via in situ hybridization strategies, but a comprehensive understanding of the cellular makeup of the pronephric kidney remains incomplete. Here, we carried out single-cell mRNA sequencing of the functional Xenopus pronephric nephron and evaluated its cellular composition through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney. This study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and associated gene expression profiles. Thus, our data highlight significant conservation in podocytes, proximal and distal tubule cells, and divergence in cellular composition underlying the capacity of each nephron to remove wastes in the form of urine, while emphasizing the Xenopus pronephros as a model for physiology and disease.

Zebrafish (Danio rerio) larva as an in vivo vertebrate model to study renal function.

There is an increasing interest in using zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition. As the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in this study whether 3- to 4-day-old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cutoff of 4.4-7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of organic anion transporter oat (slc22) multidrug resistance-associated protein mrp1 (abcc1), mrp2 (abcc2), mrp4 (abcc4), and zebrafish larva p-glycoprotein analog abcb4 was shown. In addition, nonrenal clearance of fluorescent substrates and plasma protein binding characteristics were assessed in vivo. The results of transporter experiments were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 h postfertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.NEW & NOTEWORTHY The study of renal function remains a challenge. In vitro cell-based assays are approved to study, e.g., ABC/SLC-mediated drug transport but do not cover other renal functions such as glomerular filtration. Here, in vivo studies combined with in vitro assays are needed, which are time consuming and expensive. In view of these limitations, our proof-of-concept study demonstrates that the zebrafish larva is a translational in vivo test model that allows for mechanistic investigations to study renal function.

Premature differentiation of nephron progenitor cell and dysregulation of gene pathways critical to kidney development in a model of preterm birth.

Preterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

Spatial transcriptional mapping of the human nephrogenic program.

Congenital abnormalities of the kidney and urinary tract are among the most common birth defects, affecting 3% of newborns. The human kidney forms around a million nephrons from a pool of nephron progenitors over a 30-week period of development. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlage, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single-cell RNA sequencing identified progenitor states, which were spatially mapped to the nephron anatomy, enabling the generation of functional gene networks predicting interactions within and between nephron cell types. Network mining identified known developmental disease genes and predicted targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease and enhance efforts to generate new kidney structures.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

E-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1's formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

Increasing mTORC1 Pathway Activity or Methionine Supplementation during Pregnancy Reverses the Negative Effect of Maternal Malnutrition on the Developing Kidney.

BACKGROUND: Low nephron number at birth is associated with a high risk of CKD in adulthood because nephrogenesis is completed in utero. Poor intrauterine environment impairs nephron endowment via an undefined molecular mechanism. A calorie-restricted diet (CRD) mouse model examined the effect of malnutrition during pregnancy on nephron progenitor cells (NPCs). METHODS: Daily caloric intake was reduced by 30% during pregnancy. mRNA expression, the cell cycle, and metabolic activity were evaluated in sorted Six2 NPCs. The results were validated using transgenic mice, oral nutrient supplementation, and organ cultures. RESULTS: Maternal CRD is associated with low nephron number in offspring, compromising kidney function at an older age. RNA-seq identified cell cycle regulators and the mTORC1 pathway, among other pathways, that maternal malnutrition in NPCs modifies. Metabolomics analysis of NPCs singled out the methionine pathway as crucial for NPC proliferation and maintenance. Methionine deprivation reduced NPC proliferation and lowered NPC number per tip in embryonic kidney cultures, with rescue from methionine metabolite supplementation. Importantly, in vivo, the negative effect of caloric restriction on nephrogenesis was prevented by adding methionine to the otherwise restricted diet during pregnancy or by removing one Tsc1 allele in NPCs. CONCLUSIONS: These findings show that mTORC1 signaling and methionine metabolism are central to the cellular and metabolic effects of malnutrition during pregnancy on NPCs, contributing to nephrogenesis and later, to kidney health in adulthood.

Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF.

Nephron 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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, ß-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. TGFß-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. CONCLUSIONS: 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.

A ß-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 ß-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated ß-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of ß-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/ß-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/ß-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, ß-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising ß-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

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.

Nascent nephrons during human embryonic development: Spatial distribution and relationship with urinary collecting system.

The two major components of the metanephros, the urinary collecting system (UCS) and nephron, have different developmental courses. Nephron numbers vary widely between species and individuals and are determined during fetal development. Furthermore, the development of nascent nephrons may contribute to the expansion of the proximal part of the UCS. This study investigated the distribution of nascent nephrons and their interrelationship with UCS branches during human embryogenesis. We obtained samples from 31 human embryos between Carnegie stages (CSs) 19 and 23 from the Kyoto Collection at the Congenital Anomaly Research Center of Kyoto University in Japan. Serial histological sections of the metanephros with the UCS were digitalized and computationally reconstructed for morphological and quantitative analyses. The three-dimensional (3D) coordinates for the positions of all UCS branch points, end points, attachment points to nascent nephrons (APs), and renal corpuscles (RCs) were recorded and related to the developmental phase. Phases were categorized from phase 1 to phase 5 according to the histological analysis. The UCS branching continued until RCs first appeared (at CS19). End branches with attached nascent nephrons (EB-AP[+]) were observed after CS19 during the fifth generation or higher during the embryonic period. The range of end branch and EB-AP(+) generation numbers was broad, and the number of RCs increased with the embryonic stage, reaching 273.8 ± 104.2 at CS23. The number of RCs connected to the UCS exceeded the number not connected to the UCS by CS23. The 3D reconstructions revealed RCs to be distributed around end branches, close to the surface of the metanephros. The RCs connected to the UCS were located away from the surface. The APs remained near the end point, whereas connecting ducts that become renal tubules were found to elongate with maturation of the RCs. Nascent nephrons in RC phases 3-5 were preferentially attached to the end branches at CS22 and CS23. The mean generation number of EB-AP(-) was higher than that of EB-AP(+) in 19 of 22 metanephros and was statistically significant for eight metanephros at CS22 and CS23. The ratio of the deviated branching pattern was almost constant (29%). The ratio of the even branching pattern with EB-AP(+) and EB-AP(+) to the total even branching pattern increased with CS (9.2% at CS21, 19.2% at CS22, and 45.4% at CS23). Our data suggest the following: EB-AP(+) may not branch further at the tip (i.e., by tip splitting), but branching beneath the AP (lateral branching) continues throughout the embryonic stages. Our study provides valuable data that can further the understanding of the interactions between the UCS and nascent nephrons during human embryogenesis.

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.

Analysis of structure and gene expression in developing kidneys of male and female rats exposed to low protein diets in utero.

A maternal low protein (LP) diet in rodents often results in low nephron endowment and renal pathophysiology in adult life, with outcomes often differing between male and female offspring. Precisely how a maternal LP diet results in low nephron endowment is unknown. We conducted morphological and molecular studies of branching morphogenesis and nephrogenesis to identify mechanisms and timepoints that might give rise to low nephron endowment. Sprague-Dawley rats were fed a normal protein (19.4% protein, NP) or LP (9% protein) diet for 3 weeks prior to mating and throughout gestation. Embryonic day 14.25 (E14.25) kidneys from males and females were either cultured for 2 days after which branching morphogenesis was quantified, or frozen for gene expression analysis. Real-time PCR was used to quantify expression of key nephrogenesis and branching morphogenesis genes at E14.25 and 17.25. At E17.25, nephron number was determined in fixed tissue. There was no effect of either maternal diet or sex on branching morphogenesis. Nephron number at E17.25 was 14% lower in male and female LP offspring than in NP controls. At E14.25 expression levels of genes involved in branching morphogenesis (Gfrα1, Bmp4, Gdnf) and nephrogenesis (Hnf4a, Pax2, Wnt4) were similar in the dietary groups, but significant differences between sexes were identified. At E17.25, expression of Gfrα1, Gdnf, Bmp4, Pax2 and Six2 was lower in LP offspring than NP offspring, in both male and female offspring. These findings provide new insights into how a LP diet leads to low nephron endowment and renal sexual dimorphism.

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.