ABSTRACT
Background Hnf4a is a major regulator of renal proximal tubule (PT) development. In humans, a mutation in HNF4A is associated with Fanconi renotubular syndrome (FRTS), which is caused by defective PT functions. In mice, mosaic deletion of Hnf4a in the developing kidney causes a paucity of PT cells, leading to FRTS-like symptoms. The molecular mechanisms underlying the role of Hnf4a in PT development remain unclear.
Methods We generated a new Hnf4a mutant mouse model employing Osr2Cre, which effectively deletes Hnf4a in developing nephrons. We characterized the mutant phenotype by immunofluorescence analysis. We performed lineage analysis to test if Cdh6+ cells are PT progenitors. We also performed genome-wide mapping of Hnf4a binding sites and differential gene analysis of Hnf4a mutant kidneys to identify direct target genes of Hnf4a.
Results Deletion of Hnf4a with Osr2Cre led to complete loss of mature PT cells, causing lethality in the Hnf4a mutant mice. We found that Cdh6high, LTLlow cells serve as PT progenitors and that they show higher proliferation than Cdh6low, LTLhigh differentiated PT cells. We also found that Hnf4a is required for PT progenitors to develop into differentiated PT cells. Our genomic analyses revealed that Hnf4a directly regulates the expression of genes involved in transmembrane transport and metabolism.
Conclusion Our findings show that Hnf4a promotes the development of PT progenitors into differentiated PT cells by regulating the expression of genes associated with reabsorption, the major function of PT cells.
Significance Proximal tubule cells are the most abundant cell type in the mammalian kidney and they perform the bulk of the renal reabsorption function. Despite their importance in kidney function, the molecular mechanisms of proximal tubule development and maturation are not well understood. Here we find that, in the developing mouse kidney, Cdh6high, LTLlow cells act as proximal tubule progenitors and that Hnf4a is required for these cells to further develop into proximal tubules. Our genomic analyses show that Hnf4a directly regulate the expression of genes required for reabsorption such as transmembrane transport genes and metabolism genes. This study advances our understanding of how kidney proximal tubule cells form during development.
INTRODUCTION
The kidneys function to filter the blood, regulate osmotic levels, maintain electrolyte balance, and metabolize drugs. The functional unit of the kidney is the nephron, which is composed of the glomerulus, the proximal tubule, the loop of Henle, and the distal tubule.1 Each segment of the nephron has distinct physiological functions and morphology. The proximal tubule cells are the most populous cell type in the kidney and they carry out the bulk of reabsorption in the nephron.2–4 Under physiological conditions, proximal tubules reabsorb approximately two-thirds of glomerular-filtered water and sodium chloride as well as most of the filtered glucose and phosphate.5 Proximal tubular reabsorption of water and metabolites is essential in the regulation of body fluid composition and volume. Numerous transporter and metabolism genes are expressed in the proximal tubules in order to facilitate the function and energy demands of these highly active renal epithelial cells.6–12 Despite their importance in kidney function, the molecular mechanisms of proximal tubule development and maturation are not well understood.
Fanconi renotubular syndrome (FRTS) is defined as generalized proximal tubule dysfunction.13, 14 Symptoms of FRTS include glucosuria, phosphaturia, proteinuria, polyuria, and polydipsia.14, 15 These symptoms are consistent with a failure of the proximal tubules to reabsorb and transport filtered molecules, causing urinary wasting.16, 17 In humans, the heterozygous mutation R76W in the HNF4A gene causes FRTS with nephrocalcinosis.18 Since this mutation is located in the DNA-binding domain, it has been speculated that the mutation affects the interactions of HNF4A with regulatory DNA.18 A recent study of the FRTS HNF4A mutation in Drosophila nephrocytes confirmed that the mutation reduced binding of Hnf4a to DNA and caused nuclear depletion of Hnf4a in a dominant-negative manner, leading to mitochondrial defects and lipid accumulation.19
We have previously shown that Hnf4a is expressed in developing proximal tubules in the mouse kidney and that Hnf4a is important for proximal tubule formation.20 Mosaic loss of Hnf4a in the murine nephron lineage caused Fanconi renotubular syndrome-like symptoms, including polyuria, polydipsia, glucosuria, and phosphaturia.20 Due to the mosaic expression of Six2GFPCre in mesenchymal nephron progenitor cells, the Hnf4a mutant kidney by Six2GFPCre was a chimera of wild-type and mutant proximal tubule cells.20 This made it difficult to perform more rigorous differential gene expression analyses. In this study, we generated a new mouse model with thorough deletion of Hnf4a in the proximal segments of the nephron using Osr2IresCre and investigated the requirement of mature proximal tubules for postnatal survival.21 We also performed lineage tracing to identify proximal tubule progenitor cells in the developing kidney. To further elucidate the role of Hnf4a in proximal tubule development, we performed genome-wide mapping of Hnf4a binding sites in the murine neonate kidney and transcriptomic analysis of the Hnf4a mutant kidney. We found that Hnf4a is required for terminal differentiation of proximal tubule cells and that mature proximal tubules are required for postnatal survival. Cdh6high, LTLlow cells in the developing kidney are proximal tubule progenitor cells and loss of Hnf4a causes their developmental arrest. Our genomic analyses revealed that Hnf4a directly regulates expression of many mature proximal tubule genes, including transport and metabolism genes, consistent with the fact that active reabsorption is the major function of proximal tubules.
METHODS
Mice
All mouse alleles used in this study have been previously published: Osr2tm2(cre)Jian (Osr2IresCre);22 Hnf4atm1Sad (Hnf4ac);23 Cdh6tm1.1(cre/ERT2)Jrs (Cdh6CreER);24 Gt(ROSA)26Sortm3(CAG-EYFP)Hze (Rosa26Ai3).25 All mice were maintained in the Cincinnati Children’s Hospital Medical Center (CCHMC) animal facility according to animal care regulations. All experiments were performed in accordance with animal care guidelines and the protocol was approved by the Institutional Animal Care and Use Committee of the Cincinnati Children’s Hospital Medical Center (IACUC2017-0037). We adhere to the NIH Guide for the Care and Use of Laboratory Animals.
Tamoxifen Treatment
Tamoxifen (T5648, Sigma) was dissolved in corn oil (C8267, Sigma) at a concentration of 20mg/ml. Pregnant female mice were injected with tamoxifen intraperitoneally (4mg/40g body weight).
Immunofluorescence Staining
Embryonic, neonatal, and adult murine kidneys were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), incubated overnight in 10% sucrose/PBS at 4°C, and imbedded in OCT (Fisher Scientific). Cryosections (8-9μm) were incubated overnight with primary antibodies in 5% heat-inactivated sheep serum/PBST (PBS with 0.1% Triton X-100). We used primary antibodies for GFP (1:500, Aves GFP-1020), Jag1 (1:20, DSHB TS1.15H), Wt1 (1:100, Santa Cruz sc-7385), Biotin-LTL (1:500, Vector Labs B-1325), FITC-LTL (1:200, Vector Labs FL-1321), Hnf4a (1:500, Abcam ab41898), Hnf4a (1:500, Santa Cruz sc-8987), Lrp2 (1:100, Santa Cruz sc-515772), Ki67 (1:500, BioLegend 652402), and Cdh6 (1:200, Sigma HPA007047). Fluorophore-labeled secondary antibodies were used for indirect visualization of the target. Images were taken with a Nikon Ti-E widefield microscope equipped with an Andor Zyla camera and Lumencor SpectraX light source housed at the Confocal Imaging Core (CIC) at CCHMC.
Histology
Mouse kidneys were harvested and fixed in 4% paraformaldehyde in PBS overnight. Paraffin sections (5 μm) were stained with hematoxylin and eosin or periodic acid-Schiff reagent (American MasterTech KTPAS). Images were taken with a Nikon Ti-E widefield microscope equipped with an Andor Zyla camera and Lumencor SpectraX light source housed at the Confocal Imaging Core (CIC) at CCHMC.
ChIP-seq
ChIP-seq was performed as previously described.26 Briefly, kidneys from newborn (P0) mice were crosslinked with 1% paraformaldehyde, sonicated, incubated with Hnf4a antibody (Abcam ab41898) coupled Dynabeads Protein G (ThermoFisher). Eluted DNA was used for constructing sequencing libraries using the ThruPLEX DNA-seq kit (Takara). Libraries were sequenced on an Illumina HiSeq 2500 by the DNA Sequencing and Genotyping Core at CCHMC. ChIP-seq reads were mapped to mm9 using Bowtie.27 We performed peak calling and motif analysis using HOMER.28 Data are available at Gene Expression Omnibus under accession number GSE144824.
RNA-seq
RNA-seq was performed as previously described.20 Briefly, 1 μg total RNA was isolated from P0 Hnf4a mutant and control kidneys using the RNeasy Plus Micro kit (Qiagen 74034) followed by mRNA was isolation with NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, NEB). Fragmentation of mRNA followed by reverse transcription and second strand cDNA synthesis was done using NEBNext Ultra RNA Library Prep Kit for Illumina (E7530, NEB). Sequencing libraries were constructed using ThruPLEX DNA-seq kit (Takara R400428). Sequencing was performed as described above. RNA-seq reads were mapped to mm9 using TopHat and normalized gene expression values were calculated using Cufflinks.29 Genes that showed at least a 1.5- fold change in expression with a p-value ≤0.05 were considered differentially expressed. Data are available at Gene Expression Omnibus under accession number GSE144772.
Genomic Regions Enrichment of Annotations Tool (GREAT) analysis
GREAT analysis was performed using the online program, version 3 (great.stanford.edu).30 To associate genomic regions with genes, gene regulatory domains were defined as minimum 5.0 kb upstream and 1.0 kb downstream of the TSS, and distally up to 1000 kb to the nearest gene’s basal domain (‘basal plus extension’ option). 10,417 genomic regions from the Hnf4a ChIP-seq dataset were entered into the GREAT online program and Mouse Genome Informatics (MGI) Expression terms of genes associated with the genomic regions were assessed.
Gene Ontology Analysis
Gene ontology analysis was performed using DAVID Bioinformatics Resources (david.ncifcrf.gov) on differentially expressed genes identified from the RNA-seq analysis.31
Statistical Analyses
Statistical analysis of Kaplan-Meier survival curve was performed using GraphPad 8 Prism software. The Log-rank test was used for survival analysis. Student’s t test was performed using GraphPad 8 Prism software. P<0.05 was considered to be significant.
RESULTS
Hnf4a is required for mature proximal tubule formation
In our previous study, we utilized Hnf4a floxed alleles and Six2GFPCre to generate a mouse model with kidney-specific deletion of Hnf4a. However, Six2GFPCre displayed mosaic expression in nephron progenitor cells and allowed a subset of nephron progenitors to escape Cre-mediated recombination.20, 32, 33 Therefore, our previous Hnf4a mutant kidney was a chimera of wild-type and mutant cells leading to the FRTS- like phenotype we observed. In order to thoroughly investigate the Hnf4a loss-of- function phenotype, we utilized a less mosaic Cre that specifically targets the proximal segments of the nephron. We generated a new mouse model with nephron-specific deletion of Hnf4a using a mouse line expressing Cre recombinase under the Osr2 promoter (Osr2IresCre)22 bred with Hnf4a floxed mice (Hnf4ac/c).23 We have recently shown that Osr2IresCre is expressed in the proximal and medial segments of the S- shaped body (SSB) of the developing nephron and that the medial segment of SSB develops into proximal tubules and loops of Henle.21 This Cre, therefore, targets all nephron segments except for the distal tubule. Osr2IresCre achieved almost complete deletion of Hnf4a in the kidney (Figure 1A). Lotus Tetragonolobus Lectin (LTL) is known to bind to glycoproteins on the surface of the proximal tubules specifically.34, 35 Deletion of Hnf4a in the nephron led to loss of differentiated proximal tubule cells with high LTL staining (LTLhigh) in postnatal day 0 (P0) kidneys (Figure 1A). The Hnf4a mutant kidneys showed a decrease in the level of Lrp2 (Low-density lipoprotein-related protein 2), a proximal tubule-specific endocytic receptor protein also known as Megalin (Figure 1A).20, 36 A few LTLlow, Lrp2low cells persisted in the Hnf4a mutant kidney (Figure 1A, yellow arrowheads). We reasoned that these cells might represent immature proximal tubules or proximal tubule progenitor cells. A distinctive feature of the mature proximal tubules is the apical brush border. The brush border is composed of microvilli which increase the surface area of the proximal tubule to facilitate reabsorption.9, 10, 37 The absence of the proximal tubule brush border has been associated with proximal tubule dysfunction in patients, highlighting the importance of brush border formation.38 We analyzed brush border formation using the Periodic acid-Schiff (PAS) stain39 and found that Hnf4a mutants showed a lack of brush border formation (Figure 1B). Considering that brush border formation only occurs in post-mitotic, differentiated cells40, our result suggests that the Hnf4a mutant kidney lacks terminally differentiated proximal tubule cells.
Loss of Hnf4a in the nephron leads to postnatal lethality
To examine the effects of loss of LTLhigh differentiated proximal tubules on postnatal kidney development, we analyzed the histology of Hnf4a mutant kidneys at P0, P7, and P14. At P0, the Hnf4a mutant kidney was similar in size to the control (Figure 2A). At P7, the Hnf4a mutant kidney was slightly smaller with a thinner cortex than the control kidney (Figure 2B). At P14, the medullary region of the Hnf4a mutant kidney was severely damaged, cysts formed in the cortical region, and hydronephrosis was apparent (Figure 2C). Increased filtrate flow through the renal tubules can lead to renal pelvic dilation and nonobstructive hydronephrosis in nephrogenic diabetes insipidus.41–43 It is likely that hydronephrosis seen in the Hnf4a mutant kidney is caused by increased filtrate flow through the nephron tubules due to lack of reabsorption in the proximal tubule. Survival analysis of the Hnf4a mutant mice showed that ∼60% of Hnf4a mutants were deceased by P14, likely due to kidney dysfunction (Figure 2D). No Hnf4a mutants survived to weaning age (P28). These results show that the lack of mature proximal tubules causes postnatal lethality, highlighting the importance of the mature proximal tubule function for survival.
Cdh6high, LTLlow cells in the developing kidney are proximal tubule progenitor cells
It has been previously suggested that Cdh6-expressing cells in the developing murine kidney are presumptive proximal tubule cells.44 It was reported that, in the mouse embryonic kidney, Cdh6 was expressed in the medial segment of the SSB and that LTL+ proximal tubules were still positive for Cdh6 although its expression was downregulated compared to Cdh6+ cells in the nephrogenic zone. Based on these observations, it was proposed that Cdh6-expressing cells in the nephrogenic zone were destined to become proximal tubules.44 Consistent with this, we found that there were two distinct populations of Cdh6+ cells in the wild type developing murine kidney: Cdh6high and Cdh6low cells (Figure 3A). The majority of Cdh6high cells were Hnf4a+ and had no or low LTL staining (red arrowheads and orange arrowheads, respectively, in Figure 3A), suggesting that these Cdh6high, LTLlow cells are prospective, immature proximal tubule cells. Cdh6low cells were also positive for Hnf4a and had strong LTL staining (yellow arrowheads in Figure 3A), suggesting that these Cdh6low, LTLhigh cells are differentiated proximal tubule cells. We found that, in the Hnf4a mutant kidney, Cdh6low, LTLhigh cells were absent and the number of Cdh6high, LTLlow cells were increased, suggesting that the loss of Hnf4a prevents Cdh6high, LTLlow cells from developing into Cdh6low, LTLhigh cells (Figure 3B).
In order to definitively test if Cdh6high cells are proximal tubule progenitor cells, we performed lineage analysis using a tamoxifen-inducible Cre recombinase under the Cdh6 promoter (Cdh6CreER) and a Cre-inducible Rosa26Ai3 reporter.24, 25 Pregnant dams were injected with tamoxifen at E14.5 or E16.5 to label Cdh6high cells and their descendant cells with the Rosa26Ai3 reporter. Embryos were harvested at E18.5. We found that all Rosa26Ai3 labeled cells were Hnf4a+ and most were also LTL+, indicating that Cdh6high cells in the developing kidney are proximal tubule progenitor cells (Figure 4).
Hnf4a has been shown to inhibit proliferation in hepatocytes and promote terminal differentiation.45 Many models of cellular differentiation show an inverse relationship between proliferation and differentiation.46–49 Terminal differentiation commonly involves exiting the cell cycle and entering a postmitotic state.46, 50 To determine whether the transition from Cdh6high, LTLlow proximal tubule progenitors to Cdh6low, LTLhigh differentiated proximal tubule cells coincides with cell cycle exit, we examined Ki67 expression in Cdh6high and Cdh6low cell populations (Figure 5, A and B). Ki67 is present in actively proliferating cells and absent in resting cells.51–54 We found that Cdh6high proximal tubule progenitor cells were highly proliferative while only few Cdh6low cells showed Ki67 expression (Figure 5A). When quantified, Cdh6high cells had a 10-fold higher proliferative rate than Cdh6low cells, indicating an expansion of the progenitor cell population before they exit the cell cycle and undergo terminal differentiation into mature proximal tubule cells (Figure 5B). This suggests that the number of proximal tubule cells in the newborn kidney is largely determined by the proliferation of Cdh6high proximal tubule progenitor cells.
Hnf4a gene regulatory network reveals the roles of Hnf4a in regulating proximal tubule development
To further elucidate the role of Hnf4a in the proximal tubule transcriptional program, we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) with P0 murine kidneys to identify Hnf4a-bound genomic regions. Two Hnf4a ChIP-seq replicates yielded 10,417 reproducible binding sites (peaks) (Supplemental Table 1). Our motif analysis showed high enrichment of the canonical Hnf4a binding DNA sequence within these Hnf4a peaks (Figure 6A), indicating that Hnf4a-bound genomic regions were successfully enriched in our ChIP-seq samples. We also found that DNA motifs for other nuclear receptors including the estrogen-related receptor alpha (ESRRA), the retinoid X receptor (RXR), the peroxisome proliferator- activated receptor (PPAR), and hepatocyte nuclear factor 1 beta (HNF1B) were also enriched within the Hnf4a-bound genomic regions, suggesting that these nuclear receptors share common target genes with Hnf4a to regulate proximal tubule development (Figure 6A). The majority of the Hnf4a peaks were found within 50kb of transcription start sites (TSS) (Figure 6B) and 30% of the peaks were located in promoter regions (Figure 6C). Genomic Regions Enrichment of Annotations Tool (GREAT) analysis of MGI expression annotations of genes associated with Hnf4a binding sites showed enrichment within the developing renal proximal tubules (Figure 6D),30 consistent with the fact that Hnf4a is specifically expressed in proximal tubules in the kidney. Multiple peaks were identified near the promoters of proximal tubule genes (Supplemental Table 1). In particular, we found Hnf4a peaks near genes such as Slc34a1 and Ehhadh, genes linked to FRTS in human patients (Figure 6E).55, 56
We conducted transcriptomic analysis (RNA-seq) of P0 Hnf4a mutant kidneys to complement our candidate target gene list with differential gene expression data (Supplemental Table 2). In the Hnf4a mutant kidney, 442 genes showed a significant decrease in expression (fold change ≥ 1.5; p-value < 0.05), including Slc34a1, Ehhadh, and Ass1, which are highly expressed in proximal tubules (Figure 7A, Supplemental Table 2).7, 8 As previously mentioned, mutations in SLC34A1 and EHHADH are associated with FRTS in patients.55, 56 Gene ontology (GO) analysis of these 442 downregulated genes showed enrichment of genes associated with metabolism and transport (Figure 7B). We found that 196 genes were significantly upregulated in the Hnf4a mutant kidneys (fold change ≥ 1.5; p-value < 0.05), including Cdh6, the gene marking proximal tubule progenitors (Figure 7A, Supplemental Table 2). GO analysis of the 196 upregulated genes showed enrichment of genes associated with phospholipid homeostasis and cholesterol transport (Figure 7C). In order to determine which genes are directly regulated by Hnf4a, we compared the differentially expressed genes in the Hnf4a mutant with the 7,823 genes associated with Hnf4a binding sites to find overlapping genes (Figure 7D, Supplemental Table 3). There were 245 genes in common between the significantly downregulated genes and the Hnf4a binding sites and 81 common genes between significantly upregulated genes and the Hnf4a binding sites (Figure 7D, Supplemental Table 3). GO analysis of the 245 downregulated genes showed enrichment of genes associated with transport and metabolism (Figure 7E). GO analysis of the 81 upregulated genes showed enrichment of genes associated with transport, metabolism, and response to thyroid hormone and ischemia (Figure 7F). Our genomic and transcriptomic analyses suggest that Hnf4a regulates proximal tubule maturation via activation of transport and metabolism genes, consistent with the functions of the proximal tubule.
DISCUSSION
In our previous study, we identified two populations of LTL+ cells in the developing mouse kidney: LTLlow and LTLhigh. Based on our results, we concluded that these two populations represent presumptive proximal tubules and differentiated proximal tubules, respectively.20 In order to further examine the presumptive proximal tubule population, we sought to identify a marker of proximal tubule progenitors. Previously, Cdh6 had been proposed as a marker for prospective proximal tubule cells.44 However, it had not been definitively shown that Cdh6+ cells develop into proximal tubule cells.44 To address this, we performed lineage analysis of Cdh6+ cells in the developing kidney and found that these cells all became Hnf4a+ proximal tubule cells (Figure 4). This experiment provides strong evidence that Cdh6+ cells are proximal tubule progenitors in the developing kidney. Cdh6+ proximal tubule progenitors are highly proliferative, while LTLhigh, Hnf4a+ proximal tubule cells proliferate less frequently (Figure 5). This suggests that expansion of proximal tubule progenitors determines the number of proximal tubule cells. Identification of proximal tubule progenitors will allow us to further investigate the developmental mechanisms of proximal tubule development.
We have previously reported that mosaic deletion of Hnf4a by Six2GFPCre in the developing mouse kidney causes a significant reduction in proximal tubule cells, phenocopying FRTS.20 The paucity of proximal tubules is consistent with reduced expression of proximal tubule genes, including the genes encoding glucose and phosphate transporters. However, it was unknown which genes were directly regulated by Hnf4a in proximal tubules. In this study, we performed Hnf4a ChIP-seq on newborn mouse kidneys and RNA-seq analysis of Hnf4a mutant kidneys by Osr2IresCre. From intersection of the ChIP-seq and RNA-seq datasets, we identified 245 Hnf4a direct target genes that were downregulated in the Hnf4a mutant during kidney development (Figure 7D). Among these 245 targets, the most enriched were genes associated with transmembrane transport, suggesting the role of Hnf4a in proximal tubule development correlates with the major function of the proximal tubule, active reabsorption (Figure 7E, Table 1A). The genes associated with fatty acid metabolism were also enriched in these 245 direct target genes (Table 1B). Taking into account that proximal tubule cells are highly active in metabolism and their energy demands are primarily met by fatty acid oxidation11, 57–59, our results suggest that Hnf4a regulates metabolic reprogramming during proximal tubule development. Interestingly, only 3% of the Hnf4a bound genes showed differential expression in the Hnf4a mutant kidney. Since many proximal tubule genes are upregulated postnatally,60 it is possible that Hnf4a alone is not sufficient to induce expression of the majority of its target genes and co-factors are required.
Motif analysis of genomic regions bound by a given transcription factor provides a list of other transcription factors that physically or genetically interact with the target transcription factor, sharing common target genes. Known motif analysis of our Hnf4a ChIP-seq datasets revealed that the DNA motifs for ESRRA, RXR, PPAR, and Hnf1b were enriched within the Hnf4a-bound genomic regions in the developing mouse kidney. The binding motifs of ESRRA and RXR are quite similar to the Hnf4a binding motif (Figure 6A), which could suggest that there is cooperative binding among these nuclear receptor transcription factors to activate a proximal tubule-specific transcriptional program. In contrast, similar binding motifs could imply competitive binding, since overlapping DNA motifs can lead to competition between transcription factors to activate or repress context-specific transcriptional programs.61 Recent studies in zebrafish have implicated retinoic acid signaling in the formation of proximal tubules, further supporting RXR as a potential co-regulator of proximal tubule development.62, 63 Both PPARa/g and Hnf1a/b have been implicated in proximal tubule development and function, indicating that they are good candidate co-regulators of proximal tubule development.64–66 PPAR transcription factors are known binding partners of RXR and one study predicted 17 common targets between Hnf4a and PPARa.67 Hnf1b is expressed in all nephron segments68–72 and Hnf1b deficiency in the nephron lineage of the mouse kidney leads to defects in nephron formation, particularly the proximal tubules, loops of Henle, and distal tubules.72, 73 Hnf1b is known to interact with Hnf4a and regulate common target genes.67, 69, 74 It has also been shown that expression of Hnf1b and Hnf4a, along with Emx2 and Pax8, can convert fibroblasts into renal tubular epithelial cells, strongly suggesting that Hnf1b is a co-regulator of proximal tubule development.75 Further investigation is needed to elucidate the interactions among these transcription factors and their roles in proximal tubule development.
In conclusion, we examined the molecular mechanisms of Hnf4a-regulated proximal tubule development. We found that proximal tubule development was arrested in the absence of Hnf4a. The Hnf4a mutant kidney cannot generate mature proximal tubules. Loss of proximal tubule cells in the Hnf4a mutant mice caused postnatal lethality, highlighting the importance of functional proximal tubules for survival. In the Hnf4a mutant kidney, there is an increase in Cdh6high, LTLlow presumptive proximal tubule cells. We definitively showed that Cdh6+ cells in the developing kidney are proximal tubule progenitors. These results suggest that Hnf4a is required for proximal tubule progenitors to differentiate into mature proximal tubule cells. Genome-wide analysis of Hnf4a binding sites in the kidney and transcriptomic analysis of the Hnf4a mutant kidney indicate that Hnf4a directly regulates expression of multiple genes involved in transmembrane transport and metabolic processes in the proximal tubule.
Author contributions
S.S.M. performed mouse experiments. S.S.M. and E.C. performed ChIP-seq. E.C. performed RNA-seq. S.S.M. and J.P. designed the experiments, analyzed the data, and cowrote the manuscript. S.S.M made the figures. All authors approved the final version of the manuscript.
Disclosures
None.
Supplemental Material
Supplemental Table 1. Genome-wide mapping of Hnf4a binding sites in the mouse kidney at P0 (ChIP-seq)
Supplemental Table 2. Differential gene analysis of the Hnf4a mutant kidney at P0 (RNA-seq)
Supplemental Table 3. Intersection of ChIP-seq and RNA-seq
Acknowledgments
The authors thank Steve Potter for critically reading the manuscript. We also thank the Confocal Imaging Core (CIC) and the DNA Sequencing and Genotyping Core (DSGC) at CCHMC. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health F31 DK120164 to S.S.M. and R01 DK120847 to J.P.
Footnotes
The authors declare no conflict of interest.