BASIC RESEARCH www.jasn.org Hnf4a Is Required for the Development of Cdh6-Expressing Progenitors into Proximal Tubules in the Mouse Kidney Sierra S. Marable,1,2 Eunah Chung,1,2 and Joo-Seop Park 1,2,3 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Hepatocyte NF 4a (Hnf4a) is a major regulator of renal proximal tubule (PT) development. In humans, a mutation in HNF4A impairs PT functions and is associated with Fanconi renotubular syndrome (FRTS). In mice, mosaic deletion of Hnf4a in the developing kidney reduces the population of PT cells, leading to FRTS-like symptoms. The molecular mechanisms underlying the role of Hnf4a in PT develop- ment remain unclear. Methods The gene deletion tool Osr2Cre removed Hnf4a in developing nephrons in mice, generating a novel model for FRTS. Immunofluorescence analysis characterized the mutant phenotype, and lineage analysis tested whether Cadherin-6 (Cdh6)–expressing cells are PT progenitors. Genome-wide mapping of Hnf4a binding sites and differential gene analysis of Hnf4a mutant kidneys identified direct target genes of Hnf4a. Results Deletion of Hnf4a with Osr2Cre led to the complete loss of mature PT cells, lethal to the Hnf4a mutant mice. Cdh6high, lotus tetragonolobus lectin-low (LTLlow) cells serve as PT progenitors and demon- strate higher proliferation than Cdh6low,LTLhigh differentiated PT cells. Additionally, Hnf4a is required for PT progenitors to differentiate into mature PT cells. Genomic analyses revealed that Hnf4a directly reg- ulates the expression of genes involved in transmembrane transport and metabolism. Conclusions Hnf4a promotes the differentiation of PT progenitors into mature PT cells by regulating the expression of genes associated with reabsorption, the major function of PT cells. JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2020020184 The kidneys filter the blood, regulate osmotic lev- volume. Numerous transporter and metabolism els, maintain electrolyte balance, and metabolize genes are expressed in the proximal tubules in order drugs. The functional unit of the kidney is the to facilitate the function and energy demands of nephron, which is composed of the glomerulus, these highly active renal epithelial cells.6–12 Despite the proximal tubule, the loop of Henle, and the their importance in kidney function, the molecular distal tubule.1 Eachsegmentofthenephronhas mechanisms of proximal tubule development and distinct physiologic functions and morphology. maturation are not well understood. 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 Received February 17, 2020. Accepted July 6, 2020. physiologic conditions, proximal tubules reabsorb Published online ahead of print. Publication date available at approximately two thirds of glomerular-filtered www.jasn.org. water and sodium chloride as well as most of the Correspondence: Dr. Joo-Seop Park, Cincinnati Children’s filtered glucose and phosphate.5 Proximal tubular Hospital Medical Center, Location R1566, ML7007, 3333 Burnet reabsorption of water and metabolites is essential in Avenue, Cincinnati, OH 45229. Email: [email protected] the regulation of body fluid composition and Copyright © 2020 by the American Society of Nephrology JASN 31: ccc–ccc, 2020 ISSN : 1046-6673/3111-ccc 1 BASIC RESEARCH www.jasn.org Fanconi renotubular syndrome (FRTS) is defined as gen- Significance Statement eralized proximal tubule dysfunction.13,14 Symptoms of FRTS include glucosuria, phosphaturia, proteinuria, polyuria, and Proximal tubule cells are the most abundant cell type in the mam- polydipsia.14,15 These symptoms are consistent with a failure malian kidney, and they perform the bulk of the renal reabsorption of the proximal tubules to reabsorb and transport filtered mol- function. Despite the importance of these cells in kidney function, the molecular mechanisms of proximal tubule development and 16,17 ecules, causing urinary wasting. In humans, the heterozy- maturation are not well understood. Experiments reveal that, in the gous mutation R76W in the gene encoding Hepatocyte NF 4a developing mouse kidney, Cadherin-6-expressing cells act as (HNF4A) causes FRTS with nephrocalcinosis.18 Because this proximal tubule progenitors and they require Hnf4a to further de- mutation is located in the DNA binding domain, it has been velop into mature proximal tubules. Genomic analyses show that speculated that the mutation affects the interactions of Hnf4a directly regulates the expression of genes required for re- absorption, such as transmembrane transporter genes and me- 18 HNF4A with regulatory DNA. A recent study of the FRTS tabolism genes. This study advances understanding of how kidney HNF4A mutation in Drosophila nephrocytes confirmed that proximal tubule cells form during development. 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 regulations. All experiments were performed in accordance We have previously shown that Hnf4a is expressed in de- with animal care guidelines, and the protocol was approved veloping proximal tubules in the mouse kidney and that Hnf4a by the Institutional Animal Care and Use Committee of the – is important for proximal tubule formation.20 Mosaic loss of CCHMC (IACUC2017 0037). We adhere to the National In- Hnf4a in the murine nephron lineage caused FRTS–like symp- stitutes of Health Guide for the Care and Use of Laboratory toms, including polyuria, polydipsia, glucosuria, and phos- Animals. phaturia.20 Because of the mosaic expression of Six2GFPCre in mesenchymal nephron progenitor cells, the Hnf4a mutant Tamoxifen Treatment kidney by Six2GFPCre was a chimera of wild-type and mutant Tamoxifen (Sigma; T5648) was dissolved in corn oil (Sigma; cells.20 This made it difficult to perform more rigorous differ- C8267) at a concentration of 20 mg/ml. Pregnant female mice ential gene expression analyses. In this study, we generated a were injected with tamoxifen intraperitoneally (4 mg/40 g body wt). new mouse model with thorough deletion of Hnf4a in the IresCre proximal segments of the nephron using Osr2 and inves- Immunofluorescence Staining tigated the requirement of mature proximal tubules for post- Embryonic, neonatal, and adult murine kidneys were fixed in natal survival.21 We also performed lineage tracing to identify 4% paraformaldehyde in PBS, incubated overnight in 10% proximal tubule progenitor cells in the developing kidney. sucrose/PBS at 4°C, and imbedded in OCT (Fisher Scientific). To further elucidate the role of Hnf4a in proximal tubule de- Cryosections (8–9 mm) were incubated overnight with pri- velopment, we performed genome-wide mapping of Hnf4a mary antibodies in 5% heat-inactivated sheep serum/PBS binding sites in the murine neonate kidney and transcrip- with 0.1% Triton X-100. We used primary antibodies for tomic analysis of the Hnf4a mutant kidney. We found that GFP (1:500; Aves GFP-1020), Jag1 (1:20; DSHB TS1.15H), Hnf4a is required for terminal differentiation of proximal tu- Wt1 (sc-7385, 1:100; Santa Cruz), Biotin-LTL (B-1325, bule cells and that mature proximal tubules are required for 1:500; Vector Labs), FITC-LTL (FL-1321, 1:200; Vector postnatal survival. Cadherin-6-expressing (Cdh6high), lotus Labs), Hnf4a (ab41898, 1:500; Abcam), Hnf4a (sc-8987, tetragonolobus lectin-low (LTLlow) cells in the developing kid- 1:500; Santa Cruz), LDL-related protein 2 (Lrp2; sc-515772, ney are proximal tubule progenitor cells, and loss of Hnf4a 1:100; Santa Cruz), Ki67 (652402, 1:500; BioLegend), Slc12a1 causes their developmental arrest. Our genomic analyses (18970–1-AP, 1:500; Proteintech), Slc12a3 (HPA028748, revealed that Hnf4a directly regulates expression of many ma- 1:300; Sigma), Ass1 (16210–1-AP, 1:500; Proteintech), ture proximal tubule genes, including transporter and metabo- Slc5a2 (HPA041603, 1:100; Sigma), Miox (HPA039562, lism genes, consistent with the fact that active reabsorption is the 1:500; Sigma), Cdh6 (MAB2715-SP, 1:100; R&D Systems), major function of proximal tubules. and Cdh6 (HPA007047, 1:200; Sigma). Fluorophore-labeled secondary antibodies were used for indirect visualization of the target. Images were taken with a Nikon Ti-E wide-field METHODS microscope equipped with an Andor Zyla camera and Lumen- cor SpectraX light source housed at the Confocal Imaging Mice Core (CIC) at CCHMC. All mouse alleles used in this study have been previously pub- IresCre tm1Sad lished: Osr2tm2(cre)Jian (Osr2 ),22 Hnf4a Histology c tm1.1(cre/ERT2)Jrs CreER (Hnf4a ),23 Cdh6 (Cdh6 ),24 and Mouse kidneys were harvested and fixed in 4% paraformalde- tm3(CAG-EYFP)Hze Ai3 Gt(ROSA)26Sor (Rosa26 ).25 All mice were hyde in PBS overnight. Paraffinsections(5mm) were stained maintained in the Cincinnati Children’s Hospital Medical with hematoxylin and eosin or periodic acid–Schiff reagent Center (CCHMC) animal facility according to animal care (American MasterTech KTPAS). Images were taken with a 2 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH A LTL Lrp2 Hnf4a Hoechst LTL Lrp2 Hnf4a IresCre/+ ;Osr2 Control c/+ Hnf4a IresCre/+ mutant ;Osr2 c/c Hnf4a Hnf4a B PAS Control mutant Hnf4a Figure 1. Hnf4a deletion by Osr2Cre leads to loss of mature proximal tubule (PT) cells. (A) Loss of Hnf4a in the nephron inhibits formation of LTLhigh, mature PT cells and causes a decrease in expression of Lrp2, a PT-specific gene in the newborn (P0) kidney. Yellow arrowheads mark LTLlow, Lrp2low cells that persist in the mutant. Image is representative of n53. Scale bar, 100 mm. (B) Periodic acid–Schiff (PAS) staining of control and Hnf4a mutant kidneys at P0 shows that Hnf4a mutants lack brush border. Black arrowheads mark brush border. Image is representative of n53. Scale bar, 50 mm. JASN 31: ccc–ccc, 2020 Hnf4a in Renal Proximal Tubules 3 BASIC RESEARCH www.jasn.org Nikon Ti-E wide-field microscope equipped with an Andor newborn mice were crosslinked with 1% paraformaldehyde, Zyla camera and Lumencor SpectraX light source housed at sonicated, and incubated with Hnf4a antibody (ab41898)– the CIC at CCHMC.