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Pax2 and Pax8 Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult

Ann M. Laszczyk,1 Atsuko Y. Higashi,1 Sanjeevkumar R. Patel ,2 Craig N. Johnson,3 Abdul Soofi,1 Saji Abraham,2 and Gregory R. Dressler1

1Department of Pathology, University of Michigan, Ann Arbor, Michigan 2Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 3Department of Cell and , University of Michigan, Ann Arbor, Michigan

ABSTRACT Background As the glomerular filtrate passes through the nephron and into the renal medulla, electro- lytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 continues. The collecting tubules and renal medulla are derived from Pax2- positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established. Methods To examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2, Pax8, or both in adult mice and examined the resulting and changes in expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells. Results Mice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2, as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyl- transferase complex to the promoter. Conclusions These data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine.

JASN 31: 1212–1225, 2020. doi: https://doi.org/10.1681/ASN.2019090962

The kidney regulates salt and water homeostasis and BP while continuously filtering waste products from the bloodstream into the urine. Reabsorption Received September 25, 2019. Accepted February 29, 2020. of water from the glomerular filtrate is essential for A.M.L., A.Y.H., and S.R.P. contributed equally to this work. urine concentration and maintaining proper hy- Published online ahead of print. Publication date available at drationinland-dwellingvertebrates.Failureto www.jasn.org. concentrate urine results in a diabetes insipidus Correspondence: Dr. Gregory R. Dressler, Department of Pa- characterized by excessive urination, thology, University of Michigan, BSRB 2049, 109 Zina Pitcher, electrolyte imbalance, volume depletion, and exces- Ann Arbor, MI 48109-0650. Email: [email protected] sive thirst. The regulation of water reabsorption is Copyright © 2020 by the American Society of Nephrology

1212 ISSN : 1046-6673/3106-1212 JASN 31: 1212–1225, 2020 www.jasn.org BASIC RESEARCH controlled by families of aquaporins (AQPs) and urea trans- Significance Statement porters (UTs), located at various points along the nephron and the collecting ducts.1 AQP2 and Arginine Vasopressin Pax2 plays an essential role in kidney development, and although (AVPr) are among the most well studied proteins that regulate subsets of epithelial cells in the adult kidney continue to express water reabsorption within the inner medullary collecting duct Pax2 and the related Pax8 protein, their function in adult kidneys has not been defined. The authors examined phenotypes and al- (IMCD) epithelia. UTs have also been implicated in urine tered gene expression patterns in adult mice lacking Pax2, Pax8, concentration. The Tonicity enhancer binding protein To- or both, showing that Pax2 and Pax8 regulate multiple trans- nEBP can regulate the expression of AQP2 and UTA1 (solute membrane ion and water channels in the adult renal medulla, carrier family 14 member 2, Slc14a2) in response to water including aquaporins and urea transporters. Inner medullary col- deprivation,2,3 but the mechanisms of action and its effects lecting duct cells respond to high-salt levels by upregulating Pax8, leading to increased activation of such transporters through specific on other transporters remains unknown. In addition to methylation of histones, defining a mechanism for regulating urine TonEBP, nuclear transcription factors expressed in the renal concentration. These findings point to a novel and redundant role medulla include the Pax2 and Pax8 proteins, whose roles in for Pax proteins in regulating salt and water homeostasis in the adult development are well characterized but whose functions in kidney. adult renal epithelia are unclear. fi fi The paired-box containing Pax genes were rst identi ed as Although the necessity for Pax2 in development is well estab- Drosophila regulators of in , but have since been lished, the need for continued expression of Pax2 or Pax8 in associated with mammalian development, congenital abnor- adult kidneys has not been examined. 4,5 malities, and cancer. The Pax2/5/8 subfamily of regulatory In this report, we examined the functions of Pax2 and Pax8 genes is expressed during embryonic development of a variety in the adult mouse kidney using conditional mutant alleles of tissues, including the kidney, the eye, the , the , and a tamoxifen-inducible Cre recombinase driver. Deletion the central nervous system, and B lymphocytes. Pax2, 5, and 8 of either Pax2 or Pax8 did not produce a discernible phenotype proteins share an identical DNA-binding paired domain and in healthy adult mice, whereas a double knockout (KO) of fi have signi cant in the carboxy-terminal octapep- both Pax2 and Pax8 generated mice with severe polyuria and tide, the partial homeodomain, and the activation domains, dehydration, consistent with the overlapping expression pat- suggesting that all of these genes were derived from duplica- terns of these two Pax proteins in the inner and outer renal 6 tion of an ancestral precursor. Genetic mutations in mouse medulla. The histology of the renal epithelium from cortex to and humans indicate an essential requirement for Pax2 in the papilla did not exhibit obvious structural abnormalities; how- development of the kidney, the , and the optic ever, gene expression analyses revealed significant differences 7,8 9 nerve, whereas Pax8 is required for thyroid development, in families of transporters needed for water reabsorption and 10 and Pax5 is required for B cell differentiation. Thus, urine concentration. Among the most affected proteins were fi fi although there is signi cant overlap in tissue-speci cgene AQPs and UTs, which were directly activated by Pax proteins expression patterns, individual Pax2, 5, or 8 mutants have in cell culture and contained Pax binding elements in the tran- fl distinct phenotypes that re ect unique functions in a subset scriptional promoter regions. Our data point to a novel and of tissues that express the proteins. redundant role for Pax proteins in regulating salt and water Kidney development is initiated by two cell types within the homeostasis in the adult kidney. intermediate mesoderm, the metanephric mesenchyme and the ureteric bud epithelium, both of which express Pax2. The invasion of metanephric mesenchyme by the ureteric METHODS bud epithelia initiates an inductive interaction that promotes aggregation and proliferation of Six 2–positive nephron progenitor cells around the ureteric bud tips.11,12 The mouse embryonic stem cells carrying a conditional Pax2 These nephron progenitors also express high levels of Pax2, floxed allele were obtained from the University of California, Da- which becomes downregulated as the glomerular, proximal vis Knockout Mouse Project repository (clone EPD0847_2_B03). tubule, and distal tubule epithelia matures. The differentiating Mice were rederived by blastocyst injection and germline trans- renal epithelia now begins to express Pax8, which persists into mitters were crossed to Flip recombinase expressing mice to delete adult kidneys. Pax2 germline-null mice have complete renal the lacZ marker and the neomycin cassette. The Pax8 floxed allele agenesis, as the initial inductive interactions required for kid- was kindly provided by Dr. M. De Felice16 and deletes 3 and ney development never occur.7,13,14 However, Pax8 mutants 4 of the paired domain to generate a null allele. The Rosa26- do not have any overt renal phenotypes, presumably because CreER allele was from The Jackson Laboratory (no. 008463, of the redundancy with Pax2 at early times in development.15 B6.129-Gt(ROSA)26Sortm(Cre.ERT2)TyiIJ). Mice were bred to ho- In adults, Pax2 expression remains in the ureteric bud derived mozygosity for Pax2 and Pax8 floxed alleles and were heterozy- epithelia of the collecting ducts and the inner and outer me- gous for the CreER allele. For each experiment, cohorts of six dulla, whereas it is off in the proximal and distal tubules. Pax8 adultmiceat8–10 weeks of age were injected intraperitoneally remains on in most all renal epithelial cells of the adult kidney. with 50 mg/kg of tamoxifen in corn oil for three consecutive days

JASN 31: 1212–1225, 2020 Pax Genes and Urine Concentration 1213 BASIC RESEARCH www.jasn.org andagainwithin4weeks.Bothmaleandfemalemicewereused (Genomatix Software GmbH) and extracted 1000 bp up- and kept according to National Institutes of Health guidelines. For stream and 100 bp downstream from all transcription start water and urine measurements, mice were housed blindly in met- sites of the top 100 genes downregulated. These sequences abolic cages for 24 hours at the Michigan Mouse Metabolic Phe- were searched for common represented motifs using default notyping Center. All procedures were approved by the University parameter in an unbiased manner. Committee on Use and Care of Animals at the University of Michigan. Immunostaining After euthanasia with CO2, kidneys were collected, fixed in 4% Antibodies formaldehyde or Carnoy fixative, paraffin embedded, and sec- Rabbit antibodies to Pax2 and PTIP have been previously vali- tioned 5-mm thick. Slides were numbered and selected for dated.17,18 Rabbit IgG (011–000–003) was from Jackson similar areas of the kidney. Sections were permeabilized with ImmunoResearch (West Grove, PA), anti–b-actin (A-1978) was TBST (5% Triton X-100 in Tris-buffered saline) and blocked from Sigma (St. Louis, MO), anti-H3K4me3 (ab8580) and anti- with 1% BSA (50–109–0681; Proliant Biologicals, Boone, IA) Pol II CTD (ab5408) were from Abcam (Cambridge, UK), in TBST. The primary antibodies, as listed above, were di- anti–Ash2-like (Ash2L, A300–107A) and anti–retinoblastoma- luted in TBST and incubated overnight at 4°C. Secondary binding protein 5 (RBBP5, A300–109A) were from Bethyl Labo- antibodies conjugated to Alexa Fluor 594 (A-11012 or ratories (Montgomery, TX); and anti-Pax8 antibodies (sc-81353 R37121; Invitrogen, Carlsbad, CA) were diluted 1:500 in mouse and sc-16279 goat) were from Santa Cruz Biotechnology TBST with 1% horse serum containing fluorescein-labeled (Dallas, TX). Antibodies used for immunostaining were Pax8, lotus tetragonolobus lectin (LTL) or dolichos biflorus agglu- 1:300 (10336–1-AP; Proteintech, Rosemont IL); SLC14a2, 1:50 tinin (DBA) at 1:500 (FL-1321 or FL-1031; Vector Labora- (ab95365; Abcam); AQP1, 1:100 (AQP-001; Alomone Labs, Jer- tories, Burlingame, CA) and sections were incubated for usalem, Israel); AQP2, 1:100 (AQP-002; Alomone Labs); AQP3, 1 hour at room temperature. Nuclei were labeled with 1:100 (AQP-003; Alomone Labs); and AQP4, 1:100 (AQP-004; 496-diamidino-2- phenylindole (D1306; Life Technologies, Alomone Labs). Carlsbad, CA). Images were taken at 403,203,or103 on an Olympus IX73 inverted microscope fitted with an Gene Expression Analyses Olympus DP80 digital camera. Total RNA was extracted from three independent adult kid- For frozen sections, tissues from adult animals were fixed neys of each genotype using TRIzol reagent (Life Technolo- with 4% PFA for 3 hours at 4°C, followed by sucrose substi- gies) and RNeasy Mini Kit (Qiagen). RNAs were numbered tution, and embedded in tissue-Tek O.C.T. compound (Sakura and submitted blindly for microarray expression analysis by Finetek). Blocks were sectioned using a cryostat (6 mm). Sec- the University of Michigan Comprehensive Cancer Center tions were treated with 0.1% Triton X-100 for 10 minutes at Affymetrix and Microarray Core Facility using Affymetrix room temperature, and subsequently blocked with 5% normal Mouse Gene 2.1 ST Arrays, as described.19 The microarray goat serum in PBS-0.05% Tween for 1 hour at room data were analyzed using Bioconductor packages in R version temperature. 3.4.0. Probe sets were limited to those defined as “main” by the Affymetrix. Robust multiarray average was used to fitlog2 Cell Culture and Transfection expression values.20 We further removed probe sets that had HEK293 cells were cultured in DMEM (450 mg/dl glucose) a variance across all samples ,0.025 because they are, by def- supplemented with 10% FBS and 100 U/ml penicillin and inition, uninteresting to us and likely unexpressed genes in 100 mg/ml streptomycin in 5% CO2/95% air at 37°C. Then, the tissue. We then used weighted21 linear models22 similar 10-cmplatesweretransfectedwith1–2 mg of DNA and 12 ml to fitting t-statistics to each probe set through the limma of Fugene 6 as described (Roche Molecular Biochemicals). At package of bioconductor. From there we selected probe 40 hours after transfection, cells were harvested, and chroma- sets with a $1.5-fold change and an adjusted P value of tin or whole-cell lysates were prepared as described.26,27 #0.05, using the Benjamini–Hochberg technique.23 Expres- Mouse mIMCD3 cells (American Type Culture Collection, sion values for each gene was calculated using the robust Manassas, VA) were cultured as recommended by the sup- multiarray average method and fitted to weighted linear plier. Cells were grown in 300 mOsm/kg control medium until models in R using the Affymetrix package of bioconductor.24 confluent, and treated at various times (4–24 hours) with ex- The complete excel file with all probe sets is available in perimental medium hyperosmotic (500 mOsm/kg) by the ad- Supplemental Table 1 and can be accessed at the Gene Ex- dition of NaCl or urea. pression Omnibus (accession number GSE141455). Gene Ontology and functional expression analyses of the genes Real-Time RT-PCR whose expression were altered in Pax2-null embryos, com- RNA was prepared from cells using the TRIzol RNA Isolation pared with controls, was carried out using Toppgene.25 For system (Life Technologies). Briefly, 1 mg of total RNAwas reverse- transcription factor motif analyses, we utilized the gene to transcribed into complementary DNA using random hexamers promoter function in the Genomatix Genome Analyzer and the SuperScript First Strand kit (Life Technologies) as

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A B C D

25 Pax2 Cdh1 50 Pax2 LTL 25 Pax2 DBA 25 Pax2 DBA

E F G H

25 Pax8 Cdh1 50 Pax8 LTL 25 Pax8 DBA 25 Pax8 DBA

Figure 1. Expression of Pax2 and Pax8 in newborn and adult kidneys. Immunostaining for Pax2 (red [A–D]) and Pax8 (red [E and F]) is shown with either cadherin1 (Cdh1), LTL, or DBA in green. Newborn kidneys show strong Pax2 in the nephrogenic zone ([A] arrows) and in collecting ducts (arrowheads), whereas Pax8 is in all proximal, distal, and collecting tubules (E). The adult cortex is negative for Pax2 in LTL-positive proximal tubules (B) but remains Pax2-positive in LTL-negative collecting ducts ([B] arrow), whereas LTL-positive epi- thelia exhibit strong Pax8 expression (F). The outer medulla shows strong Pax2 in DBA-positive collecting ducts ([C] arrows), whereas Pax8 is strong in DBA-positive and DBA-negative epithelial cells of the outer medulla (G). Cells in the renal papilla show strong Pax2- positive (D) and Pax8-positive (H) nuclei in all epithelial nuclei. described by the manufacturer. Complementary DNAwas diluted with ROX mastermix (Bio-Rad) in a Prism 7900 Sequence 20-fold and quantitative real-time PCR performed with the iTaq Detection system (Applied Biosystems). Eleven primer sets SYBR Green master mix (Bio-Rad) in a 7900HT Real-Time PCR within the SLC14A2 gene, spanning 2 kb upstream of ATG system (Applied Biosystems, Foster City, CA). Each reaction was to 1 kb downstream, were used (Supplemental Table 3). ChIP performed in triplicate. b-Actinwasusedasanendogenouscon- data are presented as percent input after subtraction of non- trol to normalize values. Primers pairs for PCR for b-actin (Life specific binding to rabbit IgG. In all experiments, nonspecific Technologies), and AQPs and UTs are listed in Supplemental binding was ,0.005% of input. Table 2. At least one primer for Pax2 or Pax8 RT-PCR maps to the deleted domains in the mutants. Annealing temperature was 60°C. Results were analyzed with the Applied Biosystems Prism RESULTS 7900 system software and mRNA expression levels were calculated using the DDCTmethod, as performed previously.28 The normal- Generation of Pax2 and Pax8 KOs in Adult Kidneys ized expression of each transcript in cells grown in 300 mOsm/kg Although Pax2 expression in the kidney has been well de- media was set to 1.0 and expression in the other cells is relative to scribed in development, a direct comparison of Pax2 and each respective control. Pax8 in adults was lacking. Thus, we compared the expression of Pax2 and Pax8 in newborn, developing kidneys and in 8- Chromatin Immunoprecipitation and Real-Time PCR week-old adults (Figure 1). As published previously, Pax2 pro- Mouse mIMCD3 cells in triplicate were treated with control tein expression is easily detected in the nephrogenic zone, in media or media supplemented with NaCl to raise osmolality to Six Homeobox 2–positive progenitor cells, also known as cap 500 mOsm/kg for 24 hours. DNA was crosslinked for 10 min- mesenchyme, surrounding the Pax2-positive ureteric bud tips utes by the addition of formaldehyde to a final concentration (Figure 1A). As the nephron develops further, Pax2 is down- of 1%, followed by quenching in 0.125 M glycine for 5 minutes. regulated in more proximal and distal, but remains on in the Chromatin was prepared and chromatin immunoprecipita- ureteric bud–derived collecting ducts. In contrast, nuclear tion (ChIP) was performed as described previously.27,28 The Pax8 protein is observed in tubular epithelial cells in the kid- precipitated DNA was reconstituted in sterile water and real- neys of newborn mice (Figure 1E). In adult kidneys, Pax2 is time PCR quantitation of precipitated genomic DNA relative still easily detected in the DBA-positive cortical collecting to inputs was performed in triplicate using IQ SYBR GREEN ducts, and in the outer and inner medulla, where the collecting

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A B Water Consumption 80 p<0.002

60

40

20 volume (ml/24h) volume 0 Cre- P2 P28P8

C Urine Output 60 p<0.01

40

20 volume (ml/24h) volume 0 Cre- P2 P28P8

D Urine Osmolality 2000

1500 p<0.02 1000 E mOsm/ml 500 0 Cre- Cre+

F Pax2 Pax8 2

1 mRNA (. units) mRNA (rel.

0 Cre- P2 P28P8 Cre- P2 P28P8 Cre- vs. P2, p<0.003 Cre- vs. P2, n.s. Cre- vs. P8, n.s. Cre- vs. P8, p<.001 Cre- vs. P28, p<.001 Cre- vs. P28, p<.001

Figure 2. Gross phenotypes of Pax2/8 double mutant kidneys. (A) Histology of adult kidneys with the indicated genotypes after ta- moxifen administration shows no major structural abnormalities of pathology. Slight dilation of IMCDs is seen in the Pax2/8 KOs (right lower panel), which may reflect increased urine output. (B) Water consumption as measured in single-housed metabolic cages of mice 2 with no Pax2/8 deletion (Cre ) or deletions of either Pax2 (P2), Pax8 (P8), or both genes (P28). A significant increase in water con- sumption was observed in Pax2/8 double KOs (*P,0.001; n56 per genotype). (C) Urine outputs as collected from metabolic cages over 24-hour periods shows significant increase in urine excretion only in Pax2/8 double KOs (*P,0.001; n56 per genotype). (D) Osmolality 1 2 was measured in urines from Pax2/8 double KOs (Cre )andcontrols(Cre ). (E) Immunostaining for Pax2 or Pax8 (green) in kidneys from Pax2fl/fl;Pax8fl/fl mice with or without RosaCreEr after tamoxifen administration. (F) Quantitative RT-PCR for Pax2 or Pax8 mRNA from 2 three total kidney RNAs per cohort after tamoxifen treatment of control mice (Cre ), Pax2, Pax8, or Pax2/8 double KOs. ducts converge to form the renal papilla (Figure 1, B–D). Nu- the renal papilla (Figure 1, F–H).Thus,Pax2andPax8are clear Pax2 protein is low to undetectable in the LTL-positive coexpressed in the nuclei of collecting ducts and in the inner proximal tubules and in the parietal epithelia of the glomeruli. and outer medulla, suggesting some potential for redundancy, Pax8 continues to be expressed in the LTL-positive proximal whereas only Pax8 remains highly expressed in proximal tubules as well as the DBA lectin–positive collecting ducts and tubules.

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C Controls Pax2/8 KOs Slc4a1 A Slc14a2 Slc5a2 Slc34a3 Slc10a2 6 Slc5a12 Slc6a19 Slc4a1 Slc7a7 Slc6a12 Slc17a4 4 Slco1a6 Slc22a4 Slc22a1 Slc1a1 Slc25a19

–log10(P.Value) Slc25a42 2 Slc13a2 Histogram Slc2a5 600 Slc16a4 Slc26a10 Slc5a6 Count 0 Slc5a11 0 –4 0 4 Slc6a13 Value Slc16a13 –4 –2 0 2 4 Slc23a3 logFC Slc7a9 Controls Pax2/8 KOs Go: Molecular Function B D Genes in ID Name pValueFDR B&H FDR B&Y Bonferroni Genes from Input Annotation 1 GO:0005215 transporter activity 5.4E-14 4.7E-11 3.4E-10 4.7E-11 52 1294 secondary active transmembrane 2 GO:0015291 2.7E-12 1.1E-9 8.7E-9 2.3E-9 21 236 transporter activity Histogram 3 GO:0022857 transmembrane transporter activity 8.5E-12 2.4E-9 1.8E-8 7.7E-9 42 1014

600 GO: Biological Process

ID Name pValue FDR B&H FDR B&Y Bonferroni Genes from Input Genes in Annotation Count 0 –4 0 4 1 GO:0006820 anion transport 7.2E-12 2.2E-8 1.8E-7 2.2E-8 33 641 Value 2 GO:0015711 organic anion transport 4.0E-11 6.1E-8 5.3E-7 1.2E-7 27 462

3 GO:0006811 ion transport 1.0E-10 1.0E-7 9.2E-7 3.2E-7 53 1627 Mouse Phenotype Genes from Genes in ID Name pValue FDR B&H FDR B&Y Bonferroni Input Annotation 1 MP:0009643 abnormal urine homeostasis 4.3E-8 5.9E-5 5.0E-4 1.1E-4 26 472 2 MP:0001762 polyuria 5.8E-8 5.9E-5 5.0E-4 1.5E-4 12 98

3 MP:0001756 abnormal urination 6.6E-8 5.9E-5 5.0E-4 1.7E-4 15 164

V$PAX2.01 Matrix Common to 2.0 P-value Match Total E Family #sequences V$PAX2 6.21E-22 607 458 V$ZFHX 0.000036 1063 507 1.0 bits V$SORY 0.00023 6807 609 V$GATA 0.00044 2104 582 V$HOXF 0.0019 3118 580 0.0 2X Down-regulated - 241 unique genes Genes Down - Common TF motifs 5101520 from MaxBase 11.1 (Genomatix) 2X Up-regulated - 466 unique genes

Figure 3. Gene expression analysis of Pax2/8 mutant kidneys. (A) Volcano plot of Affymetrix gene array comparisons between control (n53) and Pax2/8 KOs (n53) using total kidney RNA. (B) Heat map and clustering of 241 genes downregulated (blue) and 466 genes upregulated (red) in Pax2/8 KOs. Gene symbols and fold change are available in Supplemental Table 1. (C) Heat map of genes en- coding the solute carrier family of transmembrane transporters that are downregulated in Pax2/8 KOs. (D) Gene Ontology analyses of genes downregulated in Pax2/8 KOs, using the Toppgene suite.25 The gene list reduced in KOs shows strong correlation with ion transporters and urine concentration. (E) Common transcription factor motifs (TFs) found in the genes downregulated in Pax2/8 KOs and in genes upregulated in Pax2/8 KOs. The top 5 TFs are shown, the most significant by far being the Pax2 family found in genes downregulated. The Pax2 DNA-binding matrix common to most genes downregulated in the mutants is shown. V$IRFF, interferon regulatory factors; V$MYT1, C2H2 zinc finger proteins; V$SORY, Sox/Sry HMG Box proteins; V$SP1F, GC-Box factor Sp1; V$ZFHX, homeodomain.

To study the function of Pax2 and Pax8 in adult kidneys, but leaves the rest of the mRNA intact. We utilized a Rosa26- we obtained embryonic stem cells carrying a conditional CreEr driver to delete the Pax2 and Pax8 conditional floxed floxed alleles of Pax2 from the University of California, Davis alleles after tamoxifen injection. Given the potential for re- Knockout Mouse Project repository29 and rederived germline dundancy between Pax2 and Pax8, we first deleted the genes chimeras. The LacZ and Neo cassettes were then deleted by individually and also generated double mutants that were fl/fl fl/fl crossing mice to a Flip recombinase expressing strain. Activa- Pax2 :Pax8 :Rosa26CreER with tamoxifen at 8–10 weeks tion of Cre recombinase will delete exons 2 and 3 of the Pax2 of age. Control cohorts also receive tamoxifen but did not mRNA, which encodes most of the DNA-binding paired do- carry the Rosa26CreER allele. Adult mice that had deleted main. The Pax8 floxed allele was obtained from Marotta et al.16 either Pax2 or Pax8 appeared healthy; however, within days and deletes exons 3 and 4 of the DNA-binding paired domain, of receiving tamoxifen, the Pax2/8 double mutants (KOs)

JASN 31: 1212–1225, 2020 Pax Genes and Urine Concentration 1217 BASIC RESEARCH www.jasn.org

Medulla & Cortex Outer Medulla Inner Medulla

Figure 4. Regulation of UTA1 and UTA2 in Pax2/8 mutants. Adult kidney sections from control mice or mice carrying floxed alleles of either Pax2, Pax8, or Pax2 and Pax8 after tamoxifen treatment were immunostained with antibodies against Slc14a2 (red) or the brush border protein villin (green). Low-power images on the left show staining in the outer medulla (UTA2) of controls and single mutants, but not in the Pax2/8 double KOs. Villin marks the proximal tubule rich cortex. Higher-powered images through the inner medulla are shown on the right, with little detectable apical UTA1 in the Pax2/8 double KOs. Representative sections from three independently derived kidneys are shown for each genotype. exhibit severe polyuria, as evidenced by excessive urination IMCDs could be observed and likely reflects the increased and thirst. Histologic sections did not reveal any remarkable urine output measured in the Pax2/8 KOs. Additional repre- gross abnormalities in the Pax2/8 KOs (Figure 2A), with intact sentative histologic sections and higher-powered magnifica- epithelial tubules in the cortex and medulla and no evidence of tions are shown in the supplemental data (Supplemental fibrosis or other pathologies. Slight increase in the dilation of Figure 1, A and B). Water consumption and urine excretion

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Control Pax8 Pax2 Pax2/8 AQP1 LTL DAPI

AQP2 LTL DAPI

AQP3 LTL DAPI

AQP4 LTL DAPI

Figure 5. Differential regulation of AQPs in Pax mutants. Immunostaining for AQPs 1–4 as indicated (red) and LTL (green) in adult kidneys from mice with the indicated Pax gene deletions. Expression of AQP1 protein appears unaffected in all mutants, whereas Pax2/ 8 double KOs exhibit decreased expression of AQPs 2–4 in the inner and outer medulla. Representative sections are shown from immunostainings done with three independent kidneys for each genotype. was measured overnight in metabolic cages for controls, single although the ROSA26Cre driver is reported to exhibit some Pax2 or Pax8 KOs, and Pax2/8 double KOs (Figure 2, B and C). leakiness, varied efficiency, and potential off-target effects, the Only the Pax2/8 KOs exhibited a significant five- to ten-fold fact that our single Pax2 or Pax8 mutants did not show a increase in water consumption and a ten-fold increase in urine robust phenotype upon Cre activation indicates that the dou- production compared with controls or single mutants. Urine ble Pax2/8 KO phenotypes were specific and not attributable osmolality was also significantly reduced in the Pax2/8 KOs, to off-target effects. indicating a severe urine concentration defect (Figure 2D). Because Cre recombination only deletes selected exons, we Gene Expression Analyses in Pax2/8 Double KOs also examined the efficiency of Cre-Lox–mediated Pax gene To more precisely define the genes and proteins affected in excision by immunostaining for Pax2 or Pax8 in kidneys from the Pax2//8 KOs, we isolated RNA from whole kidneys and mice carrying both floxed alleles with or without tamoxifen compared relative gene expression by Affymetrix microarrays (Figure 2E). Nuclear Pax2 or Pax8 staining was nearly com- (Figure 3). The complete data set is available from the Gene pletely absent throughout the kidneys of mice carrying all four Expression Omnibus (accession number GSE141455). Princi- floxed alleles when given tamoxifen. Using primers that map pal component analyses showed good separation for controls to the deleted domains, quantitative RT-PCR for Pax2 or Pax8 and Pax2/8 KOs, with .680 genes significantly altered at least in controls, single, and double KOs also confirmed the effi- two-fold (Figure 3, A and B, Supplemental Table 1). Among cient deletion of Pax2 and/or Pax8 mRNAs (Figure 2F). Thus, the genes whose levels were significantly reduced in the Pax2/8

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A B shN shPTIP C D PTIP –––+ Pax8 –+++ Pax8 PTIP 0.75 0.4 PTIP 0.3 Fp PRS Pax 0.50 TK GFP 0.2 % Input Bp GFP 0.25 % Input 0.1 b-Actin 0.00 0.0 1234 1234 1234

E Ash2L F Rbbp5 G H3K4me2 H H3K4me3 0.4 0.4 20 20

0.3 0.3 15 15

0.2 0.2 10 10 % Input % Input % Input % Input 0.1 0.1 5 5

0.0 0.0 0 0 1234 1234 1234 1234

Figure 6. Mechanism of Pax8-activated gene expression. Human HEK293 cells carrying an integrated Pax reporter gene (A) driven by Pax Responsive Sequences (PRS) were used to characterize Pax8-mediated transcription activation. (B) Cells were treated with negative control short hairpin RNA(shN) or PTIP KO short hairpin RNA (shPTIP) and transfected with Pax8 or an shPTIP-resistant rescue plasmid (lane 4). Note that PTIP KOs fail to activate the GFP reporter gene in the presence of Pax8. (C–H) Chromatin from cells transfected as in (B) was immunoprecipitated with antibodies against the indicated proteins. Quantitative PCR was done with primers against the PRS sequence to determine protein occupancy. Note that Pax8 binds regardless of PTIP KOs. However, Pax8 recruits PTIP and components of the Mll3/4 complex (Ash2L and Rbbp5) to imprint high levels of H3K4me2 and H3K4me3. All ChIP assays were performed in triplicate, with error bars representing one SD from the mean.

KOs were the solute carriers (Slcs; Figure 3C), which encode To confirm some of the observations obtained from differ- transmembrane ion and UTs. Gene Ontology analyses con- ential gene expression analyses, we performed quantitative firmed that the number one molecular function downregulated RT-PCR for several genes and immunostaining for Slc14a2 in Pax2/8 KOs was ion transport, with abnormal urine homeo- and selected AQPs. Affymetrix arrays tend to underestimate stasis or polyuria the most significant phenotypic correlation. the fold change, as confirmed by quantitative RT-PCR for For the top 100 genes downregulated in Pax2/8 mutants, we used genes using total RNAs isolated from the Pax2/8 double KOs Genomatix software to examine transcription start sites for and single mutants (Supplemental Figure 2). The Slc14a2 gene common transcription factor binding motifs and found the encodes multiple splice forms that generate different UT pro- number one motif was the Pax2/5/8 consensus binding site teins. The SLc14a2 antibody (ab95365) recognizes the (V$Pax2; P,6.21E-22). The second most common identified carboxy-terminal that is common to UTA1 and UTA2 motif was for the zinc finger homeobox family of proteins proteins encoded by Slc14a2 (Figure 4). In control animals, (V$ZFHX), yet this motif had a much lower statistical P value strong Slc14a2 staining is seen in thin epithelial tubules of the of 3.6E-5. For genes whose expression was higher in the Pax2/8 inner medulla, which is likely to represent UTA2 in the thin KOs, no correlation with polyuria or transmembrane ion chan- descending limb. Weaker staining was observed along the api- nels was observed, nor did the transcription start sites for genes cal surface of cells in the inner medulla, which would likely upregulated contain significant Pax binding motifs. These data correspond to UTA1. The single Pax2 and Pax8 KOs exhibited suggest that many of the affected transmembrane ion channels similar UTA2 staining as controls in the outer medulla, but whose expression is reduced may be direct targets activated by had reduced apical staining in the inner medulla (Figure 4). Pax2, Pax8, or most likely both proteins, because they share an The Pax2/8 double KOs did not show UTA2 staining in the identical DNA-binding paired domain. Note that the Affymetrix outer medulla and had no detectable UTA1 staining in the probe set for Pax8 still hybridized to the remaining mRNA se- inner medulla, consistent with the polyuria phenotype quences downstream of the deletion encompassing exons 3 and observed. 4. Also of note, there were no changes in the expression levels of We also observed reduction in expression of select AQP arginine vasopressin (Avp), the receptors Avpr1a and Avpr1b, or proteins in the renal medulla and in subsets of tubules (Fig- the AVP-induced gene Avpi1 in kidney RNAs isolated from con- ure 5). The Pax2/8 double KOs had a clear reduction of AQPs trol or Pax2/8 double KOs (Supplemental Table 3). However, we 2–4expressioninthemedulla,whereasAQP1wasnotaf- did see a two-fold decrease in the Avpr2 receptor upon loss of fected. Additional staining for AQPs 1–4confirmed localiza- Pax2 and Pax8. tion of AQP1 and AQP2 to apical surfaces in controls, whereas

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A B Urea transporter mRNA C Aquaporin mRNA 20 10 Pax 8 UTA1 Aqp2 50 UTA2 Aqp3 15 8 * 50 UTA3 Aqp4 Pax 2 UTA4 6 10 ** Legend 4 * * 50 β -Actin 5 2 Relative Expression 37 Relative Expression (normalized to β -actin) 0 4 8 12 17 24 (normalized to β -actin) 0 0 0 4 8 12 16 20 24 0 4 8 12 16 20 24 NaCl Exposure Time (Hours) Time (hours) Time (hours)

D 5.20 kb Forward strand 78.207Mb 78.208Mb 78.209Mb 78.210Mb 78.211Mb

primer pairs 11 10 9 8 7 6 5 4 3 2 1

< Slc14a2-201 protein coding

< Slc14a2-202 protein coding

< CCDS29361.1 protein coding

EFUTA1 UTA3 G Pax8 (G) 0.15 Pax 8 15 * 15 * 300 50 500 10 10 0.10 β-Actin

37 5 5 % Input 0.05 300 500 Osmolality Relative Expression Relative Expression 0 0 (normalized to β -actin) (normalized to β -actin) 0.00 300 500 300 500 1234567891011 Osmolality Osmolality

H PTIP I Ash2L J Rbbp5 0.15 0.15 0.15 300 300 300 500 500 500 0.10 0.10 0.10 % Input % Input 0.05 % Input 0.05 0.05

0.00 0.00 0.00 1234567891011 1234567891011 1234567891011

K H3K4Me3 L Pol II M Pax2 1.5 0.15 0.15 300 300 300 500 500 500 1.0 0.10 0.10 % Input % Input % Input 0.5 0.05 0.05

0.0 0.00 0.00 1234567891011 1234567891011 1234567891011

Figure 7. Pax8-dependent osmoregulation of Slc14a2. (A) Protein lysates from IMCD cells cultured with 500 mM NaCl were isolated at various times after salt addition and blotted for Pax8 and Pax2. (B) Expression of UTs was measured post salt addition. (C) As in (B), AQP mRNAs were measured in response to high-salt addition in IMCD cells. (D) A schematic of exons 1 and 2 of the Slc14a2 gene on mouse chromosome 18 is shown with the position of primer pairs used for ChIP indicated. (E and F) IMCD cells cultured under 300 or 500 mM

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Pax2/8 double KOs had a complete loss of AQP2 staining in suggesting an alternate mechanism of activation in response the inner and outer medulla (Supplemental Figure 3). How- to high-salt conditions in this mouse cell line. Surprisingly, ever, AQP3 and AQP4 could still be detected on apical surfaces Pax2 did not activate the UTA1 and UTA3 mRNAs in IMCD of inner medullary epithelial cells in Pax2/8 double KOs, al- cells (Supplemental Figure 5), which suggests the Pax2 and 8 though this was clearly reduced compared with the controls proteins are not entirely redundant. (Supplemental Figure 4). Nevertheless, the clear reduction of We next examined the promoter region of Slc14a2 around AQPs 2–4 and UTA1 and UTA2 implicates these proteins in the transcription start site for UTA1 and UTA3 (Figure 7D) by the urine concentration defect. ChIP, using IMCD cells cultured under normal and high-salt conditions (Figure 7, E–M). Primer pairs spanning the Slc14a2 Mechanism of Osmoregulation by Pax8 in IMCD Cells transcriptional start site were tested for binding to various The Pax2 and Pax8 proteins have identical DNA-binding proteins of interest. As predicted, 500 mM NaCl strongly ac- paired domains and show significant homology within the tivated Pax8 above baseline and resulted in increased UTA1 carboxy terminus. Genetic studies also suggest that the and UTA3 expression (Figure 7, E and F). ChIP experiments Pax2/5/8 family have redundancy in functions and can, at least showed increased Pax8 binding to primer pair 4 and 6 under in part, substitute for one another.30 Previous work indicated high-salt conditions (Figure 7G). Furthermore, increased Pax2 recruits the adaptor protein PTIP, which is part of an Pax8 expression resulted in the recruitment of PTIP, Ash2L, Mll3/4 histone methyltransferase complex.27 Given the wide- and Rbbp5, which led to increased H3K4me3 and RNA Pol II spread expression of Pax8 in adult renal epithelia, it was crit- (Figure 7, H–L). However, we did not detect Pax2 binding by ical to determine whether Pax8 functions similarly to Pax2. ChIP to the Slc14a2 promoter in this mIMCD cell line, con- Thus, we transfected Pax8 into HEK293 cells that carried an sistent with the inability of Pax2 to activate Slc14a2 by trans- integrated Pax responsive GFP reporter gene. Because fection. Taken together, the data show that Pax8 directly binds HEK293 cells express neither Pax2 or Pax8, this enabled us to the promoter region of Slc14a2 and regulates expression in to examine precisely what happens at the Pax-responsive ele- response to high-salt conditions to maintain salt and water ment that binds the paired domain (Figure 6). We examined homeostasis. expression of the reporter in the presence or absence of Pax8 and PTIP, using short hairpin RNAs against PTIP. ChIP re- vealed that Pax8 can bind to the PRS sequence regardless of DISCUSSION PTIP. However, PTIP was necessary to recruit elements of the MLL complex (Ash2L and Rbbp5) and for high levels of H3K4 In this report, we demonstrate overlapping expression of Pax2 di- and trimethylation (Figure 6, C–H). Thus, Pax8-mediated and Pax8 within the cortical collecting ducts and in the inner reporter gene activation was very similar to that described for and outer medulla of the adult kidney. Given the identical Pax2, consistent with the conservation of the DNA-binding DNA-binding paired domains and significant homologies and transactivation domains. throughout the carboxy-terminal transactivation domain, For a more physiologically relevant cell culture model, we we proposed that these two proteins have similar biochemical next utilized IMCD cells that do express both Pax2 and Pax8. properties and functions. This was confirmed in cell culture Prior work suggested that Pax2 was necessary to protect IMCD models and genetically by creating adult loss-of-function mu- cells from the high-osmolality environment that these cells are tations with conditional alleles and tamoxifen-induced Cre exposed to during urine concentration.31 We noted that cul- recombinase in mice. The most striking phenotype was turing mouse IMCD cells in 500 mM NaCl strongly increased the inability to concentrate urine, resulting in a diabetes the amounts of Pax2 and Pax8 proteins within 4–8hours insipidus–like phenotype in the Pax2/8 double KOs. Nephro- (Figure 7A). High-salt conditions also increased the expres- genic diabetes insipidus (NDI) is characterized by the insen- sion of UT mRNAs UTA1 and UTA3 more than ten-fold after sitivity of the water channel AQP2 to the antidiuretic hormone 16 hours (Figure 7B). However, AQPs 2–4 increased approx- Avp in the collecting ducts. Upon water deprivation, vasopres- imately four-fold within 4 hours after high-salt culture sin promotes post-translational modifications and transloca- (Figure 7C), which preceded the strong increase of Pax2 and tion of AQP2 to the apical membrane of principal cells Pax8 mRNA and suggests activation by alternative mecha- to promote osmotic reabsorption of water.32,33 Congenital nisms. Transfection of Pax8 into IMCD cells under normal NDI is often because of mutations in the AVPR or AQP2 salt conditions also strongly activated the UTA1 and UTA3 genes and can be managed.34 Coincidentally, Avpr2 is down- mRNAs, but not the AQPs (Supplemental Figure 5), again regulated more than two-fold in the Pax2/8 double KOs

NaCl show increase in Pax8 protein and increased UTA1 and UTA3 expression (*P,0.01). (G–M) ChIP experiments using chromatin form IMCD cells cultured with 300 or 500 mM NaCl and immunoprecipitated with the indicated antibodies. Note binding of Pax8 near primer pairs 4 and 6, the recruitment of PTIP, Ash2L, and Rbbp5, and the increase in H3K4me2 and RNA Pol II. ChIP PCR reactions were run in triplicate for two independent chromatin sets. Error bars are one SD from the mean.

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(see Supplemental Table 3), but RNA levels of Avp or the Av- is associated with AQP2 and AQP3 downregulation in the pr1a and Avpr1b receptors are unchanged. Thus, the loss of cortical collecting ducts.44,45 The upregulated genes suggest AQP and UT protein expression is most likely not because of a more complex changes in response to the loss of Pax2/8 that lack of Avp signaling. are likely not direct targets, activated or repressed by Pax pro- NDI is also associated with lithium use,35 routinely pre- teins at promoters or enhancers. Given the severe dehydration scribed and effective for bipolar disorders. Lithium is also a phenotype, mice were euthanized within a few weeks after Pax potent inhibitor of GSK3, which activates NFAT5 (TonEBP), a deletion. Thus, if they were kept alive longer then additional positive regulator of UTs and AQPs.2,3 More recently, molec- pathology could develop. ular changes linked to lithium use have been described, which Despite many Pax2 and Pax8 mutations described in the include cell cycle progression arrest, altered inositol and PG adult population, urine concentration defects have not been signaling,36 and increased NF-kB signaling.37 The Pax2/8 KO specifically associated with Pax2/8 loss of function. Numerous mice also exhibit an increase in NF-kB target transcripts, such loss-of-function mutations in the DNA-binding paired do- as Ccl2, 12, 17, and Cxcl10, suggesting that NF-kBsignaling main of Pax2 have been described and are associated with may be upregulated in our mutants. There is an increase in congenital abnormalities, often described as papillorenal syn- Nfkb2 (NF-kB,subunit2)butnotinNfkb1inthePax2/8 drome, which consists of ocular colobomas, renal dysplasia or double KOs. NF-kB can bind to and repress the AQP2 pro- hypoplasia, reflux, and progressive renal failure.8 These are moter,38 which could contribute to the loss of AQP2 expres- most likely attributable to abnormal kidney development be- sion, but seems unlikely to be the sole reason. The data with cause of Pax2 haploinsufficiency. Given the expression of Pax2 Pax2/8 double mutants show an absence or reduction of in the progenitor cells of the nephron and in the branching AQP2–4 proteins in the outer and medulla, which had not ureteric buds, reduced Pax2 gene dosage could affect the num- been previously linked to Pax proteins. Thus, in addition to ber of nephrons formed or the branching pattern of the ure- defects in apical localization, the AQPs may thus be affected by teric bud. Nevertheless, patients that are heterozygous for mutations or loss of Pax transcription factors. Pax2 are likely homozygous for wild-type Pax8, with no de- The single Pax2 or Pax8 KOs appeared generally healthy, fects in urine concentration described to date. Similarly, for although the Pax8 KOs did show slight increases in water con- Pax8 loss of function, no renal defects have been described sumption and urine output, which did not approach statistical in most patients, presumably because of heterozygosity and significance. Because the single mutants were subject to redundancy of function with Pax2. Pax8 heterozygous indi- tamoxifen-induced Cre recombinase activation, they also viduals do have congenital hypothyroidism, which is not com- served as controls for nonspecific effects of the recombinase plemented by Pax2 because of the lack of Pax2 expression in compared with the double KOs. Hundreds of genes were al- the developing thyroid.9,46,47 Thus, our mouse genetics and tered in the Pax2/8 double KOs, many of which were trans- conditional deletion of Pax2/8 revealed a novel regulatory membrane ion transporters or channels. Immunostaining of function in the adult kidney that was not evident from either control and mutant sections confirmed the reduced levels of germline-null mouse mutations or clinically identified Pax2/8 the UT UTA1, whose loss had been previously correlated with heterozygous mutations. The direct regulation of UTA1 by urine concentration defects.39 We also noted the loss of select Pax8 was confirmed in cultured IMCD cells, which respond AQPs 2–4, but surprisingly not AQP1. Of these, mutations in to increased osmolality by upregulating Pax8 levels. Pax8 the vasopressin regulated AQP2 water channel have the most bound near the transcription start site of the UTA1 severe urinary concentration defect.40 However, mice carrying (Slc14a2) gene and recruited the histone methyltransferase AQP3 mutations survive but also have increased fluid con- complex via the adaptor protein PTIP, a mechanism similar sumption, decreased urine osmolality, and could respond to to that of Pax2.27,28 vasopressin.41 In summary, despite the clear need for Pax2 in kidney de- In addition to the downregulation of many solute carriers, velopment, the function of Pax proteins in the adult kidney loss of Pax2/8 increases expression of cell cycle control genes, have not been examined to date. Our data shows a critical need such as cyclins A2, B1, B2, and G1, and cell cycle checkpoint for Pax2 or Pax8 in regulating selective transporters in the kinases including Cdk1, Bub1 and 1b, Chek1, and Plk1. Fur- renal medulla that are responsible for maintaining salt and thermore, transcripts for proliferation markers such as MKI67 water balance. Increased expression of Pax genes is responsive and Sox9 are also increased. Because renal proliferation is rare to high osmolality and may help to maintain the viability of in normal kidneys,42 it is possible the cells may be dedifferen- renal epithelial cells in a high-salt environment.31 tiating or responding to some sort of disruption to epithelial homeostasis. Similarly, there is an increase in the kidney injury marker Lcn2 (or NGAL) and in the immediate early response genes Fos, Jun, and Nr4a1, which can also cause dedifferenti- ACKNOWLEDGMENTS ation and may indicate cellular stress such as hypoxia.43 Ad- ditionally, TGF-b,TGFb-induced, and gremlin are elevated We thank the University of Michigan Transgenic Core for blas- suggesting potential upregulation of TGF-b signaling, which tocyst injections and rederivation of Pax2 floxed mice, the University of

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Michigan Cancer Center microarray core facility for Affymetrix analyses, of aquaporin-2 expression in renal collecting duct principal cells. JAm Jeff Sands for antibodies against UTA1, and Nathan Qi and the Michigan Soc Nephrol 17: 1521–1531, 2006 Mouse Metabolic Phenotyping Center. 3. Miyakawa H, Woo SK, Dahl SC, Handler JS, Kwon HM: Tonicity- responsive enhancer binding protein, a rel-like protein that stimulates Dr. Laszczyk designed and executed experiments and prepared transcription in response to hypertonicity. Proc Natl Acad Sci U S A 96: figures. Dr. Higashi bred mice, designed and executed experiments, 2538–2542, 1999 and collected and prepared tissues. Dr. Patel designed cell culture 4. Grimley E, Dressler GR: Are Pax proteins potential therapeutic targets assays and prepared figures. Dr. Johnson did the Affymetrix analyses in kidney disease and cancer? Kidney Int 94: 259–267, 2018 and figures. Dr. Soofi managed mouse breeding, genotyping, and 5. Mahajan P, Leavey PJ, Galindo RL: PAX genes in childhood oncogenesis: Developmental biology gone awry? Oncogene 34: 2681–2689, 2015 histology. Dr. Abraham executed cell culture and chromatin immu- 6. Underhill DA: PAX proteins and fables of their reconstruction. Crit Rev noprecipitation experiments. Dr. Abraham planned experiments, Eukaryot Gene Expr 22: 161–177, 2012 prepared figures, analyzed data, and wrote the manuscript. 7. Torres M, Gómez-Pardo E, Dressler GR, Gruss P: Pax-2 controls multiple steps of urogenital development. Development 121: 4057–4065, 1995 8. Bower M, Salomon R, Allanson J, Antignac C, Benedicenti F, Benetti E, et al.: Update of PAX2 mutations in renal coloboma syndrome and es- DISCLOSURES tablishment of a locus-specific database. Hum Mutat 33: 457–466, 2012 9. Macchia PE, Lapi P, Krude H, Pirro MT, Missero C, Chiovato L, et al.: Dr. Dressler reports grants from the National Institute of Diabetes and Di- PAX8 mutations associated with congenital hypothyroidism caused by gestive and Kidney Diseases of the National Institutes of Health, during the thyroid dysgenesis. Nat Genet 19: 83–86, 1998 conduct of the study. All other authors have nothing to disclose. 10. Urbánek P, Wang ZQ, Fetka I, Wagner EF, Busslinger M: Complete block of early B cell differentiation and altered patterning of the pos- terior midbrain in mice lacking Pax5/BSAP. Cell 79: 901–912, 1994 FUNDING 11. Dressler GR: The cellular basis of kidney development. Annu Rev Cell Dev Biol 22: 509–529, 2006 12. Dressler GR: Advances in early kidney specification, development and This work was supported in part by National Institutes of Health grants patterning. Development 136: 3863–3874, 2009 DK073722 and DK054740 (to Dr. Dressler), and a Young Scientist Fellowship 13. Ranghini EJ, Dressler GR: Evidence for intermediate mesoderm and of the Japan Society for the Promotion of Science (to Dr. Higashi). kidney progenitor cell specification by Pax2 and PTIP dependent mechanisms. Dev Biol 399: 296–305, 2015 14. Soofi A, Levitan I, Dressler GR: Two novel EGFP insertion alleles reveal SUPPLEMENTAL MATERIAL unique aspects of Pax2 function in embryonic and adult kidneys. Dev Biol 365: 241–250, 2012 15. Bouchard M, Souabni A, Mandler M, Neubüser A, Busslinger M: This article contains the following supplemental material online at Nephric lineage specification by Pax2 and Pax8. Genes Dev 16: http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019090962/-/ 2958–2970, 2002 DCSupplemental. 16. Marotta P, Amendola E, Scarfò M, De Luca P, Zoppoli P, Amoresano A, et al.: The paired box transcription factor Pax8 is essential for function Supplemental Figure 1. (A) Histological sections from control and and survival of adult thyroid cells. Mol Cell Endocrinol 396: 26–36, 2014 fi Pax2/8 double KOs. (B) Higher magni cations of histological sec- 17. Dressler GR, Douglass EC: Pax-2 is a DNA-binding protein expressed in tions from control and Pax2/8 double KOs. embryonic kidney and Wilms tumor. Proc Natl Acad Sci U S A 89: Supplemental Figure 2. Quantitative RT-PCR for selected genes 1179–1183, 1992 identified by Affymetrix arrays. 18. Lechner MS, Levitan I, Dressler GR: PTIP, a novel BRCT domain- containing protein interacts with Pax2 and is associated with active Supplemental Figure 3. Immunostaining of control and Pax2/8 chromatin. Nucleic Acids Res 28: 2741–2751, 2000 double KOs with antibodies against AQP1 and AQP2. 19. Zhang P, Cai Y, Soofi A, Dressler GR: Activation of Wnt11 by trans- Supplemental Figure 4. Immunostaining of control and Pax2/8 forming growth factor-b drives mesenchymal gene expression through double KOs with antibodies against AQP3 and AQP4. non-canonical Wnt protein signaling in renal epithelial cells. JBiol – Supplemental Figure 5. Transfection of Pax8 or Pax2 in IMCD cells. Chem 287: 21290 21302, 2012 20. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf Supplemental Table 1. Excel spreadsheet of gene expression U, et al.: Exploration, normalization, and summaries of high density fi changes identi ed by Affymetrix arrays. oligonucleotide array probe level data. Biostatistics 4: 249–264, 2003 Supplemental Table 2. List of PCR primer pairs. 21. Ritchie ME, Diyagama D, Neilson J, van Laar R, Dobrovic A, Holloway A, Supplemental Table 3. Relative expression levels of AVP and et al.: Empirical array quality weights in the analysis of microarray data. receptors. BMC Bioinformatics 7: 261, 2006 22. 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