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. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Hypertension. 2024 Jan 2;81(3):541–551. doi: 10.1161/HYPERTENSIONAHA.123.22026

KDM6A demethylase regulates renal sodium excretion and blood pressure

Xiaobin Han 1, Leah Akinseye 1, Zhongjie Sun 1,*
PMCID: PMC10922853  NIHMSID: NIHMS1952889  PMID: 38164755

Abstract

Background.

KDM6A is a specific demethylase for histone 3 lysine (K) 27 trimethylation (H3K27me3). The purpose of this study is to investigate whether KDM6A in renal tubule cells plays a role in the regulation of kidney function and blood pressure (BP).

Methods.

We first crossed Ksp-Cre+/− and KDM6Aflox/flox mice for generating inducible kidney-specific deletion of KDM6A gene.

Results.

Notably, conditional knockout of KDM6A gene in renal tubule cells (KDM6A-cKO) increased H3K27me3 levels which leads to a decrease in Na excretion and elevation of BP. Further analysis showed that the expression of Na-K-2Cl cotransporter 2 (NKCC2) and Na-Cl cotransporters (NCC) was upregulated which contributes to impaired Na excretion in KDM6A-cKO mice. The expression of aquaporin 2 (AQP2) was also increased in KDM6A-cKO mice, which may facilitate water reabsorption in KDM6A-cKO mice. The expression of Klotho was downregulated while expression of aging markers including p53, p21, and p16 was upregulated in kidneys of KDM6A-cKO mice, indicating that deletion of KDM6A in the renal tubule cells promotes kidney aging. Interestingly, KDM6A-cKO mice developed salt-sensitive hypertension which can be rescued by treatment with Klotho. KDM6A deficiency induced salt-sensitive hypertension likely through downregulation of the Klotho/ERK (extracellular signal-regulatd kinase) signaling and upregulation of the WNK (with-no-lysine kinase) signaling.

Conclusion.

This study provides the first evidence that KDM6A plays an essential role in maintaining normal tubular function and BP. Renal tubule cell specific KDM6A deficiency causes hypertension due to increased H3K27me3 levels and the resultant downregulation of Klotho gene expression which disrupts the Klotho/ERK/NCC/NKCC2 signaling.

Keywords: KDM6A, H3K27me3, NKCC2, NCC, Klotho, ERK, FGFR1, WNK, SPAK

Graphical Abstract

graphic file with name nihms-1952889-f0007.jpg

INTRODUCTION

Epigenetic modification of histone alters gene transcriptional states and regulates various biological processes including development, cell differentiation, disease progression, and aging1–4. Trimethylation of histone 3 lysine 27 (H3K27me3) is modified by epigenetics and has been directly linked to lifespan and aging5–7. H3K27me3 is primarily a transcriptionally repressive mark catalyzed by the polycomb repressive complex 2 (PRC2) and removed by KDM6A (also known as UTX). PRC2 is responsible for establishing and maintaining histone H3K27 methylation during cell differentiation and proliferation. Interestingly, an opposite effect of histone modification, particularly for H3K27me3, on aging and lifespan has been observed in different aging models5–8. Epigenetic changes in aged individuals parallel those seen in patients with chronic kidney disease (CKD) manifested by accelerated kidney aging9. H3K27me3 is increased in acute kidney injury (AKI)10. Elevated H3K27me3 levels were also found in fibrotic kidneys in mice with unilateral ureteral obstruction (UUO)11. It is not known, however, whether KDM6A/H3K27me3 regulates sodium excretion and blood pressure.

Klotho is an aging-suppressor gene that is primarily expressed in the kidney tubule epithelial cells12–15. Mutation of the klotho gene leads to multiple aging phenotypes and shortened lifespan while overexpression of Klotho extends the life span in mice12–14. Klotho is a transmembrane protein which serves as a co-receptor for fibroblast growth factor receptor 1 (FGFR1) in the regulation of N-Pi co-transporters. Klotho is also found in the cytosols15, 16. Renal tubule cells secrete Klotho which serves as a paracrine factor and regulates adjacent cell function17. However, it is not fully understood whether Klotho regulates sodium transporters in renal tubule cells.

In this study, we investigated whether epigenetic modification of histone H3K27 affects kidney function and blood pressure by creating an inducible renal tubule cell-specific deletion of KDM6A gene mouse model. Here we reported that renal tubule cell-specific deletion of KDM6A downregulated Klotho gene expression which upregulated NKCC2, NCC and AQP2 expression and increased BP. Deletion of KDM6A from renal tubule cells impaired ERK and activated the WNK signaling which led to upregulation of sodium transporters, such as NCC, to the apical membrane of tubule cells, increasing renal sodium reabsorption.

MATERIALS AND METHODS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animals.

All animal research was conducted according to guidelines provided by the National Institutes of Health and the Institute of Laboratory Animal Resources, National Research Council. The project, of which this study was a part, was approved by the University of Tennessee Health Science Center’s Animal Care and Use Committee (IACUC). The floxed Klotho mice (KLflox/flox) were generated in a C57BL/6 background. Ksp-cadherin-Cre/ERT2 mice in a C57BL/6 background were kindly provided by Dr. Dorien J.M. Peters (Leiden University Medical Center)18. All mice were maintained on a standard diet (7912, Harlan Teklad, Madison, WI, USA). First, we crossed KDM6Aflox/+ to Ksp-Cre+/− to obtain heterozygous Ksp-Cre+/−/KDM6Aflox/+. The Ksp-Cre+/−/KDM6Aflox/+ male and female were crossed to obtain Ksp-Cre+/−/KDM6Aflox/flox mice (Figure S1A). KDM6Aflox/flox or Ksp-Cre+ mice were used as controls (wild-type equivalent control). For this study, mice were randomly divided into four cohorts: Wild type (WT) mice treated with vehicle (corn oil); WT mice treated with tamoxifen; Ksp-Cre+/−/KDM6Aflox/flox mice treated with vehicle; and Ksp-Cre+/−/KDM6Aflox/flox mice treated with tamoxifen. Mice at the age of 4–6 months were used for the study. Tail clips were collected to genotype the mice using REDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich, St. Louis, MO, USA) for DNA extraction and PCR amplification. Mice were genotyped for the KDM6Aflox allele using forward primer 5′-GGTCACTTCAACCTCTTATTGGA-3′ and reverse primer 5′-ACGAGTGATTGGTCTAATTTGG-3′ (325 bp product for the KDM6Aflox floxed allele and 355 bp product for WT allele), and for the Ksp-Cre+ using forward primer-1 5′-CATTCTCTCCCACTGGAATGGA-3′, forward primer-2 5’-ACAGAGTGGGGTTTGTGTCTG-3’, and reverse primer 5′-AACTGTCCCCTTGTCATACCC-3′ (507 bp product for the Ksp-Cre, and 388 bp for wild type mice). We collected 72 hours of urine and daily water intake from all groups of mice using metabolic cages for measuring urine sodium levels using atomic absorption spectrometer/Na-K analyzer (Perkin Elmer, USA). For salt-sensitive hypertension, we collected 72 hours of urine and daily water intake during the time after the animal received high-salt diet (HS) for two weeks and first Klotho treatment from all groups of mice using metabolic cages. Water balance was calculated by daily water intake minus daily urinary output. Blood pressure was measured using tail cuff (Kent Scientific) in awake mice without using anesthesia and confirmed by carotid artery cannulation in anesthetized mice at the endpoint of study as we described recently19–24. To study salt-sensitive hypertension, mice were fed with high salt (8% NaCl g/g) (Teklad) and Klotho (R&D) treatment (0.5 μg/mouse/3 times, i.p.) was performed in both control and KDM6A-cKO mice fed in a blinded manner with HS diet for up to 7 weeks. Both male and female mice were used.

Immunofluorescent staining.

The immunofluorescence staining was performed as we described recently25–32. Briefly, fresh kidney tissues were fixed in 4% paraformaldehyde overnight at 4°C after the mice were euthanized. Fixed kidneys were treated with 30% sucrose overnight and embedded in OCT (optimal cutting temperature) on dry ice and then stored at −80°C before use. Cryo sections (10 μm) were cut and air dried before storing at −80°C. We cut the kidney in a longitudinal direction and collected 20 sections from the middle kidney. Each kidney section contains cortex and medulla region of the kidney. Immunofluorescence staining was performed in multiple sections to detect expression of KDM6A, NCC, NKCC2, AQP2, and Klotho as we described previously25, 29, 33. Slides were rehydrated in phosphate buffered saline for 10 min and blocked with 5% goat serum (Invitrogen) in TBS containing 0.1% Triton-x 100 for 2 hours at room temperature, and then primary antibodies (Klotho antibody 1:50 R&D, FGFR1 and KDM6A (UTX) antibodies 1:100, Cell Signaling, NKCC2 antibody 1:100, LSBio, AQP2 antibody 1:200, Invitorgen, NCC antibody 1:100, StressMarq) were applied to slides and incubated overnight at 4°C. Secondary fluorescent labeled antibodies (AF555 or AF546 1:1000, Invitrogen) were applied following two PBS washes (10 min each) and incubated for 1 hour. Slides were rewashed twice for 10 min with PBS then dehydrated with series (50%, 75%, 90%, and 100%) ethanol for 5 min before sealing with an antifade reagent with DAPI (Invitrogen). Images were captured using Olympus IX73 fluorescent microscopy and Zeiss 710 2-photon confocal microscopy.

Kidney RNA isolation and real-time reverse transcriptase (RT)-qPCR.

Total RNA was isolated from the whole kidney of the mouse at the end point of the study using a RNeasy Mini Kit (Qiagen, Germany)34–36. For quantitative real-time RT-PCR, 1.0 μg total RNA isolated from kidney of mice was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) by following the manufacturer’s instructions. Real-time PCR (qPCR) reactions contained 2 μl of cDNA (equivalent to 50 ng of total RNA), 300 nM each primer, and 1× iQ SYBR Green supermix (Bio-Rad, Hercules, CA, USA) in a total of 20 μl reaction volume performed with CFX96 Real-Time PCR Detection Systems (Bio-Rad). Relative expression values were evaluated with the 2−ΔΔCt method using GAPDH as housekeeping gene (forward primer: 5’-CACCACCAACTGCTTAGCC −3’, and reverse primer: 5’-TGGCATGGACTGTGG-TCA-3’). Klotho: 5’- AGCGATAGTTACAACAAC-3’ (forward) and 5’-GCATTCTCTGATATTATAGTC-3’ (reverse), KDM6A: 5’-CTGACAGCGGAGGAGAGGGA-3’ (forward) and 5’-CCAATAAGAGGTTGAAGTGACCT-3’ (reverse). NCC: 5’-CTTCGGCCACTGGCATTCTG-3’ (forward) and 5’-GATGGCAAGGTAGAGATTGG-3’ (reverse). NKCC2: 5’-GGCTTGATCTTTGCTTTTGC-3’ (forward) and 5’-CCATCATTGATCGCTCTCC-3’ (reverse).

Western blot analysis.

Western blot analysis was performed as we described previously31, 37–39. Briefly, samples from mouse kidneys and mouse renal tubule cells were prepared in T-per tissue extraction buffer and M-per mammalian extraction buffer (ThermoFisher), respectively. Nuclear and cytoplasmic proteins were isolated by using a NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, Rockford, IL). Protein contents in the samples were quantified and stored at −80°C until use. For electrophoresis, 40μg kidney protein and 20μg renal tubule cell lysates were loaded onto Expressplus Page 4–12% gel (GenScript, USA). Proteins were separated at 120 V for 60 minutes and transferred to nitrocellulose membrane using Trans-Blot Turbo (BioRad). Membranes were blocked with 5% BSA blocking buffer in TBST (Thermo Scientific, Rockford, IL) for 2 hours and then incubated with primary antibody (Klotho, 1:500, R&D; KDM6A, FGFR1, p-ERK, tERK, H3K27me3, H3 (histone 3) 1:1000, Cell Signaling; tNCC and pNCC, 1:1000, Dr. DH. Ellision’s lab, NKCC2, 1:1000, LSBio; αENaC, 1:1000, StressMarq) with gentle agitation overnight at 4 °C. After 3 washes with TBST (15 min once and 2× 5 min), membrane was incubated with secondary antibody in 5% BSA blocking buffer at room temperature for 1 hour. Membrane was then washed 2 times (10 min) and subjected to ECL (BioRad) and analyzed with BioRad ChemiDoc, MP imaging system. Western blotusing β-actin or α-Tubulin (Cell Signaling) was used as loading control.

Statistical analysis

We evaluated the differences between two groups by unpaired t-test and multiple groups by one-way analysis of variance. All values are expressed as means ± SEM. All computations were performed using GraphPad Prism 8 (GraphPad Software Inc. La Jolla, CA, USA). A value of P< 0.05 was considered statistically significant.

RESULTS

Generation of conditional renal tubule cell-specific deletion of KDM6A mouse model - KDM6A-cKO mice

To generate the KDM6A-cKO mice, we crossed Ksp-Cre+ mice that express Cre recombinase in renal tubule cells under the control of the tamoxifen-inducible mouse cadherin 16 (Ksp) promoter with KDM6Aflox/flox mice that were generated by introduction of LoxP sites into the KDM6A locus on either side of exons 340. We treated Ksp-Cre/KDM6Aflox/flox mice with a low dose of tamoxifen (10 mg/kg body weight) to induce Cre recombination for 7 days (Figure S1A). Efficiency of KDM6A deletion was determined by real-time PCR (Figure 1A), Western blot analysis (Figure 1B), and immunofluorescence staining of kidney sections (Figure 1C). We found that expression of KDM6A was significantly reduced in the renal tubules of KDM6A-cKO mice. Renal tubule-specific deletion of KDM6A did not affect body weight in either male or female mice (Figure S1B and 1C).

Figure 1. Genetic removal of KDM6A from renal tubule cells increases H3K27me3 levels and causes hypertension.

Figure 1.

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(A) mRNA levels of the KDM6A determined by real-time PCR (qPCR) (n=3). (B). Western blot analysis of KDM6A protein expression (n=3). (C) Immunofluorescence to detect KDM6A in the kidney of sections (n=3). Scale bar, 50 μm. (D) Western blot analysis of H3K27me3 levels (n=3). (E) Systolic blood pressure of male and female control and KDM6A-cKO mice (n=10) and comparison of changes in systolic blood pressure of male vs female mice. (F) Systolic blood pressure of control and KDM6A-cKO mice measured by carotid artery cannulation (n=3–4). Values represent the mean ± SEM. *p<0.05 vs control by 2-tailed Student’s t test and one-way ANOVA followed by Fisher’s LSD ot-hoc test (E).

Genetic removal of KDM6A from renal tubule cells increases H3K27me3 levels and causes hypertension

To study the phenotype of renal tubule specific deletion of KDM6A, we first examined the levels of H3K27me3 in the kidney of KDM6A-cKO mice. We found that the level of H3K27me3 was increased approximately 5-fold in the kidney of KDM6A-cKO mice (Figure 1D). This finding indicated that renal tubule-specific deletion of KDM6A resulted in specific loss of histone demethylase function of KDM6A in renal tubule cells. Thus, KDM6A is an important demethylase that determines the H3K27me3 levels in renal tubule cells. We further demonstrated that blood pressure was significantly increased in both male and female KDM6A-cKO mice compared to their respective KDM6A-con and wild-type mice (Figure 1E). To eliminate the effect of tamoxifen on blood pressure, we also measured the blood pressure of tamoxifen-treated wild-type mice in this study. Blood pressure was similar in the wild-type mice treated with vehicle or tamoxifen, suggesting that tamoxifen treatment per se had no impact on the blood pressure in KDM6A-cKO mice. We performed a simple linear regression analysis and found that deletion of KDM6A caused elevation of blood pressure to a similar degree in male and female mice (Figure 1E), suggesting no sex difference in KDM6A-cKO-induced hypertension. Female mice usually have relatively lower resting BP than male mice do. However, the sex difference in BP in mice varies with strain and age41. In this study, the baseline BP was similar in male and female C57BL/6 mice at the age of 6 months (Figure 1E). A real-time monitoring of BP using the telemetry system may be used to further explore the phenomenon in different age groups. Finally, elevation of blood pressure in KDM6A-cKO mice was confirmed by measuring blood pressure with carotid artery cannulation methodology (Figure 1F).

Renal tubule cell-specific conditional knockout of KDM6A (KDM6A-cKO) upregulates expression of sodium transporters and impairs sodium excretion

To study the potential mechanisms of increased blood pressure in KDM6A-cKO mice, we performed a metabolic cage study and collected 72 hours of urine to measure the sodium levels in mice fed with normal chow and water. Water intake and urine output were increased in female KDM6A-cKO mice compared to female KDM6A-con mice (Figure 2A). Water intake was increased significantly in male KDM6A-cKO mice, but urine output was not different between male KDM6A-cKO and KDM6A-con mice (Figure 2B). It should be mentioned that water intake does not reflect fluid or Na balance. KDM6A-cKO did not alter body weight in either male or female mice during the study (Figure 2A and 2B). However, the urine sodium level was reduced by about 25% in both male and female KDM6A-cKO mice (Figure 2C) without sex difference, suggesting that sodium retention occurs in KMD6a-cKO mice. Interestingly, the immunofluorescent and real-time PCR analysis of NCC gene expression indicated that the NCC level was upregulated in the kidney of KDM6A-cKO mice compared to KDM6A-con mice (Figure 2D and 2E).

Figure 2. Renal tubule cell-specific conditional knockout of KDM6A (KDM6A-cKO) impairs sodium excretion.

Figure 2.

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(A) Daily water intake, urine output, and body weight of female mice (n=3). (B) Daily water intake, urine output and body weight of male mice (n=3). (C) Daily output of urine sodium in male and female mice (n=3). (D) Immunofluorescence to detect NCC in the kidney of sections (n=3), DAPI is in blue and NCC is in red. Scale bar, 50 μm. (E) qPCR of NCC mRNA (n=3). Values represent the mean ± SEM. *p<0.05 vs control by 2-tailed Student’s t test (C and E) and one-way ANOVA followed by Fisher’s LSD ot-hoc test (A and B).

Additionally, expression of NKCC2 in the ascending limb (Figure 3A–C) and AQP2, a water channel, in the collecting ducts (Figure 3E) was significantly increased in KDM6A-cKO mice compared to KDM6A-con mice. Upregulation of NKCC2 and AQP2 was confirmed by immunofluorescent staining (Figure 3D and 3F). In this study, we used a proximal tubule marker LTL (lotus tetragonolobus lectin) (green) to differentiate the proximal tubule from collecting ducts stained with AQP2 (red) in the kidney section (Figure 3F).

Figure 3. Loss of function of KDM6A upregulates expression of NKCC2.

Figure 3.

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(A) qPCR of NKCC2 mRNA (n=3). (B) Western blot analysis of NKCC2 (n=3). (C) Quantitation of immunoblotting for NKCC2 (n=3). (D) Immunofluorescence to detect NKCC2 in the kidney of sections (n=3), DAPI is in blue and NKCC2 is in red. Scale bar,100μm. (E) Western blot analysis and quantitation of AQP2 (n=3). (F) Immunofluorescence to detect AQP2 in the kidney of sections (n=3), DAPI is in blue, LTL is in green, and AQP2 is in red. Scale bar, 50μm. Values represent the mean ± SEM. p<0.05 vs control by 2-tailed Student’s t test (A, C and E).

Gain of function of H3K27me3 downregulates Klotho in the kidney and promotes kidney aging

Real-time PCR analysis showed that Klotho mRNA expression was reduced by more than 50% in the kidney of KDM6A-cKO mice compared to KDM6A-con mice (Figure 4A). Klotho protein expression was also decreased by more than 50% in the kidney of KDM6A-cKO mice (Figure 4B and 4C). Immunofluorescent staining showed that expression of Klotho was detected in both distal tubule (NCC positive) and proximal tubule cells, which was downregulated by deletion of KDM6A (Figure 4D).

Figure 4. Gain of function of H3K27me3 suppresses Klotho expression.

Figure 4.

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(A) qPCR of Klotho mRNA (n=3). (B) Western blot analysis of Klotho (n=3). (C) Quantitation of immunoblotting for Klotho (n=3). (D) Immunofluorescence to detect Klotho in the kidney sections (n=3), LTL is in green, DAPI is in blue, and Klotho is in red. Scale bar, 50μm. (E) Western blot analysis of p53, p21, and p16. β-actin was used as loading control. (n=3). (F) Quantitation of p53, p21, and p16, respectively. Values represent the mean ± SEM. p<0.05, p<0.01 vs control by 2-tailed Student’s t test.

Notably, the aging markers including p53, p21, and p16 were upregulated in the kidney of KMD6a-cKO mice compared to KDM6A-con mice (Figure 4E,F), suggesting that renal tubule cell-specific deletion of KDM6A accelerates kidney aging.

ERK signaling is diminished in the kidney of KDM6A-cKO mice

To study the mechanism by which the sodium transporters are upregulated in the kidney of KDM6A-cKO mice, we examined the expression of FGFR1, a co-receptor of Klotho for FGF23 (fibroblast growth factor 23) signaling in the kidney. We found that expression of FGFR1 was significantly increased in the kidney of KDM6A-cKO mice (Figure 5A and 5B). FGFR1 staining was stronger in the renal tubule cells of KDM6A-cKO mice compared to KDM6A-con mice (Figure 5C). Next, we investigated the ERK signaling by measuring the ERK activation. We showed that phosphorylated ERK was significantly decreased in the kidney of KDM6A-cKO mice compared to KDM6A-con mice (Figure 5D), suggesting that ERK signaling was inhibited due to KDM6A-cKO. We also demonstrated that expression of ENaC (epithelial sodium channel) was upregulated in the kidney of KDM6A-cKO mice compared to KDM6A-con mice (Figure 5E). Moreover, NCC, NKCC2, and AQP2 proteins were increased in the apical membrane of corresponding renal tubules in the kidney of KDM6A-cKO mice (Figure 5F).

Figure 5. ERK signaling is diminished in the kidney of KDM6A-cKO mice.

Figure 5.

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(A) Western blot analysis of FGFR1 (n=3). (B) Quantitation of immunoblotting for FGFR1 (n=3). (C) Immunofluorescence to detect FGFR1 in the kidney sections (n=3), LTL is in green, DAPI is in blue, and FGFR1 is in red. Scale bar, 50μm. (D) Western blot analysis and quantitation of p-ERK and t-ERK. (n=3). (E) Western blot analysis and quantitation of βENaC (n=3). (F) Immunofluorescence to detect NCC, NKCC2, and AQP2 in the kidney sections (n=3). DAPI is in blue, and NCC is in red. Scale bar, 50μm. (n=3). Values represent the mean ± SEM. p<0.05 vs control by 2-tailed Student’s t test.

KDM6A deficiency causes salt-sensitive hypertension which is eradicated by Klotho treatment

We then treated the hypertensive KDM6A-cKO mice (males and females) with an 8% high salt diet (HS) and monitored the blood pressure changes for the indicated period with or without Klotho interventions (Figure 6A and 6B). Notably, blood pressure of hypertensive KDM6A-cKO mice (males and females) was further largely elevated by the high salt diet compared to KDM6A control mice whose blood pressure was not affected by treating the high salt diet. This new finding indicates that KDM6A-KO causes salt-sensitive hypertension. Interestingly, Klotho treatments (3 days) normalized the blood pressure in KDM6A-cKO mice without affecting the blood pressure of KDM6A control mice. High salt-induced hypertension recurred 2 weeks later after withdrawal of Klotho treatment, and hypertension was reduced by repeating Klotho treatment (Figure 6A and 6B). These results suggest that KDM6A-induced salt-sensitive hypertension may be mediated by downregulation of Klotho levels.

Figure 6. KDM6A deficiency causes salt-sensitive hypertension which is eradicated by Klotho.

Figure 6.

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(A) Systolic blood pressure of male mice treated with high salt diet with or without Klotho treatment (n=6). (B) Systolic blood pressure of female mice treated with high salt diet with or without Klotho treatment (n=6). (C) Daily urine sodium output of male mice treated with high salt diet with or without Klotho treatment (n=3). (D) Daily water balance of male mice treated with high salt diet with or without Klotho treatment (n=3). (E) Daily urine sodium output of female mice treated with high salt diet with or without Klotho treatment (n=3). (F) Daily water balance of female mice treated with high salt diet with or without Klotho treatment (n=3). Values represent the mean ± SEM. *p<0.05, **p<0.01 vs control by 2-tailed Student’s t test.

Notably, urinary sodium excretion was significantly lower in high salt-treated KDM6A-cKO mice than in KDM6A controls, which was largely increased after Klotho treatments (Figure 6C and 6E). Daily water balance (water intake - urine output) was significantly higher in the HS-treated KDM6A-cKO mice compared to KDM6A controls, which was decreased after Klotho treatments (Figure 6D and 6F).

To investigate the potential mechanisms of the regulation of kidney function and blood pressure by Klotho, we measured ERK and WNK signaling in the kidney of these mice. ERK signaling was downregulated, while WNK signaling (p-SPAK, phosphor SPS1—related proline/alanine-rich kinase) was upregulated, in the kidney of KDM6A-cKO mice compared to KDM6A-con mice (Figure S2A–C). Klotho treatment abolished the changes in ERK and SPAK activities in KDM6A-cKO mice (Figure S2A–C). Accordingly, KDM6A deficiency increased expression of NKCC2, which was reduced by Klotho treatment (Figure S2D and S2E). Renal tubule deletion of KDM6A resulted in increased expression of NCC, which, however, was not affected by Klotho treatment (Figure S2F–H). Interestingly, Klotho treatment was able to reduce apical surface levels of NCC, NKCC2, and AQP2 in KDM6A-cKO mice compared to KDM6A-con mice (Figure S2I). Taken together, these findings indicate that Klotho reduces blood pressure in salt-sensitive hypertensive KDM6A-cKO mice, likely through regulation of sodium transporter(s), namely NCC and/or NKCC2 in renal tubule cells via ERK and WNK signaling pathways.

DISCUSSION

This study provides the first evidence that epigenetic upregulation of H3K27me3 mark in the renal tubule cells impairs sodium excretion and results in hypertension in a mouse model, in which the KDM6A gene was specifically deleted from the renal tubule cells. KDM6A-KO causes salt-sensitive hypertension as high salt diet largely exacerbated hypertension in KDM6A-KO mice but did not affect BP in control mice. The action of H3K27me3 is at least partly mediated by repression of the Klotho/ERK signaling, leading to upregulation of NCC, NKCC2, and AQP2 levels in renal tubule cells which increase reabsorption of sodium and water by the kidney (Figures 1–5).

This study demonstrated that elevated H3K27me3 levels due to KDM6A-cKO downregulated Klotho expression (Figure 4). This finding was supported by our recent report that H3K27me3 is physically associated with the promoter region of the Klotho gene in the kidney in aged mice42. Inhibition of PRC2 decreased H3K27me3 recruitment to the Klotho promoter region leading to increased Klotho expression in renal tubule cells42. However, whether downregulation of Klotho mediates KDM6A deficiency-induced hypertension is an important mechanistic question which has not been addressed. Treatment with Klotho protein completely and repeatably rescued the impairment in sodium excretion and elevation of BP in KDM6A-KO mice (Fig. 4), suggesting that Klotho is a critical mediator of KDM6A deficiency-induced hypertension. Given the increasing importance being placed on Klotho deficiency in the pathogenesis of aging15, 17, it would be practical to consider the epigenetic regulation of Klotho and Klotho-related pathways when exploring strategies for therapeutic interventions. Klotho is an aging-suppressor protein which is mainly produced in renal tubule cells17, 43. Consistent with downregulation of Klotho, the expression of aging markers including p53, p21, and p16 were upregulated in the kidney of KDM6A-cKO mice (Figure 4), suggesting that KDM6A-cKO mice may undergo an aging-like process in the kidney.

NCC is exclusively expressed in the distal convoluted tubules (DCT) cells which controls sodium reabsorption and regulates blood pressure. We found that KDM6A-cKO upregulated NCC levels which may contribute to increased sodium reabsorption and elevated BP. Although treatment with Klotho did not affect the upregulation of total NCC or phosphorylated NCC (pNCC) in KDM6A-cKO mice, it effectively reduced the overabundance of NCC in the apical membrane (Figure S2I). Additionally, expression of NKCC2 in the thick ascending limb was upregulated due to KDM6A deficiency (Figure 3) which may contribute to impairment in sodium excretion. This may be mediated by downregulation of Klotho as Klotho treatment rescued KDM6A deficiency-induced upregulation of NKCC2 levels (Figure S2). The thick ascending limb of the loop of Henle reabsorbs 25–30% of the sodium mediated by the apical NKCC2. Loss of function due to mutations in the NKCC2 gene results in severe salt and volume loss and lower blood pressure in humans (Bartter syndrome type 1) and a mouse model of Bartter syndrome44. On the other hand, KDM6A-cKO upregulated AQP2 expression in the collecting duct (Figure 3), which may facilitate water reabsorption. The excessive accumulation of AQP2 on the apical membrane may also be mediated by downregulation of Klotho because Klotho treatment abolished KDM6A deficiency-induced overabundance of AQP2 on the apical membrane (Figure S2I). Taken together, these findings suggest that epigenetic modulation of H3K27me3 levels in the renal tubule cells regulate sodium reabsorption and blood pressure, at least in part, via downregulation of Klotho expression which upregulates NCC, NKCC2, and AQP2 expression in the apical membrane of renal tubule cells throughout the nephron. A limitation of this study is that it did not assess the potential contribution of proximal tubules to KDM6A-cKO associated Na excretion. A further study is warranted for investigating KDM6A, H3K27me3 and Na transporters in proximal tubules in KDM6A-cKO mice.

Studies have shown that inhibition or deletion of FGFR1 from renal distal tubules causes hypertension45, 46. Klotho is a co-receptor of FGFR1 for FGF23/ERK signaling47, which regulates reabsorption of phosphate and calcium in the proximal tubule and distal tubule18, 48. Surprisingly, we found that FGFR1 expression was upregulated, while the ERK signaling was diminished in the kidney of KDM6A-cKO mice (Figure 5). This finding suggests that H3K27me3-mediated Klotho deficiency impairs the ERK signaling in the renal tubule cells of KDM6A-cKO mice. Klotho treatment rescued KDM6A deficiency-induced inactivation of ERK. Furthermore, expression of ENaC (epithelial sodium chloride transporter), a target of the ERK signaling in the collecting duct49, 50, was also upregulated in the kidney in KDM6A-cKO mice. Notably, NCC, NKCC2, and AQP2 were enriched onto the apical membrane of their corresponding renal tubule cells (Figure S2I), possibly due to the ERK inactivation and/or activation of the WNK signaling in the renal tubule cells. ERK signaling along the nephron plays an important role in electrolytes balance51. Activation of ERK signaling by phorbol esters (PE), a functional analogue of diacylglycerol (DAG), decreases NCC surface expression in the distal tubule cells through NCC ubiquitination52, while activation of ERK by lithium downregulates AQP2 expression in the collecting duct of rat kidney53. However, the precise mechanisms that promote NCC abundance to the apical membrane of DCT cells in the KDM6A-cKO mouse model remain to be determined.

Recent studies have shown that the WNK signaling plays a critical role in regulating blood pressure through regulation of sodium transporters in renal tubule cells54–57. The well-studied SLC12 ion cotransporters regulated by WNK signaling in the kidney are NKCC2 and NCC55, 58–60. Activation of the WNK-SPAK-OSR1 pathway promotes phosphorylation of membrane NKCC2, and triggers membrane translocation of NCC58, 61. Indeed, the WNK signaling was activated as evidenced by increased p-SPAK in KDM6A-cKO mice (Figure S2). The activation of the WNK signaling may be mediated by downregulation of Klotho in KDM6A-decicient mice which can be abolished by Klotho treatment. Although Klotho treatment did not attenuate total NCC or pNCC levels, it largely decreased the apical membrane abundance of NCC (Figure S2). Klotho treatment abolished upregulation of p-SPAK which may contribute to the attenuating effect of Klotho on KDM6A deficiency-induced NCC accumulation on the apical membrane. Hence, the crosstalk between Klotho and WNK signaling may play a role in sodium reabsorption and blood pressure regulation in KDM6A-cKO mice.

Supplementary Material

Supplemental Publication Material

Perspectives.

We show for the first time that KDM6A is essential to the maintenance of normal kidney function and blood pressure because renal tubule cell-specific knockout of KDM6A impairs renal Na excretion and increases blood pressure. The effect of KDM6A knockout is mediated by upregulation of H3K27me3 in the renal tubule cells which downregulates expression of Klotho leading to diminished ERK signaling and increased WNK signaling which may contribute to accumulation of NCC, NKCC2 and AQP2 in the apical membrane of renal tubule cells (Graphical Abstract). These changes ultimately resulted in an increase in increased Na reabsorption and elevated blood pressure.

Novelty and Relevance.

What is new?

  • This study reported for the first time that kidney-specific deletion of KDM6A gene impairs renal sodium excretion causes hypertension.

  • This study revealed that the Klotho/ERK/NCC/NKCC2 signaling pathway mediates KDM6A deficiency-induced hypertension.

2. What is relevant?

  • Deficiency of the histone demethylase KDM6A impairs renal sodium excretion leading to hypertension.

  • This study demonstrated that treatment with Klotho may be an effective therapeutic strategy for salt-sensitive hypertension due to KDM6A deficiency.

3. Clinical/Pathophysiological Implications

This study provides the first evidence that the Klotho/ERK/NCC/NKCC2 signaling pathway may mediate KDM6A deficiency-associated impairment in sodium excretion and hypertension. Downregulation of Klotho decreased ERK activity which leads to upregulation of NCC, NKCC2 and AQP2 expression in the tubular apical membrane. The results from this study suggests that Klotho may be a new therapeutic target for sodium retention and salt-sensitive hypertension.

ACKNOWLEDGEMENTS

This work was supported by NIH R01 AG062375, DK138872 and HL154147.

Non-standard Abbreviations and Acronyms

KDM6A

a specific demethylase for histone 3 lysine (K) 27 trimethylation

NKCC2

Na-K-2Cl cotransporter 2

NCC

Na-Cl cotransporters

ERK

extracellular signal-regulatd kinase

WNK

with-no-lysine kinase

FGFR1

fibroblast growth factor receptor 1

OCT

optimal cutting temperature

H3K27me3

Trimethylation of histone 3 lysine 27

PRC2

polycomb repressive complex 2

p-SPAK

phosphor SPS1—related proline/alanine-rich kinase

Footnotes

DISCLOSURE

None

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