Sex-specific modulation of renal epigenetic and injury markers in aging kidney

Abstract

Sex differences in renal physiology and pathophysiology are now well established in rodent models and in humans. Epigenetic programming is known to be a critical component of renal injury, as studied mainly in male rodent models; however, not much is known about the impact of biological sex and age on the kidney epigenome. We sought to determine the influence of biological sex and age on renal epigenetic and injury markers, using male and female mice at 4 mo (4M; young), 12 mo (12M), and 24 mo (24M; aged) of age. Females had a significant increase in kidney and body weights and serum creatinine levels and a decrease in serum albumin levels from 4M to 24M of age, whereas minor changes were observed in male mice. Kidney injury molecule-1 levels in serum and renal tissue greatly enhanced from 12M to 24M in both males and females. Circulating histone 3 (H3; damage-associated molecular pattern molecules) levels extensively increased with age; however, males had higher levels than females. Overall, females had markedly high histone acetyltransferase (HAT) activity than age-matched males. Aged mice had decreased HAT activity and increased histone deacetylase activity than sex-matched 12M mice. Aged females had substantially decreased renal H3 methylation at lysine 9 and 27 and histone methyltransferase (HMT) activity than aged male mice. Antiaging protein Klotho levels were significantly higher in young males than age-matched females and decreased substantially with age in males, whereas epigenetic repressor of Klotho, trimethylated H3K27, and its HMT enzyme, enhancer of zeste homolog 2, increased consistently with age in both sexes. Moreover, nuclear translocation and activity of proinflammatory transcription factor nuclear factor-κB (p65) were significantly higher in aged mice. Taken together, our data suggest that renal aging lies in a range between normal and diseased kidneys but may differ between female and male mice, highlighting sex-related differences in the aging process.

NEW & NOTEWORTHY Although there is evidence of sex-specific differences in kidney diseases, most preclinical studies have used male rodent models. The clinical data on renal injury have typically not been stratified by sex. Our findings provide convincing evidence of sex-specific differences in age-regulated epigenetic alterations and renal injury markers. This study highlights the importance of including both sexes for better realization of underlying sex differences in signaling mechanisms of aging-related renal pathophysiology.

INTRODUCTION

Aging refers to the systemic evidence of structural changes in organs with an increase in physiological dysfunction, with kidneys notably among the fastest aging organs. Irreversible physiological changes occur during renal aging that resemble diseased kidneys. In addition, the growing aging population (>65 yr) has resulted in an increased number of elderly individuals with chronic kidney disease (CKD), which increases the likelihood of its interaction with other age-related clinical factors, such as stress, diabetes, and cardiovascular disease (CVD). It has been shown that the incidence of CKD is higher in women compared with men; however, most of the patients with end-stage renal disease are men (1). Several meta-analyses and studies involving experimental animal models of renal injury have demonstrated that adult males exhibit greater renal function and structural abnormalities, which contribute to the accelerated progression of CKD and CVD risk compared with females (2, 3). Sex differences are evident in transcriptomic profiles of aged kidney cells of renin lineage, where aged female and male mice showed differential expression of 159 and 503 genes, respectively, compared with sex-matched young mice (4). Intriguingly, global transcription profiling of estrogen (E2) activity has identified kidneys as the most E2-sensitive nonreproductive organ with the third-largest number of E2-regulated genes (5). This underscores the role of biological sex in the incidence and progression of CKD in males and females, although the underlying molecular mechanisms remain unresolved.

Epigenetic programming of the epigenome is an important connection between genetics and the environment. Epigenetic dysregulation is involved in a wide variety of age-related chronic diseases, such as inflammation, type 2 diabetes, and neurodegenerative diseases (6, 7). The epigenetic alterations can modify the disease phenotype by directly targeting the genes in response to various environmental signals such as oxidative stress, inflammation, and metabolic and hormonal stimuli, including sex hormones (8). Epigenetic mechanisms like DNA methylation and histone modifications, including methylation (me) and acetylation (ac), have been closely linked to lifespan regulation (6). For instance, epigenetic processes regulate antiaging protein Klotho and its downstream target, nuclear factor-κB (NF-κB), a transcriptional regulator of proinflammatory genes, which are associated with the prevention of premature aging and CKD (9–11); however, sex-specific regulation of Klotho and NF-κB in renal aging is not known. The epigenome is responsible for orchestrating sex-specific gene expression during development and establishes a predisposition to sex-specific diseases later in life (12). A combination of epigenetic alterations, histone modifications, and DNA methylations are involved in X chromosome inactivation to ensure dosage compensation of the X chromosome between males and females (13). In addition, inactive X chromosome accumulates widespread epigenetic variability with age, which might contribute to the aging process in women (14). On the contrary, sex steroid hormones, such as E2 and testosterone, regulate epigenetic modifications under normal and disease conditions (8, 15).

In the kidneys, epigenetic modifications impact sex-specific gene expression (16, 17), which can potentially contribute to the sex differences observed in the progression and outcome of renal injury with aging. However, information regarding sex-defined molecular processes, including epigenetic patterns in the aging kidney, is limited. Most of the preclinical studies in renal epigenetics linked to injury are historically performed in male rodents or failed to stratify by sex, despite the epidemiological evidence of sex differences in outcomes of all stages of CKD (18). We hypothesized that the renal epigenetic mechanisms and injury markers will be differentially regulated in a sex- and age-dependent manner, with changes in older females reflecting the loss of estrogen. In this study, we have analyzed renal injury markers and epigenetic modifiers implicated in renal diseases and compared the results across age and sex.

METHODS

Mouse Tissue and Serum

Kidneys (whole) and serum (collected from the same animal) of C57BL/6JN male and female mice of 4 mo (4M; young), 12 mo (12M), and 24 mo (24M; aged) of age were obtained from the National Institute of Aging (NIA) Aged Rodent Tissue Bank. Animals were euthanized by CO₂ asphyxiation; then, unfixed tissue and organs were harvested, flash frozen in liquid nitrogen, and stored at −80°C.

Cytosolic, Nuclear, and Histone Extraction and Immunoblot Assay

Cytoplasmic and nuclear extracts (CE and NE) and total histones were extracted from frozen renal tissues, as previously described (19). The protein concentration of the extracts was measured with the Bradford protein detection kit (Bio-Rad, Hercules, CA). The CE (50–60 µg), NE (30–40 µg), or histones (10 µg) were mixed with sample loading buffer and separated by using 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis essentially as described earlier (11). Protein bands were visualized by using the SuperSignal West Femto Chemiluminescent kit (Thermo Scientific, Rockford, IL) using the ChemiDoc MP imaging System (Bio-Rad), and densitometry analysis was performed by using ImageJ software (National Institutes of Health, Bethesda, MD) (20). The catalog number and the dilutions of the antibodies used in Western blot were as follows: kidney injury molecule-1/T cell immunoglobulin and mucin domain 1 (KIM-1/TIM-1) (AB78494; 1:1,000) from Abcam (Cambridge, MA); histone 3 (H3) lysine 9 acetylation (H3K9ac) (No. 9649; 1:1,000), H3 lysine 27 acetylation (H3K27ac) (No. 8173; 1:1,000), enhancer of zeste homolog 2 (EZH2) (No. 4905; 1:1,000), H3K27 trimethylation (me3) (H3K27me3) (No. 9733S; 1:1,000), NF-κB (p65) (No. 8242 T; 1:1,000), TATA binding protein (TBP) (No. 44059; 1:1,000), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (No. 7074; 1:5,000) from Cell Signaling Technology (Danvers, MA); Klotho (AF1819; 1:1,000) from R&D Systems (Minneapolis, MN); and histone 3 (H3; sc-517576; 1:500), β-actin (sc-47778-HRP; 1:5,000), and HRP-conjugated anti-mouse IgG (sc-516102; 1:5,000) from Santa Cruz Biotechnology (Santa Cruz, CA).

Total Histone Acetyltransferase, Histone Deacetylase, and Histone Methyltransferase HMT (H3K9) and HMT (H3K27) Activity Assay

In renal nuclear extracts (10 µg), total histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities were measured using colorimetric EpiQuik HAT activity/inhibition assay and Epigenase HDAC activity/inhibition assay kits, respectively, from Epigentek (Farmingdale, NY) following the manufacturer’s instructions, as described in our previous studies (11, 19). Total histone methyltransferase (HMT) (H3K9) and HMT (H3K27) activities were measured using colorimetric EpiQuick Histone Methyltransferase Activity/Inhibition Assay Kits from Epigentek.

Quantification of Global H3 Lysine Methylation

Following Epigentek’s protocol, renal H3 methylation (me) at lysine 9 and 27 was quantified in histone extracts using EpiQuik Global H3-K9me and H3-K27me Methylation Assay Kits (colorimetric). The absorbance was read at 450 nm, and methylated histone levels were calculated using a standard curve.

Circulating Histone H3 Quantification

The EpiQuik Circulating Total Histone H3 Quantification Kit (colorimetric) was used to measure total histone H3 in the serum according to the manufacturer’s protocol. In brief, H3 in serum was captured on wells coated with anti-histone H3 antibody and recognized by a secondary antibody-color development system. The intensity of absorbance was measured at 450 nm, and the amount of H3 was quantitated using a standard control.

Mouse Kidney Injury Molecule-1 Immunoassay

Serum KIM-1 levels were measured utilizing the Mouse TIM-1/KIM-1 Immunoassay kit from R&D Systems, following the manufacturer’s protocol. In brief, standards, control, and samples were added to the microplate precoated with mouse KIM-1 monoclonal antibody, which was recognized by a secondary antibody-color development system. The intensity of the absorbance was measured at 450 nm, and the quantity was calculated from the standard curve.

Serum Creatinine and Albumin Quantification

Serum creatinine (sCr) concentration and albumin levels were measured utilizing the creatinine assay kit and the quantichrom BCG albumin assay kit, respectively, from Bioassay Systems (Hayward, CA) following the manufacturer’s instructions.

NF-κB (p65) Transcription Factor Binding Assay

NF-κB (p65) DNA binding activity in the renal nuclear extracts was determined by using the NF-κB (p65) Transcription Factor Assay Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s protocol. Briefly, nuclear NF-κB (p65) binds to the NF-κB response element (a double-stranded DNA sequence immobilized to the wells of a 96-well plate), and the DNA:protein complex is detected by the NF-κB (p65) primary antibody, which is recognized by a secondary antibody-color development system, and the absorbance was read at 450 nm.

Statistical Significance

All values are expressed as means ± SE, and results were analyzed using the statistical package from GraphPad Prism 7 (GraphPadSoftware, La Jolla, CA). Two-way ANOVA was used with the factors sex (P_sex), age (P_age), and their interaction (P_sex*age), and the results are reported in the table with each graph. A post hoc analysis was performed using Tukey’s multiple-comparisons test, and results are presented as the impact of age (*) and sex (#) on each graph. P ≤ 0.05 was accepted as statistically significant.

RESULTS

Sex- and Age-Related Changes in Body Weight, Kidney Weight, and Renal Injury Markers

Sex differences were observed in body weight (BW) and kidney weight (KW) of mice. The BW and KW were significantly lower in 4M (young) and 12M (adult) females than in age-matched males; however, 24M (aged) females had a significant increase in KW compared with 24M males (Fig. 1, A and B). In female mice, BW and KW increased consistently with age, but minor changes were observed in the KW of male mice, and BW decreased in 24M male mice compared with 12M males. Overall, females had a significantly higher KW/BW ratio at 12M and 24M compared with age-matched males (Fig. 1C).

Figure 1.

Body weight (BW), kidney weight (KW), and renal injury markers in male and female mice. A: BW; B: KW; C: BW/KW ratio. n = 14/group. D: serum creatinine; E: serum albumin; F: circulating histone 3 (H3) levels; G: serum KIM-1 levels; H: renal KIM-1 protein levels. n = 6/group. Bar represents biological replicates along with means ± SE. Two-way ANOVA is provided in the tables and Tukey’s post hoc analysis is denoted on the graph. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (impact of age, same sex); #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 (impact of sex in age-matched counterparts).

In females, sCr levels significantly increased at 24M of age and were considerably higher than those in age-matched males (Fig. 1D). Serum albumin levels were higher in young females than young males but decreased gradually with age in both sexes (Fig. 1E). Circulating H3 [damage-associated molecular pattern (DAMP) molecules] levels showed significant interaction with age and sex and increased significantly in 12M and 24M mice compared with sex-matched 4M mice (Fig. 1F). Proximal tubular injury marker KIM-1 levels increased greatly in the serum of aged male and female mice compared with 4M and 12M sex-matched mice (Fig. 1F). Also, renal KIM-1 protein levels were augmented in 24M male and female mice (Fig. 1G).

Sex- and Age-Mediated Changes in Renal Epigenetic Enzymes and Histone Modifications

Overall, female mice had significantly higher HAT activity in all age groups compared with age-matched males (Fig. 2A). In both sexes, HAT activity initially increased with age and significantly decreased in 24M mice compared with 12M mice. On the contrary, HDAC activity was substantially higher in aged male and female mice (Fig. 2B). Western blot and densitometry analysis demonstrated no differences in the renal H3K9ac and H3K27ac levels with age or sex (Fig. 2C).

Figure 2.

Renal histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity and histone 3 acetylation (H3ac) levels in mice. Renal nuclear extracts were used to measure HAT activity (A) and HDAC activity (B) by using a colorimetric assay kit. n = 7 or 8/group. C: Western blot and densitometric analysis of H3K9ac and H3K27ac. Histone 3 (H3) was used as the loading control. n = 7 or 8/group. Bar represents biological replicates along with means ± SE. Two-way ANOVA is provided in the tables and Tukey’s post hoc analysis is denoted on the graph. *P < 0.05, **P < 0.01, and ***P < 0.001 (impact of age, same sex); ###P < 0.001 (impact of sex in age-matched counterparts).

The enzymatic activities of renal HMTs, HMT (H3K9) and HMT (H3K27), increased with age in male mice; however, in females, after an increase at 12M, it decreased at 24M (Fig. 3, A and B). Sex differences were observed, as aged females had lower HMT activity than aged male mice. There was significant interaction between age and sex in total methylation levels of H3K9me and H3K27me (Fig. 3, C and D). In males, H3K9me and H3K27me levels increased with age, and in females, it decreased at 24M after an initial increase, exhibiting sex differences in aged mice.

Figure 3.

Renal histone methyltransferase (HMT) H3K9 and HMT (H3K27) activity and histone 3 (H3) methylation (me) levels in mice. Renal nuclear extracts were used to measure HMT (H3K9) activity (A) and HMT (H3K27) activity (B) using a colorimetric assay kit. n = 7 or 8/group. C and D: renal H3me levels at lysine 9 (H3K9me) and lysine 27 (H3K27me) were quantified using colorimetric assay kits. n = 7 or 8/group. Bar represents biological replicates along with means ± SE. Two-way ANOVA is provided in the tables and Tukey’s post hoc analysis is denoted on the graph. *P < 0.05 (impact of age, same sex); #P < 0.05 (impact of sex in age-matched counterpart).

Sex- and Age-Associated Epigenetic Regulation of Renal Klotho and NF-κB (p65) Activation

Sex differences were observed in Klotho protein levels, as young males had significantly higher levels than age-matched females. However, Klotho protein levels decreased significantly in males with aging, but females exhibited minimal changes (Fig. 4, A and B). Trimethylation (me3) of H3K27, an epigenetic repressor of Klotho, consistently augmented with age in both the sexes, and 24M mice had significantly higher levels of H3K27me3 compared with 4M sex-matched mice (Fig. 4, A and C). Similarly, the HMT enzyme EZH2 (enhancer of zeste homolog 2), responsible for catalyzing H3K27 trimethylation and global transcription repression, increased consistently with age in male and female mice (Fig. 4, A and D). In females, significant increases in EZH2 levels were noticed at 12M and 24M of age compared with 4M mice, whereas in males, it was observed at 24M as compared with 4M mice.

Figure 4.

Renal Klotho, H3K27me3, and enhancer of zeste homolog 2 (EZH2) protein levels and nuclear factor-κB (NF-κB) (p65) localization and activity in mice. A–D: Western blot and densitometric analysis of Klotho protein in cytoplasmic extract (CE) and H3K27me3 and EZH2 proteins in nuclear extracts (NE). β-Actin and histone 3 (H3) were used as loading controls for CE and NE, respectively. n = 7 or 8/group. E: Western blot showing renal NF-κB (p65) localization in CE and NE. β-actin and TBP were used as loading controls of CE and NE, respectively. F: densitometric analysis showing ratio of nuclear vs. cytoplasmic localization of NF-κB (p65). G: DNA binding activity of NF-κB (p65) in NE (n = 6). Bar represents biological replicates along with means ± SE. Two-way ANOVA is provided in the tables and Tukey’s post hoc analysis is denoted on the graph. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (impact of age, same sex); ###P < 0.001 (impact of sex in age-matched counterparts).

The renal cytoplasmic level of NF-κB (p65) protein was significantly higher and nuclear levels were lower in young mice, which was reversed in aged male and female mice (Fig. 4E). The ratio of NF-κB (p65) in the nuclear to cytoplasmic fraction was lower in young mice and significantly increased in both sexes of aged mice (Fig. 4F). In females, DNA binding activity of nuclear NF-κB (p65) was significantly higher in 12M and 24M mice compared with 4M mice. However, in males, a significant increase in NF-κB (p65) DNA binding was observed only at 24M compared with 4M male mice (Fig. 4G).

DISCUSSION

Our results demonstrate sex differences in some of the age-associated renal injury markers and epigenetic modifiers implicated in renal disease. In females, body and kidney weights and sCr levels significantly increased with aging, whereas minor changes were observed in males. On the contrary, serum albumin levels decreased and KIM-1 protein levels increased in serum and renal tissue of aged male and female mice. KIM-1, a transmembrane glycoprotein, is principally expressed in the renal proximal tubular cells and directly involved in tubular cell damage (21). During renal injury, excess KIM-1 protein is released into the circulation and in the urine, and circulating KIM-1 is implicated as a biomarker of acute and CKD (22). We observed an increase in serum KIM-1 levels in aged male mice that did not correlate with changes in sCr levels as seen in aged female mice, suggesting sex differences in renal injury biomarkers. An earlier study in the male mouse model of CKD has reported an increase in plasma and urinary KIM-1 levels but no change in plasma creatinine (22). Moreover, sCr levels decline more significantly in older males than in older females due to sex-specific muscle mass decline in humans (23). Also, urinary KIM-1 increases with age in males and females and has been suggested as a biomarker for the prediction of kidney age in healthy people (24).

Histones and their posttranslational modifications have intranuclear functions in chromatin remodeling and gene transcription. However, histones are released in the extracellular spaces due to cell injury or death and act as DAMP molecules, signifying their role in the regulation of sterile inflammation (25). We observed increased levels of circulating H3 with aging and 12M and 24M males had higher levels than age-matched females. In animal models of organ injury, including kidneys, elevated serum histone levels have been reported (26, 27); however, sex-specific changes in aging kidneys have not been stated previously. Furthermore, dying renal tubular epithelial cells have been shown to release histones into the circulation (27). These histones induce NF-κB and mitogen-activated protein kinase signaling via direct interaction with Toll-like receptors.

A multitude of epigenetic modifications including histone acetylation and methylation are involved in the activation of signaling pathways that result in renal fibrosis and inflammation. Histone acetylation is a reversible process, catalyzed by HATs, and deacetylation is catalyzed by HDACs, and the balance between the enzymes is essential in gene regulation (28). The present data exhibit sex differences in renal HAT activity, with female mice having higher activity in all age groups compared with age-matched male mice. We further analyzed renal H3K9 and H3K27 acetylation levels, as these are implicated in renal injury in male mice; however, conflicting results have been observed, like a progressive increase in histone 3 acetylation (H3ac) and HAT activity leading to an increase in inflammatory and fibrotic gene expression (29) or decrease in renal H3ac in injured kidneys (19, 30). An earlier study has shown that many H3ac marks increase or decrease with age (31). Interestingly, no changes were observed in H3K9ac and H3K27ac levels with age or sex in this study. We observed an increase in renal HDAC activity with aging in both male and female mice. The majority of the studies and our earlier study, conducted in male rodents, have demonstrated that increased HDAC activity positively correlates with the causation and progression of renal injury (11, 32). The histone methylation at H3K9 and H3K27 has been observed in male rodent models of renal injury (33–35) and in our previous study (19). Interestingly, our present results document sex differences in H3K9me and H3K27me levels and their enzymes in aged mice. Overall, males exhibited an increase in histone methylation with aging, but in females, it decreased significantly in aged mice after an increase in 12M mice. Histone methyltransferases catalyze specific methylation sites on histones and their aberrant expression or activity is linked with the development and progression of various kidney diseases in human and animal models (33, 36). However, the implications of histone acetylation or methylation in renal injury of female rodents have not been studied to the best of our knowledge.

Klotho is strongly expressed in the renal distal tubular cells, which is mainly responsible for the regulation of phosphate excretion (9). Klotho protein exists in various forms with varied functions: a full-length transmembrane Klotho (∼130 kDa), a truncated soluble Klotho (65 kDa), and a secreted Klotho (65 kDa), and our results showed full-length α-Klotho (130 kDa) and soluble α-klotho (65 kDa) in the renal tissues. Sex differences were observed in Klotho levels, with higher expression in young male mice compared with young females, and it decreased significantly with age in males. Previous studies have reported sex differences in renal Klotho levels in the cortex of 2M mice (37) and enhanced renal Klotho gene expression by testosterone via the androgen receptor-mediated pathway (38). A positive relationship between testosterone and Klotho levels in healthy adult men has been documented (39). Conflicting results have been observed with the effect of E2 on Klotho; notably, E2 treatment of aromatase knockout mice decreased renal Klotho expression at both the mRNA and protein levels (40). Conversely, Klotho mRNA levels in the hippocampus positively correlated with circulating E2, and an ovariectomy-mediated decrease in Klotho was reversed by E2 replacement (41).

Epigenetic alterations that are involved in the repression of Klotho during aging may be partly attributed to upregulation of H3K27me3 (10). Methyltransferase EZH2 is responsible for the generation of H3K27me3 and is part of the protein mechanism forming the aging epigenome (42). An increase in the expression of renal EZH2 and H3K27me3 has been reported in kidney biopsy of patients with injury and in male C57BL6 mice with renal injury (33, 35). This study exhibits a significant increase in H3K27me3 and EZH2 protein levels with aging in both sexes. In contrast, we saw a decrease in total (mono, di, and tri) H3K27me and activity of total HMT (H3K27) enzymes in aged female mice (Fig. 3, B and D), signifying sex-specific differential expression of HMTs with aging. Indeed, there are specific HMTs that add the mono-, di-, or tri-methyl group to H3K27, suggesting that specific methyl mark is dynamically controlled under different circumstances (43). Moreover, Klotho prevents nuclear translocation of NF-κB, which is essential for proinflammatory signaling of NF-κB (9). An earlier study in diabetic mice has shown reduced renal Klotho levels linked to increased NF-κB activation and inflammation (44). We also observed similar results with reduced Klotho levels in aged mouse kidneys coinciding with an increase in nuclear translocation of NF-κB and its DNA binding activity. Conversely, NF-κB binds to the Klotho promoter and mediates the loss of Klotho in response to inflammation (45).

A limitation of this study is that renal function assays like glomerular filtration rate and glomerular injury markers, such as urinary albumin and creatinine levels, could not be performed, as renal tissues were obtained from the NIA facility; henceforth, the results from kidney tissues cannot be associated with renal function in these mice. As urinary levels of KIM-1 have been suggested as a predictive biomarker for kidney age (24) and urinary Klotho has been proposed as a novel biomarker in the early detection of the deterioration of renal function (46), assessing their urinary levels in this work would have strengthened the study. Another limitation is that the whole kidney was utilized to draw mechanistic insights and it does not reflect cell-specific information.

In conclusion, utilizing kidney tissues from both male and female mice across various age groups in our study offers compelling evidence of sex-specific alterations in the aging renal epigenome, accompanied by distinct modulation of renal injury markers. Addressing sex differences in the renal epigenetic regulatory pathways is an important and overlooked area. Further studies are needed to investigate sex-specific molecular processes in aging kidneys to better understand sex disparities in age-related renal pathologies.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by National Institute on Aging Grant R03AG075396 and Tulane University Carol Lavin Bernick Faculty Grant (to P.K.).

DISCLOSURES

Heddwen L. Brooks is the Editor-in-Chief of the American Journal of Physiology-Renal Physiology and was not involved and did not have access to information regarding the peer-review process or final disposition of this article. An alternate editor oversaw the peer-review and decision-making process for this article. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

P.K. conceived and designed research; G.A.A-S. and P.K. performed experiments; H.L.B. and P.K. analyzed data; P.K. interpreted results of experiments; P.K. prepared figures; P.K. drafted manuscript; G.A.A-S., H.L.B., and P.K. edited and revised manuscript; G.A.A-S., H.L.B., and P.K. approved final version of manuscript.