Role Of The Renal Androgen Receptor in Sex Differences in Ammonia Metabolism

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Autumn N. Harris et al

Abstract

There are sex differences in renal ammonia metabolism and structure, many of which are mediated by testosterone. The goal of the present study was to determine the role of the renal expression of testosterone’s canonical receptor, androgen receptor (AR), in these sexual dimorphisms. We studied mice with kidney-specific AR deletion [KS-AR-knockout (KO)] generated using Cre/loxP techniques; control mice were Cre-negative littermates (wild type). In male but not female mice, KS-AR-KO increased ammonia excretion, which eliminated sex differences. Although renal structural size typically parallels ammonia excretion, KS-AR-KO decreased kidney size, cortical proximal tubule volume density, and cortical proximal tubule cell height in males—neither were altered in females and collecting duct volume density was unaltered in both sexes. Analysis of key protein involved in ammonia handling showed in male mice that KS-AR-KO increased both phosphoenolpyruvate carboxykinase (PEPCK) and Na þ -K þ -2Cl cotransporter (NKCC2) expression and decreased Na þ/H þ exchanger isoform 3 (NHE3) and electrogenic Na þ -bicarbonate cotransporter 1 (NBCe1)-A expression. In female mice, KS-AR-KO did not alter these parameters. These effects occurred even though KS-AR-KO did not alter plasma testosterone, food intake, or serum Na þ, K þ, or HCO3 significantly in either sex. In conclusion, AR-dependent signaling pathways in males, but not females, kidneys regulate PEPCK and NKCC2 expression and lead to the sexual differences in ammonia excretion. Opposing effects on NHE3 and NBCe1-A expression likely limit the magnitude of ammonia excretion changes. As AR is not present in the thick ascending limb, the effect of KS-AR-KO on NKCC2 expression is indirect. Finally, AR mediates the greater kidney size and proximal tubule volume density in males compared with female mice.

NEW & NOTEWORTHY Sexual dimorphisms in ammonia metabolism involve androgen receptor (AR)-dependent signaling pathways in male, but not female, kidneys that lead to altered proximal tubule (PT), phosphoenolpyruvate carboxykinase, and thick ascending limb Na þ -K þ -2Cl cotransporter expression. Adaptive responses in Na þ/H þ exchanger 3 and electrogenic Na þ -bi- carbonate cotransporter 1-A expression limit the magnitude of the effect on ammonia excretion. Finally, the greater kidney size and PT volume density in male mice is the result of PT androgen signaling through AR.

key words: acid-base; ammonia; androgen receptor; sex differences; testosterone

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INTRODUCTION

The presence of sex differences in many aspects of mammalian biology has been well established with sexual dimorphisms in structure and/or function have been identified in nearly every organ, including the kidney (1–3). Sex affects many aspects of kidney function and disease. Sex differences in blood pressure are widely recognized and thought to be related to sex-specific variations in nitric oxide, the renin-angiotensin-aldosterone system, and inflammation (4, 5). There are also differences in the expression of many renal epithelial cells Naþ transporters (6, 7). Understanding these differences can identify new disease mechanisms and/or new and improved therapeutic opportunities. The kidney regulates acid-base homeostasis, which is critical for normal health (8, 9). Renal ammonia Fn1 1 metabolism has a major role in acid-base homeostasis; ammonia excretion is the predominant component of basal net acid excretion, and changes in ammonia metabolism account for >80% of the response to exogenous stimuli (10–16). Dysregulation of acid-base homeostasis directly affects essentially every major organ system, accelerates the progression of chronic kidney disease, and correlates with increased mortality (17–19).

Our studies have shown that there are important sex effects on both ammonia metabolism and renal structure. The male kidney excretes less ammonia than the female kidney and does so despite the male kidney being larger and having greater cortical volume than the female kidney (20). This difference in ammonia excretion correlates with variations in multiple renal aspects, including several enzymes involved in ammonia generation and recycling, in proximal tubule size, in the thick ascending limb of the loop of Henle (TAL) Naþ -Kþ - 2Cl- cotransporter (NKCC2) expression, and in collecting duct intercalated cell size and Rhesus (Rh) glycoprotein expression (20). Finally, there are sex differences in the response to acid loading (21). Many of these sex differences are reversed by orchiectomy through a mechanism abrogated by physiological testosterone replacement, suggesting that they involve testosterone-dependent signaling pathways (22).

The canonical pathway through which testosterone mediates biological effects involves binding to the androgen receptor (AR) (23, 24). AR protein and mRNA expression have been observed in both male and female kidneys for decades (25–28). We recently showed that AR protein is expressed specifically in the proximal tubule and that expression was not detectable in either the TAL or collecting duct (22), where there are known sex differences in proteins involved in ammonia handling. Our studies further showed that proximal tubule AR expression was present in both male and female kidneys, with expression being approximately twice as much in the male kidney than in the female kidney (22). These observations suggest that AR may mediate some, but not all, of the effects of sex on renal structure and on ammonia generation and transport. They also raise the question as to the role of AR in the female kidney.

Thus, the purpose of the present study was to determine the role of renal AR in mediating these identified sexual dimorphisms in renal ammonia metabolism and renal structure. Because AR activation has numerous biological effects, which may indirectly alter acid-base homeostasis and thereby ammonia handling, interpreting studies using pharmacological AR inhibition and/or global AR deletion is complicated. Therefore, the present study used mice with kidney-specific AR deletion [KS-AR-knockout (KO)] generated using Cre-loxP techniques. We compared Cre-positive mice with their Cre-negative littermates to determine the effect of KS-AR-KO. Both male and female mice were evaluated to allow understanding of the role of AR in both sexes.

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METHODS

Animals

We used mice with loxP sites flanking exon 2 of the AR gene (ARflfl/flfl ), which have been previously described (29–31). We induced whole kidney AR deletion by breeding mice expressing Cre-recombinase under control of the Pax-8 promotor (Pax8Cre; 32). KS-AR-KO mice were ARflfl/flfl, Pax8-Cre þ; control mice were ARflfl/flfl, Pax8-Cre. All mice were on the C57/Bl6 background strain. We genotyped all mice using DNA extracted from tail clip samples. Mice used in these experiments were adult male and female mice of -4 mo of age. Mice were maintained on a standard rodent diet (18% protein, Harlan Teklad, Madison, WI) with free access to water. The University of Florida and the North Florida/South Georgia Veterans Health System Institutional Animal Care and Use Committees approved all animal protocols. Animal breeding was performed in the University of Florida College of Medicine Cancer and Genetics Transgenic Animal Core Facility by trained personnel.

Metabolic Cage Experiments

Mice were placed in metabolic cages for 3 days and fed a powdered normal diet (18% protein, Harlan Teklad) mixed 1:1 with water. Daily food consumption was measured, and daily 24-h urine samples were collected. Urine samples were collected in tubes containing water-equilibrated mineral oil to prevent evaporation and gas molecule loss. Daily urine volume and pH were recorded. Urine samples were stored at - 20℃ until analyzed further.

Blood Analysis

Blood was obtained by cannulation of the abdominal aorta in mice anesthetized with isoflurane, drawn into a heparinized syringe, and immediately analyzed for Naþ, K þ, and bicarbonate concentrations using a Siemens Microanalytic Blood Gas Analyzer (RAPIDLab 348 Analyzer, Siemens, Munich, Germany). Serum testosterone was measured in The University of Virginia School of Medicine Center for Research in Reproduction Ligand Assay and Analysis Core (IBL America mouse testosterone ELISA, Minneapolis, MN)

Urinary Analysis

Urine ammonia was measured using a modification of a commercially available kit (A7553, Pointe Scientific, Canton, MI), as previously described (20, 33). Urine pH was measured using a micro-pH electrode (ROSS semi-micro pH, Orion 8103BN, Thermo Fisher Scientific, Waltham, MA). Titratable acid excretion was determined using previously described standard techniques (20, 33).

Antibodies

Table 1 shows the antibodies used in this study, their suppliers, and their catalog/identification numbers.

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Protein Preparation

Animals were anesthetized with inhalant isoflurane, and kidneys were rinsed by in vivo cardiac perfusion with PBS (pH 7.4) containing 6,000 U/L of Na-heparin and 120 mg/L of lidocaine. The right kidney was removed rapidly, and the cortex, outer medulla, and inner medulla were isolated on a cold stage under a dissecting microscope. Samples were snap-frozen in liquid nitrogen and stored frozen at - 80 ℃ until use. Tissues were homogenized in the T-PER tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL) using microtube pestles (USA Scientific, Ocala, FL), and protein was extracted according to the manufacturer’s recommendations. An aliquot was used for total protein quantification using a bicinchoninic acid (BCA) assay, and the remainder was stored frozen at 80℃until use.

Immunoblot Procedures Ten micrograms of renal protein were electrophoresed on a 10% PAGE ReadyGel (Bio-Rad, Hercules, CA). Gels were transferred electrophoretically to nitrocellulose membranes, blocked with 5 g/dL nonfat dry milk diluted in Blotto buffer (50 mM Tris, 150 mM NaCl, 5 mM Na2EDTA, and 0.05% Tween 20, pH 7.6), and incubated at 4 C overnight with the primary antibody in nonfat dry milk. Loading and transfer equivalence were assessed with Ponceau S staining and by evaluating the expression of the housekeeping protein b-actin. After being washed, membranes were exposed to secondary antibody (goat anti-rabbit IgG, Cell Signaling Technology, Beverly, MA) conjugated to horseradish peroxidase at a dilution of 1:5,000. Sites of antibody-antigen reaction were visualized using enhanced chemiluminescence (SuperSignal West Pico Substrate, Pierce) and a Kodak Image Station 440CF digital imaging system. In selected experiments, blots were stripped. Band density was normalized such that the mean density in a region (cortex or outer medulla) in male wild-type (WT) tissue was 100. Both sexes (male and female) and both genotypes (WT and KO) were loaded on the same gel for these experiments. The absence of saturation was confirmed by examining pixel intensity distribution in all immunoblots.

Immunoblot Procedures

Ten micrograms of renal protein were electrophoresed on a 10% PAGE ReadyGel (BioRad, Hercules, CA). Gels were transferred electrophoretically to nitrocellulose membranes, blocked with 5 g/dL nonfat dry milk diluted in Blotto buffer (50 mM Tris, 150 mM NaCl, 5 mM Na2EDTA, and 0.05% Tween 20, pH 7.6), and incubated at 4C overnight with the primary antibody in nonfat dry milk. Loading and transfer equivalence were assessed with Ponceau S staining and by evaluating the expression of the housekeeping protein b-actin. After being washed, membranes were exposed to secondary antibody (goat anti-rabbit IgG, Cell Signaling Technology, Beverly, MA) conjugated to horseradish peroxidase at a dilution of 1:5,000. Sites of antibody-antigen reaction were visualized using enhanced chemiluminescence (SuperSignal West Pico Substrate, Pierce) and a Kodak Image Station 440CF digital imaging system. In selected experiments, blots were stripped. Band density was normalized such that the mean density in a region (cortex or outer medulla) in male wild-type (WT) tissue was 100. Both sexes (male and female) and both genotypes (WT and KO) were loaded on the same gel for these experiments. The absence of saturation was confirmed by examining pixel intensity distribution in all immunoblots.

Tissue Preparation for Immunohistochemistry

Mice were anesthetized with inhalant isoflurane. The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) containing 6,000 U/L of Na-heparin and 120 mg/L of lidocaine followed by periodate-lysine-2% paraformaldehyde, cut transversely into several 2- to 3-mm-thick slices, and then immersed for 24–30 h at 4℃ in the same fixative. Kidney samples from each animal were embedded in polyester wax made with polyethylene glycol 400 distearate (Polysciences, Warrington, PA) with 10% 1-hexadecanol and 2-mm-thick sections were cut and mounted on gelatin-coated slides.

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Immunohistochemistry

Immunolocalization for Rh family B glycoprotein (Rhbg), Rh family C glycoprotein (Rhcg), Naþ /Hþ exchanger isoform 3 (NHE3), glutamine synthetase (GS), NKCC2, and AR was accomplished using previously described immunoperoxidase procedures (20, 22, 34–37). Briefly, sections were dewaxed in ethanol, rehydrated, heated in Trilogy (Cell Marque, Rocklin, CA) to 88 C for 30 min and then to 96 ℃ for 30 min, cooled for 30 min, and rinsed in PBS. Endogenous peroxidase activity was blocked by incubating sections in 3% H2O2 in distilled water for 45 min. Sections were blocked for 15 min with serum-free protein block (Dako Cytomation) and then incubated overnight at 4 C with the primary antibody. Sections were washed in PBS, incubated for 30 min with polymer-linked, peroxidase-conjugated horse anti-rabbit IgG (ImmPRESS, Vector Laboratories, Burlingame, CA), washed again with PBS, and then exposed to diaminobenzidine for 5 min. Sections were washed with distilled water, dehydrated with xylene, mounted, and observed by light microscopy. Comparisons of labeling were made between sections from the same immunohistochemistry experiment. Sections were examined on a Zeiss Axio Imager A2 microscope equipped with DIC optics and photographed using Axiocam 305 color digital camera and Zen 2.3 software (Zeiss).

Immunolocalization for phosphoenolpyruvate carboxykinase (PEPCK) was accomplished using modified immunoperoxidase procedures as previously described (20, 22, 38– 40). Briefly, sections were dewaxed in ethanol, rehydrated, heated in Trilogy (Cell Marque, Rocklin, CA) to 96 C for 60 min, cooled for 30 min, and rinsed in PBS. Endogenous peroxidase activity was blocked by incubation of the sections in 3% H2O2 in methanol for 45 min. Sections were treated with 0.5% Triton X-100 in PBS for 15 min. Sections then underwent several washes in PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin, followed by blocking for 15 min with serum-free protein block (DAKO Cytomation) and then incubated overnight at 4 C with primary antibody. Sections were washed in PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin followed by PBS and incubated for 60 min with polymer-linked, peroxidase-conjugated goat anti-rabbit IgG (MACH2, Biocare Medical, Concord, CA), washed again in PBS, and then exposed to diamino-benzidine for 5 min.

Negative Control

Each immunohistochemistry experiment included a section that was exposed to the immunolabeling procedures without primary antibodies to ensure that the label was due to primary antibody binding only.

Morphometric Analysis

The volume density of the proximal tubule in the cortex and outer stripe of the outer medulla as well as the collecting duct in the cortex and inner stripe of the outer medulla was determined using standard point-counting techniques as previously described (20–22, 41).

Quantitative Immunohistochemistry

Quantitative immunohistochemistry was performed as we have previously described and validated (20, 22, 33, 37, 42). Proximal tubule segments were studied in sections labeled under identical conditions in the same immunolabeling experiment by an observer blinded to the treatment group. The specific proximal tubule segments measured were the initial proximal convoluted tubule (PCT), defined as PCT segments continuous with Bowman’s capsule, the proximal straight tubule (PST) in the medullary ray, and the PST in the outer stripe of the outer medulla. We used high-resolution digital micrographs taken of randomly selected fields of the renal cortex and outer stripe of the outer medulla using a Zeiss Axio Imager A2 microscope equipped with an Axiocam 305 color digital camera and Zen 2.3 software (Zeiss) using no imaging enhancement techniques. Using ImageJ software (v, 1.34j, National Institutes of Health, Bethesda, MD), we measured pixel intensity across a straight line drawn from the tubule lumen through an individual cell. These data were then analyzed using custom-written software executed in Microsoft Excel 2016. Net intensity at each pixel on the line was then determined as the difference between absolute intensity and mean background pixel intensity measured outside the cell. Total cellular expression was determined by integrating net pixel intensity across the entire cell. Cell height was determined as the distance in pixels between the apical and basolateral edges of the cells and converted to absolute length using calibrated determination of individual pixel size. A minimum of 15 individual cells from at least four photomicrographs from each kidney were analyzed. Data from all cells examined of a given proximal tubule segment type were averaged to yield a single data point per animal for statistical analysis.

We determined to type A intercalated cell size and cell-specific protein expression as previously described (20, 22). Briefly, we obtained high-power digital micrographs of the inner stripe of the outer medulla of tissue sections that had undergone immunohistochemistry for the intercalated cell-specific marker Rhbg or Rhcg. Individual intercalated cells were circumscribed using ImageJ software (v. 1.34j, National Institutes of Health). Cell size was determined as the number of pixels within the outlined regions and converted to the area using calibrated measurement of pixels per micrometer. Immunolabel intensity at each pixel was determined as the difference between absolute pixel intensity and mean background intensity, and single-cell immunolabel intensity was determined by integrating net pixel intensity within the cell using custom-written software executed in Microsoft Excel 2016.

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RESULTS

Verification of Kidney-Specific AR Deletion

AR pharmacological blockage or global deletion induces a wide variety of phenotypic changes (43), which could indirectly alter renal structural and ammonia responses. To avoid these issues, we generated KS-AR-KO mice using CreloxP techniques. We confirmed the effectiveness of renal AR deletion using immunohistochemistry (Fig. 1). In WT mice, immunohistochemistry showed nuclear AR immunolabel limited to the proximal tubule; this was similar to our findings in the normal mouse kidney (22). In the KO kidney, there was no detectable AR immunolabel present in either sex (Fig. 1). These findings show the generation of mice with renal AR deletion.

statistical analysis using SPSS software (26.2) and Microsoft Excel 2016.

Because testosterone has numerous extrarenal effects, which could possibly indirectly alter renal acid-base mechanisms, we considered the possibility that KS-AR-KO could alter plasma testosterone or estradiol levels. However, plasma testosterone and estradiol concentrations did not differ significantly between WT and KO mice in either sex (Table 2).

Physiological Characterization of Kidney-Specific AR Deletion Body weight was greater in WT male mice than in WT female mice despite no sex difference in food intake, as previously reported (22), consistent with known extrarenal effects of sex on body weight. However, KS-AR-KO did not alter daily food intake or bodyweight in either sex (Table 2).

Kidney weight was significantly greater in male WT mice than in female WT mice (Fig. 2), similar to what we have previously reported in normal mice (22). KS-AR-KO significantly decreased kidney weight in male KS-AR-KO mice, to the extent that male KS-AR-KO kidney weight did not differ significantly from female WT or KS-AR-KO kidney weight (Fig. 2). In female mice, KS-AR-KO did not alter kidney weight significantly. Thus, the effect of sex on kidney weight requires AR expression in the male kidney.

Neither plasma Naþ, K þ, nor bicarbonate concentrations differed significantly between WT and KS-AR-KO mice for either sex (Table 2). Urea clearance, used as a marker of glomerular filtration rate, did not differ significantly between WT and KS-AR-KO mice in either sex (Table 2).

Net Acid Excretion

Net acid excretion involves two processes: ammonium (NH4þ ) excretion and titratable acid excretion. In male mice, KS-AR-KO increased ammonia excretion significantly, which eliminated the sex difference in ammonia excretion (Fig. 2). Ammonia excretion was not significantly altered in female mice by KS-AR-KO. Titratable acid excretion was not significantly altered by KS-AR-KO in either sex (Table 2). Urine pH was significantly decreased by KS-AR-KO in male mice but not in female mice (Table 2); however, the effect of KS-AR-KO on ammonia excretion persisted after statistical consideration of urine pH (P < 0.001 by ANOVA). Thus, proximal tubule AR mediates sex differences in ammonia excretion in male mice, but its deletion had no significant effect on ammonia excretion in female mice.

Effect of Kidney-Specific AR Deletion on Proximal Tubule Structure

Proximal tubules account for a greater proportion of cortical volume in the male kidney than in the female kidney, and this is testosterone dependent (20, 22). Thus, we determined the effects of KS-AR deletion in male and female mice on proximal tubule structure. Using PEPCK immunolabel as a proximal tubule-specific marker, immunohistochemistry showed qualitatively that KS-AR-KO decreased the proportion of the cortex that was proximal tubule (Fig. 3A). Quantitative analysis, using formal point counting techniques (41), showed that KS-AR-KO significantly decreased proximal tubule volume density in the cortex of males, which parallels the change in kidney weight (Fig. 3B). In contrast, in the outer stripe of the outer medulla, KS-AR-KO did not alter proximal tubule volume density in male mice significantly (Fig. 3B). In female mice, KS-AR-KO did not qualitatively alter the proportion of the cortex that was proximal tubule (Fig. 3A) and quantitatively did not alter proximal tubule volume density in either the cortex or outer medulla (Fig. 3B). The sex difference in cortical proximal tubule volume density present in WT mice was not present in KS-AR-KO mice. Thus, in the male kidney, AR mediates the sex differences in proximal tubule volume density in the cortex. In the outer medulla, AR expression does not appear to influence proximal tubule volume density in either sex.

These changes in proximal tubule structure in male KSAR-KO mice appear to involve changes in cell size. In male mice, KS-AR-KO decreased proximal tubule cell height significantly in both the PCT and cortical PST, i.e., throughout the cortex (Fig. 3C). Cell height in the PST in the outer medulla was not altered significantly (Fig. 3C). These results parallel the volume density findings. Kidney-specific AR deletion in female mice did not alter proximal tubule cell height in either the PCT, cortical PST, or PST in the outer medulla, which parallels the lack of effect on volume density and total kidney size in the female kidney (Fig. 2B).

Kidney weight, cortical proximal tubule volume density, and cell height in the PCT and cortical PST were all greater in the WT male kidney than in the WT female kidney, which parallels our previous observations (20, 22). In male KS-AR-KO mice, this sex difference no longer existed, and each of these parameters did not differ significantly from that observed in either WT or KS-AR-KO female mice. Thus, sex differences in kidney weight, cortical proximal tubule volume density, and cell height in the PCT and cortical PST are dependent on AR expression.

Effect of Kidney-Specific AR Deletion on Collecting Duct Structure

There are important sex differences in the collecting duct, with collecting duct volume density being greater in the proportion of the cortex or outer medulla that was collecting duct in either male or female mice (Fig. 4A). Quantitative morphometric analysis showed that KS-AR-KO did not alter collecting duct volume density significantly in either the cortex or outer medulla in either sex (Fig. 4B). Thus, renal AR does not mediate the sexual dimorphism in collecting duct volume density.

Ammoniagenic Enzyme Expression The results in Net Acid Excretion show that kidney-specific AR deletion increases ammonia excretion in male mice even though it decreases proximal tubule volume density and cell height in the cortex. This indicates that the effects on ammonia excretion cannot be attributed simply to proximal tubule hypertrophy leading to increased ammonia genesis and instead are the result of separate and specific effects on ammonia genesis and/or ammonia transport. Thus, we next examined key proteins involved in ammonia genesis and in ammonia transport.

PEPCK is a key proximal tubule enzyme required for ammonia genesis and is found exclusively in the proximal tubule in the kidney (16, 44). In male mice, KS-AR-KO increased cortical PEPCK expression significantly; there was no effect of KS-AR-KO in female mice (Fig. 5A). The increase in PEPCK expression in male KS-AR-KO mice eliminated the sex difference in WT mice (Fig. 5A). Examination of PEPCK immunolabels expression showed that male KS-AR-KO mice had greater PEPCK immunolabel intensity in the PCT and cortical PST than did WT mice (Fig. 5B). In the outer stripe of the outer medulla, there was no detectable difference in PEPCK immunolabel intensity (Fig. 5B). Quantitative immunohistochemistry of male mice showed that KS-AR-KO increased PEPCK immunolabel intensity significantly in both the PCT and cortical PST but not in the outer stripe of the outer medulla (Fig. 5C). Thus, in male mice, but not in female mice, proximal tubule AR expression decreases PEPCK expression in cortical proximal tubule segments, but not in the PST in the outer medulla.

The proximal tubule has an ammonia recycling capability involving GS (37, 42, 45, 46). In neither male nor female mice did KS-AR-KO significantly alter GS expression (Fig. 5D). Thus, renal AR does not appear to modulate GS expression.

Effect of Kidney-Specific AR Deletion on NBCe1 Expression

NBCe1 is a proximal tubule integral membrane protein with a major role in filtered bicarbonate reabsorption that also regulates ammonia metabolism (47–49). Two splice variants of NBCe1, NBCe1-A, and NBCe1-B, are present in the mouse kidney (50, 51). Using a pan-NBCe1 antibody, which recognizes both NBCe1 variants, we found that KS-AR-KO significantly decreased cortical NCBe1 expression in the male, but not female, kidney (Fig. 6A).

Effect of Kidney-Specific AR Deletion on Ammonia Transporters Renal ammonia excretion involves coordinated NH3/ NH4þ transport by specific membrane proteins (10, 16, 52). This involves transporters in the proximal tubule (NHE3), TAL of the loop of Henle (NKCC2), and collecting duct immunolabel was not detectable in outer medullary proximal tubule segments (Fig. 6C), as previously reported (48). We then used quantitative immunohistochemistry analysis to quantify segment-specific changes in NBCe1-A expression. This showed that KS-ARKO in male mice decreased NBCe1-A expression in the PCT and cortical PST, i.e., throughout the cortex (Fig. 6D). Thus, AR expression regulates cortical proximal tubule NBCe1-A expression in the male kidney but not in the female kidney.

(Rhbg and Rhcg). Sex differences in ammonia excretion correlate with differences in the expression of each of these (6, 20), and testosterone regulates the expression of both NHE3 and NKCC2 (22).NKCC2 mediates TAL ammonia reabsorption (10, 16, 52). In male mice, KS-AR-KO increased NKCC2 protein expression using immunoblot analysis significantly; in female mice, there was no significant change (Fig. 8). This change in expression in male mice likely contributes to altered medullary ammonia shunting and thereby facilitates the effect of KS-AR-KO to increase ammonia excretion in male mice.

Rh glycoproteins Rhbg and Rhcg are the primary proteins that mediate collecting duct ammonia secretion, and their expression generally parallels changes in ammonia excretion (10, 16, 52). However, KS-AR-KO did not alter significantly Rhbg protein expression in either male or female KS-AR-KO mice in either the cortex or inner stripe of the outer medulla (Fig. 9). The effects on Rhcg expression were similar. Rhcg immunohistochemistry, evaluated qualitatively and using quantitative immunohistochemistry, showed no significant changes in Rhcg immunolabel intensity in intercalated cells of the outer stripe of the outer medulla in either sex (Fig. 10). Thus, renal AR does not appear to modulate either Rhbg or Rhcg expression.

DISCUSSION

The present study provides important new information regarding the mechanism of how AR modulates sexual dimorphisms in the kidney. Because AR has numerous extra-renal roles, which could complicate understanding its role in the kidney, we created the first KS-AR-KO mouse. KS-AR-KO in male mice increased ammonia excretion, yet decreased total kidney size, proximal tubule volume density, and proximal tubule cell height, eliminating previously identified sex differences. The expression of multiple key proteins (PEPCK and NKCC2) involved in ammonia handling was increased by KS-AR-KO, likely contributing to the increased ammonia excretion. Simultaneously, the expression of other proteins (NHE3 and NBCe1-A) were decreased, which will tend to blunt changes in ammonia excretion. KS-AR-KO had no detectable effect in female mice or in the collecting duct of either sex. Table 3 shows these effects. These findings suggest that renal ammonia handling and proximal tubule structure involve renal AR-dependent signaling pathways in the cortex of the male kidney but not the female kidney and not in the outer medulla of either sex.

The first major finding in this study is that AR expression mediates sex differences in renal size. Previous studies in humans, rats, and mice have shown that the male kidney is larger than the female kidney (3, 56, 57). The first evidence that this involved a testosterone-dependent signaling pathway was the observation that excess androgen administration qualitatively increased rat proximal tubule size (57). Further evidence in adult males suggests that there is a dose-dependent increase in kidney volume, with an estimated 4.04-cm3 increase per 100 mg of weekly testosterone supplementation (58). We have recently shown that male mice have greater kidney and proximal tubule size than do female mice (20, 22) and that these differences were testosterone dependent (22). Consistent with these previous findings, the present study demonstrates that in male mice, renal AR expression regulates both whole kidney and proximal tubule size. Moreover, KS-AR-KO results in male kidney size being the same as in females with intact AR expression. Thus, in male mice, testosterone acting through renal AR-dependent signaling pathways can entirely account for the sexual dimorphism in the kidney and proximal tubule size.

The second major finding is that sex-based differences in ammonia excretion are mediated through AR-dependent pathways in male mice. Our previous studies showed that female mice excrete approximately twice as much urinary ammonia as do male mice (20–22), use different response pathways to respond to acid loading (21) and that the difference in basal ammonia excretion involves a testosterone-dependent mechanism (22). The present study extends these findings by showing that KS-AR-KO eliminates the sexual dimorphism in ammonia excretion. Alterations in dietary fixed acid loads can alter ammonia excretion, but changes in ammonia excretion in mice with AR deletion cannot be attributed to this mechanism as KS-AR-KO did not alter food intake. Most changes that occur in ammonia metabolism, such as occurs with metabolic acidosis or hypokalemia, result in changes in kidney and proximal tubule size that parallel ammonia excretion (59–61). However, male mice with renal AR deletion have increased ammonia excretion but decreased total kidney and proximal tubule size, indicating that the effect on ammonia excretion is not a non-specific response to changes in renal mass. This incongruent renal size and ammonia response in response to renal AR deletion parallels findings in our previous studies using a gonadectomy model that evaluated the effect of testosterone (22). Thus, in male mice, testosterone acting through renal AR in males regulates ammonia excretion through pathways unrelated to effects on renal size, proximal tubule size, and endogenous acid loads related to food intake.

The majority of ammonia generation occurs in the proximal tubule. Our previous work has identified sexual dimorphisms in proximal tubule ammonia generation that are dependent on testis-derived testosterone (22). The results of the present study further extend these findings by showing that renal AR in male mice regulates the expression of the ammonia-generating enzyme PEPCK but not the ammonia recycling enzyme GS. This effect on PEPCK expression is not a nonspecific effect related to changes in proximal tubule size, since the effects of renal AR deletion on proximal tubule size are the opposite of its effects on PEPCK expression. Moreover, the finding that renal AR deletion does not alter GS expression indicates that GS is regulated through mechanisms distinct from those used for PEPCK.

Ammonia produced in the proximal tubule is secreted preferentially into the luminal fluid through an apical NHE3-dependent mechanism (54, 55). Previous studies have shown that loss of testosterone decreases NHE3 expression (22, 62). The findings of the present study further extend these findings by showing that renal AR deletion in male mice decreases the expression of NHE3. Thus, testosterone acting through AR activation appears to increase cortical proximal tubule NHE3 expression. However, it is important to recognize that these effects do not parallel ammonia excretion and instead would be predicted to minimize the observed changes in ammonia excretion. Instead, because NHE3 is also critical for filtered NaCl and water reabsorption, these effects may contribute to the greater proximal tubule NaCl reabsorption present in males (6, 7).

Ammonia reabsorption in the TAL establishes the interstitial ammonia gradient necessary for collecting duct ammonia secretion (63, 64). Previous studies from our group have shown that NKCC2 expression is greater in female mice than in male mice (20–22), that there is sexual dimorphism in the response of NKCC2 to acid loading (21), and that the difference in basal NKCC2 expression involves a testosterone-dependent mechanism (22). The present study shows that loss of renal AR in male mice increased NKCC2 expression. The magnitude of this effect was almost identical to the difference we have identified between male and female mice (20, 21) and in the current study eliminated the sexual dimorphism of NKCC2 expression. Furthermore, the identified difference between male mice with intact renal AR and AR deletion parallels our previously identified differences between male mice with orchiectomy compared with intact and sham-operated controls (24). Thus, testosterone acting through AR appears to mediate the sexual dimorphism in NKCC2 expression. However, this regulation appears to be indirect, as there was no detectable AR expression in the TAL in either our previous report (22) or in the present study. One possible indirect mechanism explaining this observation is that in males, AR deletion leads to decreased proximal tubule size that in combination with decreased proximal tubule NHE3 expression increases increased solute delivery to the TAL, which stimulates NKCC2 expression.

Collecting duct ammonia secretion is the major determinant of urinary ammonia excretion, and the Rh glycoproteins Rhbg and Rhcg are the primary collecting duct ammonia transporters (16, 65–68). We have previously shown sexual dimorphisms in collecting duct volume density and expression of the Rh glycoproteins, with female mice having greater collecting duct volume density, intercalated cell size, and Rhbg and Rhcg expression (20). The present study found no detectable changes in collecting duct volume density or Rhbg and Rhcg expression in response to KS-AR-KO in either male or female mice. This parallels the lack of effect of testosterone deficiency, induced by orchiectomy, on collecting duct volume density, intercalated cell size, and Rhbg and Rhcg expression (22). We conclude that the sexual dimorphism in the collecting duct occurs through mechanisms not involving testosterone and AR-dependent signaling pathways.

The outer medulla appears to respond differently to sex, testosterone, and AR than does the cortex. In contrast to the renal cortex, in the outer medulla, there is no sexual dimorphism in proximal tubule volume density or proximal tubule height (20), no effect of orchiectomy-induced testosterone deficiency on these parameters (22), and no effect of renal AR deletion (present study). This lack of effect of sex, testosterone, and AR expression is not due to the absence of AR expression, as we have shown AR expression throughout the entire proximal tubule, including in the outer medulla (22). Understanding the mechanisms underlying this axial difference in the proximal tubule response to sex, testosterone, and AR will be an important avenue for future investigation.

A final observation is that AR deletion did not have detectable effects on the female kidney. AR is present throughout the entire proximal tubule in both sexes, and although expressed at lower levels in females, the absolute difference in expression was only - 50% (22). In part, the lack of effect of AR deletion could indicate decreased AR activation by circulating testosterone, which is, as shown in the present study, substantially less in females than in males. However, immunohistochemistry indicates substantial nuclear AR localization in the female kidney (Ref. 22 and present study), which typically indicates activation of AR-dependent signaling processes. These observations suggest the possibility that other sex-dependent differences impair AR-dependent signaling mechanisms in the female proximal tubule.

Perspectives and Significance

A better understanding of the mechanism and biological implications of sex’s effect on renal structure and ammonia metabolism is critical for optimizing our ability to care for both men and women with acid-base disturbances. Despite the recent advances in our understanding of the effect of sex on ammonia metabolism and the role of renal AR, additional studies are needed. There are certainly testosterone-independent effects of sex, the mechanism of which is incompletely understood at present. Whether this involves direct effects of other sex steroid hormones, such as estrogens or progesterone, indirect effects of proximal tubule AR activation or effects of sex chromosomes that are independent of gonadal hormones will be important issues for future studies to address.

cistanche relieves kidney problems and enhances kidney function

ACKNOWLEDGMENTS

We thank Dr. Sharon W. Matthews and Chao Chen (the University of Florida, College of Medicine Electron Microscopy Core Laboratory) for excellent tissue processing for our immunohistochemical experiments. We thank The University of Virginia School of Medicine Center for Research in Reproduction Ligand Assay and Analysis Core for measurement of serum testosterone levels. We thank Dr. Gail Prins for supplying anti-AR antibodies and for their expertise in AR.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.N.H. and I.D.W. conceived and designed research; A.N.H., R.A.C., H.-W.L., and J.W.V. performed experiments; A.N.H., R.A.C., H.-W.L., J.W.V., and I.D.W. analyzed data; A.N.H., R.A.C., J.W.V., and I.D.W. interpreted results of experiments; A.N.H. prepared figures; A.N.H. drafted the manuscript; A.N.H., R.A.C., H.-W.L., J.W.V., and I.D.W. edited and revised the manuscript; A.N.H., R.A.C., H.-W.L., J.W.V., and I.D.W. approved the final version of the manuscript.

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