Effect of sex chromosome complement versus gonadal hormones on abundance of renal transporters

Abstract

Sex differences in renal tubular salt and water transporters, channels, claudins and regulatory factors are evident all along the nephron. The influence of sex hormones on physiologic dimorphisms has been established in studies removing, inhibiting or restoring sex hormones and their receptors. The influence of the sex chromosome complement (SCC, XY vs. XX) on renal transporter abundance and activity is an open question. We employed the Four Core Genotypes (FCG) mouse model (in which the testis determining SRY gene is deleted from the Y chromosome and inserted onto an autosomal chromosome) to compare abundance of more than fifty renal transporters and regulators in: FXX gonadal females, FXY gonadal females, MXX Sry males, and MXY XYSry males using semi-quantitative immunoblots. In addition to establishing the significant influence of gonadal hormones, we show, for the first time, that SCC contributes to sexual dimorphisms in abundance of renal transporters including: NHE3, SGLT1 and 2, AQP1, mNKAα1 and β1, NCC, and ENaC β and γ subunits. The findings in this FCG model analysis provide the foundation for future studies of the role of sex hormones vs. chromosomes on physiologic parameters including filtration and flow, on transporter covalent modifications, and trafficking in both heath and disease.

Graphical Abstract

In the USA, 1 in 7 people have chronic kidney disease (CKD) provoked mainly by diabetes and hypertension. Medications for these chronic diseases target the kidneys (1), which are key to homeostasis of circulating volume and blood pressure set-point (2). Hypertension and diabetes pre-menopause are less common in females vs. males implicating sex chromosomes or gonadal hormones as causal (3). In rodents, females reabsorb a smaller fraction of the glomerular filtrate in proximal tubules due to smaller transport area and lower abundance of transporters. The subsequently larger fractional delivery of salts and water downstream is handled by a greater abundance of distal sodium transporters culminating in equivalent urine excretion between the sexes (4–6). Physiologic relevance of sex differences can be attributed to the need for females to adapt to the challenges of pregnancy and lactation (increased reabsorption of nutrients, salts, and water), while maintaining blood volume and blood pressure homeostasis (7, 8).

All along the nephron, baseline sex differences in abundance of tubular salt and water transporters, channels, claudins and regulatory factors (collectively termed “transporters” herein) are evident (4, 5). While the influence of sex hormones on physiologic dimorphisms has been established in studies removing, inhibiting or restoring sex hormones and their receptors (4, 9, 10) the contribution of sex chromosome complement (SCC, XY vs. XX) to baseline differences in tubular transporter abundance and activity is an open question. The Four Core Genotypes (FCG) mouse model is well suited to address this question (11). FCG mice are produced by crossing XX gonadal female mice with XY-Sry^- gonadal male mice in which the testis determining SRY gene is deleted from the Y chromosome and inserted onto an autosome. The offspring fall into four core genotypes: FXX: gonadal females, FXY^-: gonadal females, MXX: XXSry gonadal males, and MXY: XY^-Sry gonadal males (11). The FCG model can independently test for sex chromosome effects (XX vs. XY) and gonadal effects (female vs. male phenotype), as well as physiological interactions between SCC and gonadal phenotype. Herein, we determine transporter abundance along the renal tubule as a function of SCC and gonadal phenotype.

Methods

Animals.

Cryo-recovered Four Core Genotype mice [B6.Cg-Tg(Sry)2Ei Sry^dl1RlbT(XTmsb4x-Hccs;Y)1Dto/ArnoJ; Stock# 010905] were purchased from the Jackson Laboratory (Bar Harbor, Maine). XY^-Sry males were bred with wild-type XX female C57BL/6J mice to produce FCG pups. Genotyping of the offspring was performed by polymerase chain reaction (PCR) as recommended by JAX using DNA from tail samples. Males were defined as mice with testes, and females as mice with ovaries. Mice were housed at the Icahn School of Medicine at Mount Sinai (ISMMS) with free access to tap water and standard chow (LabDiet #5001, Richmond, IN) containing 0.4% Na⁺ and 1.18% K⁺. Mice were euthanized at 12–15 weeks of age in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Kidneys were removed, snap-frozen in liquid nitrogen, stored at −80°C then shipped on dry ice to Keck School of Medicine of USC (Keck USC). Animal protocols were approved by the Institutional Animal Care and Use Committee at the ISMMS.

Semiquantitative Immunoblotting.

At Keck USC, kidneys (n=6 mice/group) on ice were decapsulated, dissected into cortex and medulla, and homogenized as recently described in detail (12) and in Supplement. Homogenate protein concentrations were determined, samples immediately denatured in Laemmli sample buffer, to equivalent protein concentrations, and heated 20 min at 60°C. Sample loading was verified on Coomassie stained gels (Fig. S1). Samples processing and immunodetection protocols are described in Table S1. Signals were quantified with the Odyssey Infrared Imaging System (LI-COR) and accompanying Image Studio software and displayed as arbitrary density units. Uncut blots images provided in Supplement.

Statistical analysis.

Protein abundance data from n=6 mice/group are reported as means ± SEM. Statistical analyses were performed using GraphPad Prism 8.4 (Boston, MA). Statistical significance of protein abundance between genotypes was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. P value assessments were limited to: 1) within phenotypes: (FXX and FXY) vs. (MXX and MXY), 2) within genotypes: FXX vs. FXY and MXX vs. MXY; and 3) interactions between phenotypes and genotypes.

Glossary.

AQP1, aquaporin 1; AQP2, aquaporin 2; AQP2pS256, aquaporin 2 phosphorylated at S256; AQP3, aquaporin 3; AQP4, aquaporin 4; AR, androgen receptor; BK β4, calcium-activated potassium channel beta4 subunit; c = cortical; ENaCα, epithelial sodium channel subunit alpha; ENaCβ, epithelial sodium channel subunit beta; ENaCγ, epithelial sodium channel subunit gamma; ER, estrogen receptor; F = female; Kir4.1, inwardly rectifying potassium channel; M = male; m = medullary; Muc1, mucin-1; NaPi2, sodium-phosphate cotransporter type 2; NBCe1, electrogenic sodium bicarbonate exchanger 1; NCC, sodium-chloride cotransporter; NCCpS71, NCC phosphorylated at S71; NCCpT53, sodium-chloride cotransporter phosphorylated at T53; NDCBE1, sodium-driven chloride bicarbonate exchanger 1; NHE3, sodium/hydrogen exchanger isoform 3; NHE3p, NHE3 phosphorylated at S552; NHERF1, NHE3 regulatory cofactor 1; NKCC2, sodium-potassium-2 chloride cotransporter; NKCC2pS91, NKCC2 phosphorylated at S91; NKAα1, alpha1 subunit of sodium-potassium ATPase; NKAβ1, beta1 subunit of sodium-potassium ATPase; OAT1, organic anion transporter 1; OAT2, organic anion transporter 2; OSR1pS325, oxidative stress responsive kinase 1 phosphorylated at S325; ROMK, renal outer medullary potassium channel; SGK1, serum and glucocorticoid-induced protein kinase 1; SCC, sex chromosome complement; SGLT1, sodium glucose cotransporter 1; SGLT2, sodium glucose cotransporter 2; SPAK, Ste20-related proline alanine rich kinase; SPAKpS373, Ste20-related proline alanine rich kinase phosphorylated at S373; TRPV4, transient receptor potential cation channel subfamily V member 4; UMOD, uromodulin; V-ATPaseβ1, vacuolar-type ATPase subunit beta 1; WNK4, with-no-lysine protein kinase 4.

Results

The impact of sex chromosome complement (SCC) and gonadal phenotype on protein abundance of more than 50 renal transporter proteins was investigated in cortical and medullary homogenates by semiquantitative immunoblotting (protocols in Table S1). Fig. 1, 2, and 3 compare transporter specific abundance of all four core genotypes: immunoblots in Panel A and summary data in Panel B. Fig. S2-S6 provide transporter profiles as bar graphs comparing FXX vs. MXY, FXY vs. MXY, FXX vs. MXX, MXX vs. MXY and FXX vs. FXY.

Figure 1. Transporter abundance between FCG strains – proximal tubule through medullary thick ascending limb.

A: Semi-quantitative immunoblots of transporters in homogenates of renal cortex (c) and medulla (m), n = 6/group (protocols in Table S1). Molecular weight (MW, kD) on the right. B: Relative abundance in arbitrary density units with means ± SEM analyzed by two-way ANOVA with Tukey correction for multiple comparisons; P-values indicated for phenotype and sex chromosome impact as well as interaction of these two variables on transporter abundance.

Figure 2. Transporter abundance between FCG strains - cortical thick ascending limb to collecting duct.

A: Semi-quantitative immunoblots of transporters in homogenates of renal cortex (c) and medulla (m), n = 6/group (protocols in Table S1). Molecular weight (MW, kD) on the right. B: Relative abundance in arbitrary density units with means ± SEM analyzed by two-way ANOVA with Tukey correction for multiple comparisons; P-values indicated for phenotype and sex chromosome impact as well as interaction of these two variables on transporter abundance.

Figure 3. Transporter abundance between FCG strains - collecting duct (continued), regulators and other proteins of interest.

A: Semi-quantitative immunoblots of transporters in homogenates of renal cortex (c) and medulla (m), n = 6/group (protocols in Table S1). Molecular weight (MW, kD) on the right. B: Relative abundance in arbitrary density units with means ± SEM analyzed by two-way ANOVA with Tukey correction for multiple comparisons; P-values indicated for phenotype and sex chromosome impact as well as interaction of these two variables on transporter abundance.

Figure 1 compares transporters expressed from the proximal tubule through medullary thick ascending limb. NHE3 was lower in FXX vs. MXY as previously reported (5). NHE3 was not lower in XX vs. XY SCC in gonadal females (p= 0.16) nor males (p=0.10). Claudin 2 tended to be greater in gonadal females vs. males of both SCC (p=0.06) in contrast to a previous report of greater claudin 2 in MXY vs. FXX (5), perhaps a strain difference. cSGLT2, cSGLT1, mNKCC2pS87, NKAα1, and AQP1 were more abundant in gonadal females vs. males by two-way ANOVA. In contrast, cOAT1 was greater in males vs. females of both SCC as previously reported (13) but SCC had no impact on cOAT1. cOAT2 was lower in MXX vs. MXY, but not different in gonadal females vs. males. Interactions between SCC and gonadal hormone effects are evident for cSGLT1, cSGLT2, mNKA α1, mNKAβ1 and mAQP1: abundance was greater in FXX vs. FXY for cSGLT1, mNKA α1, and mNKAβ1, also lower in MXX vs. MXY for SGLT2. Statistically significant differences between genotypes were not evident for: cNHE3p, cNaPi2, cNBCE1, mNHE3, mNHE3p, mSGLT1 or mNKCC2.

Figure 2 compares transporters from cortical thick ascending limb (cTAL) through collecting duct (CD). The cotransporters NKCC2 (in cTAL), and NCC (in distal convoluted tubule, DCT) exhibit similar patterns of their total and phosphorylated (activated) forms: higher abundance in gonadal females than males, and no SCC differences. Along the cortical CD, ENaC α, β,and γ subunits and their cleaved forms are more abundant in gonadal females than males. The heteromeric basolateral inward rectifying K⁺ channel Kir4.1/5.1 positively impacts ENaC activity (14) and abundance of cKir4.1 was higher in females than males consistent with greater ENaC. Likewise, the junction proteins claudin 7 and claudin 8, which modulate paracellular sodium back leak (15, 16), are more abundant in gonadal female vs. male. In the CCD, interactions between SCC and gonadal hormone on abundance were evident for ENaC β and γ subunits and claudin-7.

Potassium and acid base transport are closely regulated along the CCD. Variability in abundance of the potassium secretory channel ROMK is insignificant across FCG. Among mechanosensitive channels expressed along the CCD (17, 18), TRPV4 was more abundant in gonadal males than females, while the β4 subunit of the BK channel was more abundant in female than male phenotypes. The CCD intercalated cell anion transporters exhibited distinct patterns: pendrin (HCO3^- /Cl^- antiport) was more abundant in female vs. male phenotypes, while NDCBE (Na⁺ -HCO₃^- cotransporter) and cortical V-ATPase β1 subunit were more abundant in gonadal males than females.

Figure 3 summarizes FCG differences in water and protein transporters and select transporter regulators. CD apical cAQP2 and basolateral mAQP4 were more abundant in gonadal females than males; mAQP4 exhibited a significant SCC-gonadal hormone interaction. The cotransporter regulatory kinases WNK4, SPAK and OSR1 and the aldosterone responsive cSGK1 were more abundant in females than males. Increases in abundance are evident in FXX vs. MXX for NKCC2p and NCCp and their kinases SPAK and OSR1. The proximal tubule endocytic receptors megalin and cubilin were more abundant in gonadal females than males, consistent with lower protein excretion in females (19), as was the multifunctional transmembrane protein cMUC1 (20).

Discussion

Numerous studies have identified sex-specific differences in renal function in health and disease (3). This short report surveys the impact of SCC and gonadal hormones on renal transport protein abundance without further investigation of physiological correlates. The heatmap (Fig. 4) illustrates that gonadal phenotype (FXY vs. MXY, FXX vs. MXX) has a strong impact on transporter abundance. However, we show for the first time that SCC also contributes to sexual dimorphisms in abundance of important renal transporters: NHE3, SGLT1 and 2, AQP1, mNKAα1 and β1, NCC, and ENaC β and γ subunits. Other transporters do not exhibit abundance dependence on either SCC or gonadal phenotype: cNaPi2, cNBCE1, mNHE3, mNKCC2 ROMK, AQP2 or AQP3. A recent study of the origins of sex biased gene expression in mice (employing gonadectomy, renal AR and ER tubular knock-out (KO)) provides insights (10): sex differences were predominately a function of testicular androgens, with little impact of ovariectomy or ER KO. In males androgens and their receptors increase kidney volume, increase salt and water reabsorption along the proximal tubule, and regulate ammonia metabolism and renal structure (4, 10). However, 17β-estradiol (E2) has been reported to enhance both ENaC-mediated transepithelial currents in CCD cells (21) and α-ENaC subunit mRNA abundance (22), consistent with G-protein coupled estrogen receptor expression in CCD (4).

Figure 4. Fold difference in transporter abundance normalized to phenotype or genotype.

Transporters in cortex (c) and medulla (m) organized from proximal tubule to collecting duct followed by transporters expressed in both c and m. FXX vs. MXY: Collective impact of SCC (XX vs. XY) and gonadal phenotype (male vs. female) on relative abundance of transporters; MXY = 1. FXY vs. MXY: Selective impact of gonadal phenotype in mice with same SCC (XY) on relative abundance; MXY = 1. FXX vs. MXX: Selective impact of gonadal phenotype in mice with same SCC (XX) on relative abundance. MXX = 1; MXX vs. MXY: Selective impact of SCC in mice with same gonadal phenotype (male) on relative abundance; MXY = 1. FXX vs. FXY: Selective impact of SCC (XX vs. XY) in mice with same gonadal phenotype (female) on relative aundance; FXY = 1

Although multiple (12–14) copies of the testis-determining Sry gene are present on chromosome 3 in the FCG mouse, there is no evidence for masculinization of anogenital distance in XY females relative to XX females, suggesting that sex chromosome differences are not due to differences in circulating levels of androgens prenatally (23). In fact, no differences in testosterone levels in XY versus XX, nor estradiol levels in XX versus XY are detected in adults (24, 25). However, whether high levels of Sry transgene expression early in life in FCG males alters responsiveness to sex steroids later in life remains uncertain (25). In liver, there is no evidence that that transgenic insertion of the Sry gene alters gene expression in the vicinity of the transgene (23), but the impact of the Sry transgene in kidney has yet to be determined.

In interpreting abundance results, it is key to acknowledge that differences in one region of the renal tubule attributed to SCC and/or gonadal hormones can provoke secondary effects in another region of the tubule via impact on tubular composition, flow, kinase abundance, transporter phosphorylation, transporter trafficking, or resultant systemic fluid and electrolytes levels, all independent of SCC and/or gonadal phenotype in that region. Additionally, the SCC may contribute to sex-specific differences via pathways not addressed here, e.g. the purinergic P2Y4 receptor, implicated in salt and water transporter regulation (26), is located on the X chromosome (27).

Some findings warrant comment. Na,K-ATPase (NKA) α and β subunits generate the driving force for most all transepithelial Na⁺ and K⁺ transport. The α and β subunits are similarly more abundant in gonadal females than males as reported in rats (5), yet SCC also differentially impacts abundance (interaction p < 0.001: FXX>FXY and MXX<MXY). A similar pattern of gonadal differences and SCC interactions is evident for AQP1. Proximal tubule NHE3, which reabsorbs a significant fraction of the glomerular filtrate, exhibits lower abundance in FXX vs. MXY (5), but, shown herein, FXX is not lower than FXY or MXX. Cortical NHE3 in FXX is localized to the base of the microvilli where activity is low compared to NHE3 in MXY located in the body of the microvilli (5, 28). Apical mNHE3 and mNKCC2 abundance showed little impact of sex hormones or SCC; in contrast cNKCC2, cNKCC2p, cNCC, cNCCp, and their kinases cWNK and cSPAK show gonadal sex differences (females > males) as previously reported (5, 29, 30) highlighting regional differences.

Functionally, fluxes depend on transporter abundance, their covalent modifications, distribution between intracellular and surface membranes, and driving forces (i.e., concentration and electrical potential gradients). Increases in flow can enhance transporter trafficking to/from the membrane in the proximal tubule (31) and activate mechanosensitive channels downstream (18, 32, 33). Males have a higher glomerular filtration rate than females, a difference that should, per se, lead to greater transporter activation in males. The adaptive responses of mice fed high salt diet (HSD) also illustrate the point: while proximal sodium transporters’ abundance and Na⁺ reabsorption were depressed during HSD, and distal transporters were likewise depressed during HSD, distal Na⁺ reabsorption actually increased (predicted and confirmed by diuretic responses), secondary to increased delivery from proximal nephron. The opposite was observed in response to low salt diet (34).

Potassium secretion in the distal nephron is mediated by both the principal cell ROMK channel, and the Ca²⁺, voltage and stretch-activated BK channel (in both principal and intercalated cells). Whereas ROMK mediates constitutive potassium secretion, the intercalated cell BK channel, comprised of α and β4 subunits, effects flow-induced K⁺ secretion (FIKS)(18). Both ROMK and BK channel-mediated potassium secretion require ENaC-mediated sodium reabsorption (35) which can be activated itself by flow (34, 36, 37). Abundance of ROMK is similar across FCG mice, consistent with previous report (5). During pregnancy, to satisfy the need for positive potassium balance during fetal growth, apical localization of ROMK along DCT and CNT is reduced even though ROMK mRNA is unchanged (38), indicative of multiple layers of regulation. In contrast to ROMK, abundance of CD BKα is greater in females vs. males (39). This difference, coupled to greater BKβ4 in females, is expected to enhance FIKS and urinary potassium losses in non-pregnant females vs. males, secondary to lower proximal sodium and volume reabsorption and greater distal delivery in females. In fact, baseline plasma potassium is significantly lower in female vs. male rats (5).

Further analyses of the relative contributions of gonadal hormones vs. sex chromosome complement to renal transport protein physiology may enable us to better understand the impact of these factors on female vs. male biology, including salt and water balance and regulation of blood pressure, in conditions such as pregnancy, lactation and menopause in females (4, 40), low testosterone in males (40), as well as in long-term gender-affirming hormone therapy.

Supplementary Material

Supplemental Materials location and list:

https://figshare.com/articles/journal_contribution/Supplementary_data_for_AJP_Renal_i_Effect_of_sex_chromosome_complement_versus_gonadal_hormones_on_abundance_of_renal_transporters_i_/28432412?file=52427735

Data sharing

All data are included in the manuscript and/or supporting information.