Sexual Dimorphic Pattern of Renal Transporters and Electrolyte Homeostasis

Abstract

Compared with males, females have lower BP before age 60, blunted hypertensive response to angiotensin II, and a leftward shift in pressure natriuresis. This study tested the concept that this female advantage associates with a distinct sexual dimorphic pattern of transporters along the nephron. We applied quantitative immunoblotting to generate profiles of transporters, channels, claudins, and selected regulators in both sexes and assessed the physiologic consequences of the differences. In rats, females excreted a saline load more rapidly than males did. Compared with the proximal tubule of males, the proximal tubule of females had greater phosphorylation of Na⁺/H⁺ exchanger isoform 3 (NHE3), distribution of NHE3 at the base of the microvilli, and less abundant expression of Na⁺/Pi cotransporter 2, claudin-2, and aquaporin 1. These changes associated with less bicarbonate reabsorption and higher lithium clearance in females. The distal nephrons of females had a higher abundance of total and phosphorylated Na⁺/Cl^- cotransporter (NCC), claudin-7, and cleaved forms of epithelial Na⁺ channel (ENaC) α and γ subunits, which associated with a lower baseline plasma K⁺ concentration. A K⁺-rich meal increased the urinary K⁺ concentration and decreased the level of renal phosphorylated NCC in females. Notably, we observed similar abundance profiles in female versus male C57BL/6 mice. These results define sexual dimorphic phenotypes along the nephron and suggest that lower proximal reabsorption in female rats expedites excretion of a saline load and enhances NCC and ENaC abundance and activation, which may facilitate K⁺ secretion and set plasma K⁺ at a lower level.

Keywords: Na transport; distal tubule; gender difference; potassium; proximal tubule.

Graphical abstract

Figure 1.

Lower fractional sodium reabsorption in female versus male rats. Rats were challenged with an i.p. bolus of warmed saline equivalent of 6%–7% of their body weight and placed in metabolic cages for hourly urine collection. (A) Fraction of injected Na⁺ and volume excreted over 5-hour collection period. (B) Accumulated excretion of Na⁺ and volume. Data are expressed as mean±SEM, n=4 per group. *P<0.05. (C) NHE3 preferentially distributes to the base of the microvilli in females. Indirect immunofluorescence microscopy of NHE3 distribution and abundance in kidney samples from untreated male and female Sprague Dawley rats processed side by side on the same slide; NHE3 detected with polyclonal anti-NHE3 (green) and microvilli identified using monoclonal anti-villin (red). In males, the microvilli, primarily yellow, indicate colocalization of NHE3 with villin in the body of the villi, whereas in the females relatively more NHE3 is located at the base of the microvilli, revealing more red- (villin) stained microvilli. (D) NaPi2 exhibits similar distribution and lower abundance in female versus male rats. NaPi2, detected with polyclonal anti-NaPi2 (1:50, green), exhibited similar distribution in the body of microvilli overlaying villin in both sexes, but signal indicated lower abundance in females than males when viewed with the same settings. For (C and D), three different sets of rat kidneys were analyzed with similar results. Bar=20 µm. (E) Proximal tubule HCO₃⁻ reabsorption (JHCO₃⁻), a measure of NHE3 activity, is lower in female than male rats. JHCO₃⁻ was evaluated by in vivo stationary microperfusion via continuous measurement of luminal pH (see Supplemental Material for details). Number of perfused tubules: males=32, and females=25. Values represent individual measurements and mean±SEM. *P<0.05 versus males. (F) CLi, a measure of volume flow leaving the proximal tubule, is greater in female than male rats. CLi was calculated according to the conventional expression: CLi = [U/P]_Li × [Vu/body wt] (ml. min−¹. kg BW⁻¹). [U/P]_Li denotes the urine-to-plasma concentration ratio of lithium and [Vu/body wt] is the rate of urine flow divided by the body weight. Values represent individual measurements and mean±SEM (n=4). *P<0.05 versus males.

Figure 2.

Sexual dimorphic pattern of transporters, channels, regulators and cytoskeletal proteins along the proximal nephron. Immunoblots of renal homogenates. Both 1 and 1/2 amounts of protein were loaded (only one amount is displayed), see antibodies in Supplemental Table 1 for details. Density values, normalized to male group=1.0, displayed as mean±SEM (n=6). MW (molecular weight) indicates the lane loaded with prestained protein ladders (BioRad or Thermo Scientific). kD indicates apparent MW of the stained markers. *P<0.05 versus male. (A) Renal cortex samples, (B) evidence for shorter microvilli in females. Villin abundance and distribution in renal cortex samples from females and males collected, fixed, and processed at same time and viewed on same slide with identical settings. Villin was detected using anti-villin (1:100). Two different sets of rat kidneys were analyzed with similar results. Bar=25 µm. (C) Renal medulla samples. (D) Profile of transporters, structural and associated proteins, and regulators expressed along the proximal nephron summarized from (A and C). AQP1–35, -24, aquaporin 1 forms with apparent mobilities of -35 and -24 kD; Cldn2, claudin-2; DPPIV, dipeptidyl peptidase IV; fl, full length; HO-1, heme oxygenase-1; KO, SPAK^−/−; NaPi2, Na⁺/Pi cotransporter isoform 2; NBCe1, Na⁺/HCO₃⁻ cotransporter 1; NHE3, Na⁺/H⁺ exchanger isoform 3; NHE3pS552, Na⁺/H⁺ exchanger isoform 3 phosphorylated at Ser 552; NHERF1, Na⁺/H⁺ exchanger regulatory factor 1; NKA, Na⁺,K⁺ - ATPase α1 and β1 subunits; NKCC2, Na⁺-K⁺- 2 Cl⁻ cotransporter isoform 2; NKCC2pT96T101, NKCC2 phosphorylated at Thr 96 and 101; SPAK, STE20/SPS1-related proline alanine- rich kinase.

Figure 3.

Sexual dimorphic pattern of transporters, channels and regulator proteins along the distal tubule and collecting duct. Immunoblots of renal homogenates. Both 1 and 1/2 amounts of protein were loaded (only one amount is displayed), see antibodies in Supplemental Table 1 for details. Density values, normalized to male group=1.0, displayed as mean±SEM (n=6). *P<0.05 versus male. (A) Renal cortex samples probed for thick ascending limb and distal tubule cotransporters, kinases, and claudins. (B) Renal cortex and medulla samples probed for CD channels; “A” indicates samples from AngII-infused rats, which possess more abundant levels of activated ENaC subunits. (C) Profile of transporters, channels, claudins, and regulatory kinases expressed along the distal nephron and CD summarized from (A and B). AQP2–23, -37, aquaporin 2 forms with apparent mobilities of −23 and −37 kD; Cldn10, claudin 10; ENaC, epithelial Na⁺ channel; fl, full length; KO, SPAK^−/−; KS-SPAK, kidney specific SPAK; NCC, Na⁺- Cl⁻ cotransporter; NCCpT53 and NCCpS71, NCC phosphorylated at Thr 53 and Ser 71, respectively; ROMK, renal outer medullary K⁺ channel partial glycosylated (pg) and fully glycosylated (fg) forms; SPAK, STE20/SPS1-related proline alanine- rich kinase.

Figure 4.

Physiologic correlates of sex-specific differences in renal transporters. Rats were fasted overnight with water, then fed a meal containing 0% K⁺ for 3 hours. Overnight, urine was collected in metabolic cages. At 3 hours, plasma was collected. Aldosterone was measured in plasma. Values represent individual measurements and mean±SEM (n=5–12). *P<0.05 versus males. The last two panels summarize abundance of renal cortical NCC and NCCp from Figure 3A as a function of plasma [K⁺]. Female rats (○, ◇, △) exhibit higher NCC and NCCp abundance along with lower plasma [K⁺] than male rats (●, ◆, ▲); NCC (●, ○), NCCpS71 (◇, ◆), NCCpS53 (▲, △). UKV, urinary K⁺ excretion; UNaV, urinary Na⁺ excretion; UV, urinary volume.

Figure 5.

Females respond to 3-hour 2% K meal with kaliuresis and reduced NCCp. Rats were fasted overnight and then fed either 2% K diet (F2K) or 0% KF0K diet in metabolic cages for 3 hours before they were anesthetized for blood and tissue collection and euthanized. (A) Plasma [K⁺], UKV (urinary K⁺ excretion), and UNaV (urinary Na⁺ excretion) increase in response to a K⁺-rich meal. (B) Immunoblots of renal cortex homogenates demonstrate that NCC and NCCp abundance decrease in response to a K⁺-rich meal. Both 1 and 1/2 amounts of protein were loaded (only one amount is displayed), see antibodies in Supplemental Table 1 for details. Density values, normalized to F0K=1.0, displayed as mean±SEM (n=6). *P<0.05 versus F0K group. (C) Abundance of renal cortical NCCp plotted as a function of plasma [K⁺] demonstrates that 2% K⁺-rich meal (△, ▲), versus 0% K⁺ meal (○, ●), increases plasma [K⁺] and depresses NCCpT53 (○, △) and NCCpS71 (●, ▲). cl, cleaved; fg, fully glycosylated; fl, full length; pg, partially glycosylated.

Figure 6.

Sexual dimorphic pattern of transporters, channels and regulators along the nephron in C57BL/6 mice, summarized from Supplemental Figures 4 and 5. (A) Renal cortex and medulla samples probed for proximal tubule and thick ascending limb proteins. (B) Renal cortex and medulla samples probed for DCT, connecting tubule, and CD proteins. Mean±SEM.