Effects of K+-deficient diets with and without NaCl supplementation on Na+, K+, and H2O transporters' abundance along the nephron

Abstract

Dietary potassium (K(+)) restriction and hypokalemia have been reported to change the abundance of most renal Na(+) and K(+) transporters and aquaporin-2 isoform, but results have not been consistent. The aim of this study was to reexamine Na(+), K(+) and H(2)O transporters' pool size regulation in response to removing K(+) from a diet containing 0.74% NaCl, as well as from a diet containing 2% NaCl (as found in American diets) to blunt reducing total diet electrolytes. Sprague-Dawley rats (n = 5-6) were fed for 6 days with one of these diets: 2% KCl, 0.74% NaCl (2K1Na, control chow) compared with 0.03% KCl, 0.74% NaCl (0K1Na); or 2% KCl, 2%NaCl (2K2Na) compared with 0.03% KCl, 2% NaCl (0K2Na, Na(+) replete). In both 0K1Na and 0K2Na there were significant decreases in: 1) plasma [K(+)] (<2.5 mM); 2) urinary K(+) excretion (<5% of control); 3) urine osmolality and plasma [aldosterone], as well as 4) an increase in urine volume and medullary hypertrophy. The 0K2Na group had the lowest [aldosterone] (172.0 ± 17.4 pg/ml) and lower blood pressure (93.2 ± 4.9 vs. 112.0 ± 3.1 mmHg in 2K2Na). Transporter pool size regulation was determined by quantitative immunoblotting of renal cortex and medulla homogenates. The only differences measured in both 0K1Na and 0K2Na groups were a 20-30% decrease in cortical β-ENaC, 30-40% increases in kidney-specific Ste20/SPS1-related proline/alanine-rich kinase, and a 40% increase in medullary sodium pump abundance. The following proteins were not significantly changed in both the 0 K groups: Na(+)/H(+) exchanger isoform 3; Na(+)-K(+)-Cl(-) cotransporter; Na(+)-Cl(-) cotransporter, oxidative stress response kinase-1; renal outer medullary K(+) channel; autosomal recessive hypercholesterolemia; c-Src, aquaporin 2 isoform; or renin. Thus, despite profound hypokalemia and renal K(+) conservation, we did not confirm many of the changes that were previously reported. We predict that changes in transporter distribution and activity are likely more important for conserving K(+) than changes in total abundance.

Fig. 1.

Abundance of total and phosphorylated Na⁺/H⁺ exchanger isoform 3 (NHE3) and Na⁺/phosphate cotransporter type 2 (NaPi2) in renal cortex (A) and medulla (B) of rats fed K⁺-deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared with 2% KCl-fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample are loaded in adjacent lanes for each animal: 30, 15 μg for cortex and 34, 17 μg for medulla NHE3 and NHE3pS552; 30, 15 μg for cortex NaPi2. Density values were normalized to mean density of control K⁺ groups (1.00). Values are displayed as dot plots over a bar at the mean value. *P < 0.05 vs. respective 2 K group control.

Fig. 2.

Abundance of total and phosphorylated Na⁺-K⁺-2Cl⁻ cotransporter type 2 (NKCC2), Na⁺-Cl⁻ cotransporter (NCC), and abundance of their regulatory kinases Ste20/SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress response kinase-1 (OSR1) in renal cortex (A) and medulla (B) of rats fed K⁺-deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared with control 2% KCl-fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample are loaded in adjacent lanes for each animal (in cortex: 15, 7.5 μg for NKCC2, NKCC2pT96T101, and SPAK; 30, 15 μg for NCCpS71 and OSR1; 60 and 30 μg for NCC; and in medulla: 10 and 5 μg for NKCC2 and NKCC2pT96T101 and SPAK; and 6 and 3 μg for OSR1). #Anti-OSR1 antibody recognizes tubulin (39). Density values were normalized to mean density of control K⁺ groups (1.00). Values are displayed as dot plots over a bar at the mean value. *P < 0.05 vs. respective 2 K group control. C: immunoblots demonstrating the detection of SPAK in: cortex (30 μg) and medulla (15 μg) of rats fed with either control (C) or low-salt (LS) diet, in mouse testis homogenate (1 μg), in wild-type (WT), and SPAK knockout [KO (14)] total kidney homogenates (10 μg); FL-SPAK, full-length SPAK; KS-SPAK, kidney-specific SPAK.

Fig. 3.

Total abundance of ENaC subunits in renal cortex (A) and medulla (B) of rats fed K⁺-deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared with 2% KCl-fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample are loaded in adjacent lanes for each animal: 60 and 30 μg for cortex α-ENaC; 30 and 15 μg for cortex β-ENaC, (cortex γ-ENaC signal too low to quantitate); 34 and 17 μg for medulla α-, β-, and γ-ENaC. Density values were normalized to mean density of control K⁺ groups (1.00). Values are displayed as dot plots over a bar at the mean value. *P < 0.05 vs. respective 2 K group control. C: immunoblots demonstrating the detection of ENaC subunits in the cortex and medulla of rats fed control (C) vs. low-salt (LS) diets (60 μg of kidney homogenate/lane).

Fig. 4.

Total abundance of renal outer medullary K⁺ channel (ROMK), Src family protein tyrosine kinase (c-Src), and clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH) in renal cortex (A) and medulla (B) of rats fed K⁺-deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared with control 2% KCl-fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample are loaded in adjacent lanes for each animal: in cortex: 30 and 15 μg for ROMK and c-Src; 5 and 2.5 μg for ARH; in medulla: 34 and 17 μg for ROMK; 15 and 7.5 μg for c-Src; 6 and 3 μg for ARH; Density values were normalized to mean density of control K⁺ groups (1.00). Values are displayed as dot plots over a bar at the mean value.

Fig. 5.

Total abundance of water channel aquaporin-2 (AQP2) in renal cortex (A) and medulla (B) of rats fed K⁺-deficient diets with either 0.74% NaCl (0K1Na) (n = 6)- or 2% NaCl (0K2Na) (n = 5)-fed, compared with control 2% KCl-fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample are loaded in adjacent lanes for each animal: 75 and 37.5 μg for cortex and 34 and 17 μg for medulla. Both 37-kDa and 29-kDa bands were quantified. Density values were normalized to mean density of control K⁺ groups (1.00). Values are displayed as dot plots over a bar at the mean value. *P < 0.05 vs. respective 2 K group control.

Fig. 6.

Total abundance of Na⁺-K⁺-ATPase (NKA) α₁- and β₁-subunits in renal cortex (A) and medulla (B) and cortex renin (A) of rats fed K⁺-deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared with control 2% KCl-fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample are loaded in adjacent lanes for each animal: 1.0 and 0.5 μg for NKAα₁; 10 and 5 μg for NKAβ₁; and 60 and 30 μg for renin. For renin, all the bands in the box were quantified together. Density values were normalized to mean density of control K⁺ groups (1.00). Values are displayed as dot plots over a bar at the mean value. *P < 0.05 vs. respective 2 K group control.

Fig. 7.

Subcellular distribution of NCC, ROMK, and AQP2 determined by differential centrifugation of 2K1Na and 0K1Na homogenates (n = 3 each) at 17,000 g to pellet-enriched plasma membranes (PM) and centrifugation of the 17,000 g supernatants at 150,000 g to pellet-enriched intracellular membranes (ICM) (43). Equal protein amounts of PM and ICM, as indicated, were analyzed by immunoblot, and the results illustrate significant differences in the PM-to-ICM ratio. Arrows indicate specific bands of interest.

Fig. 8.

Schematic representation of transporter and regulator distributions along the rat nephron. PT, proximal tubule; Thin DL, thin descending limb; Thin AL, thin ascending limb; TALH, thick ascending limb of Henle's loop; MD, macula densa; DCT, distal convoluted tubule; CNT, connecting tubule; CD, collecting duct; gHKA, gastric H⁺-K⁺-ATPase; cHKA, colonic H⁺-K⁺-ATPase (16); BK, large conductance, calcium activated K⁺ channel (18); SPAK (39); renin (35); ENaC, epithelial Na⁺ channel; ROMK (13); ARH (9); c-Src (26); NKA, Na⁺-K⁺-ATPase; OSR1 (39).