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.

Differential regulation of Na+transporters along nephron during ANG II-dependent hypertension: distal stimulation counteracted by proximal inhibition

“…Kidney cortex and medulla were immediately dissected manually and separately diced and homogenized as described in detail previously (27), quick frozen in aliquots in liquid N 2; protein concentration was determined by BCA assay (Pierce Thermo, Rockford, IL). Cortical and medullary homogenates were denatured in SDS-PAGE sample buffer for 20 min at 60°C (27).…”

Intrarenal localization of the plasma membrane ATP channel pannexin1

“…Kidneys from three wild-type and three Panx1 Ϫ/Ϫ mice were perfused with cold PBS to remove blood, snap frozen in liquid nitrogen, and sent to us on dry ice from the laboratory of Prof. Eliana Scemes (Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY). Brain samples were taken from mice, and all tissues were processed and homogenized as described recently (21). Protein concentrations were determined using the Pierce BCA kit (Thermo Scientific), and 80 g of protein were then run on SDS-PAGE gels, transferred to polyvinylidene difluoride membranes (Immobilon-FL; Millipore, Temecula, CA), blocked (bløk-FL; Millipore), and then probed with chicken polyclonal antibodies to Panx1 at a dilution of 1:1,000 overnight.…”

The absence of intrarenal ACE protects against hypertension

“…However, Ang II infusion triggered an array of responses in wild-type mice consistent with increased sodium transport in the loop of Henle and the distal nephron: increased phosphorylation of loop of Henle NKCC2, increased abundance and phosphorylation of the distal convoluted tubule NCC, increased cleavage of ENaC α and γ subunits, and increased Transporter profiling and intrarenal RAS analysis. Whole mouse kidneys, flash frozen after removal, were thawed and homogenized, as described in detail elsewhere (55). Protein concentration was determined by BCA, and samples were denatured in SDS-PAGE sample buffer (20 minutes, 60°C), resolved by SDS-PAGE (56), transferred to polyvinylidene difluoride membranes (Millipore Immobilon-FL), blocked (Li-COR blocking buffer or bløk-FL, Millipore), and then probed with specific antibodies, as detailed in Supplemental Table 1.…”