PMC

Analysis of the relationship between plasma [K⁺] and NCC, NCCpS71, and NCCpT53 abundance in rats fed either 0%K or 2%K meals for 3 h. Data are from and .

Effects of a 3-h 2%K vs. 0%K meal on plasma and urinary electrolytes. Rats were fasted overnight and then fed either 2%K diet (15.2 ± 2.5 g consumed) or 0%K diet (14.4 ± 1.2 g consumed) in metabolic cages before death. A: individual plasma [K⁺] and [Na⁺] values, means ± SE; n = 8 per group. B: individual urinary electrolytes and volume measured in urine collected in metabolic cage + bladder urine, individual values, and means ± SE; n = 6 per group. *P < 0.05.

Time course of change in urinary Na⁺ (UNaV) and K⁺ (UKV) after a meal. Rats were fasted overnight (urine collected) and then fed a 2% potassium containing meal for 3 h. Urine was collected over 2-h intervals for 12 h. Urinary K⁺ excretion ([K⁺] × volume) and Na⁺ excretion ([Na⁺] × volume) measured by flame photometry indicated as means ± SE (n = 8). *P < 0.05.

Effects of consuming a 2%K meal vs. KCl infusion on plasma aldosterone and abundance of the aldosterone early response gene SGK1 in renal cortex. A: plasma aldosterone, measured by RIA in samples taken at death from rats fed 0%K vs. 2%K diets for 3 h (n = 8; left), and measured in uninfused, NaCl infused and KCl infused for 3 h (n = 5; right). *P < 0.01. B: immunoblots of renal cortical homogenates were run at 40 and 80 μg/lane (shown is 80 μg/lane) for SGK1 and ENaC α subunit (FL, full length; CL, cleaved). Samples from rats treated chronically with angiotensin II (AII; Ref. ) run as control. Density values were normalized to mean density of the control group (defined as 1.00) displayed under respective blots as means ± SE.

Effects of raising plasma [K⁺] to 5.5 mM by tail vein infusion on plasma [K⁺] and urinary electrolyte excretion. Rats were fasted overnight and then infused with either 150 mM KCl plus a small amount of normal saline at variable rates to raise plasma [K⁺] to 5.5 mM, mimicking the changes observed after feeding 2%K diet. Controls were infused with normal saline (NaCl infused) alone matched to the volume of saline infused in the KCl infused group, or uninfused over a period of 3 h. All groups had no food and free access to drinking water (n = 4–5/group). A: plasma [K⁺] values in rats infused with KCl, NaCl, or uninfused. B: effects of raising plasma [K⁺] on urinary electrolytes in conscious rats, n = 5 per group. *P < 0.01 vs. NaCl infused group.

Effect of 2%K vs. 0%K meal on Na⁺-Cl⁻ cotransporter (NCC), Na⁺-K⁺−2Cl⁻ cotransporter (NKCC), and STE20/SPS1-related proline alanine-rich kinase (SPAK) in renal cortex homogenates. A: immunoblots of renal cortex homogenates. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample were loaded for each animal (only one amount shown): 30, 60 μg for NCC total and NCCpS71; 40, 80 μg for NCCpT53; 10, 20 μg for NKCC total and NKCCpT96T101; 10, 20 μg for SPAK-full length (SPAK-FL); and 40, 80 μg for SPAKpS373 and OSR1pS325 (detected with the same antiserum). OSR1 was not detected at levels sufficient for quantitation. Density values, normalized to mean density of 0%K group defined as 1.00, are displayed under respective blots as means ± SE. KO indicates homogenate from a SPAK knockout mouse kidney 80 μg. B: individual normalized values for NCC and NKCC are displayed with means. *P < 0.01 vs. 0%K group.

Effects of raising plasma [K⁺] to 5.5 mM by tail vein infusion on NCC, NKCC and SPAK abundance and phosphorylation in renal cortex homogenates. A: immunoblots of renal cortex homogenate samples. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample were loaded for each animal (only one amount shown): 30, 60 μg for NCC total and NCCpT53; 15, 30 μg for NCCpS71; 40, 80 μg for NCCpS89; 10, 20 μg for NKCC total and NKCCpT96T101; 10, 20 μg for SPAK total; and 40, 80 μg for SPAKpS373 and OSR1pS325 (detected with the same antiserum). Density values were normalized to mean density of the NaCl infused group (defined as 1.00) displayed under respective blots as means ± SE. Kidney samples from wild-type (WT) and SPAK KO mice were run as controls. B: individual normalized values for NCC and SPAK displayed with means. *P < 0.01 vs. NaCl-infused group.

Effect of 2%K vs. 0%K meal on proximal tubule NHE3, cortical collecting duct renal outer medullary K⁺ channel (ROMK) and ENaC abundance in renal cortex homogenates. To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample were loaded for each animal (only one amount shown): 7.5, 15 μg for NHE3 and NHE3pS552; 30, 60 μg for ROMK and ENaC-β; and 40, 80 μg for ENaC-α. Density values, normalized to mean density of 0%K group defined as 1.00, are displayed under respective blots as means ± SE. Arrows indicate core glycosylated ROMK (core) and putative fully glycosylated ROMK (full glycos) based on expected mobility. LS indicates a sample from a rat fed nominally Na⁺ free diet for 1 wk to increase ENaC expression and cleavage.

Effect of 2%K vs. 0%K meal on subcellular distribution of NCC and NCC-p. The fraction of NCC total and NCCpS71 in plasma membranes (PM) vs. intracellular membranes (ICM; n = 8) was assessed after a differential fractionation protocol that enriches for PM vs. ICM (see methods). Samples were run at a constant amount of protein/lane (6 μg) of PM and ICM. Although they are shown as distinct panels, samples of PM and ICM from both 0%K and 2%K groups were run and transferred to the same piece of PVDF so that they could be probed and compared directly to each other. The percentage of transporters in PM vs. ICM was calculated from the signal intensity corrected for the recovery of PM and ICM membrane protein in each sample (average recovery from 34.4 ± 1.2 mg starting homogenate: 9.3 ± 1.3 mg of PM and 2.8 ± 0.8 mg of ICM), and expressed as percentage of transporter abundance in total kidney homogenates. Using this method, >90% of the NCCpS71 was localized to PM, confirming previous immunohistochemistry findings and validating this approach. After the 2%K meal, ∼5% of the NCC total redistributed from PM to ICM. Values are means ± SE. *P < 0.05 vs. 0%K group.