High dietary K+ intake inhibits proximal tubule transport

Abstract

The impact of chronic dietary K⁺ loading on proximal tubule (PT) function was measured using free-flow micropuncture along with measurements of overall kidney function, including urine volume, glomerular filtration rate, and absolute and fractional Na⁺ and K⁺ excretion in the rat. Feeding animals a diet with 5% KCl [high K⁺ (HK)] for 7 days reduced glomerular filtration rate by 29%, increased urine volume by 77%, and increased absolute K⁺ excretion by 202% compared with rats on a 1% KCl [control K⁺ (CK)] diet. HK did not change absolute Na⁺ excretion but significantly increased fraction excretion of Na⁺ (1.40% vs. 0.64%), indicating that fractional Na⁺ absorption is reduced by HK. PT reabsorption was assessed using free-flow micropuncture in anesthetized animals. At 80% of the accessible length of the PT, measurements of inulin concentration indicated volume reabsorption of 73% and 54% in CK and HK, respectively. At the same site, fractional PT Na⁺ reabsorption was 66% in CK animals and 37% in HK animals. Fractional PT K⁺ reabsorption was 66% in CK and 37% in HK. To assess the role of Na⁺/H⁺ exchanger isoform 3 (NHE3) in mediating these changes, we measured NHE3 protein expression in total kidney microsomes as well as surface membranes using Western blots. We found no significant changes in protein in either cell fraction. Expression of the Ser⁵⁵² phosphorylated form of NHE3 was also similar in CK and HK animals. Reduction in PT transport may facilitate K⁺ excretion and help balance Na⁺ excretion by shifting Na⁺ reabsorption from K⁺-reabsorbing to K⁺-secreting nephron segments.NEW & NOTEWORTHY In rats fed a diet rich in K⁺, proximal tubules reabsorbed less fluid, Na⁺, and K⁺ compared with those in animals on a control diet. Glomerular filtration rates also decreased, probably due to glomerulotubular feedback. These reductions may help to maintain balance of the two ions simultaneously by shifting Na⁺ reabsorption to K⁺-secreting nephron segments.

Keywords: Na+ reabsorption; Na+/H+ exchanger isoform 3; dietary K+; phospho-Ser552 Na+/H+ exchanger isoform 3.

Graphical abstract

Figure 1.

Effects of high K⁺ (HK) intake on urine volume flow (UV), glomerular filtration rate (GFR), and fractional Na⁺ and K⁺ excretion (FENa and FEK, respectively). Each point shows the value of one urine collection from six rats in each group (Table 2). Statistical significance between the control K⁺ (CK)- and HK-treated groups was assessed using unpaired t tests.

Figure 2.

Effects of high K⁺ (HK) intake on fluid absorption in rat proximal tubules. Animals were fed with either control K⁺ (CK; 1% KCl) or HK (5% KCl) for 7 days before the experiment. The ratio of inulin concentration (In) in the tubular fluid to that in plasma (In TF/P) was measured at 20%, 60%, and 80% of the accessible length of the proximal tubule from the glomerulus. Each point shows the value of one tubular fluid collection from 3 to 5 rats in each group (Table 3). *Significant difference compared with values at 20% of proximal tubule length in the CK- or HK-treated group (P < 0.05 by one-way ANOVA test). Values at 80% were statistically different (P < 0.05 by one-way ANOVA) between CK- and HK-treated groups.

Figure 3.

Effects of high K⁺ (HK) intake on Na⁺ absorption in rat proximal tubules. Animals were fed with either control K⁺ (CK; 1% KCl) or HK (5% KCl) for 7 days before the experiment. Na/In TF/P was measured at 20%, 60%, and 80% of the accessible length of the proximal tubule from the glomerulus. Each point shows the value of one tubular fluid collection from 3 to 5 rats in each group (Table 3). *Significant difference compared with values at 20% of proximal tubule length in the CK- or HK-treated group (P < 0.05 by one-way ANOVA test). Values at 80% were statistically different (P < 0.05 by one-way ANOVA) between CK- and HK-treated groups. In TF/P, ratio of inulin concentration in the tubular fluid to that in plasma; Na/In TF/P, ratio of Na⁺ concentration in the tubular fluid to that in plasma divided by In TF/P.

Figure 4.

Effects of high K⁺ (HK) intake on K⁺ absorption in rat proximal tubules. Animals were fed with either control K⁺ (CK; 1% KCl) or HK (5% KCl) for 7 days before the experiment. K/In TF/P was measured at 20%, 60%, and 80% of the accessible length of the proximal tubule from the glomerulus. Each point shows the value of one tubular fluid collection from 3 to 5 rats in each group (Table 3). *Significant difference compared with values at 20% of proximal tubule length in the CK- or HK-treated group (P < 0.05 by one-way ANOVA test). Values at 80% were statistically different (P < 0.05 by one-way ANOVA) between CK- and HK-treated groups. In TF/P, ratio of inulin concentration in the tubular fluid to that in plasma; K/In TF/P, ratio of K⁺ concentration in the tubular fluid to that in plasma divided by In TF/P.

Figure 5.

Effects of high K⁺ intake on total expression and surface expression of Na⁺/H⁺ exchanger isoform 3 (NHE3) protein in the rat kidney. Animals were fed with either control K⁺ (1% KCl) or high K⁺ (5% KCl) for 7 days. Kidneys were homogenized, and microsomes were prepared. A: 15 µg of microsome protein were loaded onto polyacrylamide gels, proteins were separated by electrophoresis, and gels were stained using antibody to NHE3 protein. Each lane was loaded with microsomes from a different animal. The bottom blot shows band density as a function of the amount of protein loaded. B: biotinylated proteins were isolated using Neutravidin beads. Eluates from 660 µg microsome protein were loaded onto polyacrylamide gels, proteins were separated by electrophoresis, and gels were stained using antibody to NHE3 protein. Each lane was loaded with eluate from a different animal. C: band densities were measured and normalized to the mean density of control K⁺-treated samples. Bars represent means ± SE for four pairs of animals.

Figure 6.

Effects of high K⁺ intake on total expression and surface expression of γ-epithelial Na⁺ channel (γENaC) protein in the rat kidney. Animals were fed with either control K⁺ (1% KCl) or high K⁺ (5% KCl) for 7 days. Kidneys were homogenized, and microsomes were prepared. A: 40 µg of microsome protein were loaded onto polyacrylamide gels, proteins were separated by electrophoresis, and gels were stained using antibody to γENaC protein. Each lane was loaded with microsomes from a different animal. B: biotinylated proteins were isolated using Neutravidin beads. Eluates from 1.1 mg microsome protein were loaded onto polyacrylamide gels, proteins were separated by electrophoresis, and gels were stained using antibody to γENaC protein. Each lane was loaded with eluate from a different animal. C: band densities were measured and normalized to the mean density of control K⁺-treated samples. Bars represent means ± SE for four pairs of animals.

Figure 7.

Effects of high K⁺ intake on total expression and surface expression of phosphorylated Ser552 (pS552) Na⁺/H⁺ exchanger isoform 3 (NHE3) protein in the rat kidney. Animals were fed with either control K⁺ (1% KCl) or high K⁺ (5% KCl) for 7 days. Kidneys were homogenized, and microsomes were prepared. A: 40 µg of microsome protein were loaded onto polyacrylamide gels, proteins were separated by electrophoresis, and gels were stained using antibody to pS552 NHE3 protein. Each lane was loaded with microsomes from a different animal. B: biotinylated proteins were isolated using Neutravidin beads. Eluates from 820 µg microsome protein were loaded onto polyacrylamide gels, proteins were separated by electrophoresis, and gels were stained using antibody to pS552 NHE3 protein. Each lane was loaded with eluate from a different animal. C: band densities were measured and normalized to the mean density of control K⁺-treated samples. Bars represent means ± SE for four pairs of animals.

Figure 8.

Effects of high K⁺ intake on protein expression. Animals were fed with either control K⁺ (1% KCl) or high K⁺ (5% KCl) for 7 days. Kidneys were homogenized, and microsomes were prepared. Equal amounts of protein from an individual animal were loaded onto each lane of polyacrylamide gels [Na⁺-glucose transporter 2 (SGLT2): 7 µg, claudin-2 (Cldn2): 35 µg, electrogenic Na⁺-bicarbonate cotransporter 1 (NBCe1): 35 µg, claudin-10 (Cldn10) 7 µg, and phosphoenolpyruvate carboxykinase 1 (PEPCK): 30 µg]. Samples were separated by electrophoresis, and blots were stained using antibodies to the indicated protein. Band densities were measured and normalized to the mean density of control K⁺-treated samples, as indicated by the numbers above each blot indicating means ± SE for four pairs of animals. *Statistically significant difference (P < 0.05 by t test) between high K⁺- and control K⁺-treated animals.