KCNJ10 determines the expression of the apical Na-Cl cotransporter (NCC) in the early distal convoluted tubule (DCT1)

Abstract

The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-Cl cotransporter (NCC) in the DCT. Immunostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj10(+/+) mice (WT). This 40-pS K channel is absent in homozygous Kcnj10(-/-) (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism.

Keywords: Kir.5.1; SPAK; SeSAME/EAST syndrome; WNK.

Fig. 1.

Kcnj10 is expressed in the basolateral membrane of the DCT. The experiments were performed in the kidney slice obtained from a 4-wk-old WT mouse. (A) Kcnj10 immunostaining in the mouse kidney with low magnification. (B) Parvalbumin immunostaining in the mouse kidney with low magnification. Fluorescence microscope image showing the merged staining of Kcnj10 (red) and parvalbumin (green) with the low magnification (C) and a high magnification (D).

Fig. 2.

Kcnj10 forms a 40-pS K channel in the basolateral membrane of DCT1. (A) A patch-clamp recording shows the 40-pS K channel activity in the basolateral membrane of DCT1. The experiments were performed in a cell-attached patch of DCT1 in the p8 WT mice. The DCT was bathed in a 140 mM NaCl+5 mM KCl solution, and the pipette solution contains 140 mM KCl. The holding potentials are indicated on the top of each trace, and the channel closed levels are labeled by “C.” (B) (Upper) A whole-cell recording showing the Ba²⁺-sensitive K currents and NPPB-sensitive Cl currents in a DCT1 cell of p8 WT mouse. Ba²⁺ (1 mM) and NPPB (10 μΜ) were directly added to the bath. (Lower) The bar graph summarizes the results from 10 measurements at −60 mV on DCT1 of p7/p10 mice. The K and Cl currents were measured with the perforated whole-cell recording with 140 mM KCl in the pipette and in the bath.

Fig. 3.

The disruption of Kir4.1 abolished the basolateral K conductance and caused a depolarization in DCT1. (A) (Left) A whole-cell recording showing Ba²⁺-sensitive K currents in DCT1 cells of p8/p10 WT, heterozygous (het), and knockout (KO) mice. (Right) A bar graph summarizes the results measured at −60 mV. The K currents were measured with perforated whole-cell recording with symmetrical 140 mM KCl in the bath and in the pipette. (B) (Left) A whole-cell recording showing K reversal potential in DCT1 cells of p8/p10 WT and knockout mice. (Right) The mean value of K reversal potential from eight measurements is summarized in a bar graph. For measurement of K reversal potential, the bath solution contains 140 mM NaCl+ 5 mM KCl while the pipette solution has 140 mM KCl.

Fig. 4.

The disruption of Kcnj10 affects the membrane expression of Kcnj16. Immunostaining of Kir5.1 (Kcnj16) (red) and parvalbumin (green) in p8 Kcnj10^+/+ (A and B) and p8 Kcnj10^−/− mice (C and D). A and B are merged images showing Kir5.1 and parvalbumin staining with low magnification while C and D show the double staining with a high magnification. Arrow indicates the intracellular staining of Kcnj16 in the knockout mice.

Fig. 5.

The disruption of Kcnj10 decreased Cl conductance and SPAK expression in DCT. (A) (Upper) A recording showing NPPB-sensitive Cl currents in DCT1 of WT and knockout mice. The measurements were carried out with perforated whole-cell recording with symmetrical 140 mM KCl in the bath and pipette. (Lower) The results from eight experiments measured at -60 mV are summarized in a bar graph. (B) (Upper) A Western blot showing the expression of full-length SPAK in the kidney from WT, heterozygous, and homozygous Kcnj10^−/− mice. (Lower) Results from four experiments are summarized in a bar graph.

Fig. 6.

The disruption of Kcnj10 inhibits NCC expression. Fluorescence microscope image with a low magnification shows overall NCC immunostaining in (A) p8 Kcnj10^+/+ and (B) p8 Kcnj10^−/− mice. (C) A Western blot showing the NCC expression in Kcnj10^+/+ and Kcnj10^−/− mice. (D) A bar graph summarizes results of experiments similar as those shown in C.

Fig. 7.

NCC expression is decreased in heterozygous and knockout mice. (A) (Upper) A Western blot showing the NCC expression in WT, heterozygous, and Kcnj10^−/− mice. (Lower) Results from four experiments are summarized in a bar graph. Double immunostaining images show NCC expression (red) in parvalbumin-positive (green) DCT cell of p8 WT (B), p8 heterozygous (C), and p8 homozygous Kcnj10^−/− mice (D and E).

Fig. 8.

A cell scheme illustrating the mechanism by which the basolateral K channel activity regulates the apical NCC expression in the DCT1. The solid and dotted lines, respectively, mean an enhanced or a diminished signaling.