Expression of the kidney anion exchanger 1 affects WNK4 and SPAK phosphorylation and results in claudin-4 phosphorylation

Abstract

In the renal collecting ducts, chloride reabsorption occurs through both transcellular and paracellular pathways. Recent literature highlights a functional interplay between both pathways. We recently showed that in polarized inner medullary collecting duct cells, expression of the basolateral kidney anion exchanger 1 (kAE1) results in a decreased transepithelial electrical resistance (TEER), in a claudin-4 dependent pathway. Claudin-4 is a paracellular sodium blocker and chloride pore. Here, we show that kAE1 expression in mouse inner medullary collecting duct cells triggers WNK4, SPAK and claudin-4 phosphorylation. Expression of a functionally dead kAE1 E681Q mutant has no effect on phosphorylation of these proteins. Expression of a catalytically inactive WNK4 D321A or chloride-insensitive WNK4 L319F mutant abolishes kAE1 effect on TEER, supporting a contribution of WNK4 to the process. We propose that variations of the cytosolic pH and chloride concentration upon kAE1 expression alter WNK4 kinase activity and tight junction properties.

Keywords: Blood pressure; Chloride conservation; Claudin; Collecting duct; Distal nephron; Epithelium; Intercalated cells; Kidney; Membrane protein; Paracellular proteins; Sodium conservation; Tight junctions; Transporters.

Fig. 1

Effect of expressing kAE1 WT or E681Q protein on claudin-4 phosphorylation A, Representative FeCl₃-free or -containing immunoblots showing claudin-4 phosphorylation in the presence or absence of kAE1 WT (top) or E681Q proteins (bottom). Samples from mIMCD3 cells expressing kAE1 WT or E681Q (+Dox) or not (-Dox) were run on 10 % acrylamide gels that either contain iron Cl⁻ or not (See material and Methods for further details). The claudin-4 bands were detected with rabbit anti-claudin-4 antibody and on the “iron gel” represent the non-phosphorylated fraction of claudin-4 in these lysates. B, quantification of phosphorylated claudin-4 normalized to total claudin-4. C, immunoblots showing the abundance of claudin-8 in kAE1 WT (top) or E681Q (bottom)-expressing cells. D, quantification of claudin-8 from immunoblots shown in C. E, immunoblots showing abundance of claudin-3 in kAE1 WT (top) or E681Q (bottom)-expressing cells. F, quantification of claudin-3 from immunoblots shown in E. Error bars correspond to means ± SEM, n = 4–6. Not significant (n.s.), **P < 0.01 using a Student's t-test. Normality of the data was verified using a Shapiro-Wilk test. Uncropped blots are provided in Supplementary Figures.

Fig. 2

mIMCD3 cells express endogenous WNK4, and kAE1 WT but not E681Q mutant protein expression increases WNK4 phosphorylation. A, C, Immunoblots using rabbit anti-total WNK4, rabbit anti-phospho (S1196) WNK4 or mouse anti β-Actin antibodies to detect total, phospho- WNK4 or β-Actin, in kAE1 WT- or E681Q-expressing mIMCD3 cells, respectively, in the presence or absence of kAE1 WT proteins. B, D, quantification of total and phospho-WNK4 normalized to β-actin. Error bars correspond to means ± SEM, n = 4. Not significant (n.s.), *P < 0.05, **P < 0.01 using a Student's t-test or a Welch t-test (phospho-WNK4 in WT cells). Normality of the data was verified using a Shapiro-Wilk test. Note that additional bands may appear on the actin blot, corresponding to detection of the proteins with antibodies incubated prior to the anti-actin antibody. Uncropped blots are provided in Supplementary Figures.

Fig. 3

kAE1 WT but not E681Q mutant protein expression increases SPAK/OSR1 phosphorylation A, C, Immunoblots using rabbit anti-total or phospho-SPAK/OSR1, or mouse anti β-Actin antibodies in kAE1 WT- (A) or E681Q-expressing (C) mIMCD3 cells, respectively, in the presence or absence of kAE1 WT proteins. Note that the immunoblot for housekeeping protein actin in panel C (bottom) is the same as in Fig. 2C (bottom). B, D, quantification of total or phospho-SPAK/OSR1 normalized to β-actin. Error bars correspond to means ± SEM, n = 3 to 8. Not significant (n.s.), *P < 0.05 using a Student's t-test. Normality of the data was verified using a Shapiro-Wilk test. Uncropped blots are provided in Supplementary Figures.

Fig. 4

Kinase dead WNK4 D321A or chloride insensitive WNK4 L319F abolish kAE1 effect on tight junctions. A, Immunoblot using mouse anti-HA antibody to detect both WNK4 WT or mutants and kAE1 protein in mIMCD3 cells inducibly expressing kAE1 and transiently transfected with cDNA encoding WNK4 WT, WNK4 D321A or WNK4 L319F. B, Percentage of transepithelial electrical resistance relative to mIMCD3 cells without Dox measured in cells expressing kAE1 (+ or – Dox) and transiently transfected with cDNA encoding WNK4 WT, WNK4 D321A or WNK4 L319F. C, P_Na/P_Cl ratio, P_Na and P_Cl measured for IMCD cells transfected with WNK4 WT. D, P_Na/P_Cl ratio, P_Na and P_Cl measured for IMCD cells transfected with WNK4 D321A. E, P_Na/P_Cl ratio, P_Na and P_Cl measured for IMCD cells transfected with WNK4 L319F. Error bars correspond to means ± SEM, n = 4–6. *P < 0.05, **P < 0.01, not significant (n.s.) using two-way ANOVA and a Fisher's LSD test. Uncropped blots are provided in Supplementary Figures.