Paracellular Cl- permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension

Abstract

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and K+ homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and K+ homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.

Fig. 1.

Regulated expression of WNK4 in MDCK II tet-off cell lines. Clones showing doxycycline-regulated expression of WT and mutant WNK4 were produced as described in Methods. (a) Immunoblot analysis of HA-tagged WT-WNK4, WNK4-E559K, and WNK4-Q562E. For each construct, independent clones (, , and 3) were cultured in the presence (+) or absence (-) of doxycycline. Samples were fractionated by SDS/PAGE, followed by immunoblotting for WNK4 with anti-HA antibody. HA-tagged WT and mutant WNK4 migrate as ≈150-kDa species. (b and c) Induction and localization of WT-WNK4 in MDCK II cells. dox, Doxycycline. Cells were cultured, stained for WNK4 with anti-HA antibody, and visualized by immunofluorescence microscopy (see Methods). WNK4 expression is low in the presence of doxycycline (b) and is induced in its absence (c). WNK4 localizes to points of cell-cell contact. WNK4-D318A, WNK4-Q562E, and WNK4-E559K each showed a similar staining pattern (data not shown). (d) Localization of ZO-1 and WNK4. Cells were induced and stained for ZO-1 (green) and WNK4 (red). comp, composite. Both proteins localize to cell-cell junctions. (e) As in d, WNK4 signal only. (f) As in d, ZO-1 signal only.

Fig. 2.

Expression of WT and PHAII-mutant WNK4 decreases TER. (a) TER was measured on indicated monolayers as described in Methods. Mean ± SEM from three different clones for each construct is shown. ^*, P < 0.0001 vs. matched uninduced monolayer; ^**, P < 0.0001 vs. matched uninduced monolayer and vs. uninduced WT-WNK4 monolayer. The mean TER in the uninduced state (+dox) is expressed as 100% and the induced state (-dox) is indicated as a percentage of this value. (b) Effect of PHAII-mutant WNK4-Q562E on flux of 3-kDa dextran. Monolayers were prepared in the presence (red circles) or absence (blue triangles) of doxycycline, fluorescent dextran was applied to the apical surface, and its appearance in the basolateral media was measured over time.

Fig. 3.

WT and PHAII-mutant WNK4 increase paracellular Cl^- permeability. Indicated monolayers were cultured in the presence or absence of doxycycline for 4 days; dilution potentials and TER were measured as described in Methods. For each construct, the mean ± SEM of measurements is shown. ^*, P < 0.0001 vs. matched uninduced monolayer; ^**, P < 0.0001 vs. matched uninduced monolayer and vs. induced WT-WNK4 monolayer; #, P < 0.002 vs. matched uninduced monolayer. (a) Dilution potentials. Expression of WT-WNK4 has a significant effect on the dilution potential, and the effect is larger with PHAII-mutant WNK4. (b) Relative permeability of Cl^- and Na⁺ (P_Cl/P_Na). P_Cl/P_Na of the indicated monolayers was determined. Wild-type and PHAII-mutant WNK4 markedly increases the ratio. (c) Absolute permeability for Na⁺ (P_Na) in cm/s. (d) Absolute permeability for Cl^- (P_Cl) in cm/s. WT-WNK4 markedly increases P_Cl but not P_Na. PHAII-mutant WNK4 has a larger effect. (e) WNK4 alters the paracellular permeability of preformed tight junctions. Twelve replicates of a clone containing PHAII-mutant WNK4 (WNK4-Q562E) were plated and allowed to form monolayers in the presence of doxycycline for 4 days. TER and dilution potentials (DP) were measured. Half the monolayers were then incubated for 2 more days in the presence of doxycycline, whereas the other half were incubated in the absence of doxycycline, thereby inducing WNK4-Q562E. Measurements were repeated. Data from individual wells are shown for TER measurements and mean ± SEM of dilution potentials is shown for each group. Induction of WNK4-Q562E markedly reduced the TER and altered the dilution potential of monolayers whose tight junctions have already assembled.

Fig. 4.

Effect of WNK4 on structure of tight junctions. (a-d) Transmission EM of WNK4-Q562E cells. Monolayers were grown in the presence and absence of doxycyline, and were subjected to transmission EM. The depth of tight junctions is unaffected by WNK4-Q562E induction. Similar results were seen for WT-WNK4 (data not shown). ZA, zonula adherens; TJ, tight junctions (zonula occludens). (e and f) Freeze-fracture EM of WNK4-Q562E cells. Monolayers were treated as above and subjected to freeze-fracture EM as described in Methods. The number and complexity of tight-junction strands (indicated by arrows) are unchanged by induction of WNK4-Q562E. Similar results were seen for WT-WNK4 (data not shown). WNK4 does not impart its effect on paracellular permeability by eliciting gross structural changes on the tight-junction complex.