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. 2004 Feb 17;101(7):2064-9.
doi: 10.1073/pnas.0308434100. Epub 2004 Feb 9.

WNK4 regulates apical and basolateral Cl- flux in extrarenal epithelia

Kristopher T Kahle  1 , Ignacio Gimenez, Hatim Hassan, Frederick H Wilson, Robert D Wong, Biff Forbush, Peter S Aronson, Richard P Lifton
Affiliations

WNK4 regulates apical and basolateral Cl- flux in extrarenal epithelia

Kristopher T Kahle et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the serine-threonine kinase WNK4 [with no lysine (K) 4] cause pseudohypoaldosteronism type II, a Mendelian disease featuring hypertension with hyperkalemia. In the kidney, WNK4 regulates the balance between NaCl reabsorption and K(+) secretion via variable inhibition of the thiazide-sensistive NaCl cotransporter and the K(+) channel ROMK. We now demonstrate expression of WNK4 mRNA and protein outside the kidney. In extrarenal tissues, WNK4 is found almost exclusively in polarized epithelia, variably associating with tight junctions, lateral membranes, and cytoplasm. Epithelia expressing WNK4 include sweat ducts, colonic crypts, pancreatic ducts, bile ducts, and epididymis. WNK4 is also expressed in the specialized endothelium of the blood-brain barrier. These epithelia and endothelium all play important roles in Cl(-) transport. Because WNK4 is known to regulate renal Cl(-) handling, we tested WNK4's effect on the activity of mediators of epithelial Cl(-) flux whose extrarenal expression overlaps with WNK4. WNK4 proved to be a potent inhibitor of the activity of both the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the Cl(-)/base exchanger SLC26A6 (CFEX) (>95% inhibition of NKCC1-mediated (86)Rb influx, P < 0.001; >80% inhibition of CFEX-mediated [(14)C] formate uptake, P < 0.001), mediators of Cl(-) flux across basolateral and apical membranes, respectively. In contrast, WNK4 showed no inhibition of pendrin, a related Cl(-)/base exchanger. These findings indicate a general role for WNK4 in the regulation of electrolyte flux in diverse epithelia. Moreover, they reveal that WNK4 regulates the activities of a diverse group of structurally unrelated ion channels, cotransporters, and exchangers.

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Figures

Fig. 1.
Fig. 1.
Expression of WNK4 in extrarenal tissues. (a) RT-PCR analysis of WNK4. cDNA from indicated mouse tissues was used as a template for PCR by using primers specific for WNK4 (Upper) and GAPDH (Lower) mRNA, and the products were fractionated on agarose gels. (b) Western blot analysis of WNK4. Mouse or human tissue lysates were fractionated by SDS/PAGE and subjected to Western blot analysis with anti-WNK4. A protein of ≈135 kDa, the expected size of WNK4 (2), is not exclusively expressed in kidney, but is also present in brain, testis, colon, heart, liver, prostate, and lung. All lysates are from mouse except breast, ovary, colon, melanocytes (melano.), and prostate.
Fig. 2.
Fig. 2.
Immunolocalization of WNK4 in kidney, pancreas, liver, and epididymis. Frozen sections of mouse kidney, pancreas, liver, and epididymis were stained with anti-WNK4 (red) and anti-ZO-1 (green) and analyzed by immunofluorescence microscopy. Composite images and views for WNK4 and ZO-1 alone are shown. (a) A renal tubule in longitudinal section is shown; WNK4 is confined to tubule epithelium and in this nephron segment largely colocalizes with the TJ protein ZO-1. (b) A large pancreatic duct in longitudinal section is shown; WNK4 expression is confined to pancreatic duct epithelia and largely overlaps with ZO-1. (c) A bile duct in cross section demonstrates that WNK4 localizes to epithelial cells that line the biliary tract lumen (L). WNK4 localization largely overlaps with ZO-1. (d) Image of the epididymis in cross section demonstrates WNK4 expression in the columnar epithelial cells lining the epididymal lumen (L) as well as more basal layers. WNK4 becomes more tightly membrane associated with apical progression. (Magnifications: ×600.)
Fig. 3.
Fig. 3.
Expression of WNK4 in brain, colon, and skin. Frozen sections of mouse brain, human colon, and human skin were stained with anti-WNK4 (red), ZO-1 (green in a), CFTR (green in b and c), or 4′,6-diamidino-2-phenylindole (DAPI) (blue) and analyzed by immunofluorescence light microscopy. (a) Section of brain reveals WNK4 expression is highest in endothelial cells that comprise the blood–brain barrier and overlaps with ZO-1. (b) A low-power view of colonic crypts in cross section, demonstrating WNK4 localizes to the epithelium of crypts. CFTR staining highlights the crypt apical membrane. DAPI staining highlights nuclei. (c) A high-power view of a crypt in cross section showing WNK4, CFTR, and DAPI staining. (d) A longitudinal section of a crypt under high-power demonstrates the localization of WNK4 at intercellular junctions (arrow) in this epithelium. (e) High-power view of a sweat duct in cross section demonstrates that WNK4 is expressed in cuboidal epithelial cells lining sweat duct lumens (L). The arrow demonstrates WNK4's localization along the lateral membrane between adjacent epithelial cells. (Magnifications: ×600, a, c, and e; ×400, b; and ×1,000, d.)
Fig. 4.
Fig. 4.
WNK4 is a regulator of NKCC1. (a) Effect of WNK4 on 86Rb influx mediated by NKCC1. Oocytes were injected with water or cRNA encoding NKCC1 alone or in combination with cRNA encoding WNK4 as indicated; 86Rb influx was measured as described in Methods. Results of a representative experiment are shown; bar graphs represent means ± SE of 86Rb influx for each experimental group. (b) Effect of WNK4 on total cellular NKCC1 protein. A Western blot of whole oocyte lysates expressing NKCC1 in the presence or absence of WNK4 is shown. Samples were run in duplicate. The total amount of NKCC1 produced in injected oocytes is not altered by the presence of WNK4. (c and d) Effect of WNK4 on the surface expression of NKCC1. Representative examples of immunofluoresence microscopy of Xenopus ooctyes stained with anti-NKCC1 antibody. Oocytes were injected with cRNA encoding NKCC1 alone (b) or in combination with WNK4 (c) as described in Methods. (Magnifications: ×600.)
Fig. 5.
Fig. 5.
WNK4 is a regulator of CFEX, but not pendrin. (a) Effect of WNK4 on 14C formate uptake mediated by CFEX. Oocytes were injected with water or cRNA encoding CFEX alone or in combination with WNK4; 14C formate uptake was measured as described in Methods. Results of a representative experiment are shown. (b) Effect of WNK4 on 14C formate uptake mediated by pendrin. Oocytes were injected with water or cRNA encoding pendrin alone or in combination with WNK4 as indicated; 14C formate uptake was measured as described in Methods. Results of a representative experiment are shown.
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