Abstract. Ankyrins are a family of large, membraneassociated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain

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.protein serine-threonine kinases ͉ ion transport

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.

Abstract. In nonerythroid cells the distribution of the cortical membrane skeleton composed of fodrin (spectrin), actin, and other proteins varies both temporally with cell development and spatially within the cell and on the membrane. In monolayers of Madin-Darby canine kidney (MDCK) cells, it has previously been shown that fodrin and Na,K-ATPase are codistributed asymmetrically at the basolateral margins of the cell, and that the distribution of fodrin appears to be regulated posttranslationally when confluence is achieved (Nelson, W. J., and P. I. Veshnock. 1987. J. Cell Biol. 104:1527-1537. The molecular mechanisms underlying these changes are poorly understood. We find that (a) in confluent MDCK cells and intact kidney proximal tubule cells, Na,K-ATPase, fodrin, and analogues of human erythrocyte ankyrin are precisely colocalized in the basolateral domain at the ultrastructural level. (b) This colocalization is only achieved in MDCK cells after confluence is attained. (c) Erythrocyte ankyrin binds saturably to Na,K-ATPase in a molar ratio of ",d ankyrin to 4 Na,K-ATPase's, with a kD of 2.6/~M. (d) The binding of ankyrin to Na,K-ATPase is inhibited by the 43-kD cytoplasmic domain of erythrocyte band 3. (e) ~25I-labeled ankyrin binds to the alpha subunit of Na,K-ATPase in vitro. There also appears to be a second minor membrane protein of • 240 kD that is associated with both erythrocyte and kidney membranes that binds ~25I-labeled ankyrin avidly. The precise identity of this component is unknown. These results identify a molecular mechanism in the renal epithelial cell that may account for the polarized distribution of the fodrin-based cortical cytoskeleton.

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