Splicing a Kinase and the Regulation of Salt Transport
Authors: David B. Mount
Journal: Journal of the American Society of Nephrology (2009)

Thiazide-sensitive Na+-Cl− co-transport by the aldosterone-sensitive distal convoluted tubule (DCT) is responsible for reabsorption of approximately 10% of filtered Na+ and Cl−.1 The considerable clinical importance of Na+ and Cl− reabsorption by the DCT is illustrated by the therapeutic efficacy of thiazide diuretics and the clinical phenotype of Gitelman syndrome,2 caused almost exclusively by loss-of-function mutations in the thiazide-sensitive Na+-Cl− co-transporter (NCC). A gain-in-function of NCC occurs in pseudohypoaldosteronism type II (PHA-II; also known as Gordon syndrome or hereditary hypertension with hyperkalemia); however, this disorder is caused by mutations not in NCC but in two of the four WNK (with no K/lysine) kinases, so-named for the absence of a conserved catalytic lysine.3,4 Since the seminal genetic study implicating WNK1 and WNK4 in PHA-II,5 an increasingly populous signaling complex has been uncovered, encompassing WNK1, WNK3, WNK4 and the downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1).3,4 WNK1,5 WNK3,6 WNK4,5 and SPAK7 all are coexpressed with NCC in the DCT. Elements of this pathway also regulate the Cl− and volume sensitivity of all functional members of the cation-chloride co-transporter gene family, including NCC, the two Na+-K+-2Cl− co-transporters (NKCC1 and NKCC2), and the four K+-Cl− co-transporters (KCC1 through 4).3,4 More recently, there are outlines of the interactions of this pathway with aldosterone8 and angiotensin-dependent9 regulation of NCC. Variation in the human STK39 gene encoding SPAK is also associated with differences in BP.7 Much of the work on WNK pathways exploits the coexpression of kinases with NCC in nonmammalian cells, specifically Xenopus laevis oocytes. Notably, however, key observations in this system predict the cellular and physiologic phenotype of WNK4 transgenic mice with overexpression of wild-type WNK4 and a disease-associated WNK4 mutation,10 such that insights obtained are physiologically relevant. There still are a number of controversies and discrepancies in the reported data3,4,11; however, a consensus of results indicates that WNK4 coexpression inhibits NCC, with abrogation of this effect by PHA-II–associated mutations. WNK1 in turn has no effect on NCC but abolishes the inhibitory effect of WNK4. WNK3 coexpression activates NCC3,4,11; a recent chimeric study localized the differential effect of WNK3 and WNK4 to the N-terminal 300 to 400 or so amino acids of the two kinases.11 WNK-dependent activation of SPAK or OSR1 leads to phosphorylation of a cluster of N-terminal threonines in NCC, NKCC1, and NKCC2, resulting in the activation of Na+-(K+)-Cl− co-transport.3,4 In this issue of JASN, Glover et al.12 report an interesting twist in this intriguing story with their finding of dramatic differences in the effect of renal (R-WNK3) and brain (B-WNK3) alternatively spliced isoforms, again using coexpression with NCC in Xenopus oocytes. The two isoforms of this kinase differ in inclusion of two alternative versions of exon 18 and in the presence or absence of the 11-codon cassette from exon 22 (see Figure 1 of Glover et al.12); this C-terminal variation thus affects only a tiny fraction of the approximately 1800 residues in the kinase. Despite this modest difference in primary structure, R-WNK3 strongly activates the transporter and B-WNK3 strongly inhibits. Both kinases affect expression of the transporter at the membrane; however, whereas the effect of B-WNK3 seems to be dependent on SPAK, that of R-WNK3 is not. The effect of WNK3 and WNK4 on trafficking of the transporter does not require N-terminal phosphorylation of NCC, suggesting that SPAK-dependent phosphorylation increases intrinsic activity at the membrane. Despite its many advantages, the use of Xenopus oocytes for these studies has acknowledged limitations.4 In particular, overexpressed kinases may exert effects by sequestering, binding, or otherwise interfering with the endogenous signaling pathways of the oocyte, in this case the Xenopus laevis SPAK and WNK. Ideally, studies of this pathway should use a combination of heterologous expression and cellular models of the DCT.9 Another concern is the failure to detect co-association of SPAK with WNK3 and NCC12; all of the WNK kinases contain a specific interaction motif for binding to SPAK, as does NCC.4 Why, then, is this new study12 of interest, particularly if the brain WNK3 isoform is not expressed in kidney and thus not involved in the regulation of NCC? First, as noted by Glover et al.,12 their study emphasizes the need to consider not just tissue-specific expression but also tissue-specific alternative splicing when studying physiologic phenomena in a heterologous expression system. Second, this report is perhaps the first clean separation of SPAK-dependent and SPAK-independent effects of a WNK kinase on NCC. Undoubtedly, further complexity will follow, particularly when one considers the redundant, overlapping regulation of the epithelial sodium channel as a model for the regulation of distal salt transport.1 DISCLOSURES None. The author acknowledges support by National Institutes of Health grants (DK57708 and DK070756), an American Heart Association Grant-in-Aid, and the Veterans Administration.