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【关键词】 kidney-specific
1Division of Nephrology and Hypertension, Department of Medicine,
2Heart Research Center
3Department of Physiology and Pharmacology, Oregon Health and Science University
4Portland Veterans Affairs Medical Center, Portland, Oregon
ABSTRACT
With-no-lysine kinase-1 (WNK1) gene mutations cause familial hyperkalemic hypertension (FHHt), a Mendelian disorder of excessive renal Na+ and K+ retention. Through its catalytic activity, full-length kinase-sufficient WNK1 (L-WNK1) suppresses its paralog, WNK4, thereby upregulating thiazide-sensitive Na-Cl cotransporter (NCC) activity. The predominant renal WNK1 isoform, KS-WNK1, expressed exclusively and at high levels in distal nephron, is a shorter kinase-defective product; the function of KS-WNK1 must therefore be kinase independent. Here, we report a novel role for KS-WNK1 as a dominant-negative regulator of L-WNK1. Na+ transport studies in Xenopus laevis oocytes demonstrate that KS-WNK1 downregulates NCC activity indirectly, by inhibiting L-WNK1. KS-WNK1 also associates with L-WNK1 in protein complexes in oocytes and attenuates L-WNK1 kinase activity in vitro. These observations suggest that KS-WNK1 plays an essential role in the renal molecular switch regulating Na+ and K+ balance; they provide insight into the kidney-specific phenotype of FHHt.
distal nephron; thiazide-sensitive sodium-chloride cotransporter; with-no-lysine kinases; aldosterone
WNKS (WITH-NO-LYSINE [K]) comprise a novel group of serine/threonine protein kinases that are distinct from all other members of the protein kinase superfamily (19, 21, 22). Mutations in WNK1 and WNK4 cause familial hyperkalemic hypertension (FHHt; also known as pseudohypoaldosteronism type 2 or Gordon's syndrome) (19), an autosomal dominant disorder that results from excessive renal Na+ absorption with chloride, leading to K+ retention and hypertension (2, 4, 8). WNK1 and WNK4 converge in a novel signaling pathway that regulates renal ion transport (5, 12, 17, 26, 27). Expression studies using Xenopus laevis oocytes showed that WNK4 inhibits the thiazide-sensitive Na-Cl cotransporter (NCC; gene symbol SLC12A3) (20, 2628). Full-length kinase-sufficient WNK1 ["Long" WNK1 (L-WNK1); also referred to in previous publications as "WNK1"] has no direct effect on NCC but instead suppresses WNK4-mediated NCC inhibition, thereby restoring cotransporter activity to near-baseline levels. This suppressive effect is dependent on intact L-WNK1 kinase activity (26, 27).
The WNK1 gene (PRKWNK1) is widely expressed, but in the kidney its pattern of transcription is unique. In renal distal tubule cells, the predominant PRKWNK1 message encodes a truncated variant, lacking most of the kinase domain (3, 15, 25). This isoform (KS-WNK1) appears to be kidney specific, because current data suggest that it is only expressed in the renal distal nephron. Until recently, the function of KS-WNK1 has been elusive. The first functional study of KS-WNK1 reported that it is induced by aldosterone and enhances activity of the epithelial Na channel (ENaC) (14), although others have reported that L-WNK1 also stimulates ENaC through a different mechanism (23). These observations suggest a role for WNK1 gene products in the regulation of distal nephron Na+ transport, but their relationship to the pathogenesis of FHHt remains unclear; activation of ENaC leads to hypertension with hypokalemia rather than hyperkalemia (13).
The absence of a kinase domain from KS-WNK1 indicates that its mechanisms of action may be qualitatively distinct from that of L-WNK1. One attractive hypothesis is that KS-WNK1 functions as a negative regulator of L-WNK1 catalytic activity, inasmuch as dead or "fractured" kinases frequently suppress the activity of their kinase-sufficient homologs (9). Accordingly, we tested this hypothesis using Na+ transport studies. A rat KS-WNK1 sequence was identified and subcloned into pgh19, an oocyte expression vector, as described in Supplemental Data, part I (http://ajprenal.physiology.org/cgi/content/full/00280.2005/DC1). The general structure of KS-WNK1 in humans, mice, and rats is presented in Fig. 1. In addition, the NH2-terminal sequence differences between the kidney-specific isoform and L-WNK1 are shown. Rat exon 4a contains a start codon that yields an open reading frame resulting in 30 amino acid residues that are 86 and 97% identical to the corresponding human and mouse sequences, respectively. We also noted a second in-frame start codon located 75 nucleotides upstream from this translational start site. These two start codons do not contain an in-frame stop codon between them, suggesting that an alternative translational start site may exist in the rat KS-WNK1 isoform. This longer rat KS-WNK1 sequence was reported to GenBank (accession no. DQ177457). We elected to study the shorter KS-WNK1 sequence ( in Fig. 1), because it is homologous to the mouse and human sequences. Furthermore, RT-PCR using primers specific for the longer and shorter forms of KS-WNK1 amplified both products. (see online Supplemental Data, part II). Although this RT-PCR was not quantitative, the results indicate that the shorter form is expressed at physiologically relevant levels by the kidney.
To test whether KS-WNK1 regulates NCC activity directly, X. laevis oocytes were injected with cRNAs encoding NCC alone or NCC with KS-WNK1; the 22Na influx was measured under conditions we have reported previously (26). KS-WNK1 cRNA did not affect NCC-mediated 22Na uptake significantly compared with NCC alone (P = 0.824; Fig. 2A), even though immunoblotting of total oocyte lysates confirmed that KS-WNK1 is expressed at the protein level (Fig. 2B). These results indicate that KS-WNK1 exerts no direct regulatory effect on NCC activity.
Because the regulation of NCC by L-WNK1 occurs indirectly, through a kinase-dependent mechanism involving WNK4, we reasoned that KS-WNK1 may not have the same effect on the cotransporter as its kinase-sufficient homolog. To test this hypothesis, we performed Na+ uptake experiments comparing the effects of L-WNK1 and KS-WNK1 on WNK4-mediated NCC inhibition. For these studies, we utilized WNK4-(1681222), a construct that inhibits NCC with less experimental variability than full-length WNK4 (27). We confirmed our previous results (27) indicating that L-WNK1 suppresses the ability of WNK4-(1681222) to inhibit NCC activity; in contrast, KS-WNK1 did not interfere with WNK4-(1681222)-mediated NCC inhibition (Fig. 3A). Western blotting confirmed that coexpression of KS-WNK1 with WNK4-(1681222) and flag-tagged NCC did not alter total NCC protein abundance (Fig. 3B). To exclude the possibility that KS-WNK1 might have different effects on full-length WNK4 than WNK4-(1681222), we performed additional experiments with the full-length construct, which confirmed that KS-WNK1 was incapable of restoring WNK4-suppressed NCC activity to baseline levels (P < 0.01; Fig. 3C). These data show that KS-WNK1 is functionally different from L-WNK1 with regard to NCC regulation and are consistent with the observation that L-WNK1 requires intact kinase activity to inhibit WNK4 (27).
Although our findings indicate that KS-WNK1 does not affect either NCC or WNK4 directly, we reasoned that KS-WNK1 might still influence NCC activity indirectly, by regulating L-WNK1. Consequently, we tested whether KS-WNK1 is capable of attenuating L-WNK1-mediated inhibition of WNK4. Fixed amounts of NCC, WNK4-(1681222), and L-WNK1 and varied concentrations of KS-WNK1 were coexpressed in oocytes. The results of 22Na uptake studies showed that KS-WNK1 inhibited the L-WNK1 effect in a dose-dependent manner. Thus, as KS-WNK1 concentrations increase, L-WNK1-mediated WNK4 suppression attenuates (Fig. 4A). At high concentrations of KS-WNK1, L-WNK1 had no detectable effect on WNK4-mediated NCC inhibition. These data were highly statistically significant (P < 0.001; by 1-way ANOVA post hoc test for linear trend). We confirmed that the decrease in NCC activity was not a consequence of translational interference of KS-WNK1 on L-WNK1 synthesis (Fig. 4B). Similarly, immunoblotting revealed a consistent amount of expressed NCC, when KS-WNK1 was coinjected with L-WNK1 and WNK4-(1681222). Taken together, these data indicate that KS-WNK1 inhibits L-WNK1 activity, thereby permitting WNK4 to downregulate NCC. This effect appears to be independent of any alteration in the total cellular abundance of L-WNK1 or NCC.
To address the mechanisms of L-WNK1 inhibition by KS-WNK1, we tested whether the two isoforms could interact in a protein complex. Figure 4C shows that hexahistidine-tagged L-WNK1-(1555) specifically immunoprecipitated with the myc antibody only in lysates where L-WNK1-(1555) was coexpressed with myc-KS-WNK1. The association was confirmed using untagged L-WNK1 coexpressed with myc-KS-WNK1 (Subramanya AR and Yang C-L, unpublished observations).
The catalytic activity of L-WNK1 is essential for its suppressive effect on WNK4-mediated inhibition of NCC (27). Consequently, we hypothesized that the downregulatory effect of KS-WNK1 on NCC activity mediated though L-WNK1 inhibition could occur through the alteration of L-WNK1 catalytic activity. In vitro kinase assays using purified glutathione-S-transferase (GST)-tagged L-WNK1-(1491) and GST-KS-WNK1-(284), which contains exon 4a and the fractured portion of the WNK1 kinase domain, but not the autoinhibitory domain.(Fig. 4D), indicate that GST-KS-WNK1-(284) inhibits GST-L-WNK1-(1491) autophosphorylation and phosphorylation of a generic substrate in a concentration-dependent manner. GST control protein had no effect. GST-KS-WNK1-(2148), a KS-WNK1 fusion protein containing the residues in GST-KS-WNK1-(283) plus the autoinhibitory domain, was also able to inhibit the kinase activity of GST-L-WNK1-(1491) (Subramanya AR and Zhu X, unpublished observations). These results indicate that KS-WNK1 is capable of inhibiting the kinase activity of L-WNK1 via multiple inhibitory sequences.
The current results show that KS-WNK1 downregulates NCC activity indirectly through the suppression of L-WNK1, relieving the inhibition of WNK4. The findings are physiologically relevant, as both L-WNK1 and KS-WNK1 are expressed in the distal convoluted tubule (DCT), overlapping with the site of NCC expression (3, 15). Moreover, the abundance of KS-WNK1 transcript at this site is much greater than that of the kinase-sufficient isoform; it has been estimated that KS-WNK1 comprises 90% of the PRKWNK1 message in the distal nephron (3). Taking the high KS-WNK1/L-WNK1 transcript ratio into consideration, our data implicate the kidney-specific isoform as an endogenous dominant-negative factor that suppresses L-WNK1 activity in the DCT. Furthermore, our findings suggest that the net effect of this dominant-negative regulation is to inhibit Na+ reabsorption with Cl along the distal nephron.
L-WNK1 appears to have a multifunctional role in cellular processes and biological systems. Current evidence indicates that it is essential for organogenesis, because homozygous disruption of PRKWNK1 in mice is lethal to the embryo (29). It is also implicated in signaling cascades involving epidermal growth factor and insulin (12, 22, 24). Furthermore, L-WNK1 plays a widespread role in regulating membrane insertion or removal events (11). Surprisingly, despite these diverse functions, human mutations in PRKWNK1 cause an organ-specific phenotype. FHHt is a hypertensive disease of enhanced renal Na-Cl reabsorption, impaired kaliuriesis, and resistance to aldosterone (1, 2, 7, 19), which can be corrected by thiazide diuretics (6, 16). These clinical features implicate a mechanism in which normal renal distal nephron ion transport homeostasis is dysregulated and support an increase in NCC activity as part of the process.
The dominant-negative role of KS-WNK1 in renal Na-Cl cotransport may explain the kidney-specific phenotype of FHHt. Our findings suggest that this isoform clusters in a regulatory pathway with L-WNK1 and WNK4, two other FHHt-implicated molecules, to regulate DCT ion transport. However, unlike L-WNK1 or WNK4, KS-WNK1 is exclusively expressed in the distal nephron (3). This implies that interactions among KS-WNK1, L-WNK1, and WNK4 play a pivotal role in determining proper distal nephron Na+ and K+ handling. Conversely, because the kidney-specific isoform is present in no other tissue (25), FHHt mutations that cause discordant interactions among KS-WNK1, L-WNK1, and WNK4 would yield a renal-limited phenotype.
Wilson and colleagues (19) showed that in FHHt-C, the disease subtype associated with PRKWNK1 mutations, deletions within the first intron of the gene upregulate its expression in leukocytes. It is not clear, however, whether the over-abundant message encodes L-WNK1 or KS-WNK1. The functional studies reported here support the recent hypothesis by Xu and colleagues (23) and ourselves (27) that the kinase-sufficient isoform may be upregulated in FHHt-C. Such an alteration in L-WNK1 expression might lead to an increase in the ratio of L-WNK1 to KS-WNK1. This, in turn, would allow L-WNK1 to overcome the dominant-negative effect of KS-WNK1, causing inhibition of WNK4 and an increase in NCC activity.
Kahle et al. (8) postulated that WNK4 acts as a molecular switch in the distal nephron, helping to balance K+ secretion and Na+ reabsorption. They further suggested that disease-causing WNK4 mutations might alter this balance, thereby causing hyperkalemia and hypertension by "uncoupling" the kaliuretic and Na+-retentive effects of aldosterone. However, they did not identify an underlying physiological mechanism that mediates shifts between states of Na+ retention and K+ loss. Based on our observations, and the findings of others, we propose that KS-WNK1 plays an essential role in this process. Recently, Naray-Fejes-Toth et al. (14) reported that KS-WNK1 transcription is stimulated by aldosterone and enhances ENaC-mediated Na+ transport. Our data indicate that KS-WNK1 diminishes Na-Cl cotransport via NCC. Together, these observations implicate KS-WNK1 as an aldosterone-driven factor in the late DCT that enhances electrogenic Na+ transport and suppresses electroneutral Na+ transport (Fig. 5). In other words, by decreasing the number of NCC cotransporters in the apical membrane, and increasing the density of ENaC channels, the epithelium in the late DCT (the DCT2) would effectively transform from one that primarily transports Na+ with Cl into one that transports Na+ alone. Inhibition of Na-Cl reabsorption via NCC would also enhance urinary flow to the connecting tubule and cortical collecting duct. This would effectively keep the luminal concentration of Na+ near or above the Michaelis constant for ENaC and therefore maintain high levels of electrogenic Na+ transport. The end result of these effects would be to facilitate K+ secretion, because luminal K+ concentration in the distal nephron is principally determined by transepithelial voltage (18). Thus we suggest that, in addition to WNK4, both L-WNK1 and KS-WNK1 also serve as points of regulation for aldosterone signaling, and factors that alter the relative balance of these isoforms could convert aldosterone from a Na+-retentive hormone into one that promotes kaliuresis.
GRANTS
D. H. Ellison is funded by a Department of Veterans Affairs Merit Review and National Institutes of Health (NIH) Grant RO1-DK-51496. A. R. Subramanya was supported by an American Heart Association Pacific Mountain Postdoctoral Fellowship and is currently supported by NIH Grant NRSA-F32-DK-72865.
ACKNOWLEDGMENTS
We thank David Rozansky and James McCormick for helpful suggestions and critical reading of the manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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