The klotho gene encodes a type I single-pass transmembrane protein that contains a large extracellular domain, a membrane spanning segment, and a short intracellular domain. Klotho protein exists in several forms including the full-length membrane form (mKl) and a soluble circulating form [soluble klotho (sKl)]. mKl complexes with fibroblast growth factor receptors to form coreceptors for FGF23, which allows it to participate in FGF23-mediated signal transduction and regulation of phosphate and calcium homeostasis. sKl is present in the blood, urine, and cerebrospinal fluid where it performs a multitude of functions including regulation of ion channels/transporters and growth factor signaling. How sKl exerts these pleiotropic functions is poorly understood. One hurdle in understanding sKl’s mechanism of action as a “hormone” has been the inability to identify a receptor that mediates its effects. In the body, the kidneys are a major source of sKl and sKl levels decline during renal disease. sKl deficiency in chronic kidney disease makes the heart susceptible to stress-induced injury. Here, we summarize the current knowledge of mKl’s mechanism of action, the mechanistic basis of sKl’s protective, FGF23-independent effects on the heart, and provide new insights into the mechanism of action of sKl focusing on recent findings that sKl binds sialogangliosides in membrane lipid rafts to regulate growth factor signaling.

Significance: With-no-lysine (K) (WNK) protein kinases regulate ion transport. Mutations in WNKs 1 and 4 can cause a monogenic hypertension, pseudohypoaldosteronism type II (PHA2). The WNK-regulated protein kinases STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) mediate actions of WNKs on cation chloride cotransporters. The conserved C-terminal domains (CCTs) of OSR1/SPAK recognize linear R-F-x-V/I motifs in transporters and other protein targets. We discovered that OSR1/SPAK CCTs also recognize a motif variant, R-x-F-x-V/I, found in 8 of 16 inward rectifier K ⁺ (IRK) channels. We show that WNK and OSR1 regulate IRK channels, dependent on the presence of the variant motif. WNK kinase activity-dependent regulation of ion channels reveals a previously unknown mechanism and expands knowledge of potential WNK-OSR1/SPAK actions in cardiac and renal physiology.

With-no-lysine (K)-1 (WNK1) is the founding member of family of four protein kinases with atypical placement of catalytic lysine that play important roles in regulating epithelial ion transport. Gain-of-function mutations of WNK1 and WNK4 cause a mendelian hypertension and hyperkalemic disease. WNK1 is ubiquitously expressed and essential for embryonic angiogenesis in mice. Increasing evidence indicates the role of WNK kinases in tumorigenesis at least partly by stimulating tumor cell proliferation. Here, we show that human hepatoma cells xenotransplanted into zebrafish produced high levels of vascular endothelial growth factor (VEGF) and WNK1, and induced expression of zebrafish wnk1. Knockdown of wnk1 in zebrafish decreased tumor-induced ectopic vessel formation and inhibited tumor proliferation. Inhibition of WNK1 or its downstream kinases OSR1 (oxidative stress responsive kinase 1)/SPAK (Ste20-related proline alanine rich kinase) using chemical inhibitors decreased ectopic vessel formation as well as proliferation of xenotransplanted hepatoma cells. The effect of WNK and OSR1 inhibitors is greater than that achieved by inhibitor of VEGF signaling cascade. These inhibitors also effectively inhibited tumorigenesis in two separate transgenic zebrafish models of intestinal and hepatocellular carcinomas. Endothelial-specific overexpression of wnk1 enhanced tumorigenesis in transgenic carcinogenic fish, supporting endothelial cell-autonomous effect of WNK1 in tumor promotion. Thus, WNK1 can promote tumorigenesis by multiple effects that include stimulating tumor angiogenesis. Inhibition of WNK1 may be a potent anti-cancer therapy.

Background: Mutations of PKD2, which encodes polycystin-2, cause autosomal dominant polycystic kidney disease (ADPKD). The prevailing view is that defects in polycystin-2–mediated calcium ion influx in the primary cilia play a central role in the pathogenesis of cyst growth. However, polycystin-2 is predominantly expressed in the endoplasmic reticulum (ER) and more permeable to potassium ions than to calcium ions. Methods: The trimeric intracellular cation (TRIC) channel TRIC-B is an ER-resident potassium channel that mediates potassium–calcium counterion exchange for inositol trisphosphate–mediated calcium ion release. Using TRIC-B as a tool, we examined the function of ER-localized polycystin-2 and its role in ADPKD pathogenesis in cultured cells, zebrafish, and mouse models. Results: Agonist-induced ER calcium ion release was defective in cells lacking polycystin-2 and reversed by exogenous expression of TRIC-B. Vice versa, exogenous polycystin-2 reversed an ER calcium-release defect in cells lacking TRIC-B. In a zebrafish model, expression of wild-type but not nonfunctional TRIC-B suppressed polycystin-2–deficient phenotypes. Similarly, these phenotypes were suppressed by targeting the ROMK potassium channel (normally expressed on the cell surface) to the ER. In cultured cells and polycystin-2–deficient zebrafish phenotypes, polycystin-2 remained capable of reversing the ER calcium release defect even when it was not present in the cilia. Transgenic expression of Tric-b ameliorated cystogenesis in the kidneys of conditional Pkd2-inactivated mice, whereas Tric-b deletion enhanced cystogenesis in Pkd2-heterozygous kidneys. Conclusions: Polycystin-2 in the ER appears to be critical for anticystogenesis and likely functions as a potassium ion channel to facilitate potassium–calcium counterion exchange for inositol trisphosphate–mediated calcium release. The results advance the understanding of ADPKD pathogenesis and provides proof of principle for pharmacotherapy by TRIC-B activators.

Soluble klotho (sKlotho), the shed ectodomain of α‐klotho, protects the heart by down‐regulating transient receptor potential canonical isoform 6 (TRPC6)–mediated calcium signaling. Binding to α2–3‐sialyllactose moiety of gangliosides in lipid rafts and inhibition of raft‐dependent signaling underlies the mechanism. A recent 3‐Å X‐ray structure of sKlotho in complex with fibroblast growth factor receptor (FGFR) and fibroblast growth factor 23 (FGF23) indicates that its β6α6 loop might block access to the proposed binding site for α2–3‐sialyllactose. It was concluded that sKlotho only functions in complex with FGFR and FGF23 and that sKlotho's pleiotropic effects all depend on FGF23. Here, we report that sKlotho can inhibit TRPC6 channels expressed in cells lacking endogenous FGFRs. Structural modeling and molecular docking show that a repositioned β6α6 loop allows sKlotho to bind α2–3‐sialyllactose. Molecular dynamic simulations further show the α2–3‐siaryllactose–bound sKlotho complex to be stable. Domains mimicking sKlotho's sialic acid–recognizing activity inhibit TRPC6. The results strongly support the hypothesis that sKlotho can exert effects independent of FGF23 and FGFR.—Wright, J. D., An, S.‐W., Xie, J., Lim, C., Huang, C.‐L. Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR‐FGF23 pathway. FASEB J. 33, 9182–9193 (2019). www.fasebj.org

The Na⁺-Cl⁻ cotransporter (NCC) in distal convoluted tubule (DCT) plays important roles in renal NaCl reabsorption. The current hypothesis for the mechanism of regulation of NCC focuses on WNK4 and intracellular Cl⁻ concentration ([Cl⁻]_i). WNK kinases bind Cl⁻, and Cl⁻ binding decreases the catalytic activity. It is believed that hypokalemia under low K⁺ intake decreases [Cl⁻]_i to activate WNK4, which thereby phosphorylates and stimulates NCC through activation of SPAK. However, increased NCC activity and apical NaCl entry would mitigate the fall in [Cl⁻]_i. Whether [Cl⁻]_i in DCT under low-K⁺ diet is sufficiently low to activate WNK4 is unknown. Furthermore, increased luminal NaCl delivery also stimulates NCC and causes upregulation of the transporter. Unlike low K⁺ intake, increased luminal NaCl delivery would tend to increase [Cl⁻]_i. Thus we investigated the role of WNK4 and [Cl⁻]_i in regulating NCC. We generated Wnk4-knockout mice and examined regulation of NCC by low K⁺ intake and by increased luminal NaCl delivery in knockout (KO) and wild-type mice. Wnk4-KO mice have marked reduction in the abundance, phosphorylation, and functional activity of NCC vs. wild type. Low K⁺ intake increases NCC phosphorylation and functional activity in wild-type mice, but not in Wnk4-KO mice. Increased luminal NaCl delivery similarly upregulates NCC, which, contrary to low K⁺ intake, is not abolished in Wnk4-KO mice. The results reveal that modulation of WNK4 activity by [Cl⁻]_i is not the sole mechanism for regulating NCC. Increased luminal NaCl delivery upregulates NCC via yet unknown mechanism(s) that may override inhibition of WNK4 by high [Cl⁻]_i.