Klotho is a membrane protein predominantly produced in the kidney that exerts some anti-ageing effects. Ageing is associated with an increased risk of heart failure; whether Klotho is cardioprotective is unknown. Here we show that Klotho-deficient mice have no baseline cardiac abnormalities but develop exaggerated pathological cardiac hypertrophy and remodeling in response to stress. Cardioprotection by Klotho in normal mice is mediated by downregulation of TRPC6 channels in the heart. We demonstrate that deletion of Trpc6 prevents stress-induced exaggerated cardiac remodeling in Klotho-deficient mice. Furthermore, mice with heart-specific overexpression of TRPC6 develop spontaneous cardiac hypertrophy and remodeling. Klotho overexpression ameliorates cardiac pathologies in these mice and improves their long-term survival. Soluble Klotho present in the systemic circulation inhibits TRPC6 currents in cardiomyocytes by blocking phosphoinositide-3-kinase-dependent exocytosis of TRPC6 channels. These results provide a new perspective on the pathogenesis of cardiomyopathies and open new avenues for treatment of the disease.

Cardiac hypertrophy occurs in up to 95% of patients with CKD and increases their risk for cardiovascular death. In the kidney, full-length membranous Klotho forms the coreceptor for fibroblast growth factor 23 (FGF23) to regulate phosphate metabolism. The prevailing view is that the decreased level of Klotho in CKD causes cardiomyopathy through increases in serum FGF23 and/or phosphate levels. However, we reported recently that soluble Klotho protects against cardiac hypertrophy by inhibiting abnormal calcium signaling in the heart. Here, we tested whether this protective effect requires changes in FGF23 and/or phosphate levels. Heterozygous Klotho-deficient CKD mice exhibited aggravated cardiac hypertrophy compared with wild-type CKD mice. Cardiac magnetic resonance imaging studies revealed that Klotho-deficient CKD hearts had worse functional impairment than wild-type CKD hearts. Normalization of serum phosphate and FGF23 levels by dietary phosphate restriction did not abrogate the aggravated cardiac hypertrophy observed in Klotho-deficient CKD mice. Circulating levels of the cleaved soluble ectodomain of Klotho were lower in wild-type CKD mice than in control mice and even lower in Klotho-deficient CKD mice. Intravenous delivery of a transgene encoding soluble Klotho ameliorated cardiac hypertrophy in Klotho-deficient CKD mice. These results suggest that the decreased level of circulating soluble Klotho in CKD is an important cause of uremic cardiomyopathy independent of FGF23 and phosphate, opening new avenues for treatment of this disease.

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.

Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGK«, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgk« colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGK« variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes.

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decrease unilateral ureteral obstruction-induced interstitial fibrosis, and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed- relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6-knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild type mice, but not in Trpc6-knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.

Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.soluble klotho | lipid rafts | gangliosides | sialic acids | TRPC6 M ice homozygous for a severe hypomorphic α-klotho allele manifest multiple aging-related phenotypes and die prematurely at 2-3 mo after birth (1). α-Klotho is predominantly expressed in renal tubules, parathyroid glands, and epithelial cells of the choroids plexus, but not in myocardium. Overexpression of α-klotho extends life span in mice, indicating that it is an agingsuppression molecule (2). The full-length α-klotho protein is a single-pass membrane protein with a large extracellular domain, a membrane-spanning segment, and a short intracellular carboxyl terminus (1). Membranous α-klotho associates with FGF receptors to form coreceptors for the ligand FGF23, a bone-derived circulating hormone that plays an important role in phosphate homeostasis (3).The ectodomain of α-klotho, ∼950 aa in length, is composed of two internal repeats, KL1 and KL2, each sharing amino acid sequence homology to family-1 glycosidases (4). The ectodomain (soluble klotho, sKL) is released into the systemic circulation, urine, and cerebrospinal fluid and functions as a humoral factor (2, 5). To date, more than 10 different functions of sKL have been described (2, 6-9). sKL regulates ion transporters, antagonizes Wnt and TGF-β1signaling to suppress cellular senescence, tissue fibrosis, and cancer metastasis, and inhibits insulin and insulin-like growth factor-1 (IGF1)-driven PI3K/Akt signaling contributing to extension of lifespan in mice, cardioprotection, and inhibition of tumor cell proliferation. A fundamental gap in the understanding of sKL's mechanism of action is the lack of knowledge of potential membrane receptor(s) that mediate cellular responses to sKL.Lipid rafts are highly dynamic, cholesterol-and sphingolipidrich membrane microdomains that compartmentalize cellular processes such as ...

Background: Klotho is a putative sialidase that modifies the extracellular N-glycan of TRPV5 to increase its surface expression. Results: Intracellular expression of Klotho increased TRPV5 via an N-glycosylation-dependent mechanism that requires the sialidase activity of Klotho. Conclusion: Klotho up-regulates TRPV5 surface abundance by intra-and extracellular mechanisms. Significance: We investigate a novel function of Klotho to regulate intracellular protein trafficking via enzymatic activity.

Summary Background PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca2+. While availability of the PIP2 substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive. Results Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca2+ transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP2 via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gαq are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by Gq-coupled receptors and basal input from Gq is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor. Conclusions WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP2 is itself a signaling molecule, regulation of PIP2 synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP2 on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk and a means to independently control PLC signaling and PIP2 availability.