Center of Medical Innovation, Seoul National University Hospital, Seoul, Korea.
Department of Internal Medicine, Pusan National University Hospital, Busan, Korea.
Am J Physiol Renal Physiol. 2021 Apr 1;320(4):F559-F568. doi: 10.1152/ajprenal.00044.2021. Epub 2021 Feb 22.
Hypercalciuria is one of the early manifestations of diabetic nephropathy (DN). This is partially due to a decrease in the expression of renal transient receptor potential vanilloid type 5 (TRPV5), which is responsible for renal Ca reabsorption. Soluble klotho has been previously determined to increase TRPV5 by cleaving sialic acid, causing TRPV5 to bind to membrane protein galectin-1. However, a recent study showed that soluble klotho binds to α2-3-sialyllactose, where sialic acid is located, on TRPV5, rather than cleave it. Here, we report that soluble klotho tethers TRPV5 on the membrane by binding both TRPV5 and galectin-1, thereby protecting membrane TRPV5 from diabetes-induced endocytosis. In the present study, we injected recombinant soluble α-klotho protein (rKL) into and mice for 8 wk and collected urine and kidneys. We administered rKL, AZD4547 [fibroblast growth factor (FGF) receptor type 1 inhibitor], and OTX008 (galectin-1 inhibitor) to cultured mouse distal tubular cells with or without 30 mM high-glucose (HG) exposure. mice showed increased renal Ca excretion and decreased renal TRPV5 expression. rKL treatment reversed this change. In vitro, TRPV5 expression in distal tubular cells decreased under HG conditions, and rKL successfully upregulated TRPV5 with or without FGF23. Also, immunofluorescence showed colocalization of klotho, TRPV5, and galectin-1 in distal tubule cells, suggesting that klotho binds to both TRPV5 and galectin-1. Moreover, when both FGF receptor type 1 and galectin-1 were inhibited, rKL failed to increase TRPV5 under HG conditions. Our results indicate that soluble klotho prevents TRPV5 from degradation and subsequent diabetes-induced endocytosis by anchoring TRPV5 through binding with both TRPV5 and galectin-1. Soluble α-klotho anchors transient receptor potential vanilloid type 5 (TRPV5) on the apical membrane of the distal tubule by binding both TRPV5 and a membrane-abundant protein, galectin-1. This newly discovered mechanism works even when fibroblast growth factor (FGF)23 signaling is inhibited by treatment with FGF receptor type 1 inhibitor. Therefore, we identified how soluble α-klotho increases TRPV5 without FGF23. We confirmed this mechanism by observing that soluble α-klotho fails to enhance TRPV5 when both FGF receptor type 1 and galectin-1 are inhibited.
高钙尿症是糖尿病肾病 (DN) 的早期表现之一。这部分是由于肾脏瞬时受体电位香草酸亚型 5 (TRPV5) 的表达减少，而 TRPV5 负责肾脏钙的重吸收。可溶性 klotho 先前已被确定通过裂解唾液酸来增加 TRPV5，从而使 TRPV5 与膜蛋白半乳糖凝集素-1 结合。然而，最近的一项研究表明，可溶性 klotho 结合到 TRPV5 上位于唾液酸的 α2-3-唾液乳糖上，而不是裂解它。在这里，我们报告可溶性 klotho 通过与 TRPV5 和半乳糖凝集素-1 结合来固定膜上的 TRPV5，从而防止糖尿病引起的内吞作用使膜上的 TRPV5 降解。在本研究中，我们将重组可溶性 α-klotho 蛋白 (rKL) 注射到 db/db 和 ob/ob 小鼠中 8 周，并收集尿液和肾脏。我们用 rKL、AZD4547（成纤维细胞生长因子 (FGF) 受体 1 抑制剂）和 OTX008（半乳糖凝集素-1 抑制剂）处理培养的小鼠远曲小管细胞，无论是否暴露于 30 mM 高葡萄糖 (HG)。db/db 小鼠表现出肾钙排泄增加和肾 TRPV5 表达减少。rKL 处理逆转了这一变化。在体外，HG 条件下远曲小管细胞中 TRPV5 的表达下降，而 rKL 成功地上调了 TRPV5，无论是否存在 FGF23。此外，免疫荧光显示 klotho、TRPV5 和半乳糖凝集素-1 在远曲小管细胞中存在共定位，表明 klotho 与 TRPV5 和半乳糖凝集素-1 结合。此外，当同时抑制 FGF 受体 1 和半乳糖凝集素-1 时，rKL 无法在 HG 条件下增加 TRPV5。我们的结果表明，可溶性 klotho 通过与 TRPV5 和半乳糖凝集素-1 结合来固定 TRPV5，从而防止 TRPV5 降解和随后的糖尿病引起的内吞作用。可溶性 α-klotho 通过与 TRPV5 和膜上丰富的蛋白半乳糖凝集素-1 结合，将瞬时受体电位香草酸亚型 5 (TRPV5) 锚定在远端小管的顶端膜上。这种新发现的机制即使在用 FGF 受体 1 抑制剂治疗抑制成纤维细胞生长因子 (FGF)23 信号传导时也能发挥作用。因此，我们确定了可溶性 α-klotho 如何在没有 FGF23 的情况下增加 TRPV5。我们通过观察到当同时抑制 FGF 受体 1 和半乳糖凝集素-1 时，可溶性 α-klotho 无法增强 TRPV5 来证实这一机制。
