Regulation of Renal Calcium Transport in Health and Disease

项目摘要 TRPV5介导的肾钙重吸收是维持钙稳态的关键。Klotho是一种1型 主要在肾脏产生的跨膜蛋白。Klotho存在于膜性Klotho中 形成或溶解的胞外区，进入尿液或体循环，起旁分泌或旁分泌的作用 内分泌因素。此前，我们已经发现，可溶性Klotho通过增加TRPV5的表面丰度 从通道的N-连接的糖链上去除末端唾液酸。去除唾液酸暴露于 潜在的二糖N-乙酰-乳糖胺，Galectin-1的配体，普遍存在于 细胞的外表面。与细胞外表面的Galectin-1结合导致功能性 质膜上有TRPV5。为了支持Klotho作用机制的这一新假说，我们 建议进行以下研究，将我们的体外研究结果扩展到体内，并研究病理生理学 我们的发现的相关性。AIM-1将验证可溶性Klotho调节肾钙重吸收的假说 在体内通过TRPV5，它通过推测的klotho唾液酸酶活性来实现这一点。考察……的作用 在体内，我们将产生表达可溶性Klotho in的转基因小鼠 Klotho缺乏症小鼠的背景(通过限制饮食磷酸盐和维生素D使其免于死亡) 并检测尿钙排泄、TRPV5表达、几个相关基因表达参数 TRPV5介导的钙重吸收和膜片钳记录的TRPV5通道活动 小管。我们还将定义与Klotho的唾液酸酶活性有关的可溶性Klotho的结构域，以及 体内检测携带唾液酸酶活性的突变体Klotho灭活突变的效果。慢性肾脏 疾病(CKD)是一种klotho缺乏的状态。目的2将检验TRPV5介导的肾脏缺陷的假设 慢性甲状旁腺功能亢进症继发性甲状旁腺功能亢进症与可溶性Klotho缺乏症的钙重吸收有关 肾脏疾病。慢性肾脏病患者甲状旁腺激素(PTH)升高是对战斗的反应 高磷血症和低血钙症。甲状旁腺机能亢进症导致多种不良后果 CKD。我们将检验这一假设，即可溶性klotho的减少和TRPV5的下调在 两种CKD小鼠模型的甲状旁腺功能亢进症。野生型转基因接种的效果 或灭活突变株sKlotho对血清甲状旁腺素和钙水平、尿钙排泄等矿物质的影响 将检测CKD中的代谢物。Klotho刺激肾脏作用机制的研究 钙重吸收及其在CKD矿物质代谢中的作用将对我们理解CKD的 慢性肾脏病矿物质紊乱的病理生理学机制及其对未来矿物质治疗的帮助 慢性肾脏病患者的精神障碍。

Project Summary TRPV5-mediated renal Ca2+ reabsorption is critical for maintaining Ca2+ homeostasis. Klotho is a type-1 transmembrane protein predominantly produced in the kidney. Klotho exists either in the membranous klotho form or the soluble ectodomain that is shedded into urine or systemic circulation to function as a paracrine or endocrine factor. Previously, we have found that soluble klotho increases surface abundance of TRPV5 by removing terminal sialic acids from the N-linked glycan chains of the channel. Removal of sialic acids exposes underlying disaccharide N-acetyl-lactosamine, a ligand for galectin-1 that is ubiquitously present on the external surface of cells. Binding to galectin-1 at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane. To support this novel hypothesis for mechanism of action for klotho, we propose the following studies to extend our in vitro findings into in vivo and to investigate pathophysiological relevance of our findings. Aim-1 will test the hypothesis that soluble klotho regulates renal Ca2+ reabsorption via TRPV5 in vivo and that it does so through the putative sialidase activity of klotho. To examine the role of soluble klotho in renal Ca2+ reabsorption in vivo, we will generate transgenic mice that express soluble klotho in the background of klotho-deficient mice (rescued from death by dietary phosphate and vitamin D restriction) and examine urinary calcium excretion, TRPV5 expression, several gene expression parameters related to TRPV5-mediated Ca2+ reabsorption, and patch-clamp recording of TRPV5 channel activity in the native renal tubules. We will also define domains of soluble klotho involved in klotho's putative sialidase activity in vitro and test the effect of mutant soluble Klotho carrying sialidase activity-inactivating mutations in vivo. Chronic kidney disease (CKD) is a klotho-deficient state. Aim 2 will test the hypothesis that defect in TRPV5-mediated renal Ca2+ reabsorption from soluble klotho deficiency contributes to secondary hyperparathyroidism in chronic kidney disease. Elevation of parathyroid hormone (PTH) occurs in patients with CKD as a response to combat hyperphosphatemia and hypocalcemia. Hyperparathyroidism yet causes multiple adverse consequences in CKD. We will test the hypothesis that decrease in soluble klotho and downregulation of TRPV5 plays a role in hyperparathyroidism of CKD by using two mouse models of CKD. The effect of transgenic delivery of wild-type or inactive mutant soluble klotho on serum PTH and Ca2+ levels, urinary Ca2+ excretion, and other mineral metabolites in CKD will be examined. These studies on the mechanism by which klotho stimulates renal calcium reabsorption and its role in mineral metabolism of CKD will be important for our understanding the pathophysiology of mineral disorder of CKD and may help management or deign future therapy of mineral disorder in CKD patients.