Calcium transport in kidney proximal tubule and calcium phosphate stone formation

 描述(申请人提供)：多项研究表明，磷酸钙结石形成于肾单位的早期，即近端小管(PT)和Henle环(LOH)，由于高钙(Ca2)和磷酸盐浓度以及相对较高的pH，这些条件是有利的。PT是钙重吸收的主要部位，已有报道称存在一条细胞旁途径。然而，目前尚不清楚是否存在通过跨细胞途径调节的钙离子转运。我们目前的建议将研究这一未知的调节钙进入机制，它控制着跨细胞钙转运，这在结石形成中起到了作用。我们的初步数据表明，钙敏感受体(CSR)和瞬时受体电位规范3(TRPC3)都定位于PT细胞的管腔区域。CSR是一种G蛋白偶联受体，对细胞外[Ca~(2+)]o的变化做出反应。我们的数据还表明：1)CSR与TRPC3在生理和功能上偶联；2)[Ca~(2+)]o通过CSR介导这种偶联反应，CSR通过磷脂酶C(PLC)依赖的途径向TRPC3通道发出信号。更重要的是，我们发现CSR和TRPC3的药物/遗传干扰显著减少了PT细胞中的这种钙内流，TRPC3基因缺失的小鼠表现为尿液中[钙]升高，肾脏钙化，尿液和LOH中分散晶体。根据我们的初步数据，我们假设PT腔液中增加[Ca~(2+)]和其他调节剂，如质子和氨基酸，可以通过PLC依赖的途径激活CSR-TRPC3信号，从而启动跨细胞的Ca~(2+)转运。我们进一步假设，这种促进钙转运的机制加上PT腔内液体的酸化，共同起到了防止LOH处帽状结石成核的作用。我们有以下具体目标来检验这一假设。目的1利用TRPC3基因敲除(KO)小鼠研究CSR-TRPC3信号在PT细胞钙转运中的作用，以及CSR-TRPC3信号的药理学/遗传学干扰，探讨CSR-TRPC3信号在PT细胞钙转运中的作用机制。在目的2中，我们建议研究TRPC3KO小鼠体内的钙进入/转运，并通过将体内siRNA应用于PT来干扰TRPC3KO小鼠的磷酸盐和草酸转运机制，以促进LOH处CaOx结石的形成。最后，在目标3中，我们计划挽救TRPC3 KO小鼠的表型(例如，使尿液中[Ca~(2+)]正常化)，并确定升高的[Ca~(2+)]和pH在PT中的作用及其通过酸化或碱化尿液在或不引起高钙血症的TRPC3 KO小鼠的帽状结石形成中的作用，然后测量LOH小鼠的尿液性质和钙化/结石形成程度。提出的AIMS将揭示新的机制：1)调节甲状旁腺的跨细胞钙转运；2)维持甲状旁腺腔液中的[钙]。所获得的信息将有助于理解帽状结石的形成，这可能会导致新的治疗策略的开发。

 DESCRIPTION (provided by applicant): Several studies have shown that calcium phosphate (CaP) stones are formed in the early segments of the nephron, namely the proximal tubule (PT) and the loop of Henle (LOH), where conditions are favorable due to high calcium (Ca2+) and phosphate concentrations, as well as a relatively high pH. The PT is the major site for Ca2+ reabsorption, where a paracellular pathway has been reported. However, existence of any regulated Ca2+ transport through a transcellular route is unknown. Our present proposal will study this yet unknown regulated Ca2+ entry mechanism that controls transcellular Ca2+ transport, which has a role in stone formation. Our preliminary data show that Ca2+-sensing receptor (CSR), a G protein-coupled receptor that responds to alterations in extracellular [Ca2+] ([Ca2+]o), and a transient receptor potential canonical 3 (TRPC3), a Ca2+ permeable channel, both localize at the luminal region of PT cells. Our data show also that: 1) CSR couples with TRPC3 both physically and functionally; and 2) [Ca2+]o mediates this coupling response through CSR which signals TRPC3 channels via a phospholipase C (PLC)-dependent pathway. More importantly, we found that the pharmacological/genetic disruption of both CSR and TRPC3 markedly attenuated this Ca2+ influx in PT cells and that TRPC3-null mice displayed a phenotype of elevated [Ca2+] in urine, calcification in kidney and scattered crystals in the urine and the LOH. Based on our preliminary data, we hypothesize that increased [Ca2+] and other modulators, like protons and amino acids, in PT luminal fluid can activate CSR-TRPC3 signaling via a PLC-dependent pathway, thereby initiating transcellular Ca2+ transport across the PT. We further hypothesize that such a mechanism to increase Ca2+ transport plus the acidification of the PT luminal fluid together serves to prevent the nucleation of CaP stone at the LOH. We have the following specific aims to test this hypothesis. Aim 1 proposes to determine the mechanism of CSR-mediated Ca2+ entry/transport into PT cells by determining the role of CSR-TRPC3 signaling in Ca2+ entry/transport in PT cells using TRPC3 knockout (KO) mice and the pharmacological/genetic disruption of CSR-TRPC3 signaling. In Aim 2, we propose to study the Ca2+ entry/transport in vivo in TRPC3 KO mice, and to disrupt the phosphate and oxalate transport mechanism in TRPC3 KO mice by introducing in vivo siRNA application to PT to favor the process of CaP and CaP+CaOx stone formation at LOH. Finally, in Aim 3, we plan to rescue the phenotype (e.g., normalize [Ca2+] in urine) of TRPC3 KO mice and determine the role of increased [Ca2+] and pH in PT and its contribution to CaP stone formation by acidifying or alkalinizing the urine with or without inducing hypercalcemia in TRPC3 KO mice, and then measure the urine properties and degree of calcification/stone formation in LOH. Proposed aims will unravel novel mechanisms: i) the regulated transcellular Ca2+ transport in PT; and ii) maintenance of [Ca2+] in PT luminal fluid. Information gained will help to understand the formation of CaP stone that could potentially lead to the development of new therapeutic strategies.