Calcium transport in kidney proximal tubule and calcium phosphate stone formation

 描述（申请人提供）：几项研究表明，磷酸钙（CaP）结石形成于肾单位的早期段，即近端小管（PT）和汉勒氏袢（洛），其中由于高钙（Ca 2+）和磷酸盐浓度以及相对高的pH值，条件是有利的。PT是Ca 2+重吸收的主要部位，其中已经报道了细胞旁途径。然而，通过跨细胞途径的任何调节Ca 2+运输的存在是未知的。我们目前的建议将研究这一未知的调控Ca 2+进入机制，控制跨细胞Ca 2+转运，这在结石形成中有一定的作用。我们的初步数据表明，钙敏感受体（CSR），一个G蛋白偶联受体，响应细胞外[Ca 2 +]（[Ca 2 +]o）的变化，和瞬时受体电位典型3（TRPC 3），一个Ca 2+渗透通道，都定位于PT细胞的腔区域。我们的数据还显示：1）CSR在物理上和功能上与TRPC 3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）依赖性途径向TRPC 3通道发信号。更重要的是，我们发现CSR和TRPC 3的药理学/遗传破坏显著减弱了PT细胞中的这种Ca 2+内流，并且TRPC 3缺失小鼠表现出尿中[Ca 2 +]升高、肾脏钙化和尿中分散晶体和洛的表型。基于我们的初步数据，我们假设PT腔液中[Ca 2 +]和其他调节剂（如质子和氨基酸）的增加可以通过PLC依赖性途径激活CSR-TRPC 3信号传导，从而启动跨PT的跨细胞Ca 2+转运。我们进一步假设，这种机制，以增加Ca 2+的运输加上酸化的PT管腔流体一起用于防止钙磷结石的成核在洛。我们有以下具体目标来检验这一假设。目的1提出通过使用TRPC 3敲除（KO）小鼠确定CSR-TRPC 3信号传导在PT细胞中的Ca 2+进入/转运中的作用以及CSR-TRPC 3信号传导的药理学/遗传学破坏来确定CSR介导的Ca 2+进入/转运到PT细胞中的机制。目的2：研究TRPC 3基因敲除小鼠体内Ca 2+的进入/转运，并通过在PT中引入siRNA促进洛时CaP和CaP+CaOx结石形成，从而破坏TRPC 3基因敲除小鼠体内磷酸盐和草酸盐转运机制。最后，在目标3中，我们计划拯救表型（例如，使TRPC 3 KO小鼠的尿中的[Ca 2 +]标准化），并通过在TRPC 3 KO小鼠中酸化或碱化尿（诱导或不诱导高钙血症）来确定PT中增加的[Ca 2 +]和pH的作用及其对CaP结石形成的贡献，然后测量洛中的尿性质和钙化/结石形成的程度。提出的目标将揭示新的机制：i）PT中受调节的跨细胞Ca 2+转运;和ii）PT腔液中[Ca 2 +]的维持。所获得的信息将有助于了解CaP结石的形成，这可能会导致新的治疗策略的发展。

 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.