Calcium transport in kidney proximal tubule and calcium phosphate stone formation

肾近曲小管中的钙转运和磷酸钙结石的形成

基本信息

批准号：
9765294
负责人：
Bidhan Chandra Bandyopadhyay
金额：
$ 24.59万
依托单位：
INSTITUTE FOR CLINICAL RESEARCH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-02 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9765294
关键词：
Acetazolamide Active Biological Transport Age-Years Alkalinization Amino Acids Animal Model Apical Attenuated Betaine Breast Microcalcification Calcium Calcium Gluconate Calcium Oxalate Calcium-Sensing Receptors Carbonic Anhydrase Inhibitors Cells Chronic Kidney Failure Clinical Complementary DNA Couples Coupling Crystallization Data Development Diagnosis Electrolytes Electrophysiology (science)Epidemiology Excretory function G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs Gene Delivery Genetic Henle&apos s loop Hormones Hospitalization Hypercalcemia Hypertension ITPR1 gene Image Infusion procedures Intervention Kidney Kidney Calculi Knockout Mice Lead Light Limb structure Link Liquid substance Lithium Luminal region Maintenance Measures Mediating Microfluidic Microchips Molecular Mus Nephrolithiasis Nephrons Obesity Operative Surgical Procedures Oral Oxalates Patch-Clamp Techniques Pathogenesis Pathway interactions Patients Permeability Pharmaceutical Preparations Pharmacology Phenotype Phospholipase C Phospholipases A Prevalence Process Property Protons Proximal Kidney Tubules Recurrence Regulation Reporting Research Resolution Risk Factors Role Route Signal Transduction Signaling Protein Site Small Interfering RNA Testing Therapeutic Intervention Thinness Time Tubular formation United States Urine Woman adeno-associated viral vector alkalinity base calcification calcium phosphate calcium phosphate precipitation cost experimental study extracellular in vivo inorganic phosphate lost work time luminal membrane men mouse model novel novel therapeutic intervention overexpression potassium citrate prevent public health relevance receptor response targeted delivery time interval urinary

项目摘要

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.

描述（由申请人提供）：多项研究表明，磷酸钙 (CaP) 结石形成于肾单位的早期部分，即近端小管 (PT) 和亨利袢 (LOH)，由于以下原因，这里的条件有利。高钙 (Ca2+) 和磷酸盐浓度以及相对较高的 pH 值 PT 是 Ca2+ 重吸收的主要部位，据报道存在细胞旁途径。通过跨细胞途径的 Ca2+ 转运尚不清楚。我们目前的提案将研究这种控制跨细胞 Ca2+ 转运的未知调节 Ca2+ 进入机制，该机制在结石形成中发挥作用。对细胞外 [Ca2+] ([Ca2+]o) 的变化做出反应的蛋白偶联受体，以及瞬时受体电位规范 3 (TRPC3)，Ca2+ 通透通道，我们的数据还表明：1) CSR 在物理上和功能上与 TRPC3 偶联；2) [Ca2+]o 通过 CSR 介导这种偶联反应，CSR 通过磷脂酶 C (PLC) 向 TRPC3 通道发出信号。更重要的是，我们发现 CSR 和 TRPC3 的药理学/遗传破坏显着减弱了 PT 细胞中的 Ca2+ 流入。 TRPC3 缺失小鼠表现出尿液中 [Ca2+] 升高、肾脏钙化以及尿液和 LOH 中分散晶体的表型。根据我们的初步数据，我们发现 [Ca2+] 和其他调节剂（如质子和氨基酸）增加。，在 PT 腔液中可以通过 PLC 依赖性途径激活 CSR-TRPC3 信号传导，从而启动跨 PT 的跨细胞 Ca2+ 转运，我们进一步研究了这种增加 Ca2+ 转运的机制。 PT 管腔液的酸化共同作用是防止 CaP 结石在 LOH 处成核。我们有以下具体目标来检验这一假设：目标 1 旨在确定 CSR 介导的 Ca2+ 进入/转运到 PT 细胞的机制。使用 TRPC3 敲除 (KO) 小鼠确定 CSR-TRPC3 信号在 PT 细胞中 Ca2+ 进入/运输中的作用以及 CSR-TRPC3 信号的药理学/遗传破坏。 2，我们建议研究TRPC3 KO小鼠体内Ca2+的进入/转运，并通过将体内siRNA应用于PT来破坏TRPC3 KO小鼠中的磷酸盐和草酸盐转运机制，以促进CaP和CaP+CaOx结石的过程最后，在目标 3 中，我们计划挽救 TRPC3 KO 小鼠的表型（例如，使尿液中的 [Ca2+] 正常化）并确定 LOH 的作用。在 TRPC3 KO 小鼠中，通过酸化或碱化尿液，在有或没有诱导高钙血症的情况下增加 PT 中的 [Ca2+] 和 pH 值，及其对 CaP 结石形成的贡献，然后测量 LOH 中的尿液性质和钙化/结石形成程度。揭示新机制：i) PT 中受调节的跨细胞 Ca2+ 转运；ii) PT 腔液中 [Ca2+] 的维持获得的信息将有助于了解 CaP 的形成。石头可能会导致新治疗策略的开发。