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：
9322613
负责人：
Bidhan Chandra Bandyopadhyay
金额：
$ 18.77万
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 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9322613
关键词：
Acetazolamide Active Biological Transport Age-Years Alkalinization Amino Acids Animal Model Apical Attenuated Betaine Breast Microcalcification Calcified 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（由适用提供）：几项研究表明，磷酸钙（CAP）结石是在肾单位的早期部分中形成的，即近端细胞（PT）和Henle（LOH）的环（PT）（PT）（LOH），其中由于钙（CA2+）和磷酸盐浓度较高而有利，以及磷酸盐浓度高。 PT是CA2+重吸收的主要部位，报告了细胞细胞途径。但是，尚不清楚任何通过跨细胞途径的调节Ca2+转运的存在。我们目前的建议将研究控制跨细胞Ca2+转运的这种调节的CA2+进入机制，该机制在石材形成中起作用。我们的初步数据表明，Ca2+传感器受体（CSR）是G蛋白偶联受体，对细胞外[Ca2+]（[Ca2+] O）的变化做出反应，并且瞬态受体潜在的典型的典型典型3（TRPC3），Ca2+permable通道，A均位于Pt pt细胞的腔内区域。我们的数据还显示：1）与物理和功能上都具有TRPC3的CSR夫妇； 2）[Ca2+] o通过CSR介导了这种耦合响应，该响应通过磷脂酶C（PLC）依赖性途径向TRPC3通道发出信号。更重要的是，我们发现CSR和TRPC3的药物/遗传破坏显着减弱了PT细胞中的这种Ca2+影响，TRPC3-NULL小鼠在尿液中表现出升高的[Ca2+]表型，尿液中的计算[Ca2+]，肾脏和尿液中尿液和LOH的散射晶体的计算。基于我们的初步数据，我们假设在PT腔内流体中增加了[Ca2+]和其他调节剂，例如质子和氨基酸，可以通过PLC依赖性途径激活CSR-TRPC3信号传导，从而引发了跨PT的经跨细胞CA2+转运。我们进一步假设，这种增加Ca2+转运的机制加上PT腔流体的酸化在一起，以防止LOH处的帽石的成核。我们具有以下特定目的来检验这一假设。 AIM 1提案，通过确定CSR-TRPC3信号传导在Ca2+进入/转运中使用TRPC3基因敲除（KO）小鼠和药物/药物/遗传破坏CSR-TRPC3信号传导的作用，以确定CSR介导的Ca2+进入/转运到PT细胞中的机制。在AIM 2中，我们建议研究TRPC3 KO小鼠体内的Ca2+进入/转运，并通过在LOH中引入Cap和Cap+ Caox Stone形成的PT来促进pT的PT，以促进PT的pT中，以破坏TRPC3 KO小鼠中的磷酸盐和草酸盐转运机制。最后，在目标3中，我们计划挽救TRPC3 KO小鼠的表型（例如，在尿液中[Ca2+]归一化[Ca2+]），并确定PT [Ca2+]和pH在PT中增加的作用及其对Cap Stone的作用，并通过酸化或碱化尿液中的TRPC3 KO小鼠的尿液和碱性的含量，然后在Trpc3 KO中酸化或碱性，然后在Trpc3 KO中均添加脉络，然后进行脉络级别的含量。 loh。拟议的目标将揭示新的机制：i）PT中受调节的跨细胞Ca2+转运； ii）在PT腔流体中维持[Ca2+]。获得的信息将有助于了解帽石的形成，这可能会导致新的治疗策略的发展。