The role of claudin-2 in proximal tubule calcium reabsorption

 描述（由申请人提供）：虽然肾结石已经影响人类，只要有历史记录，他们继续提出一个相当大的公共卫生问题，肾结石疾病的患病率在过去的世纪经历了上升。复发性肾结石患者通常没有任何代谢异常，除了尿钙增加。在肾脏中，近端小管（PT）负责重吸收约三分之二的过滤钙。PT钙重吸收通过被动和细胞旁途径发生的证据是丰富的，尽管这种发生的确切机制尚不清楚。细胞旁通透性的选择性在很大程度上是由紧密连接蛋白决定的，紧密连接蛋白是在上皮细胞之间的紧密连接处发现的蛋白质家族。紧密连接蛋白-2是一种阳离子选择性紧密连接蛋白，在渗漏上皮细胞中表达，包括PT和胃肠道。与野生型（WT）小鼠相比，Claudin-2敲除（Cldn-2 KO）小鼠具有增加的钙排泄分数。钙排泄增加的原因尚未仔细研究。我们推测，密蛋白-2介导的PT细胞旁钙重吸收。对于我们的第一个目标，我们计划评估Cldn-2 KO小鼠中的钙消耗表型。我们还提供了Cldn-2基因敲除小鼠发生肾钙质沉着症的证据，并提出了进一步研究这种关系的研究。细胞外液（ECF）容量收缩通过增加PT钙重吸收减少尿钙。为了检验密蛋白-2有助于PT钙重吸收的假设，具体地，我们提出了在诱导ECF体积收缩后测量WT和Cldn-2 KO小鼠中的尿钙的实验。异聚紧密蛋白相互作用可能在上皮组织的渗透性中起重要作用。在PT中，最丰富表达的密封蛋白似乎是密封蛋白-2、密封蛋白-3和密封蛋白-10a。我们的第二个目标将确定是否发生密切的相互作用的PT claudins，在体外和原位，使用邻近连接试验。此外，我们建议测量过表达PT claudins的细胞中的钙渗透性，包括研究与低尿钙相关的claudin-2的人类SNP。大量证据表明，在体外，通过MEK抑制，肾上皮细胞中的claudin- 2表达增加。我们的最终目的是检查体内MEK抑制对肾密蛋白-2表达和尿钙排泄的影响。本研究的广泛、长期目标是阐明claudin-2在PT钙重吸收中的作用，包括其作为减少尿钙和预防复发性结石形成者肾结石形成的治疗靶点的潜力。

 DESCRIPTION (provided by applicant): Although kidney stones have affected mankind for as long as history has been recorded, they continue to present a considerable public health concern, and the prevalence of kidney stone disease has experienced an upswing in the past century. Recurrent kidney stone formers frequently lack any metabolic abnormalities, with the exception of an increase in urine calcium. In the kidney, the proximal tubule (PT) is responsible for the reabsorption of approximately two-thirds of filtered calcium. Evidence that PT calcium reabsorption occurs through a passive and paracellular route is abundant, although the precise mechanism by which this occurs is not known. The selectivity of paracellular permeability is determined in large part by claudins, a family of proteins found at the tight junction between epithelial cells. Claudin-2 is a cation-selective claudin that is expressed in leaky epithelia, including the PT and gastrointestinal tract. Claudin-2 knockout (Cldn-2 KO) mice have increased fractional calcium excretion compared with wild type (WT) mice. The cause of this increase in calcium excretion has not been investigated carefully. We hypothesize that claudin-2 mediates paracellular calcium reabsorption in the PT. For our first aim, we plan to assess the calcium wasting phenotype in Cldn-2 KO mice. We also provide evidence that Cldn-2 KO mice develop nephrocalcinosis, and we propose studies to investigate this relationship further. Extracellular fluid (ECF) volume contraction reduces urine calcium by causing an increase in PT calcium reabsorption. To test the hypothesis that claudin-2 contributes to PT calcium reabsorption, specifically, we propose experiments measuring urine calcium in WT and Cldn-2 KO mice following induction of ECF volume contraction. Heteromeric claudin interactions may play a significant role in the permeability properties of epithelial tissues. In the PT, the most abundanty expressed claudins appear to be claudin-2, claudin-3, and claudin-10a. Our second aim will determine whether close interaction of PT claudins occurs, in vitro and in situ, using proximity ligation assays. In addition, we propose measurement of calcium permeability in cells overexpressing PT claudins, including investigation of a human SNP of claudin-2 associated with low urine calcium. Evidence is abundant that claudin- 2 expression is increased in renal epithelial cells by MEK inhibition, in vitro. Our final aim examines the effect of MEK inhibition, n vivo, on renal claudin-2 expression and urine calcium excretion. The broad, long term objective of this research is to elucidate the role of claudin-2 in PT calcium reabsorption, including its potential as a therapeutic target for reduction of urine calcium and prevention of kidney stone formation in recurrent stone formers.