Drosophila model of oxalate nephrolithiasis

描述（由申请人提供）：肾结石（肾结石）是常见的，通常疼痛，在许多情况下产生肾脏并发症。2005年，美国在肾结石相关治疗和并发症上花费了超过50亿美元（约占当前NIH预算的20%）。草酸钙（CaOx）结石是最常见的（~70%）。肠道草酸盐吸收和过量尿草酸盐排泄是高尿酸患者形成CaOx结石的重要致病因素。Slc 26 a6是一种产电Cl-/ox 2-交换剂，参与草酸盐的肠吸收和肾排泄。Slc 26 a6基因敲除小鼠出现高尿症和CaOx结石。然而，患有特发性肾结石的患者不具有突变人SLC 26 A6 45。总之，这些发现表明（a）调节/信号传导蛋白（而不是转运蛋白突变）可能控制Slc 26 a6介导的草酸盐转运和/或（B）草酸盐转运竞争对于CaOx结石形成是重要的（即，通过底物可用性的差异可能存在底物竞争）。 在这个提议中，我们将进一步开发一个果蝇遗传模型的CaOx石头。我们的初步数据表明，肠道草酸盐吸收和肾小管草酸盐分泌都保存在苍蝇和介导的dPrestin（Slc 26 a5/a6），哺乳动物Slc 26 a6的功能直系同源物。dPrestin的遗传敲低降低小管CaOx晶体含量。对于这个提议，我们将使用这种CaOx石头的新果蝇模型来实现3个目标。首先（目的1），我们将通过（a）dPrestin与小鼠Slc 26 a6转运活性的详细比较来进一步评估我们的果蝇模型（非洲爪蟾卵母细胞），（B）使用双折射、eYFP-Cl传感和微CT，具有肠或小管特异性dPrestin敲低，（c）使用SILAC（在细胞培养物中通过氨基酸进行稳定同位素标记）质谱以阐明上述组织特异性的蛋白质补偿，基因敲除，和（d）体外（抗体定位）和体内（标记的）定位dPrestin 转基因）。这些目标将表征体外（非洲爪蟾卵母细胞表达系统）以及体内（苍蝇中的结石形成）的功能。第二（目的2），我们假设WNK/SPAK/OSR 1信号是肠道和肾小管草酸盐转运生理的控制器。我们的数据表明（a）WNK 3通过哺乳动物Slc 26 a6激活草酸盐转运，（B）果蝇OSR 1通过dPrestin激活草酸盐转运。果蝇有一个WNK和一个OSR 1（没有SPAK），使这种遗传模型成为因果研究的理想模型。我们将使用为目标1开发的相同工具。最后（目的3），我们假设，草酸盐转运的变化，导致CaOx结石可能会发生由于Slc 26 a6/dPrestin底物竞争。生理或稳态草酸盐转运变化很可能发生，因为临界肠道草酸盐摄取或小管分泌加速，因为Slc 26 a6/dPrestin可以优先移动草酸盐，即使存在其他底物（HCO 3-，SO 42-，甲酸盐等）。这一假设将在体外用dPrestin-卵母细胞进行检验，并在体内用喂养方案进行检验。 结合上面的工具。我们的期望是，我们的实验将揭示果蝇代谢和信号传导的关键组成部分，这将使我们或其他人能够在未来以成本和时间有效的方式将研究资源集中在哺乳动物和人类CaOx结石原因上。 公共卫生相关性：草酸盐肾结石的果蝇模型肾结石是常见的、痛苦的、产生肾脏并发症并且花费> 50亿美元/年（约NIH预算的20%）。最常见的结石类型是草酸钙（约占所有结石的70%）。肠道草酸盐吸收和尿草酸盐排泄是这种人类疾病的重要因素。然而，确定尿草酸水平的机制并不完全确定。在哺乳动物中，一种蛋白质（Slc 26 a6）将草酸盐移入或移出肾脏和肠道。缺失Slc 26 a6基因的小鼠形成草酸钙结石。虽然该基因已在数千例肾结石患者中测序，但仅发现1个突变（超过90%的功能）。我们假设草酸盐转运是草酸盐结石形成的关键组成部分。对于一个简单明了的研究，需要一个简单的遗传模型，具有可以应用遗传学的简单器官。在这项研究中，我们开发了一个草酸盐肾结石（晶体形成）的果蝇遗传模型。果蝇版本的哺乳动物Slc 26 a6（dPrestin，Slc 26 a5）转运草酸盐，当基因缺失时，会导致肾小管中草酸盐晶体的形成。这个果蝇模型使我们能够确定其他基因，增加或减少草酸转运蛋白的活性，在整个动物，以及如果竞争底物改变草酸转运。使用整个果蝇肠道和肾脏，我们还将确定其他蛋白质的变化，在这个过程中使用大量蛋白质鉴定方法。随着调节基因的确定，未来的研究可以确定这些基因是否突变导致人类肾结石。重要的是，这种苍蝇模型将使我们能够确定临床治疗（通过喂食）是否有效。 苍蝇使用的药物/化合物）实际上减少草酸盐肾结石。

DESCRIPTION (provided by applicant): Kidney stones (nephrolithiasis) are common, often painful, and in many cases produce renal complications. In 2005, the US spent over $5 billion (~20% of the current NIH budget) on kidney stone related treatment and complications. Calcium oxalate (CaOx) stones are the most common (~70%). Gut oxalate absorption and excess urinary oxalate excretion are important pathogenic factors producing the CaOx stones that form in patients with hyperoxaluria. Slc26a6 is an electrogenic Cl-/ox2- exchanger involved in both intestinal absorption and renal excretion of oxalate. Slc26a6 knockout mice develop hyperoxaluria and CaOx stones. Nevertheless, patients with idiopathic nephrolithiasis do not have mutations human SLC26A6 45. Together, these findings indicate that either (a) regulatory/signaling proteins (rather than transporter mutations) likely control Slc26a6 mediated oxalate transport and/or (b) oxalate transport competition be important for CaOx stone formation (i.e., there may be substrate competition through differences in substrate availability). In this proposal, we will further develop a Drosophila genetic model of CaOx stones. Our preliminary data illustrate that both gut oxalate absorption and tubule oxalate secretion are preserved in flies and mediated by dPrestin (Slc26a5/a6), the functional orthologue of mammalian Slc26a6. Genetic knockdown of dPrestin decreases tubule CaOx crystal content. For this proposal, we will pursue 3 aims using this new Drosophila model of CaOx stones. First (Aim 1), we will further evaluate our fly model by (a) a detailed comparison of dPrestin v mouse Slc26a6 transport activity (Xenopus oocytes), (b) using birefringence, eYFP-Cl-sensing and microCT with gut- or tubule-specific dPrestin knockdown, (c) using SILAC (stable isotope labeling by amino acids in cell culture) mass spectroscopy to elucidate protein compensation for the above tissue-specific, genetic knockdowns, and (d) localize dPrestin in vitro (antibody localization) and in vivo (labeled transgenics). These Aims will characterize functions in vitro (Xenopus oocyte expression system) as well as in vivo (stone formation in flies). Second (Aim 2), we hypothesize that WNK/SPAK/OSR1 signaling is a controller of both gut and tubule oxalate transport physiologically. Our data show that (a) WNK3 activates oxalate transport by mammalian Slc26a6 and (b) Drosophila OSR1 activates oxalate transport by dPrestin. Drosophila has one WNK and one OSR1 (no SPAK), making this genetic model ideal for cause and effect studies. We will utilize the same tools developed for Aim 1. Finally (Aim 3), we hypothesize that oxalate transport changes resulting in CaOx stones may occur due to Slc26a6/dPrestin substrate competition. Physiologic or homeostatic oxalate transport changes could well occur because critical gut oxalate uptake or tubule secretion is accelerated because Slc26a6/dPrestin may preferentially move oxalate even if other substrates are present (HCO3-, SO42-, formate, etc). This hypothesis will be tested in vitro with dPrestin-oocytes and in vivo with feeding regiments in conjunction the tools above. Our expectation is that our experiments will reveal key-components of metabolism and signaling in the fly, which in the future will allow us or others to cost- and time-effectively focus research resources in mammals and human CaOx stone causes. PUBLIC HEALTH RELEVANCE: Drosophila model of oxalate nephrolithiasis Kidney stones are common, painful, produce renal complications and cost > $5 billion/y (~20% of the NIH budget). The most common stone type is calcium oxalate (~70% of all stones). Intestinal oxalate absorption and urinary oxalate excretion are important factors in this human disease. Yet, the mechanisms that determine urinary oxalate levels are incompletely defined. In mammals, one protein (Slc26a6) moves oxalate into or out of the kidney and intestine. Mice missing the Slc26a6 gene form calcium oxalate stones. While this gene has been sequenced in thousands of humans with kidney stones, only 1 mutation (more than 90% function) has been found. We hypothesize that oxalate transport is a key component to oxalate stone formation. For a straight forward and clear study, a simple genetic model, with simple organs to which genetics can be applied, is needed. For this study, we have developed a Drosophila genetic model of oxalate kidney stones (crystal formation). The Drosophila version of mammalian Slc26a6 (dPrestin, Slc26a5) transports oxalate, and when genetically deleted causes oxalate crystal formation in tubules. This Drosophila model allows us to determine other genes which increase or decrease the oxalate transporter activity in the whole animal as well as if competing substrates alter oxalate transport. Using whole Drosophila gut and kidney, we will also determine what other proteins change in this process using mass-protein identification methods. As regulator genes are identified, future studies could determine if these genes are mutated to cause kidney stones in humans. Importantly, this fly model will allow us to determine if clinical therapies (by feeding flies the drugs /compounds used) actually reduce oxalate kidney stones.