Drosophila model of oxalate nephrolithiasis

描述（由申请人提供）：肾结石（肾结石）是常见的，通常是疼痛的，在许多情况下会产生肾脏并发症。2005年，美国在肾结石相关治疗和并发症上花费了50多亿美元（约占目前NIH预算的20%）。草酸钙（CaOx）结石最常见（约70%）。肠道草酸吸收和尿液草酸排泄过量是高草酸尿患者产生草酸结石的重要致病因素。Slc26a6是一种电致Cl-/ox2-交换剂，参与草酸的肠道吸收和肾脏排泄。Slc26a6基因敲除小鼠出现高尿酸和CaOx结石。然而，特发性肾结石患者没有人类SLC26A6 45突变。总之，这些发现表明：(a)调控/信号蛋白（而不是转运体突变）可能控制Slc26a6介导的草酸转运和/或(b)草酸转运竞争对草酸石形成很重要（即，可能存在底物竞争，通过底物可用性的差异）。在本研究中，我们将进一步建立果蝇CaOx结石的遗传模型。我们的初步数据表明，果蝇的肠道草酸吸收和小管草酸分泌都被保存下来，并由哺乳动物Slc26a6的功能同源物dPrestin （Slc26a5/a6）介导。基因敲低dPrestin可降低小管CaOx晶体含量。在本研究中，我们将利用这种新的果蝇CaOx结石模型来实现3个目标。首先（目标1），我们将通过(a) dPrestin与小鼠Slc26a6运输活性（非洲非洲人卵母细胞）的详细比较，(b)使用双折射，eyfp - cl传感和microCT与肠道或小管特异性dPrestin敲低，(c)使用SILAC（细胞培养中氨基酸稳定同位素标记）质谱来阐明上述组织特异性基因敲低的蛋白质补偿，进一步评估我们的苍蝇模型。(d)在体外（抗体定位）和体内（标记）定位dPrestin

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.