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.

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Rasooly, Rebekah S
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Mayo Clinic, Rochester
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Landry, Greg M; Hirata, Taku; Anderson, Jacob B et al. (2016) Sulfate and thiosulfate inhibit oxalate transport via a dPrestin (Slc26a6)-dependent mechanism in an insect model of calcium oxalate nephrolithiasis. Am J Physiol Renal Physiol 310:F152-9
Halberg, Kenneth A; Terhzaz, Selim; Cabrero, Pablo et al. (2015) Tracing the evolutionary origins of insect renal function. Nat Commun 6:6800
Chintapalli, Venkateswara R; Kato, Akira; Henderson, Louise et al. (2015) Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities. Proc Natl Acad Sci U S A 112:11720-5
Cabrero, Pablo; Terhzaz, Selim; Romero, Michael F et al. (2014) Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis. Proc Natl Acad Sci U S A 111:14301-6
Brickshawana, Adipong; Hinson, Shannon R; Romero, Michael F et al. (2014) Investigation of the KIR4.1 potassium channel as a putative antigen in patients with multiple sclerosis: a comparative study. Lancet Neurol 13:795-806
Wang, Li-Li; Chen, An-Ping; Zhong, Nai-Qin et al. (2014) The Thellungiella salsuginea tonoplast aquaporin TsTIP1;2 functions in protection against multiple abiotic stresses. Plant Cell Physiol 55:148-61
Ito, Yusuke; Kato, Akira; Hirata, Taku et al. (2014) Na+/H+ and Na+/NH+4 activities of zebrafish NHE3b expressed in Xenopus oocytes. Am J Physiol Regul Integr Comp Physiol 306:R315-27
Miller, Joe; Chi, Thomas; Kapahi, Pankaj et al. (2013) Drosophila melanogaster as an emerging translational model of human nephrolithiasis. J Urol 190:1648-56
Li, Shanshan; Kato, Akira; Takabe, Souichirou et al. (2013) Expression of a novel isoform of Na(+)/H(+) exchanger 3 in the kidney and intestine of banded houndshark, Triakis scyllium. Am J Physiol Regul Integr Comp Physiol 304:R865-76
Linser, Paul J; Neira Oviedo, Marco; Hirata, Taku et al. (2012) Slc4-like anion transporters of the larval mosquito alimentary canal. J Insect Physiol 58:551-62

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