Bondeson, Daniel P.

Background Kidney stones (nephrolithiasis) are an expensive and painful health problem with a complex etiology with ~70% of stones composed of calcium oxalate (CaOx). The Slc26 gene family contains several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl − for oxalate (ox 2− ), as well as SO 4 2− , HCO 3 − and formate. Slc26a6 −/− mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. We developed a Drosophila genetic model for CaOx stones (dPrestin, Slc26a5/6, manipulation) allowing rapid gut, renal and whole‐body physiological assessment (min/h v. mo/y). Methods (Tissue) First, Malpighian tubules (MT, renal tubule) were dissected from wild type and dPrestin knockdown mutants and soaked in ox 2− solutions. CaOx stones were visualized by DIC microscopy. Second, we generated Drosophila expressing eYFP in the MT, MT+gut or whole body. eYFP is a halide (Cl − ) sensor, allowing quantification of Cl − /ox 2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox 2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT). Results Exposing dPrestin‐knockdowns to bath ox 2− elicited almost no CaOx crystals. However, exposing wt‐MT to ox 2− elicited many CaOx crystals in 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl − /ox 2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox 2− longer. Conclusions Our observations indicate that Drosophila and dPrestin manipulation can be used for rapid, detailed studies of gut, renal and whole organism oxalate metabolism & CaOx stone formation. [DK83007]

Nephrolithiasis is a major public health problem with a complex and varied etiology. Most stones are composed of calcium oxalate (CaOx), with dietary excess a risk factor. Because of complexity of mammalian system, the details of stone formation remain to be understood. Here we have developed a nephrolithiasis model using the genetic model Drosophila melanogaster, which has a simple, transparent kidney tubule. Drosophilia reliably develops CaOx stones upon dietary oxalate supplementation, and the nucleation and growth of microliths can be viewed in real time. The Slc26 anion transporter dPrestin (Slc26a5/6) is strongly expressed in Drosophilia kidney, and biophysical analysis shows that it is a potent oxalate transporter. When dPrestin is knocked down by RNAi in fly kidney, formation of microliths is reduced, identifying dPrestin as a key player in oxalate excretion. CaOx stone formation is an ancient conserved process across >400 My of divergent evolution (fly and human), and from this study we can conclude that the fly is a good genetic model of nephrolithiasis.