Landry, Greg M.

Background Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of end‐stage renal disease in adults affecting 1 in 800 people in the US. Knocking down bicaudal‐C (BicC) in zebrafish and Xenopus has been reported as a method used to model the cystic changes in ADPKD. Gamberi and colleagues [JASN 25: 538A, 2014] recently presented a similar knockdown in Drosophila melanogaster resulting in cystic Malpighian Tubules (MTs) homologous to the human kidney. Additionally, calcium oxalate (CaOx) kidney stones affect ~1 in 10 people in the US, with the incidence increasing ~2‐fold in PKD patients. Methods Our group has investigated the PKD phenotype in Drosophila using a Gal4/UAS‐RNAi system to knockdown the expression of BicC in MTs. We initiated CaOx crystal formation by feeding Na‐oxalate or adding to tubule bathing solution [Hirata, AJP 2012]. Results Penetrance of Uro, CapaR, and c724 drivers in the MTs were compared using UAS‐YFP, resulting in Uro::BicC expression in ~50% of cells with CapaR::BicC approaching 100% and c724 expression specific to MT stellate cells only. MT morphology was analyzed: controls [WT (0%); parental (18%)] occurrence v BicC knockdowns [Uro::BicC (25%); CapaR::BicC (15%); c724::BicC (6.3%)]. Functional tests using Ramsay secretion assays reveal a slower secretion rate in Uro::BicC, CapaR::BicC, and c724::BicC knockdowns compared to WT MTs. In CapaR::BicC knockdowns, slower secretion rates are observed even after stimulation with Drosophila ‐kinin. Since there is an increased risk for ADPKD‐related kidney stones, MT calcium oxalate (CaOx) crystallization experiments were performed to quantify crystal formation in BicC knockdown vs WT MTs. While crystals are present in all oxalate exposed MTs, BicC knockdowns using Uro::BicC and c724::BicC form more CaOx crystals than CapaR::BicC and WT. However, crystals formed by Uro::BicC and c724::BicC knockdowns have a significantly smaller total crystal area and average area/individual crystal compared to both CapaR::BicC and WT. Conclusions These results suggest that BicC knockdown parallels kidney stone increases associated with human PKD, but does not yet consistently mimic morphologic ADPKD‐induced cystic phenotypes usually observed in PKD models. Support or Funding Information DK90278, DK101405, DK101405

Zinc (Zn2+) is the second most abundant trace element, but is considered a micronutrient, as it is a cofactor for many enzymes and transcription factors. Whereas Zn2+ deficiency can cause cognitive immune or metabolic dysfunction and infertility, excess Zn2+ is nephrotoxic. As for other ions and solutes, Zn2+ is moved into and out of cells by specific membrane transporters: ZnT, Zip, and NRAMP/DMT proteins. ZIP10 is reported to be localized at the apical membrane of renal proximal tubules in rats, where it is believed to play a role in Zn2+ import. Renal regulation of Zn2+ is of particular interest in light of growing evidence that Zn2+ may play a role in kidney stone formation. The objective of this study was to show that ZIP10 homologs transport Zn2+, as well as ZIP10, kidney localization across species. We cloned ZIP10 from dog, human, and Drosophila ( CG10006), tested clones for Zn2+ uptake in Xenopus oocytes and localized the protein in renal structures. CG10006, rather than foi (fear-of-intimacy, CG6817) is the primary ZIP10 homolog found in Drosophila Malpighian tubules. The ZIP10 antibody recognizes recombinant dog, human, and Drosophila ZIP10 proteins. Immunohistochemistry reveals that ZIP10 in higher mammals is found not only in the proximal tubule, but also in the collecting duct system. These ZIP10 proteins show Zn2+ transport. Together, these studies reveal ZIP10 kidney localization, a role in renal Zn2+ transport, and indicates that CG10006 is a Drosophila homolog of ZIP10.

Nephrolithiasis is one of the most common urinary tract disorders, with the majority of kidney stones composed of calcium oxalate (CaOx). Given its prevalence (US occurrence 10%), it is still poorly understood, lacking progress in identifying new therapies because of its complex etiology. Drosophila melanogaster (fruitfly) is a recently developed model of CaOx nephrolithiasis. Effects of sulfate and thiosulfate on crystal formation were investigated using the Drosophila model, as well as electrophysiological effects on both Drosophila (Slc26a5/6; dPrestin) and mouse (mSlc26a6) oxalate transporters utilizing the Xenopus laevis oocyte heterologous expression system. Results indicate that both transport thiosulfate with a much higher affinity than sulfate Additionally, both compounds were effective at decreasing CaOx crystallization when added to the diet. However, these results were not observed when compounds were applied to Malpighian tubules ex vivo. Neither compound affected CaOx crystallization in dPrestin knockdown animals, indicating a role for principal cell-specific dPrestin in luminal oxalate transport. Furthermore, thiosulfate has a higher affinity for dPrestin and mSlc26a6 compared with oxalate These data indicate that thiosulfate's ability to act as a competitive inhibitor of oxalate via dPrestin, can explain the decrease in CaOx crystallization seen in the presence of thiosulfate, but not sulfate. Overall, our findings predict that thiosulfate or oxalate-mimics may be effective as therapeutic competitive inhibitors of CaOx crystallization.