The Establishment And Application Of A Drosophila Genetic Calcium Oxalate Nephrolithiasis Model

Calcium oxalate stone accounts for approximately 85%of renal stone diseases and the incidence of nephrolithiasis is increasing.However,there are currently no effective drug treatments available to dissolve stones directly in the kidney.In this study,we first establish the Drosophila malpighian tubule model to systematically study the molecular function of human genes identified from patients with nephrolithiasis.Then,we show that garcinia cambogia extract prevents calcium oxalate kidney stone formation in Drosophila larval renal tubules and treatment with Garcinia cambogia extract(GCE)completely removes calcium oxalate kidney stones from adult Drosophila renal tubules in vivo.Furthermore,we show that hydroxy citrate-a major component of GCE dissolves calcium oxalate stones in renal tubules ex vivo,suggesting that clinical-grade Garcinia cambogia extract could be used to dissolve calcium oxalate stones in patients with nephrolithiasis.Our study discovers a novel therapeutic strategy for the clinical treatment of nephrolithiasis.1.The establishment of a Drosophila genetic calcium oxalate nephrolithiasis modelObjective:To establishing a Drosophila model used to screen genes required for calcium oxalate kidney stone formation.Methods:(1).female and male Wild type flies were reared in fly food with different NaOx concentration,respectively,At different periods during the experiment,Malpighian tubules were dissected and a polarized light microscope was used to highlight the birefringent crystals of CaOx.(2).Wild type flies were reared on regular fly food containing different concentration of NaOx at 25?,The flies were transferred to a new vial every two days and the number of dead flies was counted every day,Survival assay was performed using GraphPad Prism5 software.Results:(1)administration of NaOx had the effect of elevating the incidence of crystal formation in a dose-dependent manner.(2)female formation CaOx stone more easy than male(3)high-dose administration of NaOx causes significant reduction of the life span of DrosophilaSummary:we chose to use female adult flies reared in 0.3%NaOx for 1week in the following experiments.2.Functional analysis of fly orthologs of candidate genes identified from patients with nephrolithiasisObjective:To systematically study the molecular function of human genes identified from patients with nephrolithiasis.Methods:(1)we first identified 30 genes that are associated with nephrolithiasis and whose fly orthologs are highly expressed in malpighian tubule,We classified these genes into 4 categories:oxalate metabolism related enzymes,vacuolar-type H+-ATPase,vitamin D associated receptor/hydroxylase and ion transporters.(2)10 virgins of Uro-Gal4/UAS-nGFP flies were crossed with 5 males of UAS-RNAi transgenic line in vials at 25?.Freshly hatched flies were transferred to fly food with different concentration of NaOx at 25? for 1 week.Malpighian tubules were dissected in PBS under dissection microscope and then subjected to the examination of renal stone in malpighian tubules under normal and polarized white light with an Olympus BX63 optical microscope.Results:knockdown of most of these genes resulted in increased calcium oxalate stone formation in malpighian tubule compared to wild type.Summary:The Drosophila malpighian tubule could be used to efficiently generate personalized in vivo models of genetic kidney stone diseases bearing patient-specific mutations and to elucidate the molecular mechanisms underlying the pathogenesis of most monogenic forms of nephrolithiasis.3.The treatment of Garcinia cambogia extract on NaOx-induced calcium oxalate kidney stone formation in Drosophila malpighian tubules.Objective:Discovery new drug for kidney stone therapy in Drosophila model.Methods:(1)Wild type flies were reared on regular fly food containing 1%NaOx and different concentration of HCA or GCE at 25? for one week.Malpighian tubules of 3rd instar larvae or adult female flies were dissected in PBS under dissection microscope and then subjected to the examination of renal stone in malpighian tubules under normal and polarized white light with an Olympus BX63 optical microscope.(2)Wild type flies were reared on regular fly food containing 0.5%NaOx at 25? for one week and renal tubule stone formation was evaluated briefly.Then,flies were transferred to fly food containing 0.1%NaOx and different concentration of HCA or GCE at 25? for 1 week.Malpighian tubules of adult female flies were dissected in PBS under dissection microscope and then subjected to the examination of renal stone in malpighian tubules under polarized white light with an Olympus BX63 optical microscope.(3)wild type or mutant flies were reared on regular fly food containing 0.3%NaOx for one week.Intact malpighian tubules were dissected in PBS under dissection microscope and transferred onto a slide.Then,100ul HCA or GCE solution was added to completely cover the MT tissue and subjected to live-imaging under polarized white light with an Olympus BX63 optical microscope.Pictures were taken every 20min and the total area of renal stones was measured using cellSens software.Renal stone dissolution rate was calculated by dividing the remaining stone area by the total stone area at the beginning.Results:(1)Garcinia cambogia extract(GCE)prevents calcium oxalate stone formation in both larval and adult renal tubules in a concentration-dependent manner.HCA supplemented in fly food has no effect on preventing stone formation in malpighian tubules,even in fly food containing 2%HCA(data not shown).(2)treatment with different concentration of Garcinia cambogia extract(GCE)for one week removed calcium oxalate kidney stones from adult renal tubules in a concentration-dependent manner.(3)hydroxycitrate(HCA)directly dissolves calcium oxalate stones in renal tubules ex vivo in a concentration-and time-dependent manner.Summary:Garcinia cambogia extract removes calcium oxalate renal stones via directly dissolving the stones by HCA in the renal tubules.