| Part I Differences in the formation of calcium oxalate crystals induced by different Slc26a6 expression in rat kidneyObjects:Solute-linked carrier 26 gene family 6(Slc26a6),which is mainly expressed in intestines and kidneys,is a multifunctional anion transporter crucial in the transport of oxalate anions.This study aimed to investigate the role of kidney SLC26A6 in urolithiasisMethods:Patients were divided into two groups:stone formers and nonstone formers Samples were collected from patients following nephrectomy.Lentivirus with Slc26a6(lentivirus-Slc26a6)sequence and lentivirus with siRNA-Slc26a6(lentivirus-siRNA-Slc26a6)sequence were transfected into rats'kidneys respectively and Slc26a6 expression was detected using Western blot and immunohistochemical analyses After administering ethylene glycol,oxalate concentration and prevalence of stone formation between the transgenic and control groups were measured using 24-h urine analysis and Von Kossa staining,respectivelyResults:Immunohistochemical and Western blot analyses indicated that stone formers had a significantly higher level of expression of SLC26A6 in the kidney compared with the control group.After lentivirus infection,the urinary oxalate concentration and rate of stone formation in lentivirus-Slc26a6-tranfected rats increased remarkably,while lentivirus-siRNA-Slc26a6-transfected rats showed few crystalsConclusion:The results showed that high expression levels of renal SLC26A6 may account for kidney stone formation.Downregulating the expression of SLC26A6 in the kidney may be a potential therapeutic target to prevent or treat urolithiasisPart ?.Effect of Slc26a6 expression on the function of rat renal tubular epithelial cellsObjects:This section aims to investigate the effects of differences in Slc26a6 expression in renal tubular epithelial cells on oxalate-induced cellular oxidation and crystal formationMethods:Lentivirus(Lentivirus-Slc26a6)carrying the Slc26a6 gene sequence and lentivirus(Lentivirus-siRNA-Slc26a6)carrying the siRNA-Slc26a6 sequence which interferes with the expression of Slc26a6 were separately constructed.Renal tubular epithelial cells(NRK-52E)were stably transfected with lentivirus to up-regulate or down-regulate the expression of Slc26a6 in cells.Slc26a6 expression was determined by qPCR,western blot and immunofluorescence.Cell viability was detected by CCK8 method Apoptosis was detected by Annexin V/PI flow cytometry.Reactive oxygen species(ROS)were measured by DCFH-DA flow-cytometry.Malondialdehyde(MDA)production were detected.The crystal adhesion was observed using an optical microscope,and the ultrastructure of the cells was observed by a transmission electron microscope(TEM).Then Lentivirus-siRNA-Slc26a6 was transfected into rat kidneys.Three groups of rats,normal control,lentivirus-vector,and lentivirus-small interfering RNA(Lentivirus-siRNA-Slc26a6)groups,were used,and after lentivirus transfection,they were fed 1%ethylene glycol(EG)and 0.5%ammonium chloride(NH4C1)for 2 weeks.Dihydroethidium(DHE);terminal deoxynucleotidyl transferase(TdT)deoxyuridine dUTP nick-end labeling(TUNEL),and von Kossa staining were performed,and nuclear factor kB(NFkB)and osteopontin(OPN)expression were measuredResults:In the vitro study,compared to the control group,downregulated Slc26a6 NRK cells showed alleviation of the cell viability decrease,cell apoptosis rate,and ROS generation after oxalate treatment.Crystal adhesion and vesicles were significantly less after oxalate exposure than in the untreated controls.Rats infected with lentivirus-siRNA exhibited attenuated SOD generation,cell apoptosis,and crystal formation in the kidneys Increased phosphorylation of NF?B and OPN was involved in the pathological processConclusion:The results of this section suggest that reducing the expression of Slc26a6 in the kidney may be a potential strategy to prevent stone formationPart III.Analysis of altered microRNA expression profiles in experimental hyperoxaluric ratsObjects:In the present study,differences in the miRNA expression profiles between experimental hyperoxaluric rats and normal rats were analyzed,in order to identify target genes and signaling pathways involved in the pathogenesis of hyperoxaluriaMethods:Ethylene glycol and ammonium chloride was fed to male hyperoxaluric rats(EXP)and normal age-matched male rats(CON).The oxalate concentration in the urine of each experimental rat was collected every 24 h and measured on day 14.Three rats exhibiting the highest concentrations were selected for micro-array analysis.Microarray analysis was performed to evaluate differences in the expression of microRNA(miRNA)in the kidney tissues from EXP and CON groups,and miRNAs that exhibited a>2-fold or a<0.5-fold alteration in expression between these groups were screened for differential expression patterns according to the threshold P-values.qPCR analysis was employed to confirm the microarray results.In order to predict the potential role of miRNAs in pathophysiological processes,gene ontology(GO),pathway and target prediction analyses were conductedResults:A total of 28 miRNAs were observed to be differentially expressed(>2-fold change)between EXP and CON groups.Among these miRNAs,20 were upregulated and 8 were downregulated.GO and pathway analyses revealed that the insulin resistance and phosphatidylinositol-bisphosphonate 3-kinase/AKT serine threonine kinase signaling pathways were potentially associated with miRNA regulation in this settingConclusion:In conclusion,the results of the present study identified differentially expressed miRNAs in hyperoxaluric rats,and provided a novel perspective for the role of miRNAs in the formation of calcium oxalate stones. |
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