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In vivo Drosophilia genetic model for calcium oxalate nephrolithiasis.

ABSTRACT: 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.

SUBMITTER: Hirata T

PROVIDER: S-EPMC3532482 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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In vivo Drosophilia genetic model for calcium oxalate nephrolithiasis.

Hirata Taku T Cabrero Pablo P Berkholz Donald S DS Bondeson Daniel P DP Ritman Erik L EL Thompson James R JR Dow Julian A T JA Romero Michael F MF

American journal of physiology. Renal physiology 20120919 11

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 th ...[more]

PMID: 22993075

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Project description:Oxidative stress, a well-known cause of stress-induced premature senescence (SIPS), is increased in patients with calcium oxalate (CaOx) kidney stones (KS). Oxalate and calcium oxalate monohydrate (COM) induce oxidative stress in renal tubular cells, but to our knowledge, their effect on SIPS has not yet been examined. Here, we examined whether oxalate, COM, or urine from patients with CaOx KS could induce SIPS and telomere shortening in human kidney (HK)-2 cells, a proximal tubular renal cell line. Urine from age- and sex-matched individuals without stones was used as a control. In sublethal amounts, H2O2, oxalate, COM, and urine from those with KS evoked oxidative stress in HK-2 cells, indicated by increased protein carbonyl content and decreased total antioxidant capacity, but urine from those without stones did not. The proportion of senescent HK-2 cells, as indicated by SA-βgal staining, increased after treatment with H2O2, oxalate, COM, and urine from those with KS. Expression of p16 was higher in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than it was in cells treated with urine from those without stones and untreated controls. p16 was upregulated in the SA-βgal positive cells. Relative telomere length was shorter in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS than that in cells treated with urine from those without stones and untreated controls. Transcript expression of shelterin components (TRF1, TRF2 and POT1) was decreased in HK-2 cells treated with H2O2, oxalate, COM, and urine from those with KS, in which case the expression was highest. Urine from those without KS did not significantly alter TRF1, TRF2, and POT1 mRNA expression in HK-2 cells relative to untreated controls. In conclusion, oxalate, COM, and urine from patients with CaOx KS induced SIPS and telomere shortening in renal tubular cells. SIPS induced by a lithogenic milieu may result from upregulation of p16 and downregulation of shelterin components, specifically POT1, and might contribute, at least in part, to the development of CaOx KS.

Project description:BackgroundThe aim of this study was to use bioinformatics approaches to screen and identify the key genes of idiopathic calcium oxalate nephrolithiasis, and explore its potential molecular mechanism.MethodsThe GSE73680 kidney stone data set was downloaded from the Gene Expression Omnibus (GEO). R software (The R Foundation for Statistical Computing) was used to screen differentially expressed genes. GeneMANIA and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases were used to analyze related genes interacting with crucial genes, and a protein-protein interaction (PPI) network was constructed. The differential genes were then subjected to the Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. The clinical data of 156 patients who received percutaneous nephrolithotomy (PCNL) therapy at our facility between January 2013 and December 2017 were retrospectively analyzed. The various parameters associated with postoperative urogenous sepsis were identified using multivariable logistic regression analysis.ResultsThe study discovered one differentially expressed gene was nucleotide-binding oligomerization domain-containing protein 2 (NOD2). GO and KEGG analysis showed that NOD2 might affect the occurrence of idiopathic calcium oxalate kidney stones by affecting inflammation, receptor expression, immune environment, necrosis, apoptosis, and other pathways. The clinical parameter of patients who participated in the study, including preoperative urinary white blood cell (WBC) count, preoperative urinary nitrite, stone diameter, operation time, WBC count, and WBC D values, were statistically different between the systemic inflammatory response syndrome (SIRS) group and the urosepsis group. According to multivariate logistic regression analysis, the preoperative urine nitrite, calculus diameter, blood WBC, and NOD2 expression 3 hours after surgery were all independently associated with the urosepsis development.ConclusionsPreoperative urinary nitrite positive status, postoperative WBC count ≥2.98×109/L 3 hours after operation, stone diameter >6 cm, and low expression of NOD2 in renal papillary tissue are more likely to cause the urinary source of idiopathic calcium oxalate nephrolithiasis after PCNL urogenous sepsis. These parameters also offer a viable treatment paradigm for the perioperative management of PCNL in treating idiopathic calcium oxalate kidney stones.

Project description:BackgroundUrinary oxalate is a major risk factor for calcium oxalate stones. Marked hyperoxaluria arises from mutations in 2 separate loci, AGXT and GRHPR, the causes of primary hyperoxaluria (PH) types 1 (PH1) and 2 (PH2), respectively. Studies of null Slc26a6(-/-) mice have shown a phenotype of hyperoxaluria, hyperoxalemia, and calcium oxalate urolithiasis, leading to the hypothesis that SLC26A6 mutations may cause or modify hyperoxaluria in humans.Study designCross-sectional case-control.Setting & participantsCases were recruited from the International Primary Hyperoxaluria Registry. Control DNA samples were from a pool of adult subjects who identified themselves as being in good health.PredictorPH1, PH2, and non-PH1/PH2 genotypes in cases.Outcomes & measuresHomozygosity or compound heterozygosity for SLC26A6 variants. Functional expression of oxalate transport in Xenopus laevis oocytes.Results80 PH1, 6 PH2, 8 non-PH1/PH2, and 96 control samples were available for SLC26A6 screening. A rare variant, c.487C-->T (p.Pro163Ser), was detected solely in 1 non-PH1/PH2 pedigree, but this variant failed to segregate with hyperoxaluria, and functional studies of oxalate transport in Xenopus oocytes showed no transport defect. No other rare variant was identified specifically in non-PH1/PH2. Six additional missense variants were detected in controls and cases. Of these, c.616G-->A (p.Val206Met) was most common (11%) and showed a 30% reduction in oxalate transport. To test p.Val206Met as a potential modifier of hyperoxaluria, we extended screening to PH1 and PH2. Heterozygosity for this variant did not affect plasma or urine oxalate levels in this population.LimitationsWe did not have a sufficient number of cases to determine whether homozygosity for p.Val206Met might significantly affect urine oxalate.ConclusionsSLC26A6 was effectively ruled out as the disease gene in this non-PH1/PH2 cohort. Taken together, our studies are the first to identify and characterize SLC26A6 variants in patients with hyperoxaluria. Phenotypic and functional analysis excluded a significant effect of identified variants on oxalate excretion.

Project description:BackgroundAs a common urological disease with a high recurrence rate, nephrolithiasis caused by CaOx may elicit a strong immunologic response. We present a CyTOF-based atlas of the immune landscape in nephrolithiasis models to understand how the immune system contributes to, and is affected by, the underlying response caused by SIRT3 knockout and CaOx inducement.Materials and methodsWe performed a large-scale CyTOF analysis of immune cell abundance profiles in nephrolithiasis. The immunophenotyping data were collected from four different mouse models, including the SIRT3 wild-type or knockout, including and excluding CaOx inducement. Unsupervised analysis strategies, such as SPADE and viSNE, revealed the intrarenal resident immune components and the immune alterations caused by SIRT3 knockout and CaOx-induced renal injury.ResultsAn overview analysis of the immune landscape identified T cells and macrophages as the main immune cell population in nephrolithiasis models. Highly similar phenotypes were observed among CD4+ and CD8+ T cell subsets, including cells expressing Ki67, Ly6C, Siglec-F, and TCRβ. Macrophages expressed a characteristic panel of markers with varied expression levels including MHC II, SIRPα, CD11c, Siglec-F, F4/80, CD64, and CD11b, indicating more subtle differences in marker expression than T cells. The SIRT3KO/CaOx and SIRT3WT/CaOx groups exhibited global differences in the intrarenal immune landscape, whereas only small differences existed between the SIRT3KO/CaOx and SIRT3KO/Ctrl groups. Among the major immune lineages, the response of CD4+ T cells, NK cells, monocytes, and M1 to CaOx inducement was regulated by SIRT3 expression in contrast to the expression changes of B cells, DCs, and granulocytes caused by CaOx inducement. The panel of immune markers influenced by CaOx inducement significantly varied with and without SIRT3 knockout.ConclusionIn a CaOx-induced nephrolithiasis model, SIRT3 has a critical role in regulating the immune system, especially in reducing inflammatory injury. The characteristic panel of altered immune clusters and markers provides novel insights leading to improved prediction and management of nephrolithiasis.

Dataset Information

In vivo Drosophilia genetic model for calcium oxalate nephrolithiasis.

Publications

In vivo Drosophilia genetic model for calcium oxalate nephrolithiasis.

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