Project description:INTRODUCTION AND OBJECTIVE: Loss of renal function is often the impetus for operative intervention in renal obstruction. Obstructive nephropathy is characterized discrete morphological and physiologic changes including tubular dilatation, apoptosis, and atrophy, as well as interstitial cellular infiltration and progressive interstitial fibrosis. We hypothesize that there are gene expression alterations correlated with obstructive nephropathy that could serve as biomarkers for early intervention. METHODS: C57BL/6 mice were subjected to Unilateral Urethral Obstruction (UUO) or sham surgery at postnatal day 21 of life, and kidneys were harvested after 1, 2, 5 and 9 days postoperatively. RNA was extracted from kidneys and comprehensive gene expression profiling was performed with Agilent microarrays. We used biological replicates: 13 UUO replicates, 9 sham replicates. 2-Channel microarrays were hybridized using universal reference in the Cy3 channel. Ingenuity pathway analysis was used to analyze the biological function and gene networks of gene expression data. RESULTS: Microarray analysis revealed more than 1800 transcripts that were up- or down-regulated over days 1 through 9 following obstruction, and included many transcripts reported previously (FOS, CD44, CLU, SPP1 and EGF). Pathway analysis revealed significant enrichment of transcripts in cell activation/differentiation, immune/inflammatory responses, cell cycle, metabolic process and transport. Interestingly, IPA network analysis showed that transcriptional regulatory pathway involving CEBPB/HNF4A is involved in obstructive nephropathy. CONCLUSIONS: Transcript profiling identified numerous gene expression changes following UUO and suggests a role for CEBPB/HNF4A pathway in obstructive nephropathy. This dataset provides a foundation for development of biomarkers for obstructive nephropathy. Time: day samples were collected post operation Somatic Modification: mice were operated on to generate urethral obstruction (Obstructed/Control) time_series_design

Project description:INTRODUCTION AND OBJECTIVE: Loss of renal function is often the impetus for operative intervention in renal obstruction. Obstructive nephropathy is characterized discrete morphological and physiologic changes including tubular dilatation, apoptosis, and atrophy, as well as interstitial cellular infiltration and progressive interstitial fibrosis. We hypothesize that there are gene expression alterations correlated with obstructive nephropathy that could serve as biomarkers for early intervention. METHODS: C57BL/6 mice were subjected to Unilateral Urethral Obstruction (UUO) or sham surgery at postnatal day 21 of life, and kidneys were harvested after 1, 2, 5 and 9 days postoperatively. RNA was extracted from kidneys and comprehensive gene expression profiling was performed with Agilent microarrays. We used biological replicates: 13 UUO replicates, 9 sham replicates. 2-Channel microarrays were hybridized using universal reference in the Cy3 channel. Ingenuity pathway analysis was used to analyze the biological function and gene networks of gene expression data. RESULTS: Microarray analysis revealed more than 1800 transcripts that were up- or down-regulated over days 1 through 9 following obstruction, and included many transcripts reported previously (FOS, CD44, CLU, SPP1 and EGF). Pathway analysis revealed significant enrichment of transcripts in cell activation/differentiation, immune/inflammatory responses, cell cycle, metabolic process and transport. Interestingly, IPA network analysis showed that transcriptional regulatory pathway involving CEBPB/HNF4A is involved in obstructive nephropathy. CONCLUSIONS: Transcript profiling identified numerous gene expression changes following UUO and suggests a role for CEBPB/HNF4A pathway in obstructive nephropathy. This dataset provides a foundation for development of biomarkers for obstructive nephropathy. Time: day samples were collected post operation Somatic Modification: mice were operated on to generate urethral obstruction (Obstructed/Control)

Project description:Gene expression profiling of kidneys from the murine model of HIV-associated nephropathy (HIVAN) identified an association between the expression of an endoplasmic reticulum (ER)-associated protein reticulon-1, RTN1, and the severity of kidney disease. Of the three known RTN1 isoforms, only RTN1A protein expression was increased in kidneys of murine models of HIVAN, diabetic nephropathy (DN), and renal fibrosis and humans with HIVAN and DN. Both mRNA and protein expression of RTN1-A in the kidneys correlated inversely with estimated glomerular filtration rate (eGFR) in patients with DN. In kidney cells, RTN1 overexpression induced ER stress/apoptosis, whereas RTN1 knockdown attenuated tunicamycin-, and hyperglycemia-induced ER stress/apoptosis. Incubation of kidney cells with high glucose media induced RTN1A expression likely through oxidative pathway, while knockdown of RTN1A inhibited high glucose-induced apoptosis. RTN1A interacts with PERK and mutation of its N- or C-terminal domain abolished its effects on ER stress/apoptosis. In vivo, knockdown of Rtn1a expression either before or after kidney injury attenuated renal fibrosis in mice with unilateral ureteral obstruction (UUO) and tubular epithelial cell-specific knockdown of Rtn1a also ameliorated ER stress and renal fibrosis in the UUO mice. Finally, knockdown of Rtn1a also attenuated proteinuria, glomerular hypertrophy, and mesangial expansion in STZ-induced diabetic mice, which were associated with suppression of ER stress markers. Taken together, these data suggest that RTN1 is a mediator of kidney disease progression that exacerbates kidney injury through ER stress and apoptosis. Animal studies: All animal studies were approved by the IACUC committee of Mount Sinai School of Medicine. HIV-1 transgenic mice, Tg26, and their littermates were generated and genotyped as described. Only male heterozygous Tg26 in the FVB/N background were used in the study, because homozygous HIV-transgenic mice are not viable for more than few weeks postnatally. UUO and folic acid-induced nephropathy models were created as described . Mice were grouped as wild type, Tg26 with mild kidney injury, Tg26, with serious kidney injury. The kidneys were collected from these mice for histology, western blot, real-time PCR analysis, and microarray studies. Kidney disease was confirmed by measurement of proteinuria, renal function, and histologic analysis. Microarray studies: Affymetrix gene expression microarrays were performed at the Mount Sinai Institution Microarray Core Facility. The Affymetrix GeneChip® Mouse Genome 430 2.0 Array was used to profile gene expression in the kidney cortex of Tg26 and WT mice 37. One-way analysis of variance test (ANOVA) was applied to the dataset to identify the genes that were differentially expressed between the two groups. P-values were corrected using Benjamini–Hochberg false discovery rate (FDR) with a threshold of 0.05.

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Molecular Basis of Renal Adaptation in a Murine Model of Congenital Obstructive Nephropathy

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