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Oxidative stress is a major cause of diabetic nephropathy. Upregulation of the key antioxidative transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), was found to prevent the development of diabetic nephropathy. The present study was designed to explore the therapeutic effect of Nrf2 induced by proteasomal inhibitor MG132 at a low dose (10 μg/kg) on diabetic nephropathy. Transgenic type 1 diabetic (OVE26) mice displayed renal dysfunction with albuminuria by 3 mo of age, at which time MG132 treatment was started. After 3-mo treatment with MG132, renal function, morphology, and biochemical changes were examined with real-time PCR, Western blotting, and immunohistochemical examination. Compared with age-matched, nontreated diabetic mice, MG132-treated diabetic mice showed significant improvements in terms of renal structural and functional alterations. These therapeutic effects were associated with increased Nrf2 expression and transcriptional upregulation of Nrf2-regulated antioxidants. Mechanistic study using human renal tubular HK11 cells confirmed the role of Nrf2, as silencing the Nrf2 gene with its specific siRNA abolished MG132 prevention of high-glucose-induced profibrotic response. Furthermore, diabetes was found to significantly increase proteasomal activity in the kidney, an effect that was significantly attenuated by 3 mo of treatment with MG132. These results suggest that MG132 upregulates Nrf2 function via inhibition of diabetes-increased proteasomal activity, which can provide the basis for the therapeutic effect of MG132 on the kidney against diabetes-induced oxidative damage, inflammation, fibrosis, and eventual dysfunction. [ABSTRACT FROM AUTHOR]

Aim: To define renal gene expression during the development of severe albuminuria in OVE26 diabetic mice. Methods: Kidney microarray analysis was performed at 2, 4 and 8 months. Data were validated by RT-PCR, in situ hybridization and immunohistochemistry. Results: Gene expression differences between control and diabetic mice increased 10-fold from 2 to 8 months. This change was most obvious for inflammatory genes. Three inflammatory genes, complement C3, VCAM1 and CD44 were upregulated more than 4-fold. Inflammatory gene expression correlated with albuminuria and C3 and CD44 increased in tubules that accumulated albumin. VCAM1 was induced in different tubules that were neither dilated nor accumulated albumin. Six of 8 genes previously reported to be markers of human advanced diabetic nephropathy and the NF-κB_IFN_x promoter module were elevated in the oldest diabetic mice. Vitamin D inhibits diabetic renal inflammation. Vitamin D and mRNA for vitamin D synthetic enzyme CYP2B1 were elevated in kidneys of young OVE26 mice. Conclusions: OVE26 mice induce inflammatory genes consistent with advanced renal disease, associated with severe albuminuria and to a greater extent than reported in other diabetic models. They provide an excellent model of diabetic nephropathy to assess the effect of induction of inflammatory proteins. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: OVE26 (OVE) mice provide a valuable model of advanced diabetic nephropathy (DN), but they take 8 months to develop moderate interstitial fibrosis and reduced glomerular filtration rate (GFR). The aim of this project was to produce a more rapid and advanced model of DN. Methods: Uninephrectomy was applied to OVE and FVB mice at 2 months of age. Albuminuria, GFR, glomerulosclerosis, interstitial fibrosis, gene expression and monocyte infiltration were evaluated as a function of diabetes and uninephrectomy. Results: Albuminuria, monocyte infiltration, mesangial matrix expansion and renal fibrosis were greatly accelerated in uninephrectomized mice. DN was more advanced 10 weeks after uninephrectomy than in untreated OVE mice at 8 months of age. Uninephrectomy had almost no effect on these characteristics in non-diabetic mice. Microarray studies indicated that the accelerated fibrosis and cell infiltration in nephrectomized OVE mice were accompanied by corresponding gene expression changes in canonical pathways for fibrosis and inflammation. Conclusion: Uninephrectomy greatly accelerates all features of diabetic renal damage. This procedure provides a 10-week period after surgery to examine very large changes in the pathology of DN. The model may be particularly useful for testing new therapies and for analysis of the progression of albuminuria and fibrosis in DN. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Diabetic patients have a high risk of pulmonary disorders that are usually associated with restrictive impairment of lung function, suggesting a fibrotic process (van den Borst B, Gosker HR, Zeegers MP, Schols AM. Chest 138: 393-406, 2010; Ehrlich SF, Quesenberry CP Jr, Van Den Eeden SK, Shan J, Ferrara A. Diabetes Care 33: 55-60, 2010). The present study was undertaken to define whether and how diabetes causes lung fibrosis. Lung samples from streptozotocin-induced type 1 diabetic mice, spontaneously developed type 1 diabetic OVE26 mice, and their age-matched controls were investigated with histopathological and biochemical analysis. Signaling mechanism was investigated with cultured normal human lung fibroblasts in vitro. In both diabetes models, histological examination with Sirius red and hemotoxylin and eosin stains showed fibrosis along with massive inflammatory cell infiltration. The fibrotic and inflammatory processes were confirmed by real-time PCR and Western blotting assays for the increased fibronectin, CTGF, PAI-1, and TNFa mRNA and protein expressions. Diabetes also significantly increased NADPH oxidase (NOX) expression and protein nitration along with upregulation of angiotensin II (Ang II) and its receptor expression. In cell culture, exposure of lung fibroblasts to Ang II increased CTGF expression in a dose- and time-dependent manner, which could be abolished by inhibition of superoxide, NO, and peroxynitrite accumulation. Furthermore, chronic infusion of Ang II to normal mice at a subpressor dose induced diabetes-like lung fibrosis, and Ang II receptor AT1 blocker (losartan) abolished the lung fibrotic and inflammatory responses in diabetic mice. These results suggest that Ang II plays a critical role in diabetic lung fibrosis, which is most likely caused by NOX activation-mediated nitrosative damage. [ABSTRACT FROM AUTHOR]

OVE26 (OVE) diabetic mice on the inbred strain FVB are a valuable model of diabetic nephropathy that excretes the highest amount of urine albumin of all diabetic mouse models. Crossing of OVE mice to C57BL6 or DBA2 mice reduced albuminuria 17-fold in F1 diabetic offspring without reducing diabetes. When comparing renal histology of OVE mice on the FVB background to F1 C57BL6 crosses, we found that the F1 kidneys had significantly smaller glomeruli, much less albumin accumulation in tubules, reduced mesangial matrix expansion, and less interstitial fibrosis. A genome scan of 108 OVE-positive N2 offspring for albuminuria revealed one significant peak on chromosome 11 and nearly significant peaks on chromosomes 9, 13, and 19. Homozygosity for the FVB genotype for peaks on chromosomes 11, 13, or 19 increased albuminuria. Homozygosity for the chromosome 9 peak reduced albuminuria. Combined homozyogosity for the peaks on chromosomes 11, 13, and 19 increased albuminuria over 12-fold and accounted for >70% of the difference between OVE mice on the FVB vs. the F1 background. These loci contain sequences important to susceptibility to diabetic albuminuria. [ABSTRACT FROM AUTHOR]

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electronmicroscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport. [ABSTRACT FROM AUTHOR]

Background/Aims: Tubulointerstitial fibrosis (TIF) is a prominent feature of progressive diabetic nephropathy. The goal of this study was to determine if hallmarks of TIF occur in the transgenic OVE26 type 1 diabetic mouse and define signaling events associated with TIF. Methods: The expression patterns of several phenotypic markers of TIF were determined in kidneys of OVE26 diabetic and control mice by immunohistochemistry and immunoblot analysis. Results: Pathological signatures of TIF are an accumulation of myofibroblasts and excessive deposition of extracellular matrix in the tubulointerstitium. Kidneys from OVE26 diabetic animals exhibited an increase in tubulointerstitial myofibroblast marker (α-smooth muscle actin), fibronectin and collagen I staining. Abundance of the pro-fibrotic cytokine TGF-β was also enhanced in diabetic tubules. As injury involving loss of epithelial cell-cell contact promotes tissue fibrosis, we examined expression of the adhesion protein, E-cadherin. The percent of E-cadherin-stained tubules was decreased in diabetic kidneys. Prominent regulators of TGF-β signaling, glycogen synthase kinase-3 (GSK-3) α and β, were also differentially expressed. Conclusions: These results indicate that TGF-β-induced TIF occurs in OVE26 diabetic mice, providing a practical in vivo model for defining novel regulatory events and treatment strategies for diabetes-induced TIF. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy is a common complication leading to heightened risk of heart failure and death. In the present report, we performed proteomic analysis on total cardiac proteins from the OVE26 mouse model of type 1 diabetes to identify protein changes that may contribute to diabetic cardiomyopathy. This analysis revealed that a surprising high proportion (12 of 20) of the altered proteins that could be identified by mass spectrometry were of mitochondrial origin. All but one of these proteins were upregulated by diabetes. Quantitative RT-PCR, performed for two of these proteins, indicated that part of the upregulation was attributed to increased messenger RNA levels. Morphological study of diabetic hearts showed significantly increased mitochondrial area and number as well as focal regions with severe damage to mitochondria. Diabetic mitochondria also showed reduced respiratory control ratio (9.63 ± 0.20 vs. 6.13 ± 0.41, P < 0.0001), apparently due to reduced state 3 rate, and diminished GSH level (5.5 ± 0.9 vs. 8.2 ± 2.5 µmol/mg protein, P < 0.05), indicating impaired mitochondrial function and increased oxidative stress. Further examination revealed increased mitochondrial DNA (1.03 ± 0.18 vs. 0.69 ± 0.13 relative copy number, P < 0.001) and a tendency to higher protein yield in OVE26 cardiac mitochondria, as well as increased mRNA level for mitochondrial transcription factor A and two mitochondrial encoded proteins. Taken together, these results show that mitochondria are a primary target in the diabetic heart, probably due to oxidative stress, and that this damage coincides with and may stimulate mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]