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Expression profiles of podocytes exposed to high glucose reveal new insights into early diabetic glomerulopathy.

ABSTRACT: Podocyte injury has been suggested to have a pivotal role in the pathogenesis of diabetic glomerulopathy. To glean insights into molecular mechanisms underlying diabetic podocyte injury, we generated temporal global gene transcript profiles of podocytes exposed to high glucose for a time interval of 1 or 2 weeks using microarrays. A number of genes were altered at both 1 and 2 weeks of glucose exposure compared with controls grown under normal glucose. These included extracellular matrix modulators, cell cycle regulators, extracellular transduction signals and membrane transport proteins. Novel genes that were altered at both 1 and 2 weeks of high-glucose exposure included neutrophil gelatinase-associated lipocalin (LCN2 or NGAL, decreased by 3.2-fold at 1 week and by 7.2-fold at 2 weeks), endothelial lipase (EL, increased by 3.6-fold at 1 week and 3.9-fold at 2 week) and UDP-glucuronosyltransferase 8 (UGT8, increased by 3.9-fold at 1 week and 5.0-fold at 2 weeks). To further validate these results, we used real-time PCR from independent podocyte cultures, immunohistochemistry in renal biopsies and immunoblotting on urine specimens from diabetic patients. A more detailed time course revealed changes in LCN2 and EL mRNA levels as early as 6 hours and in UGT8 mRNA level at 12 hours post high-glucose exposure. EL immunohistochemistry on human tissues showed markedly increased expression in glomeruli, and immunoblotting readily detected EL in a subset of urine samples from diabetic nephropathy patients. In addition to previously implicated roles of these genes in ischemic or oxidative stress, our results further support their importance in hyperglycemic podocyte stress and possibly diabetic glomerulopathy pathogenesis and diagnosis in humans.

SUBMITTER: Jain S

PROVIDER: S-EPMC3068212 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Expression profiles of podocytes exposed to high glucose reveal new insights into early diabetic glomerulopathy.

Jain Sanjay S De Petris Laura L Hoshi Masato M Akilesh Shreeram S Chatterjee Rajshekhar R Liapis Helen H

Laboratory investigation; a journal of technical methods and pathology 20101122 4

Podocyte injury has been suggested to have a pivotal role in the pathogenesis of diabetic glomerulopathy. To glean insights into molecular mechanisms underlying diabetic podocyte injury, we generated temporal global gene transcript profiles of podocytes exposed to high glucose for a time interval of 1 or 2 weeks using microarrays. A number of genes were altered at both 1 and 2 weeks of glucose exposure compared with controls grown under normal glucose. These included extracellular matrix modulat ...[more]

PMID: 21102505

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Project description:ObjectivePodocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy.Research design and methodsWe studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques.ResultsNondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an approximately 70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69-199) vs. 43 (26.8-69) mug/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice.ConclusionsPodocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

Project description:Diabetes is an increasing problem worldwide; almost 30 million people, nearly 10% of the population, in the United States are diagnosed with diabetes. Another 84 million are prediabetic, and without intervention, up to 70% of these individuals may progress to type 2 diabetes. Current methods for quantifying blood glucose dysregulation in diabetes and prediabetes are limited by reliance on single-time-point measurements or on average measures of overall glycemia and neglect glucose dynamics. We have used continuous glucose monitoring (CGM) to evaluate the frequency with which individuals demonstrate elevations in postprandial glucose, the types of patterns, and how patterns vary between individuals given an identical nutrient challenge. Measurement of insulin resistance and secretion highlights the fact that the physiology underlying dysglycemia is highly variable between individuals. We developed an analytical framework that can group individuals according to specific patterns of glycemic responses called "glucotypes" that reveal heterogeneity, or subphenotypes, within traditional diagnostic categories of glucose regulation. Importantly, we found that even individuals considered normoglycemic by standard measures exhibit high glucose variability using CGM, with glucose levels reaching prediabetic and diabetic ranges 15% and 2% of the time, respectively. We thus show that glucose dysregulation, as characterized by CGM, is more prevalent and heterogeneous than previously thought and can affect individuals considered normoglycemic by standard measures, and specific patterns of glycemic responses reflect variable underlying physiology. The interindividual variability in glycemic responses to standardized meals also highlights the personal nature of glucose regulation. Through extensive phenotyping, we developed a model for identifying potential mechanisms of personal glucose dysregulation and built a webtool for visualizing a user-uploaded CGM profile and classifying individualized glucose patterns into glucotypes.

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Expression profiles of podocytes exposed to high glucose reveal new insights into early diabetic glomerulopathy.

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Expression profiles of podocytes exposed to high glucose reveal new insights into early diabetic glomerulopathy.

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