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

Abstract

Podocyte injury has been suggested to play a pivotal role in the pathogenesis of diabetic glomerulopathy. To glean insights intomolecular 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 to 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 one and two 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 levels 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.

Defining cell-type specificity at the transcriptional level in human disease

“…Restricted gene expression is defined in this study as podocyte ''specific'' within the renal context if it shows gene expression limited to podocytes within the kidney, and ''podocyte specific within the renal glomerulus'' if it is expressed only in podocytes within the glomeruli but detectable in other extraglomerular cell lineages in the kidney. Previous high-throughput strategies have relied on mouse (Endlich et al 2002;Brunskill et al 2011;Jain et al 2011) or human (Saleem et al 2008) immortalized glomerular visceral epithelial cells, but in vitro culture leads to rapid loss of both lineagespecific phenotypes and lineage-specific gene expression. Wholetissue-based molecular profiles of renal disease are attainable Higgins et al 2004;Schmid et al 2006;Bennett et al 2007;Ju et al 2009;Hodgin et al 2010;Lindenmeyer et al 2010;Woroniecka et al 2011) since human renal tissue is routinely obtained by diagnostic fine needle biopsy, but wholetissue expression profiles have not previously been capable of identifying gene expression at the cell-lineage level.…”

Role of Podocyte B7-1 in Diabetic Nephropathy

“…Three major intracytoplasmic key players appeared to be potentially related to both HG intracellular signaling and B7-1 expression: phosphatidylinositol 3-kinase (PI3K), serine/threonine protein kinase (AKT), and glycogen synthase kinase 3 (GSK3). 16,17 AR-A014418 (0.2-2 mM) and triciribine (1-10 mM) (GSK3 and AKT inhibitors, respectively) did not affect B7-1 expression when added to podocytes cultured during HG To determine the pathway linking HG and B7-1 upregulation, we tested the effect of AKT, GSK3, and PI3K inhibitors on B7-1 expression. (Q) Although the AKT (triciribine) and GSK3 (AR-A014418) inhibitors are ineffective in downregulating B7-1 expression, the pan-PI3K inhibitor quercetin inhibits B7-1 upregulation (***P,0.001, 10 and 100 mM quercetin versus HG).…”

Endothelial Lipase Is Localized to Follicular Epithelial Cells in the Thyroid Gland and Is Moderately Expressed in Adipocytes

“…Metabolic inflammation, as measured by high leptin levels and increased TNFα concentrations at the fetalplacental interface, was associated with an upregulation of placental EL mRNA expression in obese women with gestational diabetes mellitus (Gauster et al 2011). EL protein expression was enhanced in podocytes 1 to 2 weeks after high glucose exposure and glomeruli from diabetic nephropathy patients (Jain et al 2010). These results provide evidence that EL may potentiate hyperglycemic podocyte stress and possibly the pathogenesis of diabetic glomerulopathy in humans.…”