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Peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) are ligand-activated transcription factors that regulate cell growth, inflammation, lipid metabolism, and insulin sensitivity. We recently demonstrated that PPAR[gamma] agonists limit high glucose-induced inflammation in a model of proximal tubular cells (PTC; Panchapakesan U, Pollock CA, and Chen XM. Am J Physiol Renal Physiol 287: F528-F534, 2004). However, the role of PPAR[gamma] in the excess extracellular matrix production is largely unknown. We evaluated the effect of 24- to 48-h 8 [micro]M L-805645 or 10 [micro]M pioglitazone on 25 mM o-glucose-induced markers of fibrosis in HK-2 cells. High D-glucose induced nuclear binding of activator protein-1 (AP-1) to 140.8 [+ or -] 10.9% (P < 0.05), which was attenuated with L-805645 and pioglitazone to 82.3 [+ or -] 14.4 (P < 0.01 vs. high o-glucose) and 99.3 [+ or -] 12.2% (P < 0.05 vs. high D-glucose), respectively. High D-glucose increased total production of transforming growth factor (TGF)-[beta]1 139.6 [+ or -] 6.5% (P < 0.05), which was reversed with L-805645 and pioglitazone to 68.73 [+ or -] 5.7 (P < 0.01 vs. high D-glucose) and 112 [+ or -] 13.6% (P < 0.05 vs. high D-glucose). L-805645 and pioglitazone reduced high D-glucose-induced fibronectin from 156.0 [+ or -] 24.9 (P < 0.05) to 81.9 [+ or -] 16.0 and 57.4 [+ or -] 12.7%, respectively (both P < 0.01 vs. high D-glucose). Collagen IV was not induced by high D-glucose. L-805645 and pioglitazone suppressed collagen IV to 68.0 [+ or -] 14.5 (P < 0.05) and 46.5 [+ or -] 11.6% (P < 0.01) vs. high D-glucose, respectively. High D-glucose increased the nuclear binding of NF-[kappa]B to 167 [+ or -] 22.4% (P < 0.05), which was not modifed with PPAR[gamma] agonists. In conclusion, PPAR[gamma] /agonists exert antifibrotic effects in human PTC in high glucose by attenuating the increase in AP-1, TGF-[[beta].sub.1], and the downstream production of the extracellular matrix protein fibronectin. thiazolidinediones; diabetic nephropathy; proximal tubular cells

Abstract: Objective: Ras proteins are known to affect cellular growth and function. The influence of the prenylation status of Ras on the observed changes in endothelial cell growth under high glucose conditions has not previously been examined. Methods: Human umbilical vein endothelial cells were exposed to normal or high glucose conditions for 72h. They were then examined for proliferative and hypertrophic effects, transforming growth factor β1 (TGFβ1) release, and phosphorylated p38 expression. The importance of prenylation was explored by the addition of mevalonate, isoprenoids or farnesyltransferase inhibitors to control the high glucose media and by measuring changes induced by high glucose and exogenous TGFβ1 in Ras prenylation and farnesyltransferase activity. Kidneys from diabetic rats treated with atorvastatin were also compared to specimens from untreated animals and the expression of the Ras effector p-Akt examined. Results: High glucose conditions caused a reduction in cell number. This was reversed in the presence of mevalonate or farnesylpyrophosphate (FPP), suggesting that the cell growth abnormalities observed are due to high glucose induced inhibition of the mevalonate pathway and subsequent prenylation of proteins. Endothelial cells exposed to high glucose increased their secretion of TGFβ1 and the phosphorylation of p38 both of which were reversed by concurrent exposure to FPP. A reduction in farnesyltransferase activity was observed after exposure to both high glucose and TGFβ1. Exposure to a farnesyltransferase inhibitor in control conditions mimicked the growth response observed with high glucose exposure and prenylated Ras was reduced by exposure to both high glucose and TGFβ1. Finally, interruption of the mevalonate pathway with a statin reduced the expression of p-Akt in diabetic rat kidneys. Conclusion: This study demonstrates that high glucose induced significant alterations in endothelial cell growth by inhibition of the mevalonate pathway, which subsequently mediates the increase in TGFβ1 and inhibition of Ras prenylation. [Copyright &y& Elsevier]

The centrality of the transcriptional regulator Snail in epithelial-to-mesenchymal transformation (EMT), known to occur in models of diabetic nephropathy, has not been established. Transforming growth factor beta-1 (TGFbeta1) is induced in diabetic nephropathy and induces both Snail and EMT. Hypoxia inducible factors (HIFs) are known to induce Snail, independent of TGFbeta1. Notch induction is integral to Snail induction and EMT in tumour cells, but its role in the kidney is unknown. The present study was undertaken to determine the upstream regulators of Snail in the kidney in high glucose and hypoxic conditions. HK-2 cells were cultured in normoxic, hypoxic, high glucose and combined hypoxic/high glucose conditions. The expression of HIF1alpha, NotchIC, Snail, Lysyl oxidase-like 2 (Loxl2), and Hairy and Enhancer Split-1 (Hes1) were measured. We found that hypoxia increased HIF1alpha expression; however, concurrent exposure to high glucose blunted this effect. A similar pattern was observed in Lox12 expression, suggesting that Loxl2 was downstream of HIF1alpha, which was confirmed using siRNA techniques. Snail was upregulated by hypoxia and high glucose and in combination the effect was additive, suggesting independent upstream activation pathways by the two stimuli. Hes1 was upregulated by high glucose and to a lesser extent by hypoxia, but the effect of the combined stimuli was no greater than that observed with high glucose alone. NotchIC was downregulated by both hypoxia and high glucose, and in combination the effect was additive. Therefore, this study suggests that hypoxia and high glucose induce Snail expression through distinct pathways, independent of Notch signalling., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

Anemia is a common complication of chronic kidney disease (CKD), with erythropoietin deficiency being the major contributing factor. The availability of erythropoiesis-stimulating agents (ESAs) has been a seminal advance in the treatment of anemia related to chronic kidney disease. Over the course of the last decade and a half, newer generations of ESAs have become available. The first-generation ESAs or epoetins have a relatively shorter half-life and have traditionally been administered up to 3 times per week intravenously or subcutaneously to maintain adequate hemoglobin (Hb) levels. At the turn of the century, darbepoetin alfa, a hyperglycosylated form, became available for clinical use. It conferred greater metabolic stability in vivo owing to two additional N-linked carbohydrate chains attached to the protein backbone and has a half-life 3 times longer than that of epoetin. Recently developed and undergoing phase III clinical trials is the third-generation ESA, Continuous Erythropoiesis Receptor Activator (CERA), which has a methoxy-polyethylene glycol polymer chain integrated and has a longer elimination half-life than the first- and second-generation ESAs. Its receptor binding characteristics also differ from those of previous ESAs. Its major advantage is that extended dosing intervals are possible in the management of anemia related to erythropoietin deficiency.

Transforming growth factor-beta(1) (TGF-beta(1)) functions as an important immunomodulatory cytokine in human kidney. Evidence suggests that connective tissue growth factor (CTGF) is an important downstream mediator of the profibrotic effects of TGF-beta(1). However, the role of CTGF in TGF-beta(1)-induced chemokine production remains unknown. This study was undertaken to determine whether CTGF is involved in mediating TGF-beta(1)-induced chemokine production in renal proximal tubular (HK-2) cells. Interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were measured. TGF-beta(1) induced an increase in IL-8 and MCP-1 (both P < 0.05) compared with control levels. CTGF was effectively silenced using small interference RNA (siRNA) in HK-2 cells. RT-PCR and real-time PCR confirmed a 94% reduction in CTGF mRNA. In the CTGF-silenced cells, TGF-beta(1)-stimulated IL-8 and MCP-1 secretion was not altered compared with control cells. Similarly, basal secretion of IL-8 and MCP-1 was not changed in CTGF-silenced cells. The direct effect of CTGF (20, 200, and 400 ng/ml) on IL-8 and MCP-1 was assessed at 24-, 48-, and 72-h time points and no stimulation was observed. Our studies further demonstrate that in the CTGF gene-silenced cells, CTGF partially mediates TGF-beta(1)-induced fibronectin and collagen IV secretion. These data suggest that TGF-beta(1) induced IL-8 and MCP-1 via CTGF-independent pathway. TGF-beta mediates both fibrosis and chemokine production in the proximal tubule of the kidney. However, CTGF plays a more specific role as a downstream mediator of TGF-beta(1)-induced fibrosis.