PPAR¦Ã Agonists On Proximal Tubular Epithelial Cells Of Connective Tissue Growth Factor Expression

BAKEGROUND The tubulointerstitial damage is a major contributing factor for progressive renal disease in Diabetic nephropathy. Connective tissue growth factor (CTGF) secreted by proximal tubular epithelium is a key factor regulating extracellular matrix accumulation in the tubulointerstitium. The purpose of this study is to observe the expression of CTGF in the tubulointerstitium in the progression of diabetic nephropathy in type 2 diabetic KKA^y mice as well as in cultured human proximal tubular epithelium cell line HK-2 induced by TGFβ₁ And the effect of a peroxisome proliferator activated receptor(PPAR)- γ agonist, rosiglitazone, on the expression of CTGF in the tubulointerstitium in KKA^y mice and in cultured HK-2 cell line induced by TGF β₁ were also studied.METHODS KKA^y mice aged 16 weeks with hyperglycemia and proteinuria (n=5, diabetic) and age-matched C57BL/6J (n=5 nondiabetic) were sacrificed as control before treatment. The other 20 KKA^y mice aged 16 weeks were divided into two groups: treated with rosiglitazone (30mg/kg per day, n=5) and untreated control (n=12). The experiment animals were sacrificed at 4th, 8th week of the treatment (n=5 at each time point) respectively. Body weight, blood glucose, serum creatinine and 24 hours proteinuria were measured. The kidney tissues were prepaired for the pathological analysis. Protein expression of transforming growth factor(TGF β₁), CTGF, PPARy and Fibronectin were assayed by Western blot. Kidney slides were stained for CTGF, PPARγ and a-SMA by immunohistochemistry method. In-vitro study, protein expression of TGF β₁, CTGF, PPARγ and Fibronectin were examined also by Western blot. The mRNA expression of TGF- β₁, CTGF, PPARγ, α -SMA and Fibronectin were tested by RT-PCR. At the same time the PPARγ immuno-fluorescence in HK-2 cell line was studied and the activity of PPARγ were examined by ELISA assay.RESULTS In-vivo study, the positive immunohistochemical stains for CTGF were founded in the dilated proximal tubule of diabetic mice and it was negative in proximal tubular epithelium in the nondiabetic mice. The excretion of proteinuria in the diabetic mice significantly increased than in the nondiabetic group (20.60±1.11 vs 10.36±0.70, P<0.01). In the group of diabetic mice aged 16 weeks, the protein expression of of TGF β₁, CTGF and Fibronectin in renal cortex respectively upregulated by 52%, 28% and 18% (P<0.01) than age-matched nondiabetic control and it was in the manner of age-dependent in diabetic mice(P<0.01). The excretion of proteinuria was founded significantly decreased in the 8th week of rosiglitazone treated group (44.53±1.96 vs 63.66±5.57, P<0.01). In the control study of rosiglitazone treatment and unteatment, the inhibition of protein expression of TGF β₁, CTGF and Fibronectin in the treated group was observed in time-depended manner also. It was decreased by 37%, 21% and 52% at 4th week of the treatment, respectively(P<0.01) and it was decreased by 61%, 50% and 51% at 8th week, respectively (P<0.01). It was founded that the CTGF expressed in the...