| AIM: Renal hypertrophy is an early important pathological feature of renal diseases such as diabetic nephropathy and remnant kidney. Recent studies have demonstrated that activation of intrarenal renin angiotensin system (RAS) has played a key role in the chronic progression of renal diseases. However, the exact mechanism for how RAS to play its role is still a hot research subject for the nephrologist. More recently, several studies have suggested that angiotensin II (Angll) might be a major contributor in mediating cell hypertrophy. However, the exact mechanism is still unclear. Connective tissue growth factor (CTGF) is a newly clarified fibrogenic factor, which has been shown increasing expression in many fibrotic renal diseases. However, whether CTGF mediates the Ang II induced renal cell hypertrophy, and what's the potential mechanism of Ang II stimulated renal cell hypertrophy are still a interesting subject, which has not been profoundly investigated so far. Our present study was to investigate the possible role of CTGF in mediating the effect of Ang II induced renal cell hypertrophy and its relationship with cell cyclin dependent kinase inhibitor, p27kipl expression. Meanwhile, we paid attention to the relationship between cell hypertrophy and its phenotypic change, soas to clarify the significance of CTGF mediated cell hypertrophy to renal fibrogenesis.METHODS: The human proximal tubular cell line (HK-2) was grown in Dulbeccos's Modified Eagle's Medium (DMEM) containing 10% heat inactivated fetal calf serum (FCS). After rested in serum-free medium for 24 hours, the dose and time response of CTGF mRNA and protein expression to the stimulation of Ang II were observed by RT-PCR and Western blot respectively. The effect of anti-CTGF antibody on Ang II (10-7mol L ) induced de novo protein synthesis ([3H]-leucine incorporation), total protein content (Coomassie brilliant blue G250 method) and change of cellular size (determined by scanning electronic microscope, SEM) were observed respectively. The influence of anti-CTGF antibody on cell cycle was analyzed by using the fluorescence-activated cell sorter (FACS) flow cytometer. The effect of anti-CTGF antibody on the Angll induced expression of mRNA for p27kipl and FN was observed by RT-PCR, and influence of anti-CTGF antibody on Ang II induced p27kip] protein expression was evaluated by using immunocytochemistry. To further understand the role of CTGF in mediating Ang II induced cell hypertrophy, we used the antisense oligonucleotide of CTGF (CTGF-AS) to interfere with the Ang II induced cell hypertrophy by determining the total cellularprotein content, cell cycle and cell size. The ultrastructural change induced by Ang II had also been determined by using the transmissive electronic microscope. For the in vivo study, we gave a bolus injection of streptozotocin (STZ) to male adult Sprague-Dawley rats to establish a diabetic model, which had been received a single kidney ablation previously to enhance the effect of renal hypertrophy. Three groups were included in the experiment: Control (Group C); Diabetic nephropathy (Group DN); and diabetic rats treated with irbesartan (Group DNI). The animal were sacrificed at week 1,2,4 and week 8 respectively. The serum and urine sample were collected for the examination of blood glucose, renal function, quantification of 24 hour proteinuria (24h Upro) and 24 hour albuminuria (24h Ualb). Kidney weight and kidney weight/body weight (KW/BW) were collected simultaneously. The glomerular capillary area (AG), glomerular volume (VG) and proximal tubular area (AT) were analyzed by computerized image analyzing system. The thickness of glomerular basement membrane (GBM) and tubular basement membrane (TBM) were analyzed by electronical microscope. Immunohistochemical staining for dynamic expression of CTGF, p27kipl and ct-SMA were performed by SP method. Double-staining for CTGF and p27kipl was performed to evaluate their relationship between these two factors. The relationship between immunostaining for CTGF and p27kip... |
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