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Objective To investigate the effects of adiponectin on angiotensin Ⅱ-induced extracellular matrix production of mesangial cells(MCs) and its possible signaling pathway.Methods RT-PCR and indirect immunofluorescence examination were performed to detect the adiponectin receptors in MCs.Quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA) were used to observe the effects of adiponectin on angiotensin Ⅱ-induced transforming growth factor β1(TGF-β1) and fibronectin production of MCs.Western blotting was used to measure the ratio of p-AMPK to total AMPK.Results(1)Adiponectin receptors 1 and 2 were found in MCs. (2)The up-regulated mRNA and protein expression of TGF-β1 and fibronectin in MCs induced with 10-7 mol/L angiotensin Ⅱ (Ang Ⅱ) was significantly inhibited by 10 mg/L adiponectin (P＜0.05).(3)The p-AMPK/AMPK ratio was significantly increased after incubation with adiponectin for 15 min and 30 min(vs 0 min, P＜0.05), which suggested that adiponectin could activate the AMPK signaling pathway in MCs.The activation of AMPK signaling pathway was blocked by 40 μmol/L compound C, a specific inhibitor of AMPK. (4)The inhibitory effects of adiponectinonangiotensin Ⅱ-upregulatedTGF-β1andfibronectinexpressioninMCswere significantly relieved by 40 μmol/L compound C (P＜0.05).Conclusions There are adiponectin receptors 1 and 2 in MCs.Adiponectin has inhibitory effects on the angiotensin Ⅱ-upregulated TGF-β1and fibronectin expression in MCs.AMPK signaling pathway may play an important role in the effects of adiponectin above-mentioned.
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Objective To investigate whether low-protein diet has protective effect on the progression of renal interstitial fibrosis in rats with cyclosporine A (CsA)-induced nephropathy. Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups (6 rats in each group). The rats in control group (C group) received common diet; in model group (M group) low-salt diet; in intervention group (Ⅰ group) low-salt and low-protein diet. After diet adaptation period of one week, the rats in C group received subcutaneous injection of olive oil 1 mg/kg daily for 5 weeks, while M group and Ⅰ group subcutaneous injection of CsA (diluted into 25 g/L with olive oil) 1 ml/kg for 5 weeks. All the rats were sacrificed at the end of the 5th week. The food-intake and body weight were measured daily. The creatinine clearance (Ccr) was examined before rats were sacrificed. The semi-quantitative pathological analysis on kidney sections was performed. The mRNA and protein expression of transforming growth factor-β1 (TGF-βI) and type Ⅰ collagen (Col Ⅰ) in kidney tissue was determined with real time PCR and immunohistochemical staining, respectively. Results The food-intake and body weight of rats in M and I groups were significantly lower than those in C group (P<0.05). Compared with C group, the Ccr levels in M and Ⅰ groups were significantly reduced [(0.65±0.15) ml/min, (0.40+0.13) ml/min vs (1.55±0.29) ml/min, P<0.05], the relative fibrosis areas of kidney interstitium in M and I groups were significantly increased (3.60%±0.46%, 3.26%±0.75% vs 0.44%±0.24%, P<0.05), the mRNA and protein expression of TGF-β1 in M and I group was significantly up-regulated (by 2.6 and 3.1 times in mRNA and by 1.5 and 1.6 times in protein, respectively, P<0.05), and the mRNA and protein expression of Col Ⅰ in M and I groups was also significantly up-regulated (by 3.0 and 3.5 times in mRNA and by 2.3 and 2.1 times in protein, respectively, P<0.05). There were no significant differences between M and I groups in every parameters above-mentioned except the rat body weight and Ccr. Both the body weight and Ccr in Ⅰ group were significantly lower than those in M group (P<0.05). Compared with C group, the urine osmotic pressure in M group and in I group were deceased (for M group, P>0.05; for I group, P<0.05). Compared with C group, the serum cholesterol levels in M and I groups were significantly increased (P<0.05), and the serum phosphorus level in I group was significantly decreased (P<0.05). The levels of serum albumin and serum calcium of all three groups had no statistical differences (P>0.05). Conclusion Low-protein diet has no renoprutective effects on the rat model of cyclosporin A nephropathy, on the contrary, may induce body weight loss.
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Objective To study whether Hirsutella sinensis (HS) can antagonize aristolochic acid (AA) induced fibrogenesis on human proximal tubular epithelial cells (HKC). Methods HKC were incubated with medium alone, medium with HS 10 mg/L, medium with AA-Na 40 mg/L or medium with AA-Na 40 mg/L and HS 10 mg/L, respectively. After 12 h (for mRNA) or 36 h (for protein) ,cells were lysed,and the mRNA and protein expression level of transforming growth factor-?1 (TGF-?1), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase-1 (TIMP-1)and plasminogen activator inhibitor-1 (PAI-1) of HKC were measured by RT-PCR, ELISA (for TGF-?1, TIMP-1 and PAI-1) and Western blotting (for CTGF), respectively. Results The mRNA and protein expression of TGF-?1, PAI-1, CTGF and TIMP-1 were significandy up-regulated by AA-Na 40 mg/L. Compared with the control group, the mRNA expression of TGF-?1,.CTGF, TIMP-1 and PAI-1 was up-regulated to 1.24,1.31,1.27 and 1.36 times,respectively (P
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Objective To establish the method of alloantigen-specific T lymphocyte clones. Methods PBMC of the recipient was stimulated by donor splenocytes for twice and the stimulated T lymphocytes were cloned by limited dilution. Results Three clones of T lymphocytes were obtained. By analysis of immunohistochemistry, two of the three clones were CD3+CD4-CD8+, another one was CD3+CD4-CD8-. The latter was analyzed by flowcytometry and CD3+ was 98.87%, CD4- 97.05%, CD8- 97.14%. Antigen specificity of the cloned T lymphocytes was determined by stimulation of donor splenocytes and non-related individual splenocytes. When stimulated by donor splenocytes, the cloned T lymphocytes proliferated and did not show proliferative role when stimulated by non-related individual splenocytes. Conclusion Our method of cloning alloantigen-specific T lymphocytes is successful.
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0. 05) . (2) AA in the concentrations of 80 and 160?g/ml significantly elevated LDH release rates of HK-2 and hRIFs ( P