| Objective Diabetic nephropathy is one of the important chronic microvascular complications of diabetes. However, the molecular details of diabetic nephropathy are still poorly understood. Glomerular basement membrane thickening, extracellular matrix proliferation and glomerulo- sclerosis caused by insulin-like growth factor-1 accumulation in kidney may play major roles in diabetic nephropathy. Catapol, one of the main active ingredient in rehmannia, exerted neuroprotective effects in diabetic peripheral neuropathy (a diabetic microangiopathy) in diabetic rats in our previous study, while it hasn't been reported on the research about diabetic nephropathy. The present study was performed to investigate the pathogenesis of IGF-1 and PI3K/Akt signal pathway in diabetic nephro- pathy and observe whether catapol could protect renal function through regulating the expression of IGF-1. Moreover, it could provide a clue of traditional Chinese medicine in clinical application.Methods Male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (NC), diabetic control group (DM) and diabetic group treated with catapol (DT) including 12 rats for each group. The diabetic rat models were induced by intraperitoneal injection of streptozotocin (STZ) (50mg/kg). Since 8th week point, the rats of DT group were administered with catapol (5mg/kg) by intraperitoneal injection once daily for 2 weeks, while the other two groups were injected with equal volume of normal saline. All rats were in metablic cage to collect 24h urine samples for urinary albumin excretion (UAE), urine microalbumin (MA), urine creatinine (Cre) testing and MA/Cre calculating at the 8th and 10th week. The next day after ten-week, before sacrificed, rats'blood was sampling from heart for measuring HbA1c, BUN, Scr levels and Ccr calculating. Then the kidneys were removed quickly for measuring relative kidney weight (KW/BW). The left kidney was dyed by HE, PAS and PASM to observe pathologic morphology and detected the expression of IGF-1 and Akt1 by immunohistochemistry. While the right kidney was used to detect the change of IGF-1 and Akt at both the levels of mRNA and protein.Results1. Body weight (g), Blood glucose (mmol/L) and HbA1C (%): At the 10th week, mean weights of DM group (266.77±55.47) and DT group (279.50±43.56) were significantly lower (P <0.001) than the NC group (356.22±50.73); Blood glucose and HbA1C of DM (25.37±3.56&5.92±0.72) and DT group (23.54±8.70&6.12±0.07) were significantly higher (P<0.001) than that of NC group, while the blood glucose and HbA1C were normal in the NC group (5.72±1.22&3.30±0.10), but there was no significant difference (P >0.05) between DM and DT group on body weight, blood glucose and HbA1C.2. Renal function and relative kidney weight: At the 10th week, the values of MA/Cre (mg/mmol) for DM group (17.35±3.51) and DT group (15.54±3.57) were markedly higher (P<0.001) than that of NC group (3.79±1.02); The levels of 24h urinary albumin excretion (mg/24h) for DM group (1.00±0.21) and DT group (0.81±0.34) were significant higher (P<0.05) than that of NC group (0.21±0.06); The relative kidney weight (%) in DM (6.09±0.91) and DT group (5.93±0.69) were significantly higher (P<0.001) than that in NC group (4.03±0.50). The data of DT group was lower than that of DM group, but there was no significant difference (P>0.05). Scr and Ccr levels were normal in each group (P >0.05).3. Histopathological assessment: In PAS and PASM dyed slides, diabetic non-treated rats showed proliferation of mesangial cells, with expansion of mesangial matrix, as well as thickening of ECM. Classic change in DN such as sclerotic nodule formation in glomeruli was observed. But the above pathology change obviously reduced in DT group.4. RT-PCR: In renal tissue, the expression of IGF-1 mRNA in DM group (0.57±0.17) increased significantly (P <0.05) than NC group (0.38±0.11) while there was no significant difference (P >0.05) between the DT (0.42±0.21) and NC group. The expression of Akt1 mRNA of DM (1.02±0.58) and DT group (0.89±0.34) increased significantly (P <0.05) than NC group (0.56±0.19), while the level of DT group was significantly lower (P<0.05) than DM group.5. Western blotting: Western blotting showed the activity of p-Akt was increased (P <0.001) in DM group (0.86±0.08) and DT group (0.65±0.06) compared with that of NC group (0.42±0.11), and it was markedly down-regulated (P <0.05) in DT group than DM group.6. Immunohistochemistry: In all three groups, renal glomerulus, proximal convoluted tubule and collecting tube were showing positive localization. The average optical density (AOD) of IGF-1 in DM group (10.35±0.73) was significantly increased (P <0.001) than NC group(4.81±0.62), but the AOD of DT group (5.63±1.77) was markedly lower (P<0.001) than DM group. The AOD of Akt1 in DM (15.49±2.33) and DT group (9.20±1.27) were both higher (P <0.001) than NC group (3.01±1.13), while the AOD of DT was lower (P <0.001) than DM group.Conclution1. The renal function was gradually impaired and the pathology of glomerular basement membrane thickening and extracellular matrix proliferation were observed at the 8th week after the onset of diabetes.2. The over-expression of IGF-1 in diabtic rats played a major role in DN, at least in part, by activation of PI3K/Akt signal pathway.3. Catapal could down-regulate IGF-1 and Akt activities and reduce the pathology changes in diabetic kidney to a certain extent, however there was no exact improvement in 24h urinary albumin excretion, MA/Cre and renal fuction values.
|
PDF Full Text Request