Inhibitory Effects Of Astragaloside ? On Endoplasmic Reticulum Stress-induced Renal Tubular Epithelial Cells Apoptosis In Type 2 Diabetic Nephropathy Rats

ObjectivesTo investigate the role of endoplasmic reticulum(ER)stress in kidney injury of type 2 diabetic nephropathy(DN)rats induced by high fat diet and low-dose streptozotocin(STZ),and to explore the effects of Astragaloside ?(AS-?)on it.MethodsThe rats,which were previously fed on high fat diet for 6 weeks to induce insulin resistance,were given a single intraperitoneal injection of 35 mg/kg STZ.The blood glucose(BG)concentration higher than or equal to 16.7mmol/L was confirmed to diabetic rats.And then these animal were randomly divided into DN-model and drug-treatment groups(n=8/each group).The AS-?treatment groups received intragastric AS-? at doses of 20,40 and 80 mg/kg,and the positive control rats were treated with Metformin(200 mg/kg).Drugs were given once per day and lasted for 8 weeks.And another 8 normal rats were taken as normal control group.The general condition of rats was observed and regular measurement of body weight and the level of BG in each group during the study.At the end of the animal treatment,all rats were housed in individual metabolic cages for 24 h to obtain urine for the measurement of microalbuminuria,24 h urine total protein and urine creatinine(UCr).The blood samples were taken through the abdominal aorta for the measurement of blood urea nitrogen(BUN),serum creatinine(SCr),triglyeride(TG),total cholesterol(TC).Kidney samples were harvested,weighted,counted the renal index of kidney weight to body weight.HE,PASM and Masson staining for routine renal histopathological examination by light microscopy.TUNEL staining was used to examine apoptotic nuclei in kidney sections;and the cells apoptosis in renal tubular epithelial cells,the morphological and structural characteristics of nuclei were observed by transmission electron microscopy(TEM).Immunohistochemistry(IHC)was conducted to detect the expression of ER stress related protein of GRP78 in renal tubular epithelial cells.Western bloting(WB)was performed to detect the expression levels of Bax,Bcl-2,Cleaved caspase-3 and p-PERK,ATF4,CHOP.Results1.Compared with control group,the general condition in model group was poor,animal looked depressed,hair was dinginess,weight was decreased.Compared with model group,AS-? treatment could improve the mental state and increased weight.2.Compared with control group,DN rats had lower body weights,BG in DN rats was significantly increased(p < 0.001).Compared with model group,AS-? treatments could improve the body weight.However the results had no significant difference(p > 0.05).And long-term treatment(6-8 weeks)with AS-?(40,80 mg/kg)had a hypoglycemic effect compared with the DN model rats.3.Compared with control group,the renal index of kidney weight to body weight in DN rats was significantly increased,AS-? treatments could decrease the renal index.However the results had no significant difference(p > 0.05).The levels of SCr,BUN,urinary total protein/urine creatinine ratio,microalbuminuria,TG and TC in the DN model rats were significantly higher(p < 0.001).Treatments of AS-? shown a remarkable reduction in these indexes,compared to model group.(p < 0.001 or p < 0.01 or p < 0.05).4.In the HE staining,compared with normal group,the model group showed severe pathological changes in renal tubules such as edema and hypertrophy of epithelial cells,renal tubular vacuolar lesions,tubular dilation,irregular shape,and exfoliation of epithelial cells.For the DN rats treated with AS-?(40,80 mg/kg),the degree of pathological changes was significantly reduced.The situation of edema of epithelial cells,renal tubular vacuolar lesions significantly improved.Exfoliation of epithelial cells and tubular dilation were significantly reduced.PASM staining showed that the color(dark)of renal tubular basement membrane was significantly thickened,which revealed thickening of basement membrane in DN rats(p < 0.001).Compared to model group,AS-?(40,80 mg/kg)treatments significantly reduced thickening of basement membrane in renal tubules(p < 0.01 or p < 0.001).The blue areas of Masson staining were significantly enlarged,suggesting collagen storage in the kidney of DN rats(p < 0.001).Collagen staining was seen in the renal tubules and interstitium.And administration of AS-?(40,80 mg/kg)significantly reduced the deposition of collagen(p < 0.01 or p < 0.001).5.The TUNEL-positive cells(deep brown)presented in renal tubules.The numbers of apoptotic cells in renal tubules in model group were significantly increased compared to normal group(p < 0.001).While compared to model group,AS-?(40,80 mg/kg)treatments significantly decreased the number of apoptotic cells in renal tubules(p < 0.01 or p < 0.001).6.TEM results showed the nuclei of the renal tubular epithelial cells in DN model rats showed nuclear chromosomal pyknosis,nuclear volume reduction,nuclear membrane shrinkage and other apoptotic morphological features.While compared to model group,AS-?(40,80 mg/kg)significantly ameliorated the morphological features of cells apoptosis in renal tubular epithelial cells.7.The IHC results showed that the GRP 78 expression was significantly increased in DN model rats when compared to normal control rats(p <0.001).Compared with model group,AS-(40,80 mg/kg)? could significantly decrease the expression of GRP 78(p < 0.05 or p < 0.01).8.The WB results showed that the expressions of apoptosis-related index Bax/Bcl-2,Cleaved caspase-3 and ER stress-related index p-PERK/PERK,ATF4,CHOP were significantly increased in DN model rats,compared to normal group(p < 0.01).Compared to model group,AS-?(40,80 mg/kg)treatments significantly decreased the expression of these indexes(p < 0.01 or p < 0.05).Conclusion1.Rats which were fed on high fat diet for 6 weeks and intraperitoneal injected with35 mg/kg STZ once could mimic a diabetic nephropathy model successfully.2.As-? ameliorated the metabolic parameters and renal pathophysiology in type 2diabetic nephropathy rats.3.As-? decreased the apoptosis-associated index Bax/Bcl-2,Cleaved caspase-3 and endoplasmic reticulum-associated protein GRP 78,p-PERK/PERK,ATF4 and CHOP in renal tissues of type 2 diabetic nephropathy rats.4.As-? inhibited endoplasmic reticulum stress-induced renal tubular epithelial cells apoptosis in type 2 diabetic nephropathy rats.