| Objective1. To explore the relationship between angiotensin Ⅱ type 1 receptor (AT1 receptor, AT1R) autoantibody (AT1-AA) and miRNA-155 in the kidney of DN rats.2. To investigate the effects of AT1-AA on the expression of endoplasmic reticulum stress (ERS)-related factors glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein-homologous protein (CHOP), phosphorylated c-Jun N-terminal kinase (p-JNK) and Caspase-12.3. To observe the roles of irbesartan in inhibiting ERS-related apoptotic signals in the kidney of AT1-AA positive DN rats, andMethodsAnimals experiment:36 SD male rats were randomly divided into normal control group (NC, n=6) and model group (Model, n=30). Model group rats were induced by high-sucrose and high-fat diet and intra-peritoneal injection of a small dose of streptozotocin (STZ,35mg/kg), and then followed by 8 weeks of observation. Enzyme-linked immunosorbent assay (ELISA) method was used to test the levels of serum AT1-AA from rats. The DN rats were randomly divided into DN group (DN, n=12) and irbesartan treated DN group (Irb+DN, n=12) according to the result of ELISA, and further divided into AT1-AA positive DN (APD, n=8), AT1-AA negative DN (AND, n=4), Irb+APD (n=7) and Irb+AND (n=5) subgroups. We ended the experiment after 4 weeks’ irbesartan treatment (50 mg·kg-1·d-1). The blood glucose (BG), renal function indicators and kidney hypertrophy index (KW/BW) were detected and calculated. HE staining and transmission electron microscope were applied to detect renal tubular and glomerulus pathological changes. The miRNA-155 levels were determined by real-time polymerase chain reaction (RT-PCR) method. The apoptosis of renal tubular epithelial cells and podocytes Were detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining. Western blot was employed for the expression of ERS chaperone protein GRP78 and ERS-associated apoptosis proteins CHOP, p-JNK and Caspase-12. Additionally, RT-PCR was performed to measure the mRNA levels of GRP78, CHOP and Caspase-12. The differentiation and correlation of detected factors between groups were analyzed by statistical methods.Podocytes experiment:The differentiated rat podocytes were divided into three groups:normal glucose (NG), high glucose (HG) and AT1-AA plus high glucose (AHG). We collected the cells of AHG group after incubating for 24h,48h and 72h, and the cells in NG and HG groups after 72h. The apoptotic podocytes were identified by TUNEL, the existence of GRP78, CHOP, p-JNK and Caspase-12 were assessed by immuno-cytochemical method. Then, western blot was employed for the expression of the proteins mantioned above, and RT-PCR was performed to measure mRNA levels of GRP78, CHOP and Caspase-12.ResultsAnimals experiment:1) The OD level and positive rate of ATi-AA in Model group were significantly higher than those in NC group (P<0.01 and P<0.05).2) Comparing with NC group, the expression of miRNA-155 in the kidney of DN group rats was significantly increased (P<0.01). While the level of miRNA-155 in APD group was lower than that in AND group (P<0.05).3) Comparing with NC group, the apoptosis rate of renal cells in DN group increased obviously, as well as the expression of GRP78, CHOP, p-JNK and Caspase-12 proteins and the mRNAs of them (all P<0.01). The cell apoptosis and levels of proteins and mRNAs were significantly decreased in Irb+DN group compared with DN group (all P<0.01).4) The renal cell apoptosis rate and proteins and mRNA levels in AND group were much lower than those in APD group (P<0.05 or P<0.01).5) Irbesartan could significantly reduce the apoptosis of renal cells, regulate the expression of ERS-related proteins and mRNA in DN rats, especially in APD group rats(P<0.05 or P<0.01).Podocytes experiment:The apoptotic rate of high-glucose cultured rat podocytes induced by AT1-AA was significantly higher than those without AT1-AA inducing (all P<0.01). Besides, AT1-AA time-dependently up-regulated the expression of ERS chaperone protein GRP78 and ERS-related apoptosis proteins CHOP, p-JNK and Caspase-12, as well as the levels of GRP78, CHOP and Caspase-12 mRNA.Conclusion1) The serum AT1-AA levels in DN rats may be related to the apoptosis of renal tubular epithelial cells and glomerular podocytes, and involved in the process of kidney damage.2) The miRNA-155 in the kidney of DN rats may play a role in regulating the production of AT1-AA.3) AT1-AA may induce ERS response in the kidney of DN rats, and promote renal cells apoptosis and kidney damage likely via the ERS-associated CHOP/JNK/ Caspase-12 death signaling pathways.4) Irbesartan can inhibit AT1-AA-mediated ERS response in the kidney of DN rats, and improve renal function via inhibiting ERS-associated CHOP/JNK/ Caspase-12 apoptotic signals and renal cells apoptosis.5) AT1-AA may accelerate the apoptosis of high-glucose cultured rat podocytes via CHOP/JNK/Caspase-12 signaling pathways. |
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