The Experimental Study Of Adiponectin On Alleviating The Progression Of Ischemic Renal Disease By Inhibiting Renal Cell Apoptosis

Objective:The clinical incidence of ischemic renal disease（IRD）is increasing year by year.It can cause kidney damage,loss of kidney function,and eventually end-stage renal disease（ESRD）.Adiponectin（APN）is a recently discovered collagen-like protein secreted by adipose tissue,which plays an important role in regulating energy metabolism and inflammation.However,the specific mechanism of APN on IRD is still unclear.Therefore,this study aims to explore the specific mechanism of APN affecting IRD symptoms in vitro and in vivo.Methods:PartⅠ:In vitro,we cultured HK-2 cells and used classic Co Cl₂to partially simulate the IRD hypoxia model.HK-2 cells were randomly divided into 4 groups:control group,APN treatment group alone,and Co Cl₂induced hypoxia model group,Co Cl₂and APN joint treatment group.（1）Use flow cytometry to detect the apoptosis rate of each group of cells;（2）Observe the morphological changes of each group of cells through a light microscope;（3）Use Mito SOX fluorescent dye to detect reactive oxygen species;（4）The protein expression levels of anti-apoptotic protein Bcl-2,apoptosis protein Bax,Fas,three related proteins STC-1,UCP2,UCP3 and inflammatory factors IL-1β,IL-6,and TNF-αwere detected by Western blot technology.PartⅡ:In vivo,we constructed a mouse model of IRD.The mice were divided into 4 groups:wild-type mouse control group（WT Control）,wild-type mouse model group（WT IRD）,and APN knockout mouse control group（APN-/-Control）,APN knockout mouse model group（APN-/-IRD）.（1）We used HE staining and Sirius red staining to observe whether the kidney structure has changed;（2）The expression levels of hypoxia-inducible factors HIF-1αand HIF-1βwere detected by immunohistochemistry;（3）TUNEL staining of kidney tissue sections in vitro was used to detect renal cell apoptosis.Results:PartⅠ:（1）After treating HK-2 cells with different concentrations of Co Cl₂（200,400,600,800μM）for 24 h,compared with the control group,600 and 800μM Co Cl₂caused a significant increase in the apoptosis rate of HK-2,while the effect of 200and 400μM Co Cl₂is not obvious;on the basis of Co Cl₂treatment,different concentrations of APN（1,2.5,and 5μg/ml）were used for intervention treatment for 24 h.The results showed that compared with the Co Cl₂group,the apoptosis rate of the three concentration treatment groups decreased,but the 2.5μg/ml APN treatment group had a more significant effect on reducing the apoptosis rate than the other two groups;（2）Observed under light microscope,control group cells are spindle-shaped,cell membranes are intact,and grow well;APN group cell morphology has no change;Co Cl₂group cell morphology is obviously rounded,cells shrink,and cell debris appears;In the combined treatment of Co Cl₂and APN,most of the cells in the combined treatment group recovered from round to spindle shape,but there was also a small amount of cell debris;（3）The cells in the control group and the APN alone group showed weak red Mito SOX fluorescence.After Co Cl₂treatment,the cells showed bright red fluorescence.In the Co Cl₂and APN combined treatment group,compared with the Co Cl₂group,the intensity of red fluorescence was significantly reduced;（4）Compared with the control group,after induction by Co Cl₂treatment,the expression of anti-apoptotic protein Bcl-2 decreased,the expression of pro-apoptotic protein Bax and Fas increased significantly,and the expression levels of inflammatory factors IL-1β,IL-6 and TNF-αalso increased significantly;After combined treatment with APN,compared with the Co Cl₂group,APN increased the expression of anti-apoptotic protein Bcl-2,but inhibited the expression of pro-apoptotic protein Bax and Fas,and also inhibited the expression of inflammatory factors IL-1β,IL-6 and TNF-α;the results also found,STC-1 and UCP3 participate in the process of APN playing a protective role.PartⅡ:（1）Compared with wild-type mice,IRD mice have glomerulosclerosis.After APN gene knockout,the glomeruli of mice have obvious hypertrophy,and the number of collagen fibers in the renal tubules is also significantly increased;（2）Compared with wild-type mice,the expression of hypoxia-inducible factors HIF-1αand HIF-1βin IRD mice increased.After the APN gene was knocked out,the expression of HIF-1αand HIF-1βappeared obvious Increase;（3）Compared with wild-type mice,the number of apoptotic cells in the kidney tissue of IRD mice increased.After the APN gene was knocked out,the number of apoptotic cells in the kidney tissue of the mice increased significantly.Conclusions:1.APN can change the morphological changes of HK-2 cells induced by Co Cl₂and prevent cell apoptosis.2.APN can reduce the generation of ROS.3.APN can significantly inhibit the apoptosis and inflammation of renal tubular epithelial cells,and STC-1 and UCP3 participate in the process of APN’s protective effect.4.Knockout of APN gene exacerbated the damage of kidney structure.5.Knockout of APN gene increased the expression of HIF-1αand HIF-1βin mouse kidney tissue.6.Knockout of APN gene exacerbated the apoptosis of mouse kidney tissue.