Atorvastatin Attenuates Diabetic Nephropathy Through The Inhibition Of Oxidative Stress-Induced Kidney Cells Ferroptosis

ObjectiveDiabetic nephropathy,one of the serious microvascular complications of diabetes,is considered to be a major cause of end-stage renal disease with high rates of fatal disability,At present,the pathogenesis of diabetes nephropathy is still unclear.Research shows that ferroptosis may participate in the pathogenesis of diabetes nephropathy as a new type of cell death.Atorvastatin（AT）is a 3-hydroxy-3-methylglutaryl coenzyme A（HMG-Co A）reductase inhibitor.In addition to its lipid-lowering and anti-inflammatory effects,it also has a kidney protective effect that is independent of lipid lowering in clinical practice.Whether AT provides protection by regulating ferroptosis pathways is unclear.This sthdy will investigate the protective effect of AT on diabetes nephropathy,and explore whether the possible mechanisms are associated with ferroptosis.MethodsIn vivo18 males,C57BL/6 mice（24-30 g,6 weeks）,SPF grade.They were randomly divided into two groups: the normal control group（Con group,n=6）,fed with regular feed and drinking water freely;High fat diet group,combined with streptozotocin（STZ,55 mg/kg）,was intraperitoneally injected for 5 consecutive days to establish type 2diabetes model.After 3 days of modeling,fasting blood glucose was measured using the method of collecting blood from the tail vein.The index for successful modeling was blood glucose ≥ 11.1 mmol/L.Diabetic mice were randomly divided into two groups,one group was the model group（STZ group,n=6）,and the other group was the atorvastatin treatment group（AT group,n=6）.The treatment group mice were given atorvastatin calcium（45 mg/kg/d）daily per mouse,and the weight of the mice was weighed weekly,and their blood glucose was measured.The mice were continuously given intragastric administration for 8 weeks.Samples were taken,and urine and serum were tested.Take the kidney for histological section,observe its pathological changes by HE and PAS staining,and detect the content of reactive oxygen species in the kidney by DHE staining.Immunohistochemical method was used to detect the content of MDA and 4-HNE in the kidney;Determination of ferroptosis related proteins Nrf₂,GPX₄,TFR,FTH,Fibronectin and TGFβ related proteins in kidney,expression of ROS related proteins p22 phox and gp91 phox.In vitroUsing human proximal renal tubular epithelial cells（HK-2 cells）as an in vitro model,the cells were cultured in low-sugar DMEM medium.HK-2 cells were stimulated with high glucose（30 mmol/L,HG group）and Erastin（10μmol/L,Er group）for 24 h.The oxidative fluorescence intensity in HK-2 cells was measured by DCFH-DA fluorescence staining assay.For the detection of ferroptosis related proteins,Western blot was used to determine the protein changes.ResultsThis study includes two parts: in vivo and in vitro experimentsin vivo1.Compared with Con group,STZ induced DN mice to significantly reduce their weight,and after atorvastatin treatment,their weight increased.Blood glucose results showed a significant increase,after intraperitoneal injection of STZ,the blood glucose levels in STZ group and AT group were higher than Con group.After AT treatment,the blood glucose of AT group mice slightly decreased,but there was no statistical significance.2.TG,TC,and urinary protein to creatinine ratios in DN mice were significantly increased,while TG,TC urinary protein to creatinine ratios were significantly decreased after AT treatment.3.Histological section results showed that AT significantly improved glomerular hypertrophy,tubular vacuoles,glycogen deposition,renal fibrosis and reactive oxygen species content caused by diabetes nephropathy.4.Western blot results showed that compared with Con group,the expression of ferroptosis related proteins FTH,Nrf₂,and GPX₄ in the renal tissue of DN mice decreased,while the expression of TFR increased,Fibronectin,TGFβ increased protein expression,The expression of NADPH oxidase subunits p22 phox and gp91 phox protein increased,and the expression of FTH,GPX₄,and Nrf₂ protein increased after AT treatment,as well as TFR,Fibronectin,and TGFβ the protein expression decreased,and the expression of NADPH oxidase subunits p22 phox and gp91 phox protein decreased.5.Immunohistochemical results showed that the expression of lipid oxidation markers MDA and 4-HNE in renal tissue of DN mice was significantly higher than that of Con group,and the content of MDA and 4-HNE decreased significantly after AT treatment.In vitro1.ROS staining of HK-2 cells induced by high sugar showed that HK-2 cells increased their ROS content after HG induction and decreased their ROS expression after AT treatment.Western blot results showed that compared with NG group,MDA and 4-HNE protein expressions were increased in HG group,and decreased after AT treatment.2.Western blot results of ferroptosis related proteins induced by high glucose showed that after HG induction in HK-2 cells,the expression of FTH,Nrf₂,and GPX₄ decreased,while the expression of TFR increased.AT treatment could increase the expression of FTH,GPX₄,and Nrf₂ proteins,and reduce the expression of TFR proteins.3.ROS staining of HK-2 cells induced by Erastin showed that the content of ROS in HK-2 cells increased after Erastin induction,while the content of reactive oxygen species expression decreased after AT treatment.Western blot showed that compared with NG group,the expression of MDA and 4-HNE increased in Er group,and their expression could be reversed after AT treatment.4.Western blot results of ferroptosis related proteins induced by Erastin showed that after Erastin induced HK-2 cells,the expression of FTH,Nrf₂,and GPX₄ decreased,while the expression of TFR increased.After AT treatment,the expression of related proteins could be improved.5.Fe2+fluorescence staining of HK-2 cells induced by Erastin showed that compared with NG group,the average fluorescence intensity of Er group cells increased,and after AT treatment,the average fluorescence intensity decreased.Conclusions1.Atorvastatin can reduce renal function damage,lower blood lipids,and improve renal function and pathological structure damage in mice with STZ induced diabetic nephropathy.2.Atorvastatin has obviously reduced renal tissue oxidative stress,upregulated the Nrf₂ pathway and inhibited renal ferroptosis in mice with diabetic nephropathy.In HK-2 cells in vitro,atorvastatin can inhibit renal cell ferroptosis caused by high glucose or the ferroptosis inducer Erastin to protect HK-2.3.Atorvastatin could improve renal function and renal tissue structural damage in mice with diabetic nephropathy,and the mechanism may be associated with decreasing oxidative stress and renal cell ferroptosis in renal cells.