Effect And Mechanisms Of Carnosine Attenuates Ferroptosis In Renal Tubular Epithelial Cells In Diabetic Nephropathy

Background and objective:Diabetic nephropathy(DN)is a common microvascular complication of diabetes.The traditional view is that DN mainly presents glomerular injury.Recent studies have linked ferroptosis to tubular damage in patients with diabetic nephropathy.Therefore,it is extremely important to explore the specific mechanism of DN tubular epithelial cell injury and to find effective therapeutic targets.Carnosine(CAR)is a dipeptide composed of β-alanine and L-histidine,which has strong anti-inflammatory and antioxidant effects,and can clear ROS andα-β-unsaturated aldehydes produced by excessive fatty acid oxidation in cell membrane under oxidative stress.In recent years,CAR has been shown to have a potential therapeutic effect on kidney disease.However,it is still unclear whether carnosine has a protective effect on ferroptosis of renal tubular epithelial cells in DN.Therefore,by exploring the protective effect of carnosine on STZ-induced ferroptosis of renal tubular epithelial cells in DN and its specific mechanism,this research group provides a new approach and target for the treatment of diabetic nephropathy.Methods:In vivo study: In this study,STZ-induced mice were used to construct a diabetic model.After successful modeling,the STZ mice were treated with CAR(1g /kg)and Fer-1(20g/kg,intraperitoneal injection)for a total of 16 weeks.The mice were randomly divided into 5 groups(n=6-8): NC group,NC+ carnosine group,STZ group,STZ + carnosine(1g /kg)group,and STZ + Fer-1(5 mg/kg)group.After 16 weeks,the serum and urine of mice were collected,and the indexes of hematuria metabolism and renal tubule pathological injury were evaluated,mitochondrial morphology was observed,and ferroptosis related indexes were detected.Serum CRE,BUN and 24 h urinary albumin were detected.HE staining and PAS staining were used to observe the degree of renal pathological injury.Mitochondrial damage in kidney of STZ mice was observed by transmission electron microscopy.Western blot and Real-time PCR were used to detect Cox-2,ACSL4 and GPX4.The expression level of GPX4 was detected by immunohistochemistry.The contents of MDA,GSH and Fe2+ in mouse kidney were detected.The expressions of inflammatory cytokines(IL-1β,IL-6,MCP-1 and TNF-α)were detected by Real-time PCR.The expression of NRF2 was detected by Western blot.In vitro study: We experimented with human renal tubular epithelial cells(HK2).First,the toxic dose and optimal concentration of the drug were screened by MTT assay,and then the time of hyperglycemic stimulation was screened by Western blot and Real-time PCR.The cells are divided into the following six groups: Mannitol group,control group,control + carnosine group,high glucose group,high glucose + carnosine(48 μmmol/L)group,high glucose +Fer-1(5 μmmol/L)group.We treated high-glucose induced HK2 cells with CAR and Fer-1,an ferroptosis inhibitor.The effects of carnosine on the expressions of Cox-2,ACSL4 and GPX4 of ferroptosis indexes were detected by Western blot and Real-time PCR.The effects of carnosine on the expression levels of inflammatory cytokines(IL-1β,IL-6,MCP-1 and TNF-α)were determined by Real-time PCR.The impact of carnosine on reactive oxygen species was detected by immunofluorescence assay(DCF and DHE).The ability of carnosine to bind NRF 2 in renal tubular epithelial cells was investigated by molecular docking experiments and cellular thermal displacement assay(CETSA).The ability of NRF 2 to bind to GPX 4 was tested by a Ch IP assay.Next,NRF2 si RNA was used to silence NRF2 in HK2 cells,and the effect of carnosine on ferroptosis in HK2 cells was examined.Results:In vivo study: We found that carnosine alleviated kidney injury in STZ mice.Western blot,Immunhistochemistry and Real-time PCR results confirmed that carnosine significantly inhibited the occurrence of ferroptosis in the kidney of STZ mice.And carnosine could reverse the increase in MDA and Fe2 + levels and the decrease in GSH levels in STZ mice.Real-time PCR results showed that carnosine reduced the inflammation in the kidney of STZ mice.Furthermore,we found that NRF 2 expression was decreased in STZ mouse kidneys,and carnosine treatment promoted renal NRF 2expression in STZ mice.In vitro study: The optimal concentration of carnosine of 48 μ mmol/L was selected based on the results of the MTT experiments.Western blot The optimal time point for screening for GPX4,a key protein of ferroptosis,was 36 h.KIM-1 was detected by immunofluorescence and Real-time PCR,and the results showed that KIM-1 expression was significantly increased under high glucose stimulation,and carnosine treatment reversed the increase of KIM-1 expression.Both Western blot and Real-time PCR results confirmed that carnosine significantly inhibited ferroptosis in HK2 cells under high glucose stimulation.Real-time PCR results indicated that carnosine decreased the inflammatory response of HK2 cells under high glucose stimulation.And carnosine can reverse the increase in MDA and Fe2 + levels and the decrease in GSH levels in HK2 cells stimulated with high glucose.Western blot Found that NRF2 expression was decreased in high glucose groups,and carnosine treatment significantly increased NRF2 expression.Results from molecular docking experiments and cellular thermal shift experiments revealed that NRF2 is a possible target of carnosine.Immunofluorescence results showed that carosepin could promote nuclear ectopic increase of NRF2,and Western blot results showed that carosepin could promote increased NRF2 nuclear protein expression.Ch IP experiments showed that carnosine promotes binding of NRF2 to the GPX4 promoter and induces its transcriptional expression.Knockdown of NRF2 in HK2 cells with si RNA abolished the protective effect of myosine against ferroptosis in HK2 cells under high glucose stimulation.Conclusion:(1)In vivo results showed that carnosine improved kidney damage in STZ-induced diabetic mice,and inhibited ferroptosis and inflammation.(2)In vitro results showed that carnosine inhibited ferroptosis and inflammation of renal tubular epithelial cells induced by high glucose.(3)Carnosine may alleviate diabetic nephropathy by inhibiting ferroptosis in tubular epithelial cells dependent on NRF2.