Effects And Mechanisms Of Carnosine On Podocyte Injury By Inhibiting Pyroptosis In Diabetic Nephropathy

Background and objective:Diabetic nephropathy(DN)is one of the main complications of diabetes and often develops into end-stage renal disease.The latest epidemiological data show that DN,as end-stage renal disease,is the main cause of death for diabetic patients,causing huge physical and psychological stress and economic burden on individual families and society.In the development of DN,chronic inflammation is considered to be a key factor.As there is still a lack of effective therapeutic drugs and key targets for the treatment of DN,it is of great significance to find new and more effective DN therapeutic drugs.Carnosine L-Carnosine,the scientific name β-alanyl-L-histidine,is a dipeptide composed of two amino acids β-alanine and L-histidine.Carnosine was originally found in skeletal muscle.In skeletal muscle,it is present in greater amounts than other tissues.Most studies on the physiological effects of carnosine have been conducted on skeletal muscles.However,recent studies have shown that carnosine may play a broader physiological role in other tissues,including the brain,heart,pancreas,kidney,and cancer cells.The results of previous studies have shown that carnosine can play an anti-inflammatory and anti-fibrotic effect in kidney disease.This topic further explores the protective function of carnosine on podocytes in diabetic nephropathy and its related mechanisms,and seeks potential therapeutic targets and new ideas for diabetic nephropathy.Methods:In vitro experiment:select mouse podocytes(MPC5)between the 6th and 15th generation for experiment.Firstly,the optimal therapeutic concentration of the drug was screened by MTT.The experiment is divided into five groups:the normal control group,the single administration group,the mannitol control group,the high-glucose model group,and the high-glucose addition group.Cell protein and mRNA were extracted,and the podocyte functional proteins nephrin and podocin were detected by Western blot and immunofluorescence.Western blot and Real-time PCR were used to detect the release of renal inflammatory factors IL-1β and IL-18.Western blot was used to detect the expression of inflammatory pathway proteins NLRP3 and ASC.Then Western blot and Real-time PCR were used to detect the key proteins caspase 1 and GSDMD.Finally,TUNEL staining was used to detect the level of programmed cell death.Molecular docking and CETSA experiments were used to verify the binding of carnosine and caspase 1.After silencing caspase 1 expression by siRNA transfection and confirming the effect of caspase 1 silencing,Western blot and Real-time PCR were used to detect podocyte damage(nephrin,podocin),inflammation(NLRP3,ASC,IL-1β and IL-18)and pyroptosis(GSDMD).In vivo experiment:A diabetic nephropathy model was constructed by intraperitoneal injection of STZ(50 mg/kg)in mice for 5 consecutive days,and 1 g/kg carnosine treatment was given.The experiment is divided into four groups:NC group,NC+car group,DM group,DM+car group.After successful modeling,the changes in renal function were monitored by 24h urine albumin,the morphological changes of glomeruli in each group were compared by HE and PAS staining,and the changes in foot process width and basement membrane thickness in each group were detected by transmission electron microscope.The number of podocytes(WT1)and the functional proteins nephrin and podocin of podocytes were detected by immunohistochemistry and Western blot.Similarly,immunohistochemistry and Western blot were used to detect the expression of NLRP3 and ASC.The inflammatory factors IL-1β and IL-18 were detected by Western blot.Finally,immunohistochemistry and Western blot were used to detect the changes of the carnosine target protein caspase 1 and the changes in the level of pyroptosis(GSDMD).Results:In vitro experiments:MTT shows that the optimal therapeutic concentration of carnosine is 40μM.Western blot and immunofluorescence results show that carnosine can reduce the damage of podocytes under high glucose environment.Western blot and Real-time PCR also showed that carnosine can reduce inflammation-related proteins NLRP3,ASC,and inflammatory factors IL-1β and IL-18.Finally,Western blot and Real-time PCR also showed that carnosine significantly reduced the expression of key proteins caspase 1 and GSDMD.TUNEL staining also confirmed that carnosine can reduce the occurrence of programmed necrosis.Both molecular docking and cell thermal displacement experiments(CESTA)have confirmed that caspase 1 is a possible target of carnosine.Immunohistochemistry also showed high expression of caspase 1 in kidney tissue of DN patients.Then,after carnosine treatment on the cells that silenced caspase 1,Western blot and Real-time PCR results showed that silencing caspase 1 can reduce podocyte functional damage,inflammation and pyroptosis,but carnosine cannot further play its role in antagonizing high glucose damage.In vivo experiment:24h urine albumin results show that carnosine can obviously reverse the kidney damage induced by STZ.Similarly,HE and PAS staining showed that carnosine reduced glomerular morphology changes.Projection electron microscopy results show that carnosine can alleviate the changes of podocyte foot processes and basement membrane induced by STZ.Western blot,Real-time PCR and immunohistochemistry confirmed that STZ-induced loss of podocytes’ functional protein,inflammation,and pyroptosis can be antagonized by carnosine.At the same time,TUNEL staining also showed that carnosine alleviated STZ-induced programmed death.Conclusion:(1)In vitro studies show that carnosine inhibits STZ-induced podocyte damage,inflammation,and pyroptosis.(2)In vitro studies show that after molecular docking and cell thermal displacement experiments(CESTA),it is proved that carnosine can act by targeting caspase 1.In the cells with caspase 1 silenced,carnosine could not further alleviate the levels of podocyte damage,inflammation and pyroptosis induced by high glucose.It suggests that carnosine may protect the podocytes from STZ-induced damage through a caspase 1-dependent mechanism.(3)In vivo experiments show that carnosine effectively inhibits STZ-induced mouse kidney podocyte damage,inflammation,and pyroptosis.Our study might provide a new target and strategy for the treatment of DN.