Protective Effects And Mechanisms Of Grape Seed Proanthocyanidin Extract On Mitochondria In Diabetic Kidney Disease

BackgroundDiabetic kidney disease(DKD)is one of the most serious microvascular complications of diabetes mellitus.It has overtaken glomerulonephritis as the first cause of chronic kidney disease in China.Due to the complex pathogenesis,expensive treatment and multiple adverse drug reactions of DKD,it is necessary to find new therapeutic targets.Mitochondria serve as the hub of cellular energy metabolism and are also highly susceptible to damage from various pathological factors.In the kidney,mitochondrial function plays a crucial role in providing energy for normal physiological activities,and the kidney has the second-highest mitochondrial content among human organs.Due to the high metabolism and high energy consumption,the number of mitochondria in renal tubular epithelial cells is significantly higher than in other cells of the kidney.Mitochondrial dysfunction typically appears early in the development of DKD and often precedes clinical manifestations.Therefore,it is crucial to investigate the role of mitochondrial damage in the pathogenesis of DKD.Mitochondrial biogenesis and dynamics homeostasis are essential for maintaining proper mitochondrial function.Maintaining mitochondrial biogenesis and dynamic homeostasis is essential for the normal physiological function of mitochondria.However,mitochondrial biogenesis and fusion are reduced,while mitochondrial fission is increased,resulting in a large number of small,fragmented,non-functional mitochondria in DKD.Additionally,the mitochondrial respiratory chain complexes undergo electron escape,producing significant amounts of reactive oxygen species(ROS)that can trigger apoptosis.Grape seed proanthocyanidin extract(GSPE)is a natural polyphenolic compound extracted from grape seeds that has various pharmacological effects,including antiinflammatory and antioxidant properties.GSPE can reduce endoplasmic reticulum stress and slow down kidney injury in DKD rats and improve mitochondrial function in skeletal muscle cells by activating silent information regulatorl(SIRT1)and peroxisome proliferator-activator receptor ycoactivator-1α(PGC-1α).However,it is still unclear whether GSPE’s renoprotective effects also promotes mitochondrial biogenesis and improves mitochondrial function through the SIRT1/PGC-1α pathway.In addition,p66Shc,a member of the ShcA bridging protein family,which plays an important role in regulating aging and metabolic disorders,can modulate mitochondrial structure and function by regulating cellular oxidative stress and signaling pathways.It has been demonstrated that p66Shc is closely related to mitochondrial biogenesis and dynamics.Further studies are needed to confirm whether GSPE’s nephroprotective effects can inhibit p66Shc expression,regulate mitochondrial biogenesis and dynamics homeostasis,and protect mitochondrial function.Therefore,p66Shc was hypothesized as a potential therapeutic target of GSPE to explore the protective effect of GSPE in DKD.And relevant experiments were designed to verify it.Objectives1.The aim of this study is to investigate the therapeutic effect of GSPE on renal injury in DKD rats and to elucidate the molecular mechanism of GSPE promoting mitochondrial biogenesis and exerting renoprotective effects.2.To determine whether GSPE can maintain mitochondrial biogenesis and dynamics homeostasis in human kidney proximal tubule(HK-2)cells under high glucose induction and to investigate whether it can reduce mitochondrial dysfunction.3.To explore the changes in p66Shc expression in HK-2 cells under high glucose induction and to elucidate its role in the process of oxidative stress injury and apoptosis.4.To investigate the effect of p66Shc on mitochondrial biogenesis and dynamics homeostasis in HK-2 cells and to examine the relationship between GSPE and p66Shc expression.Methods1 Animal experiments1.1 40 male SD rats were divided into control group,control treatment group,model group,and treatment group.The diabetic rat model was prepared by intraperitoneal injection of streptozocin(STZ).The control treatment group and the treatment group were gavaged with GSPE,while the control group and the model group were gavaged with an equal amount of saline and continuously treated for 12 weeks.1.2 After 12 weeks,the body weight of rats was measured.And blood glucose,blood creatinine,and urinary microalbumin were detected.Hematoxylin-eosin(HE)staining and periodic acid Schiff(PAS)staining were performed to observe the histopathological changes of kidney,and transmission electron microscopy was used to observe the ultrastructure of renal tissue.TUNEL staining was performed to assess apoptosis.1.3 Immunohistochemistry and Western blot were used to detect the expression of mitochondrial biogenesis-related proteins,SIRT1,PGC-1α,nuclear respiratory factor 1(NRF1),and mitochondrial transcription factor A(TFAM)in renal tissue.2 Cellular experiments2.1 HK-2 cells were used for subsequent experiments.Overexpression or deletion of p66Shc was achieved by transfection of plasmids or si-RNA.Transfection efficiency was detected by quantitative reverse transcription PCR(qRT-PCR)and Western blot.2.2 Fluorescence probes were used to detect intracellular and mitochondrial ROS in HK2 cells.The flow assay was used to detect changes in mitochondrial membrane potential and the apoptosis rate of each group.2.3 Western blot was performed to detect the expression of mitochondrial biogenesisrelated proteins(SIRT1,PGC-1α,NRF1,and TFAM),mitofusins 1(MFN1)and dynamin-related protein 1(DRP1)in HK-2 cells.Results1.GSPE reduced blood glucose,blood creatinine,and urinary microalbumin in DKD rats.2.GSPE decreased the proliferation of mesangial cells,mitigated structural damage to renal tubules,alleviated fusion of podocytic foot processes and glomerular basement membrane thickening in DKD rats,and improved mitochondrial fragmentation in renal tubular epithelial cells.3.GSPE promoted mitochondrial biogenesis,protected mitochondrial function and reduced cells apoptosis in DKD rats.4.GSPE downregulated the expression of p66Shc and reduced apoptosis in high glucoseinduced HK-2 cells.5.GSPE decreased intracellular and mitochondrial ROS production and reduced oxidative stress injury in HK-2 cells.6.GSPE elevated mitochondrial membrane potential in HK-2 cells and increased the activity of mitochondrial respiratory chain enzyme complexes I and III,while enhancing mitochondrial quality.7.GSPE promoted mitochondrial biogenesis and maintained mitochondrial dynamics homeostasis in HK-2 cells.ConclusionThe results of this experiment showed that the natural antioxidant GSPE could promote mitochondrial biogenesis,maintain mitochondrial dynamics homeostasis,reduce oxidative stress damage,protect mitochondrial function,reduce apoptosis,and slow down the progression of DKD by down-regulating p66Shc expression.