Metformin Regulates Mitochondrial Autophagy Through Nrf2/PINK1 Pathway And Delays The Senescence Of Renal Tubular Epithelial Cells In Diabetic Nephropathy

Objective:Kidney is an important metabolic organ of the human body.It bears a high oxidative stress under physiological conditions,and its function decreases year by year with the growth of age.Kidney aging includes a series of complex changes in kidney tissue structure,physiological function,intrinsic cellular state,molecular biology,and epigenetics.Aging kidneys are more sensitive to harmful factors,vulnerable to various diseases such as diabetes and hypertension,and lack of self-repair ability,which leads to the occurrence or deterioration of chronic kidney disease(CKD).Diabetic nephropathy is one of the primary causes of chronic kidney disease(CKD)in China and renal tubular epithelial cells senescence induced by high glucose is an important cellular event leading to interstitial injury in diabetic nephropathy.Therefore,it is of great and profound clinical significance to actively explore the molecular mechanism of renal aging for the prevention and treatment of CKD.The mechanism of renal aging is not completely understood.However,increasing evidence suggests that cellular senescence,especially non-telomeric factors such as oxidative stress,DNA damage,and mitochondrial dysfunction,may play a key role in mediating the renal aging process[1].And it’s also associated with activation of p16/Rb or ARF/p53 pathway.Mitochondria are the energy factories of cells and as well as the centers of fatty acid β-oxidation,reactive oxygen species production,innate immunity,and apoptosis.Mitochondrial dysfunction is not only a common event in the course of aging diseases,but also is considered to be an important intrinsic driving factor of aging.Mitophagy is a specific mode of autophagy.As an endogenous function of the body against oxidative stress damage,mitophagy can "selectively" identify and remove damaged and aging mitochondria,thus maintaining the relative stability of mitochondrial morphology,quantity and function,while eliminating the oxidative stress damage caused by excessive mtROS.Recent researchers have found that decreased mitophagy efficiency or dysfunction plays an important role in aging and variety of age-related diseases,such as metabolic syndrome,diabetes,cancer and neurodegenerative diseases.NF-E2-related factor 2(Nrf2)is an important transcription factor in the regulation of antioxidative stress response and is widely involved in mitochondrial regulation.The PINK 1/Parkin-mediated ubiquitin-dependent pathway is the classical pathway of mitophagy.Some studies have reported that Nrf2 regulates PINK1 transcription and alleviates oxidative stress-related cell death in neuroblastoma.Moreover,it has been suggested that the autophagy receptor protein p62 was essential for Parkin-mediated mitophagy,which is directly regulated by Nrf2.And Nrf2 has been reported to activate autophagy activity to protect kidney function.Therefore,the positive regulation of mitophagy by Nrf2 may be an important way to delay the onset and development of renal aging.Metformin(MET)is the classic drug for the treatment of type 2 diabetes,which can inhibit the accumulation of advanced glycation end products(AGEs)and mtROS in diabetic conditions,and reduce oxidative stress and inflammation.Thus,it inhibited the apoptosis of renal tubular epithelial cells.Meanwhile,metformin,as an AMP-activated protein kinase(AMPK),can directly affect the phosphorylation of Nrf2 and the expression of up-regulated antioxidant genes through the activation of AMPK[2].In addition,metformin prevents the increase of p16 and p21 levels during cellular senescence and reduces pro-inflammatory cytokines associated with age-related secretory phenotype(SASP).In conclusion,metformin has considerable potential in alleviating renal aging.In this study,we will take Nrf2/PINK1 pathway as the breakthrough point,and focus on Nrf2 and mitophagy,to further explore the mechanism of both in the aging process of renal tubular epithelial cells induced by high glucose,and observe whether metformin can delay kidney aging by improving the level of mitophagy and protecting the antioxidant stress ability of the cells in vitro and in vivo experiments.Methods:Part1:To establish a model of human renal tubular epithelial cell senescence induced by high glucose and observe the changes of mitophagy during senescence and the effects of metformin on it.Human renal tubular epithelial cells(HK-2)were selected for the experiment,and the senescent cell model was established by the intervention of high glucose in vitro.According to the needs of the experiment,the cells were divided into normal glucose control group(NG),high glucose group(HG),and high glucose+metformin group(HG+Met).After all the cells were synchronized with serum-free medium for 24 hours,the cells were treated with different treatments.The HG group was cultured with DMEM/F12 high glucose medium(D-glucose concentration was 30mmol/L)and the NG group was cultured with DMEM/F12 normal glucose medium(D-glucose concentration was 5.56mmol/L).The HG+Met group was stimulated with 1mmol/L metformin for 2h and then transferred to high glucose medium.All cells were cultured for 72 hours.The effect of high glucose exposure on human HK-2 cell viability was detected by CCK-8.The cell cycle was detected by flow cytometry.Western blot was used to detect the expression level of age-related protein P21,and β-galactosidase in situ staining(SAβ-gal)was used to detect the positive rate of senescence cells.Subsequently,Western blot was used to detect the expression levels of mitochondrial autophagy related proteins LC3、P62、PINK1、Parkin and TOMM20,and Real-Time Quantitative PCR was used to detect the mRNA expressions of P21、LC3 and P62.Acridine orange(AO)staining was used to detect autophagy associated acid vesicle organelle formation.LC3-GFP-MTR(MitoTracker Red)co-localization staining and laser confocal microscopy were used to observe the number of mitochondria autophagy spots.Combined with transmission electron microscopy to observe the changes of mitochondrial autophagy during the aging process of human renal tubular epithelial cells induced by high glucose and the effects of metformin on mitochondrial autophagy and aging.Part2:The role of Nrf2 in the regulation of mitophagy on high gluced-induced senescence of renal tubular epithelial cells and the role of metformin on it.1.According to the the first part of the experimnet,the cells were grouped.Western blot was used to detect the expression levels of Keap1 and Nrf2,to observe the change trend of Keapl/Nrf2 pathway after high glucose exposure,and whether metformin can induce the activation of Nrf2.2.The autophagy inhibitor 3-MA was applied to the cells,and the protein expression levels of LC3、PINK1、TOMM20 and P21 were detected by Western blot,in order to clarify the causal relationship between mitochondrial autophagy and senescence in the process of human renal tubular epithelial cells senescence induced by high glucose.3.siRNA-Nrf2 gene specificity was used to silence Nrf2.Based on the experimental grouping in the first part,high glucose+siRNA group(HG+siRNA)and high glucose+metformin+siRNA group(HG+Met+siRNA)were added.The expression levels of Keap1/Nrf2 pathway protein,mitochondrial autophagy related protein LC3,P62,Beclinl,PINK1,Parkin,TOMM20 and aging related protein P21 were detected by Western blot.The PINK 1 mRNA level was detected by RT-qPCR.Acridine orange(AO)staining combined with LC3-GFP-MTR(MitoTracker Red)co-localization staining and laser confocal microscopy were used to observe the number of mitochondrial autophagy spots.Real-Time Quantitative PCR was used to detect PINK1 mRNA expression.To verify that metformin activates mitochondrial autophagy through Nrf2/PINK1 pathway and plays a role in delaying cell senescence.Part3:To verify in vivo whether metformin improves mitophagy function through Nrf2,thereby delaying renal aging in diabetic mice7-week-old male db/m and db/db mice were used for in vivo experiments.After 1 week of adaptive feeding,the mice were divided into db/m group,db/db group,db/db+metformin group(metformin was added to drinking water and the concentration was 1mg/ml),with 10 mice in each group.Body weight and blood sugar were measured weekly,and blood and urine samples were collected at 24 weeks of age,then kidneys of mice were taken out,embedded and sectioned..Blood creatinine and 24-hour urine protein levels were measured.HE and Masson staining were used to test pathological changes,and Western blot was used to detect the expression of P21、LC3、P62、PINK1、Parkin、TOMM20 and Nrf2.Results:Part1:Compared with NG group,the expression of P21 and the positive rate ofβ-galactosidase staining in HG group were significantly higher than those in NG group.The results of CCK-8 showed that HK-2 cell activity decreased significantly in HG group.Cell cycle testing showed that more than 80%of cells in the HG group were stuck in the G0-G1 phase,which was significantly higher than that in NG group.In the process of human HK-2 cell senescence induced by high glucose,Western blot results showed that compared with NG group,LC3Ⅱ/Ⅰ ratio and PINK1 expression of autophagy related protein were significantly decreased in HG group,while P62 and TOMM20 expression were significantly increased.Compared with HG group,the expression of P21,LC3II/I ratio and PINK 1 were increased,and the expression of P62 and TOMM20 were decreased in HG+Met group.The results of RT-qPCR were consistent with those of Western blot.Autophagy acridine orange staining showed that the percentage of AO staining positive cells decreased in HG group.LC3-GFP-MTR co-localization staining showed that the number of orange fluorescent spots decreased significantly in HG group.Compared with the HG group,the above staining results of the HG+Met group were increased,but still lower than the NG group.Part2:Western blot assay showed that the expression of Nrf2 decreased and Keapl increased during senescence of human HK-2 cells induced by high glucose compared with NG group.After metformin treatment,Nrf2 expression in the HG group was improved,but still lower than that in the NG group.Nrf2 was knocking down by siRNA transfection,the expressions of mitophagy related proteins LC3,PINK1,Parkin and Beclinl were significantly decreased inHG group.The expression of P62 and TOMM2O increased significantly.The age-related protein P21 was significantly increased.After the intervention of metformin,LC3、PINK1、Parkin and Beclinl expressions in HG+Met+siRNA group were significantly increased,while P62 and TOMM2O expressions were decreased in the HG+siRNA group.However,they could not restored to normal level.RT-qPCR showed that the transcription level of PINK 1 in HG group was lower than that in NG group.Metformin restored HG-induced low expression of PINK1 mRNA and protein,but this effect was partially eliminated by Nrf2 siRNA.Moreover,the transcription level of PINK1 was not improved when given metformin again.Compared with HG group,autophagic acridine orange staining showed a decreased percentage of AO staining positive cells in theHG+siRNA group.LC3-GFP-MTR co-location staining showed a significant decrease in the number of orange fluorescent spots.When autophagy was blocked by 3-MA,western blot results showed that LC3Ⅱ/Ⅰ ratio and PINK 1 expression were decreased in HG group,the 3-MA group and the HG+3-MA group,and the expressions of TOMM20 and P21 were increased,compared with NG group.The trend of HG+3-MA group was the largest.Part3:Compared with db/m group,db/db mice showed increased fasting blood glucose,serum creatinine and 24-hour urine protein in vivo experiments.Moreover,compared with db/m group,renal HE staining showed glomerular hypertrophy and Masson trichrome staining revealed aggravated interstitial fibrosis of renal tubules in db/db group.Western blot results showed that the expressions of mitophagy related proteins LC3、PINK1 and Parkin were decreased and the expressions of P62 and TOMM20 were increased in the db/db group.The expression of aging related protein P21 was increased,and the mRNA expression of P21 was consistent with that of Western blot.Treatment with metformin down-regulated fasting blood glucose,serum creatinine,and 24-hour urinary protein levels in mice,and alleviated renal lesions as indicated by HE and Masson staining.Compared with db/db group,LC3、PINK1 and Parkin expressions were up-regulated,while P62,TOMM20 and P21 expressions were down-regulated.Conclusion1.During senescence of human renal tubular epithelial cells induced by high glucose,Keap1/Nr￡2 pathway is inhibited,which then leads to the inhibition of mitophagy function.2.High glucose stimulation induces senescence of human renal tubular epithelial cells by inhibiting mitophagy function.3.Nrf2 can positively regulate mitophagy-related protein PINK1 transcription under the condition of high glucose,promote mitophagy and delay cellular senescence.4.Metformin can improve the mitophagy function via Nrf2/PINK1 pathway to delay the senescence of human renal tubular epithelial cells induced by high glucose.