| Diabetic nephropathy(DN)is one of the major microvascular complications of diabetes,which is the leading cause of end stage renal disease and dialysis among the world.The pathogenesis of DN is complicated,and the Stress-induced senescence of renal tubular epithelial cells plays an important role in the development of DN.Studies have confirmed that the senescence of renal tubular cells is one of the pathological manifestations of DN.Senescent tubular cells can produce large amounts of senescence-associated secretory phenotypes including various proinflammatory and profibrotic factors,which cause local inflammatory responses and fibrosis.The inhibition of the accelerated aging of renal tubules can significantly alleviate the process of DN towards the end stage renal disease.E3 ubiquitin ligase Parkin is widely expressed in the brain,heart,kidney and other tissues and its genetic mutation is closely related to Alzheimer's disease and Parkinson's disease.Overexpression of Parkin can prolong the life span of Drosophila,indicating that Parkin may be a potential anti-senescence factor.However,the relationship of Parkin and DN renal aging remains to be comfirmed.The purpose of this study was to investigate the role of Parkin in stress-induced senescence of renal tubular cells in DN,and to find a new effective target and theoretical basis for the prevention and treatment of DN.Methods1.Clinical subjectsThe subjects of this study were patients with type 2 diabetes who were hospitalized in our department from January 2015 to June 2016,with a total of 40 patients diagnosed with DN after renal pathological test.In the control group,10 cases of normal renal tissues were obtained from the patients with primary renal tumor.Baseline clinical datas of the two groups were collected,including age,sex,height,weight,body mass index,systolic blood pressure,and diastolic blood pressure.Blood and urine spicements were collected 1 days before renal biopsy or surgery and preserved in-80 temperature.Serum and urinal biochemical parameters were further tested including serum creatinine,serum urea nitrogen,blood glucose,glycosylated hemoglobin,serum cystatin C,serum albumin,serum uric acid,serum total cholesterol,triglyceride,high density lipoprotein,low density lipoprotein,C reactive protein,urinary protein quantitation,urinary N-acetyl glucosaminidase and estimated glomerular filtration rate.Immunohistochemistry was used to detect the expression of Parkin in renal tissue of diabetic nephropathy.The positive rate of Parkin was calculated and the correlation of Parkin and clinical data or pathological damage score was analyzed.Immunofluorescence was performed to detect the relationship of Parkin with senescence markers p16,senescence associated secretory phenotype component IL-6 and TGF-? in renal tissue of DN patients respectively.2.In vitro experimentsRenal cortex is clipped from 3-4week old male C57 mice and then incubates with type II collagenase and filtrated through double screen mesh in order to obtain renal tubular cells.After two passages of conventional culture by special primary renal tubular cell culture medium,it can be used for subsequent experiments.30mmol/l high glucose was used to stimulate renal tubular cell for 72 h.The expression of senescence marker p16 and ?-H2 AX in renal tubular cells was detected by immunofluorescence,and the changes of p16,Parkin protein levels was detected by WB.PCR was also used to detect the level of Parkin mRNA,and ELISA was used to detect the secretion of IL-6 and TGF-? in the supernatant of cell culture medium.Parkin overexpressional adenovirus or Parkin si RNA was further used to transfect the renal tubular cells,and p16 and?-H2 AX was detected in glucose stimulated renal tubular cells after the upregulation or downregulation of Parkin,so is the secretion of IL-6 and TGF-? in the supernatant of cell culture medium.Results1.Expression of Parkin in renal tissue of diabetic nephropathy and its relationship with stress-induced senescence of renal tubular cells and renal interstitial injury.Immunohistochemistry showed that Parkin was mainly expressed in the renal tubular epithelial cells.The positive rate of Parkin in the renal tubular cells of the control group was 43.5±6.0% and the positive rate of Parkin in IFTA 0 group was ? 40.8±4.3%,which was similar to that of the control group.The positive rate of Parkin in IFTA 1 group was 32.8±4.8%,28.5±4.2% in IFTA 2,and was 19±4.9% in IFTA 3 group,suggesting that the positive rate of Parkin decreased with the development of DN.Correlation analysis showed that the positive rate of Parkin was negatively correlated with glomerular sclerosis,tubular atrophy,interstitial fibrosis and inflammation(P<0.05).Besides,Parkin was negatively correlated with renal function injury parameter,including urinary protein quantitation,urine NAG,urea nitrogen,serum cystatin C,serum creatinine and systolic blood pressure,and Parkin was positively correlated with eGFR(P<0.05).The results indicated that the decrease of Parkin expression was closely related to the renal structure and function damage of DN.Further tested by immunofluorescence,senescence marker p16,inflammatory cytokines IL-6 and profibrotic cytokine of TGF-? increased in the renal tissues of DN patient,compared with the control group,which was contrary to the trend Parkin expression.2.In vitro study of the relationship between Parkin and stress-induced renal tubular epithelial cellular senescence.2.1 High glocuse induced renal tubular cells accelerated senescense.In vitro,compared with the normal control group,the positive rate of p16 and ?-H2 AX of renal tubule cells in high glucose group increased significantly(P< 0.05).WB found that the protein level of p16 in the high glucose group was 2.13 times higher than that in the control group.ELISA showed that the secretion of IL-6 and TGF-? was significantly increased in renal tubular cells,suggesting that high glucose can inhibit the expression of Parkin and promote renal tubular epithelial cellular stress-induced senescence and expression of IL-6 and TGF-?in renal tubular cells.2.2 The change of Parkin expression in high glucose induced renal tubular cells.Compared with the control group,Parkin mRNA levels in renal tubular cells decreased by 60.8% and protein levels decreased by 53.1% after high glucose stimulation(P< 0.05),which was similar with the trend of Parkin in DN patients,suggesting that high glucose could inhibit the expression of Parkin in renal tubular cells.2.3 The Effects of Parkin on stress-induced senescence of renal tubular cells stimulated by high glucose.Compared with the high glucose group,the positive rates of p16 and ?-H2 AX in renal tubular cells were decreased by 32.1% and 37.8%(P< 0.05)respectively after the expression of Parkin upregulation and WB indicated that the protein level of p16 was also reduced by 31.7%(P< 0.05 vs high glucose group).After Parkin siRNA silented the expression of Parkin,we found that Parkin si RNA group p16 and ?-H2 AX positive renal tubular cell ratio increased 24.5% and 17.6%(P<0.05)compared with pure high glucose stimulation group;WB found that the level of p16 protein in this group was increased by 21.0%(P<0.05,vs high glucose group),proving that Parkin could inhibit the stress-induced senescence of renal tubular cells stimulated by high glucose.Further detection of IL-6 and TGF-? secretion in cell culture medium supernatant,we found that the secretion of IL-6 and TGF-?in high glucose+Parkin si RNA group were significantly increased compared with the high glucose group,while the IL-6 and TGF-? secretion in high glucose + Parkin overexpression group was significantly reduced compared with the high glucose group(P<0.05),indicating that Parkin can inhibit the secretion of IL-6 and TGF-? in renal tubular cells stimulated by high glucose.ConclusionE3 ubiquitin ligase Parkin gradually decreases with the aggravation of DN renal interstitial injury,and is closely related to the damage of DN renal tissue structure and function,and the stress-induced senescence of renal tubular cells;Parkin can inhibit the renal tubular epithelial cellular stress-induced senescence and proinflammatory cytokine secretion stimulated by high glucose. |
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