Preventive Effect Of Shenkang Injection Against High Glucose-induced Senescence Of Renal Tubular Cells

Background and ObjectiveDiabetic nephropathy(DN)is the leading cause of end-stage renal disease.Renal proximal epithelial tubular cells are susceptible to hypoxia-and oxidative stress-induced injury in a high-glucose(HG)metabolic environment.Injured epithelial tubular cells initially exhibit hyperplasia and hypertrophy and finally experience stress-induced premature senescence.Lots of studies have shown that renal tubular epithelial cell senescence appears in renal tissues of both DN patients and a diabetic rat model,even in the early stage.In vitro,it has also been confirmed that HG accelerates stress-induced premature senescence of renal tubular epithelial cells.All the studies prove that renal tubular cell senescence is an important biological event in tubulointerstitial injury during DN progression.Renal tubular cell senescence has also been found to be closely associated with tubular atrophy,interstitial inflammation,interstitial fibrosis,and renal function failure.Moreover,accelerated senescence of renal tubular epithelial cells confers a higher susceptibility to renal injury and impedes renal repair through inhibition of tubular cell proliferation.Because stress-induced premature senescence in renal tubular cells plays an important role in the progression of DN,prevention and treatment of renal tubular cell senescence may offer a novel therapeutic approach to the prevention and retardation of DN.Shenkang injection(SKI)is extracted from four kinds of traditional Chinese herbs(TCM),including Radix Astragali,rhubarb,Astragalus safflower,and Salvia.SKI(certification No.Z20040110)was approved by the State Food and Drug Administration of China in 1999 for the treatment of chronic kidney diseases(Permission No.YBZ08522004).Through extensive clinical trials,SKI was found to remarkably reduce serum creatinine and blood urea nitrogen and to protect residual renal function with good efficacy and safety in patients with chronic kidney diseases.Both in vitro and in vivo studies have found that SKI significantly inhibits oxidative stress-induced renal injury and prevents interstitial fibrosis.In addition,SKI effectively attenuates HG-induced mesangial cell hypertrophy through the inhibition of P21 overexpression,thus preventing HG-induced senescence in mesangial cells.Although SKI has been reported to have a beneficial role in minimizing renal injury and expediting tissue repair,little is known about its effects on renal proximal epithelial tubular cells.In this study,we investigated the potential protective effects of SKI against HG-induced renal tubular cell senescence and the underlying mechanism.Methods1.The primary renal tubular epithelial cells1.1 Renal cortex tissues were obtained form 3-week-old,male C57/BL6 mice under sterile conditions.The tissues were digested with type II collagenase for 30 min 37 ° and passed through two screen filters(followed by 250 uM and 80 uM mesh)to isolate primary renal proximal tubule epithelial cells.Then the Primary renal proximal tubule epithelial cells were cultured in 37 ?,5% CO2 incubator.1.2 Expression of specific renal tubular epithelial cell markers CK18 and villin was examined by immunofluorescence analysis.2.Aging renal tubular epithelial cell model was established by high glucose.The renal tubular epithelial cells from the second passage were cultured in serum-free medium for 24 h and then cultured in medium containing 5 m M glucose(control),mannitol,30 m M glucose(HG)for 72 h.Cell morphology and senescence marker p16Ink4,senescence-associated heterochromatin foci(SAHFs),cyclinD1,DcR2 and senescence-associated-?-Gal(SA-?-Gal)were observed by immunofluorescence.3.The protection effect of Shenkang injection against high glucose-induced senescence of renal tubular cellsExperimental groups:(1)control medium(control group),medium containing 5 mmol/L glucose;(2)mannitol medium(mannitol group),medium containing 5 mmol/L glucose and 25 mmol/L mannitol;(3)HG medium(HG group)containing 30 mmol/L glucose;(4)HG + 50 mg / L SKI group(50 mg/LSKI concentration in HG medium);(5)HG + 100 mg / L SKI group(100 mg/LSKI concentration in HG medium);(6)HG + 200 mg / L SKI group(200 mg/LSKI concentration in HG medium);(7)control+ SKI group(200 mg/L SKI treatment in control medium).Cell morphology and senescence marker p16Ink4,SAHFs,cyclin D1,DcR2 and SA-?-Gal were observed by immunofluorescence.4.The potential mechanisms of Shenkang injection against high glucose-induced senescence of renal tubular cellsExperimental groups:(1)control medium(control group),medium containing 5 mmol/L glucose;(2)mannitol medium(mannitol group),medium containing 5 mmol/L glucose and 25 mmol/L mannitol;(3)HG medium(HG group)containing 30 mmol/L glucose;(4)HG + 200 mg / L SKI group(200 mg/LSKI concentration in HG medium);(5)control+ SKI group(200 mg/L SKI treatment in control medium).MDA content and SOD activity and the expression of sirt1,klotho,PPAR-?,mTOR and P66 shc was detected by western-blot and real-time PCR technique.5.Statistical analysisStatistical analysis was performed using the SPSS software package(versi on 18.0;SPSS Inc,Chicago,IL,USA).Quantitative data are presented as mean ± standard deviation(SD).Data were analyzed using one-way analysis of variance(ANOVA)to evaluate inter-group differences.P<0.05 was considered statistically significant.Results1.Exposure to HG for 72 h,but not to mannitol,resulted in a flatter and larger cell morphology compared to control culture medium.HG-induced senescent cells showed the emergence of senescence associated heterochromatin foci(SAHF),up-regulation of P16INK4 and cyclin D1,increased SA-?-gal activity,and elevated expression of Dc R2.SKI treatment potently prevented these changes in a dose-independent manner.SKI inhibits HG-induced senescence among renal tubular epithelial cells in a dose-independent manner.2.SKI treatment prevented HG-induced up-regulation of MDA,as well as down-regulation of SOD.SKI has anti-oxidative stress effect during HG-induced senescence.3.SKI treatment prevented HG-induced up-regulation of pro-senescence molecules mammalian target of rapamycin(mTOR)and p66 Shc,as well as down-regulation of anti-senescence molecules klotho,sirt1,and peroxisome proliferator-activated receptor-?(PPAR-?)in renal tubular epithelial cells.ConclusionSKI as a classic Traditional Chinese Medicine prescription may be a novel strategy for protecting against HG-induced renal tubular cell senescence in the treatment of diabetic nephropathy.