A Study Of The Mechanisms And Effects Of AOPP On Renal Tubulointerstitial Fibrosis In Diabetic Nephropathy

Background and objectiveDiabetic nephropathy(DN)is one of the most common vascular complications of diabetic patients,its incidence is increasing year by year globally,and has become a global health problem that threatens people's health.Advanced oxidation protein products(AOPP)are protein products formed during oxidative stress.Studies have shown that AOPP accumulation in diabetic animal experiments can promote renal fibrosis.The protein kinase C(PKC)system,also known as the phosphoinositide signaling pathway,plays a key role in mitochondrial damage,activation of oxidative stress,and promotion of fibrosis in a variety of cells.Researchs have confirmed that AOPP accumulation in DN can trigger podocyte damage by activating PKC signaling pathway,and PKC plays an important role in DN fibrosis.The pathogenesis of DN is complex and has not been fully elucidated,and there is currently no effective and specific treatment.Therefore,it is of great practical significance to further explore the pathogenesis of DN and new treatment strategies to prevent its progress.Podocyte injury and glomerular fibrosis are key factors in the development of DN.In recent years,more and more studies have shown that renal tubular interstitial fibrosis(Tubulointerstitial fibrosis,TIF)plays an important role in DN,and its mechanism research has been paid more and more attention.The TIF mechanism is complex and has not been fully elucidated.The purpose of this study was to explore the effects of AOPP in the pathogenesis of renal Tubulointerstitial fibrosis in diabetic nepropathy and its mechanism.Methods1.Total 33 renal biopsy samples were obtained from type 2 diabetic patients and 10 renal biopsy samples were obtained from patients with thin basement membrane nephropathy.The blood and urine samples of the patients were collected,Scr,BUN,blood glucose,urine NAG,UAER and UACR were analyzed.Immunohistochemical analysis of renal AOPP,pPKC?,pPKC?,Fibronectin expression.Masson's trichrome stain analysis TIF.The correlations between renal AOPP,pPKC? and biological parameters including serum creatinine,urinary NAG and eGFR were analyzed in DN patients.2.Human renal proximal tubular epithelial cells(HK-2)were cultured in Dulbecco's Modified Eagle's medium(DEME)/F12 containing 10%fetal bovine serum(FBS),penicillin(200U/ml),and streptomyc(200?g/ml).HK-2 cells were grown to 80%-90%confluence and made quiescent by incubation overnight in a serum-free medium before experimentation.The cell line was divided into 5 groups as follows:(1)HK-2 cells were treated with MAN,NG(normal glucose),HG(30mmol/L glucose)for 72 hours;(2)HK-2 cells were cultured in different concentrations of glucose(5,15 and 30 mmol/L)for 72 hours;(3)HK-2 cells were cultured under 30mmol/L glucose-DEME/F12 respectively for 12,36,and 72 hours;(4)HK-2 cells were cultured in high glucose(30 mmol/L)for one week,supplemented with different concentrations of AOPP(50,100,and 200 ?g/mL)for 72 hours;(5)HK-2 cells were cultured in high glucose(30 mmol/L)for one week,supplemented with AOPP(200 ?g/mL)for 72 hours.PKC inhibitors(Go6983,Myr and Rottlerin)were used to treat cells for 72 hours.After the above experiments finished,cells were collected and Western blot was used to detect the protein expression of AOPP,CD36,Fibronectin,pPKC? and pPKC?.3.Six groups with six rats each were studied:(1)normal control group(NC);(2)diabetes mellitus group(DM);(3)diabetic rats treated with RSA group(DM_RSA);(4)diabetic rats treated with advanced oxidation protein products group(DM_AOPP)or(5)diabetic rats treated with advanced oxidation protein products and Myr group(DM_AOPP+Myr)or(6)diabetic rats treated with advanced oxidation protein products and Rottlerin group(DM_AOPP+Rottlerin).After 12 weeks,the expressions of renal AOPP,CD36,pPKCr? pPKC? Fibronectin were detected by immunohistochemistry;Sirius red staining was used to observe the pathological changes of renal tissue.Scr,BUN,urinary NAG,UAER,UACR,Ccr and other biological parameters were analyzed.Results1.Vacuolated TECs were found in DN patients' renal biopsy tissues and AOPP was expressed mainly in these tubules,AOPP expression was enhanced in renal biopsy tissues of DN patients with concurrent increased expression of pPKC? and Fibronectin.The level of AOPP and pPKC? were positively correlated with Scr and urinary NAG,but negatively correlated with eGFR.2.HG time and dose dependently increased the level of AOPP,CD36,pPKC?and Fibronectin.With the increase of AOPPs concentration,CD36,pPKC? and Fibronectin were upregulated.Myr and Go6983 downregulated CD36,pPKC? and Fibronectin.3.AOPPs challenge further increased the level of Scr,BUN,urinary NAG,UAER,UACR and Ccr in diabetic rats.Significantly,these alterations were mitigated by Myr but not Rottlerin.Myr decreased the level of pPKC?,and Fibronectin in tubules of AOPPs-challenged diabetic rats.Increased collagen accumulation was decreased by Mry but not by Rottlerin.ConclusionsAOPP aggravate tubulointerstitial fibrosis through PKC? in early diabetic nephropathy.