| Objective: As one of the major microvascular complications of type 2 diabetes,diabetic kidney disease(DKD) has become the leading cause of end-stage renal disease(ESRD).Albuminuria is currently the main clinical diagnostic criterion for DKD,and it also plays a key role in promoting the development of DKD.Earlier studies showed that the damage of urine protein to the kidney is mainly caused by the deposition of urinary protein on podocytes and mesangial cells,but recent studies have found that excessive albumin causes renal tubular interstitial fibrosis and renal tubular epithelial cells to shrink and apoptosis plays a key role in the injury process.Autophagy has been proven to have kidney protection in many studies.Normal lysosomal function is a necessary condition for the completion of autophagy,but how the autophagy-lysosomal pathway changes under the pathological condition of diabetic nephropathy and its specific mechanism is still not fully elucidated.Berberine(BBR)is an isoquinoline alkaloid extracted from the Chinese herbal medicine Coptis chinensis.It has been reported that BBR can improve podocyte damage in diabetic nephropathy,but its effect on renal tubular damage caused by protein overload is still not very clear.In this study,an animal model of spontaneous type 2 diabetes,Zucker Diabetic Fatty(ZDF)rats and human renal tubular epithelial cells(HK-2) was used as the research object.HK-2 cells were treated with albumin,the main component of urine protein,in order to explore: 1.Effect of berberine on proteinuria and renal tubular injury in DKD rats.2.Effect of albumin overload on autophagy and apoptosis of renal tubular epithelial cells and the intervention effect of berberine.3.Effect of albumin overload on lysosomal function of renal tubular epithelial cells and the effect intervention of berberineMethods: 1.26 ZDF rats were used as research objects,and 10 ZL rats of the same age were used as control group.After 6 weeks of feeding on a high-fat diet,the blood glucose of each group was monitored and 24 h urine was collected to measure urine microalbumin(UMA).Blood glucose ≥16.7mmol/l was taken as the criterion of T2 DM,blood glucose ≥16.7mmol/l and 24 h UMA significantly higher than the NC group is the criterion of DKD.Then DKD rats were randomly divided into diabetic nephropathy group(DKD)and berberine intervention group(DKD+BBR).After 3 weeks of berberine intervention,the rats were sacrificed.Biochemical method was used to measure liver function,serum creatinine,urea nitrogen and other indicators in each group.Biochemical method was used to determine 24-hour UMA and renal tubular injury indicators in each group.2.(1)Take the kidney tissue and HK-2 cells as the research objects,and intervene the cells with different concentrations of bovine serum albumin(BSA) for 24 hours.CCK-8 was used to determine the proliferation activity of HK-2 cells.Western blot was used to determine the level of p62 and ratio of LC3II/LC3 I,which represent activity of autophagy.Western blot was used to detect the phosphorylation levels of AMPK and mTOR.(2)Regulatory pathway of autophagy: control group,albumin overload group(5mg/ml BSA),AMPK activator group(5mg/ml BSA+AICAR),m TOR inhibitor group(5mg/ml+ Rapamycin),Western blot was used to detect the levels of p62 and ratio of LC3II/LC3I;Western blot was used to detect the phosphorylation levels of AMPK and mTOR.(3)Drug intervention cell experiment: control group,albumin overload group(5mg/ml BSA),berberine intervention group(BSA+BBR),berberine intervention+ AMPK inhibitor Compound C group(BSA + BBR + CC).Apoptosis detection kit was used to measure the apoptosis of each group.Western blot was used to detect the ratio of LC3Ⅱ/LC3Ⅰ and the level of p62.Western blot was used to detect the phosphorylation levels of AMPK and mTOR.3.The kidney tissue and HK-2 cells were used as the research objects.The cell experiment group was set as the control group,albumin overload group(5mg/ml BSA),berberine intervention group(BSA+BBR),berberine intervention+AMPK inhibitor Compound C group(BSA+BBR+CC).Total protein was extracted,and western blot was used to determine the phosphorylation level of transcription factor EB(TFEB),the main regulator of lysosomal function.Western blot was used to test the expression level of lysosome-associated membrane proteins1(LAMP1),cathepsin B(CTSB)and acid phosphatase(ACP).Enzyme activity assay kit was used to measure the activity of lysosomal-associated hydrolase CTSB,ACP.Results: Ⅰ.Effects of berberine on kidney-related indexes in diabetic nephropathy rats 1.The DKD model was successfully established: at 13 weeks of age,the random blood glucose of ZDF rats ≥16.7mmol/L and 24h-UMA was significantly higher than NC group.Compared with NC group,the fasting blood glucose level in the DKD group was significantly increased(P <0.05)without significant change in body weight.Compared with the NC group,the 24 h UMA level in the DKD group was significantly increased,and renal tubule injury indicators N-acetyl-beta-D-glucosaminidase(NAG)and β-galactosidase(GAL)also increased significantly(P <0.05).2.Effect of berberine on kidney injury: Compared with DKD group,the 24 h UMA level in the berberine intervention group decreased significantly(P <0.05).Berberine also improved the renal tubular injury indicators NAG and GAL.Berberine is safe and has no effect on liver function and renal function in rats(P> 0.05).Ⅱ.Effect of albumin overload on autophagy and apoptosis of renal tubular epithelial cells and the intervention effect of berberine 1.Compared with NC group,DKD group had decreased LC3Ⅱ/LC3 Ⅰ ratio,increased p62 expression,decreased AMPK phosphorylation level and increased mTOR phosphorylation level(P < 0.05).2.Compared with DKD group,berberine could up-regulate LC3Ⅱ/LC3 Ⅰ ratio,down-regulate p62 expression,up-regulate AMPK phosphorylation level and down-regulate m TOR phosphorylation level(P < 0.05).3.Albumin overload could lead to up-regulation of p62 protein expression,down-regulation of LC3Ⅱ/LC3Ⅰ ratio,decrease of AMPK phosphorylation level,and increase of mTOR phosphorylation level in HK-2 cells(P < 0.05).4.Compared with albumin overload group(5 mg/ml BSA),both AMPK agonist and m TOR inhibitor could up-regulate LC3Ⅱ/LC3Ⅰ ratio,down-regulate p62 protein expression level,up-regulate AMPK phosphorylation level,and down-regulate mTOR phosphorylation level in HK-2 cells(P < 0.05)5.Berberine could up-regulate LC3Ⅱ/LC3Ⅰ ratio,down-regulate p62 protein expression level,up-regulate AMPK phosphorylation level,and down-regulate mTOR phosphorylation level in albumin-intervened HK-2 cells(P < 0.05).Addition of Compound C,an AMPK inhibitor,to berberine-treated HK-2 cells revealed decreased LC3Ⅱ/LC3Ⅰ ratio,increased p62 expression,decreased AMPK phosphorylation levels,and increased mTOR phosphorylation levels(P < 0.05).6.After 5 mg/mL albumin intervention,HK-2 cell apoptosis was significantly increased,and berberine intervention could reduce albumin-induced apoptosis.The addition of Compound C to berberine-treated cells reversed the anti-apoptotic effect of berberine(P < 0.05).Ⅲ.Effect of albumin overload on lysosomal function of renal tubular epithelial cells and the effect intervention of berberine 1.Compared with rats in NC group,rats in DKD group had increased phosphorylation level of TFEB,which is key factor in the regulation of lysosomal function.Berberine intervention also decreased the expression of lysosomal membrane protein LAMP1,decreased expression of lysosomal hydrolases ACP and CTSB,and decreased activities of ACP and CTSB enzymes,2.Berberine intervention could decrease the elevated TFEB phosphorylation level,up-regulated the expression levels of LAMP1 and lysosomal hydrolases ACP and CTSB,and increased the enzyme activities of ACP and CTSB in DKD rats.3.Berberine could down-regulate the elevated TFEB phosphorylation level,up-regulate the expression levels of LAMP1 and lysosomal hydrolases ACP and CTSB,and increase the enzyme activities of ACP and CTSB in albumin overload HK-2 cells.4.Addition of Compound C,an AMPK inhibitor,to berberine-intervened HK-2 cells could increase berberine-inhibited TFEB phosphorylation levels,inhibited berberine-elevated LAMP1,ACP,and CTSB expression.The ACP and CTSB enzyme activities up-regulated by Berberine was reversed by berberine.Conclusion: 1.Berberine reduced 24-hour UMA and alleviated tubular injury in DKD rats.2.Albumin overload can inhibit autophagy,resulting in increased apoptosis of renal tubular epithelial cells.Berberine can improve the autophagy level of DKD rats by activating the AMPK / mTOR pathway,and alleviate the inhibition of autophagy and apoptosis of renal tubular epithelial cells caused by albumin..3.Albumin overload lead to damage to lysosomal function of renal tubular epithelial cells.Berberine can reduce the damage of proteinuria to the kidney by up-regulating the activity of TFEB and up-regulating the lysosomal function of HK-2 cells under albumin overload. |
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