Berberine Attenuates Podocytes Injury Induced By High Glucose Through AMPK-mTOR Pathway

Diabetic nephropathy is one of the most serious complications of diabetes, meanwhile, it is an important factor in the death and disability of diabetes. Proteinuria is a major clinical concern of diabetic nephropathy and is caused by disruption to the glomerular filtration. Podocyte is one of the glomerular filtration barrier members, playing an important role in the integrity of the glomerular function. In recent years, several studies show that podocytes injury involved in the profession of proteinuria under diabetic nephropathy conditions. Mechanism of podocytes injury has not yet been fully elucidated, more and more evidences show that autophagy defect is an important cause of early podocytes injury in DN.It was reported that AMPK-m TOR signaling pathway was involved in autophagy defective of podocyte, suggesting that podocytes injury caused by abnormal pathway may be the key point of DN. So the medicine regulating this pathway may activate autophagy in podocytes, reduce podocytes injury and and ultimately delay the development of DN.Berberine is an isoquinoline alkaloid, animal experiments and clinical studies show that berberine has significant hypoglycemic effect and it may be effective in treating diverse chronic diseases, including diabetes and its complications. Our previous studies had shown that berberine can significantly improve the kidney function. Meanwhile, it also improved proteinuria in early DN, attenuating the pathological changes of glomeruli of type II diabetes and further validates that the above pharmacological effects of berberine may be associated with AMPK activation. But berberine attenuates podocytes injury whether through AMPK-m TOR signaling pathway still unsure.In this study, we established the model of high glucose-induced podocytes injury to study the effect of AMPK-m TOR signaling pathway on autophagy and apoptosis of podocytes and the berberine protect the podocytes under diabetic conditions whether through AMPK-m TOR pathway. Methods:1.Effects of high glucose on the podocytes injury, autophagy and related signaling pathways : podocytes were cultured in vitro conditions and randomly divided into three group:normal glucose(5.6mmol/L glucose),mannitol(5.6mmol/L glucose+19.4mmol/L mannitol),high glucose(25mmol/L glucose). They were cultured for 24 h and then collected, transmission electron microscopy was used to watch the autophagy formation; Flow cytometry was used to detected the apoptosis in each group; The activity of Caspase-3 in each group was detected by Caspase-3 activity kit; Western Blot was used to detect the protein expression of Beclin-1, LC3 II, Caspase-3, Nephrin, Podocin, AMPK, P-AMPK, m TOR, P-m TOR, ULK, P-ULK.2.Effects of regulating AMPK-m TOR signaling pathway on podocytes injury and autophagy induced by high glucose: AMPK agonist AICAR, inhibitor Compound C and m TOR inhibitor rapamycin were given when podocytes were cultured in high glucose. After cultured for 24 h, Caspase-3 activity kit was used to detect the Caspase-3 activity of podocytes; Western blot was used to detect the pression of Nephrin, Podocin, Beclin-1, LC3 II, ULK1, P-ULK1, AMPK and P- AMPK protein.3.Effects of berberine on podocytes injury and autophagy induced by high glucose: Podocytes were cultured in vitro conditions and randomly divided into four groups:Normal glucose,high glucose,Berberine high-dose group,Berberine low-dose group, the cells were cultured for 24 h and collected. Transmission electron microscopy was used to watch the autophagy formation and podocytes injury; Flow cytometry was used to detected the apoptosis in each group; The activity of Caspase-3 in each group was detected by Caspase-3 activity kit; Western blot was used to detect the protein expression of Nephrin, Podocin, Beclin-1, LC3 II, ULK1, P-ULK1. 4.To explore the protective mechanism of berberine on podocytes: Podocytes Aim: were cultured in vitro conditions and randomly divided into five groups:Normal glucose, high glucose, Berberine high-dose group, high glucose+Compound C, Berberine high-dose group+Compound C, the cells were cultured for 24 h and collected. Transmission electron microscopy was used to watch the autophagy formation; Flow cytometry was used to detect the apoptosis in each group; The activity of Caspase-3 in each group was detected by Caspase-3 activity kit; Western blot was used to detect the protein expression of Nephrin, Podocin, Beclin-1, LC3 II, ULK1, P-ULK1, AMPK, P-AMPK, m TOR, P-m TOR. Results:1. The electron microscopy showed that the autophagosomes of high glucose group were increased compared with the normal group.Western blot showed that the expression of Nephrin, Podocin were decreased in high glucose group compared with the normal group; The Caspase-3 activity were significantly increased in high glucose group than normal group;The expression of Beclin-1, LC3 II, P-ULK1/ULK1, P-m TOR / m TOR were increased, P-AMPK/ AMPK decreasing in high glucose group compared with the normal group.2. Western blot results showed that when AICAR or rapamycin was given on the basis of high glucose group, Caspase-3 activity was decreased, Nephrin, Podocin expression was significantly increased; Beclin-1, LC3 II, P-ULK1/ULK1 expression was further increased compared with high glucose group; when Compound C was given on the basis of high glucose group, results were completely the opposit.3. The electron microscopy results showed that berberine could increase the number of autophagosomes, decrease the apoptosis of podocytes compared with high glucose group; the expression of Nephrin, Podocin, Beclin-1, LC3 II,P-ULK1/ULK1 was increased by BBR; The activity of Caspase-3 were also decreased in BBR group than in high glucose group. The effect of high-dose group was better than the low-dose group.4. Caspase-3 activity test results showed that when Compound C was given on the basis of berberine group, Caspase-3 activity increased significantly compared with Berberine group. The expression of Nephrin, Podocin, Beclin-1, LC3 II, P-ULK1/ ULK1, P-AMPK/AMPK were decreased; P-m TOR/m TOR expression was increased compared with Berberine group. Conclusion:1. High glucose induced podocytes injury and increased level of podocytes autophagy when it stimulated podocytes for 24 h. Activating AMPK-m TOR signaling pathway can regulate podocytes autophagy and then reduce podocytes injury.2. Berberine activated podocytes autophagy under high glucose conditions, reduced podocyte apoptosis.3. Berberine enhanced autophagy and reduced podocytes injury induced by high glucose maybe by regulating AMPK-m TOR signaling pathway.