Mechanism By Which Exosomes Derived From Diabetic Nephropathy Mesangial Cells Promote Podocyte Injury And The Effect Of Berberine

Diabetic nephropathy(DN)is one of the most serious microvascular complications of diabetes mellitus(DM).This disease is due to the damage of renal tubules,glomeruli or interstitium caused by the continuous hyperglycemia and hemodynamic factors in DM state.Podocytes and glomerular mesangial cells(GMCs)are the two indispensable types of cells in the glomerulus,and their abnormal functional changes have played a key role in the development of DN.Exosomes are small vesicles with lipid bimolecular structure secreted by cells and can carry a variety of molecules,such as proteins,lipids,DNA,mi RNA,and micro RNA.Therefore,their role in cellular communication has drawn attention.Previous research showed that exosomes released by high glucose(HG)-induced GMCsmay carry TGF-β1cytokines and affect podocyte function.However,it is not clear how the exosomes released by HG-induced GMCs affect the whole process of DN.The PI3K/Akt/FOXO signaling pathway plays a role in cell survival and apoptosis.FOXO is phosphorylated by Akt on Thr24,Ser256,and Ser319,promoting their nuclear export and transcriptional activation.The evidence indicates that F0X01 has anti-tumor effect because it has anti-proliferative and pro-apoptotic activities in a variety of cancers.Berberine(BBR)is a natural chemical extracted from Coptis Chinensis.Studies have shown that BBR has a variety of pharmacological activities,including hypoglycemic,hypolipidemic,anti-inflammatory and antioxidant properties,and improve insulin resistance.These above results indicate that the exosomes stimulated by HG may affect the function of podocytes,and the PI3K-AKT-FOXO1-Bim/m TORC1 signaling pathway may be an important therapeutic target for podocytes in the process of functional damage.Therefore,this study intends to isolate the exosomes released by GMCs andco-culture them with podocytes to detect the functional changes of cells and the expression of PI3K-AKT-FOXO1-Bim/m TORC1 signaling pathway protein.Exosomes released from GMCs were injected into mice through tail vein.The renal function and the expression of PI3K-Akt signaling pathway protein and WT-1(a marker protein of podocyte)were observed.In vitro experiments were carried out to study the mechanism of podocyte injury caused by exosomes released from GMCs,the effect of exosomes released from GMCs on renal function in mice,and the important role of BBR in improving podocyte and renal injury,so as to prove that exosomes play an important role in signal transduction between GMCs and podocytes,which provides modern scientific basis for BBR in the treatment of DN and identification of DN New drug targets provide innovative ideas.Aim: The effect of exosomes released by GMCs on podocyte functional injury was clarified.BBR has a protective effect on HG-induced GMCs-derived exosomes to damaged podocytes.The effect of exosomes released by GMCs on injury in mice,and BBR had a protective effect on HG-induced GMCs-derived exosomes in mice kidney.The specific mechanism of the protective effect of BBR on HG-induced GMCs-derived exosomes protect podocytes and kidney injury may be related to the PI3K-AKT-FOXO1-Bim/m TORC1 signaling pathway.And to explore new research directions and ideas for BBR for DN treatment drugs,and lay the foundation for clinical DN treatment.Methods:GMCs-derived exosomes were isolated by ultracentrifugation,and the expressions of exosomes marker proteins CD63,TSG101,and endoplasmic reticulum molecular chaperone protein Calnexin were detected by Western blot;observe the morphological characteristics of exosomes using transmission electron microscopy;detect the particle size percentage of exosomes using a Malvern particle size analyzer;observe the uptake of exosomes(labeled green fluorescence)by podocytes using a laser confocal microscope.Annexin V-FITC/PI apoptosis kit determines the optimal concentration of exosomes for podocyte stimulation.GMCs and podocytes were co-cultured and divided into control group,high glucose model group and GW4869(exosomal inhibitor)group.After each group was given corresponding stimulation for24 h,Annexin V-FITC/PI double staining was used to detect the podocyte apoptosis rate in each group,and Western blot was used to detect the expression of podocyte marker proteins.The isolated exosomes and BBR were applied to podocytes in groups,and the podocyte apoptosis was detected using the Annexin V-FITC/PI apoptosis kit;the migration function test was used to detect the podocyte migration ability;the expression of nephrin,podocin,WT-1,PI3 K,AKT,P-AKT,FOXO1,P-FOXO1,Bim and m TORC1 proteins were detected by Western blot method.Kidney function parameters were observed to observe mouse kidney injury status;HE staining and PAS staining were used to observe kidney pathological changes,immunohistochemical method was used to observe the distribution and expression of WT-1 protein in renal cortex,the expression of PI3 K,AKT and Bim protein in renal cortex were detected by Western blot method.Results:1.Exosomes successfully isolated from GMCs culture supernatant Western blot results showed that CD63 and TSG101 protein were highly expressed in exosomes,but Calnexin protein was not expressed at all,which proved that the isolate was pure exosomes without cell components.It can be seen under transmission electron microscopy that the exosomes have a lipid bilayer structure with a diameter of40-160 nm.The results of particle size distribution of exosomes detected by particle size analyzer show that the maximum particle density range of exosomes is about 100 nm.2.GMCs-derived exosomes are taken up by podocytes Laser confocal microscopy observations showed that PKH67(green)-labeled GMCs-derived exosomes were present in the cytoplasm of podocytes.This finding suggests that GMCs-derived exosomes can be taken up by podocytes.3.Optimal conditions for the effects of GMCs-derived exosomes on podocytes Annexin V-FITC/PI double staining method was used to detect the podocyte apoptosis rate.The results showed that when exosomes of 50 μg/ml stimulated podocytes for 24 hours,the podocyte apoptosis rate was the highest,which could be used as the final stimulation condition.4.High glucose-induced exosomes derived from GMCs participate in podocyte injury Co-culture of podocytes with GMCs showed that compared with the normal group,the podocyte apoptosis rate in the HG model group was obviously increased,and the expression levels of nephrin,podocin,and WT-1 proteins were significantly down-regulated on podocytes.Compared with the model group,the above indicators were significantly improved in the GW4869(exosomal inhibitor)group.5.BBR improves podocyte function damage induced by high glucose-induced GMCs-derived exosomes Apoptosis detection by Annexin V-FITC/PI double staining method indicated that contrasted with the control group,there was no significant difference in the rate of podocyte apoptosis in the NG-treated GMCs-derived exosomes group,and in the HG-treated GMCs-derived exosomes group,the rate of podocyte apoptosis was significantly increased.The BBR high and low dose groups and the TGF-β1 receptor inhibitor group could effectively reduce the podocyte apoptosis rate to varying degrees.The podocyte migration ability test results showed that compared with the control group,the podocyte migration ability in the NG-treated GMCs-derived exosomes group was not significantly different,the podocyte migration ability in the HG-treated GMCs-derived exosomes group was significantly increased,and the BBR high and low dose groups and the TGF-β1 receptor inhibitor group can effectively reduce podocyte migration ability to varying degrees.Western blot results indicated that contrasted with the control group,nephrin,podocin and WT-1 protein expression on podocytes in NG-treated GMCs-derived exosomes group were not significantly different,and in the HG-treated GMCs-derived exosomes group,the expression of podocyte marker protein was significantly reduced.The BBR high and low dose groups and the TGF-β1 receptor inhibitor group could effectively upregulate the expressions of nephrin,podocin and WT-1 on podocytes to varying degrees.The results show that BBR improves podocyte injury by reducing the rate of podocyte apoptosis,reducing podocyte migration ability,and increasing the expression of podocyte marker proteins.6 The effect of BBR on podocyte injury induced by high glucose-induced GMCs-derived exosomes may be related to the PI3K-AKT-FOXO1-Bim/ m TORC1 signaling pathway Western blot results showed that PI3 K,P-AKT/AKT,P-FOXO1/FOXO1,Bim and m TORC1 protein expression levels were not significantly different in the NG-treated GMCs-derived exosomes group compared with the control group,the expressionlevels of PI3 K,P-AKT/AKT,Bim and m TORC1 protein in the HG-treated GMCs-derived exosomes group increased significantly,the expressionlevels of P-FOXO1/FOXO1 protein was significantly decreased,BBR high and low dose groups and TGF-β1receptor inhibitor group can effectively down-regulate the expression levels of PI3 K,P-AKT/AKT,Bim and m TORC1 protein expression levels up-regulated P-FOXO1/FOXO1 protein.7 Effect of high glucose-induced exosomes derived from mesangial cells on renal function parameters in mice Contrast with the control group,there was no obvious changes in blood urea nitrogen(BUN),total urine protein(TUP),N-acetyl-d-glucosaminidase(NAG),urinary creatine(Ucr)in the NG-treated GMCs-derived exosomes group,while BUN,NAG,Ucr and total urine protein levels in the HG-treated GMCs-derived exosomes group were significantly higher.of berberine(50 mg/kg,100 mg/kg,200 mg/kg)and enalapril positive drug control groupdecreased the above indexes in varying degrees.8 Effects of high glucose-induced exosomes derived from mesangial cells on histopathology of mouse kidney The results showed that the kidney tissues of the control group and the NG-treated GMCs-derived exosomes group had no obvious lesions,the glomerular structure was clear,and no swelling and atrophy were seen.In the HG-treated GMCs-derived exosomes group,the glomerular volume was significantly reduced,the mesangial matrix was significantly increased,the basement membrane was diffusely thickened,and the mesangial area was thickened.Berberine(50 mg/kg,100 mg/kg,200 mg/kg)administration group and enalapril-positive control group can improve the above-mentioned pathological changes,reduce glomerular basement membrane thickening and extracellular matrix deposition.9 Effect of high glucose-induced mesangial cell-derived exosomes on the distribution and expression of WT-1 protein in mouse renal cortex Immunohistochemical results showed that the expression level of WT-1 protein in the glomeruli of the control group and the NG-treated GMCs-derived exosomes group was high and uniformly distributed,compared with the NG-treated GMCs-derived exosomes group,the expression of WT-1 protein were significantly reduced in the HG-treated GMCs-derived exosomes group.Compared with the HG-treated GMCs-derived exosomes group,the BBR(50 mg/kg,100 mg/kg,200 mg/kg)administration group and the enalapril-positive control group up-regulated the expression level of WT-1 protein.10 PI3K/AKT signaling pathway is involved in glomerular injury induced by HG-treated GMCs-derived exosomes Western blot results indicate that contrast with the control group,the expression levels of PI3 K,p-AKT,and Bim proteins in the renal cortex of NG-treated GMCs-derived exosomes group had no significant changes,while the expression levels of PI3 K,p-AKT and Bim protein were significantly increased in the HG-treated GMCs-derived exosomes group(P<0.01),compared with the HG-treated GMCs-derived exosomes group,the BBR(100 mg/kg,200 mg/kg)administration group and enalapril positive control group was significantly reduced the expression levels of PI3 K,p-AKT,and Bim.Conclusions:1.HG-treated GMCs-derived exosomes can affect podocytes and mice kidney function.2.BBR can improve the injury of podocytes and mice kidney induced by HG-induced GMCs-derived exosomes.3.BBR may improve podocyte damage through regulating the PI3K-AKT-FOXO1-Bim/m TORC1 signaling pathway.