Paeoniflorin Binds To VEGFR2 To Restore Autophagy And Inhibit Apoptosis For Podocyte Protection In Diabetic Kidney Disease Through PI3K-AKT Signaling

Background:In recent years,the incidence and prevalence of diabetic kidney disease(DKD)in China have been on the rise.The presence of albuminuria in the early stage of DKD indicates that the glomerular filtration barrier(GFB)is damaged,and podocytes are the main components of GFB.The changes of podocytes morphology,function and the number are all involved in the occurrence and development of DKD.Notably,Podocytes cannot regenerate once dying.Autophagy,which can maintain cell homeostasis and avoid cell death,is likely to be one of the therapeutic targets for protecting podocytes.Paeoniflorin(PF)is the main active ingredient extracted from the classical herb Paeonia suffruticosa.PF has received extensive attention in medical research because it has less toxic and side effects,and a broad-spectrum protective effect on the main organs of the body.In animal models of acute and chronic kidney disease,PF showed significant renal protection.In previous studies,our group also found that PF can improve urinary albumin excretion and renal pathological changes in diabetic mice,including glomerular basement membrane(GBM)thickening,foot process fusion,and mesangial region widening.Unfortunately,the research focus is limited to macrophages in the renal inerestitum,specific mechanism of action of PF in preventing and treating podocyte dysfunction,especially in early DKD,remains to be further studied.The purpose of this study was to observe the protection of PF on podocyte injury,podocyte autophagy and apoptosis in DKD,and to explore the specific mechanism of drug action on podocytes in DKD for providing a strong scientific basis for the application of PF in the treatment of DKD.Methods:Part 1:In this study,the data set GSE30122,which included the glomerular RNA-seq data(GSE30528)and the tubular RNA-seq data(GSE30529),was used to screen the most significant cell death-related pathways enriched by KEGG or GO for further clinical observation.Afterwards,we collected the clinical and pathological data of DKD patients diagnosed by renal biopsy in the First Affiliated Hospital of Anhui Medical University,as well as non-diabetic nephropathy controls,including the adjacent renal tissue of renal cancer patients without diabetes and renal dysfunction,and minimal change disease(MCD)patients as normal controls without proteinuria and controls with macroalbuminuria,respectively.The DKD patients were further divided into DKD1group with urinary albumin/creatinine ratio(ACR)of 30-299 mg/g in DKD patients,DKD2 group with urinary albumin/creatinine ratio?300 mg/g.The study observed the disease specificity of the target cell death form on glomeruli in DKD,after excluding differences in clinical baseline data among groups.Meanwhile,we analyzed the clinicopathological data of the two subgroups of DKD to select the differential indicators,which then were incorporated with the marker protein expressions of target cell death form into stepwise regression,binary logistic regression,and ROC curve analysis to evaluate the clinical and diagnostic significance of key influencing factors.Part ?:Observation of the PF pharmacokinetics.We choosed intraperitoneal injection due to the low bioavailability of paeoniflorin.We constructed a rat model of intraperitoneal and intravenous administration of PF,and then collected blood of rats at time points for mass spectrometry to detect the plasma concentrations of PF to calculate the bioavailability of PF.Meanwhile,this study also constructed a mouse model of intraperitoneal administration of PF,collected blood and kidney tissue samples at time points,detected the concentration of PF by mass spectrometry to observed the distribution of PF in kidney tissue.Part?:Established streptozocin(STZ)-induced diabetic mouse models to take blood,urine and kidney tissue samples after PF treatment.The renoprotective effect of PF on DKD was observed by physiological indicators,blood and urine biochemical indicators,and renal pathological examination.The protective effect of PF on podocytes was evaluated by detecting the marker proteins of the number and function of podocytes by electron microscopy,immunohistochemistry and immunoblotting methods in vitro and in vivo.Furthermore,the effect of PF on the target cell death form was observed by immunohistochemistry,immunofluorescence,western blotting and tunel assay of the marker proteins in vitro and in vivo.Based on the molecular formula of PF,the target of PF was predicted by network computer,and verified by molecular docking,thermal shift experiment and SPR.Then,through the string website,the interacting protein of the target was searched.Finally,si RNA knocked down the target protein in podocytes in vitro to observe the effect of PF on downstream signaling pathways and survival in podocytes,thus verifying that PF acted through the target protein.Results:Part 1:The differences and clinical significance of renal tissue autophagy in patients with DKDThis part of the study began with a bioinformatics analysis of genes in DKD patients'renal tissues.We analyzed the genes from GSE30122 and found that the differential expression genes in the glomerulus were enriched in immune cells,oxidative stress,complement,extracellular matrix secretion and multiple signaling pathways(including Ras,PI3K-Akt and Wnt signaling pathways).It was interesting that genes related to autophagy activation and regulation exhibited more significant changes in the glomerulus than in the tubulointerstitium,suggesting that the changes of autophagy during the progression of DKD may be mainly glomerular.The tubulointerstitial differential expression genes were related to the regulation of immune cells.Therefore,this study focused on glomerular autophagy activity,and used P62protein expression as a marker to measure autophagy activity.Analysis of clinical data among DKD patients showed that differences in biochemical indexes were mainly reflected in lipid metabolism,such as total cholesterol(CHO),low-density lipoprotein(LDL)and apolipoprotein B(Apo B),Apolipoprotein A1(Apo A1),all of which significantly increased in DKD patients with massive proteinuria.The urine indexes were also increased,including U-TRF,U-Ig G,U-Cyc,U-?2-MG,U-?1-MG,U-ALB/Cr,U-TP/Cr,U-ALB/TP.The pathological data showed that the degree of glomerular lesion,IFTA and interstitial inflammation were significantly aggravated.There was no significant difference in arterial hyaline and arteriosclerosis among all groups.Immunohistochemical(IHC)results showed that compared with NC group and MCD group,the level of P62 protein in glomerular of DKD patients was significantly increased,and the level of P62 protein was the highest in DKD patients with a lot of proteinuria.There was no significant difference in P62 protein expression between NC group and MCD group.Immunofluorescence double staining results suggested that there were many P62⁺/WT-1⁺positive cells in the glomerulus,suggesting that the autophagy inhibition occurred mainly in podocytes during the progression of DKD.The correlation analysis between P62 and clinicopathological data showed that the level of P62 protein in glomerulus was positively correlated with blood indicators(CHO,LDL,Apo A1,Apo B),urine indicators(TRF,cyc,Ig G,?2-MG,ALB/Cr,TP/Cr),pathological grade,IFTA,and interstitial inflammation,respectively.It was negatively correlated with serum ALB and U-Cr.Stepwise regression analysis screened out that Apo A1 and P62 protein expression on glomeruli were associated with the progression of albuminuria in DKD.P-value of p62 in the logistic regression analysis was less than0.05.The results of ROC curve analysis showed that accuracy of the P62 protein expression on the glomerulus was 0.967,the specificity was 0.709,and the AUC was0.905.Part ?:Pharmacokinetics of PFThe peak plasma concentration of PF after intraperitoneal injection was within 5minutes,indicating that the drug absorption rate by intraperitoneal injection is very fast,and then decreased rapidly.Gradual stabilization existed after 8 hours.This suggests that the drug may be rapidly metabolized or excreted,or rapidly distributed from the blood to various tissues and organs.The bioavailability of PF was 32.9%,which was significantly higher than the oral bioavailability mentioned in the previous literature.In addition,30 min after intraperitoneal injection of PF,the drug concentration in the kidneys was much higher than the blood drug concentration,and then persisted after that for nearly 24 hours.The renal drug concentration was 7.8-33.9 times than that in the blood.This result indicated that PF is rapidly distributed to tissues after absorption,while the concentration in the kidney was always higher than that in the blood,suggesting that the kidney is likely the main pharmacological target of PF.Part?:Paeoniflorin can regulate podocyte autophagy and apoptosis through VEGFR2-mediated PI3K/AKT signaling pathwayIn order to evaluate the effect of PF on DKD,we first established a STZ-induced diabetic mouse model,and detected the effects of 3 different doses(50/100/200 mg/kg)of PF on the physiological and biochemical indicators of the mice in the DKD group.The 24 h urine volume and albumin excretion were significantly higher than those in the NC group,while the urine volume in the PF administration group at 12 weeks was lower than that in the DKD group.There was no difference in liver functions,renal functions,and blood glucose.The results of renal pathological analysis showed that compared with the NC group,the area of the mesangial matrix in the DKD group was significantly increased,and the volume of the glomerulus increased,which was relieved after PF treatment.The results of electron microscopy showed that compared with the NC group,the GBM in the DKD group was significantly thicker,most of the foot processes were fused,while the above pathological changes were significantly recovered after PF administration.The results of WB and IHC showed that compared with the NC group,the protein expression levels of WT-1 and nephrin in the DKD group were significantly decreased.From the increased expression levels of WT-1 and nephrin in the PF group,it can be seen that PF treatment can relieve the number and functional damage of podocytes in DKD.In vitro,MTT assay observed that the proliferation of podocytes gradually slowed down after high glucose stimulation for 48h,while PF could improve the inhibition of podocyte proliferation.The protein expression levels of podocyte markers and functional proteins(WT-1 and nephrin)were significantly decreased after HG stimulation,but significantly increased after PF treatment.High glucose stimulation induced a marked decrease in podocyte adhesion and migration,while PF partially restored them.The above all suggest that in vitro,PF can improve the inhibition of podocyte proliferation and podocyte function damage induced by high glucose.The double immunofluorescence staining of mice renal tissues indicated that,compared with the NC group,the P62⁺/WT-1⁺cells in the DKD group increased and decreased after PF treatment,suggesting that autophagy was absent during podocyte injury and PF can improve it.The results of electron microscope observed that there were typical double-membrane autophagosomes in the podocytes of the control group,while the number of autophagosomes in the podocytes of DKD mice decreased sharply,increased significantly after PF treatment.The results of WB indicated that compared with the NC group,the protein expression levels of Beclin-1,ATG5,and ATG7 and the ratio of LC3 II/I in the DKD group were decreased,and the protein expression of P62was enhanced,and the co-administration of PF partially reversed this change.TUNEL staining and immunofluorescence double staining of cleaved-caspase 3 and synaptopodin showed that compared with the NC group,the apoptotic cells of the glomerular podocytes in the DKD group were significantly increased,and PF treatment could also significantly inhibit the podocytes apoptosis,which was consistent with the changes of cleaved-caspase 3 protein expression detected by WB and IHC.In vitro,we also observed that PF can improve autophagy inhibition and apoptosis activation stimulated by high glucose.In addition,after autophagy inhibitors treatment,PF could not exert a protective effect on high glucose-stimulated podocyte injury.It was inferred that PF exerted a protective effect on high glucose-induced podocyte injury by increasing autophagy flux.It was determined by computer network prediction that the target of PF on podocytes may be the vascular endothelial growth factor receptor.Vascular endothelial growth factor 2(VEGFR2)as the target of PF on podocytes was determined through the thermal shift experiment and observation of protein expression changes in tissues,and combining with previous literature.Molecular docking and SPR verified that PF could directly bind to VEGFR2 in vitro.The phosphorylation of VEGFR2 and VEGFR2 in mice kidney tissue was significantly decreased after PF administration compared with the DKD group.The direct binding of VEGFR2 to PIK3CA was predicted by the string website and verified by co-immunoprecipitation(CO-IP).At the same time,PF was also observed to regulate the phosphorylation of PI3K/AKT signaling pathway in in vitro and in vivo.After VEGFR2 knockdown on podocytes,it was observed that autophagy inhibition and apoptosis activation induced by high glucose were improved,while PF treatment did not show obvious additive effects,suggesting that PF regulates PI3K/AKT signaling pathway to improve podocyte autophagy and apoptosis.Conclusion:1.The inhibition of autophagy was mainly manifested in podocytes in the glomerulus,and the expression change of P62 in the glomerulus,a marker of autophagy activation,might be a predictor of DKD progression to macroalbuminuria.2.Intraperitoneal injection can improve the bioavailability of PF,which can be enriched into kidney tissue after entering the body.The results suggested that kidney may be the target organ of PF.3.PF directly binded to VEGFR2 for regulating autophagy and apoptosis of podocytes by affecting the downstream PI3K/AKT signaling pathway,and finally exerted renoprotection.