Experimental Investigation And Functional Magnetic Resonance Imaging Of Intervention Of VCP979 On Chronic Kidney Disease

Part I Effect of VCP979 on injuryed renal tubular epithelial cellsObjective: Tubular epithelial cells are the primary site of response after injury.The p38 mitogen-activated protein kinase(MAPK)pathway is an essential intracellular signal transducer responding to cellular stimuli in tubular epithelial cells.VCP979 is a new small molecular selective p38 MAPK inhibitor with strong affinity.The purpose of this study is to investigate the effect of VCP979 on HK-2 cells,which are damaged by high glucose,and the effect of VCP979 on epithelial-mesenchymal transition(EMT)of HK-2 cells.Materia Is and Methods:(1)The biological safety of VCP979 in cells is detected with CCK-8,and safety of VCP979 in animals is confirmed by H&E staining of the main organ slices of rats after VCP979 administration;(2)In the injuried human renal tubular epithelial cells(HK-2 cells)induced by high glucose,the expression of GPX4 and x CT,which are associated with intracellular ferroptosis are measured after VCP979 intervention,to verify the effect of VCP979 on renal tubular epithelial cell injury;(3)In the model of EMT in HK-2 cells induced by TGF-β1,the expression of Ecadherin、α-SAM and Vimentin are measured after VCP979 intervention,to verify the effect of VCP979 on EMT.Results:(1)The results of CCK-8 shows that VCP979 below 30 μM does not affect cell activity,and the results of H&E staining verifies that VCP979 below 150 mg/kg does not make injury to rats.(2)HK-2 cells produce a large amount of reactive oxygen species(ROS)with high glucose induction,which further caused ferroptosis.ROS generation of is reduced by 34 % by VCP979,and the expression of GPX4 is increased by 39 %,confirming that VCP979 mitigates ferroptosis.(3)The morphology of HK-2 cells changed to long spindle shaped with TGF-β1 induction,which confirmed that HK-2 cells undergo EMT process.VCP979 could obviously reverse he morphology of HK-2 cells to oval shape,and increases the expression of epithelial marker E-cadherin by 46 %,and decreases the expression of mesenchymal cell marker α-SAM and Vimentin by 27 % and 26 %,respectly.These results confirm that VCP979 attenuate EMT induced by TGF-β1.Conclusion:(1)VCP979 below 30 μM is safe for cells and VCP979 below 150 mg/kg is safe for rats.(2)VCP979 could effectively reduce the high-glucose-induced ROS generation and attenuate ferroptosis,and improve the inury caused by high glucose.(3)VCP979 could increase the expression of epithelial marker E-cadherin,and decrease the expression of mesenchymal cell marker,suggesting VCP979 could alleviate EMT in HK-2 cells induced by TGF-β1.Part II Evaluation of the effect of VCP979 on diabetic nephropathy in rat modelObjective: Oxidative stress is the key event in the pathogenesis of diabetes nephropathy,which can promote renal tissue injury by activating p38 MAPK.In part I,we have confirmed that VCP979 could improve the injury caused by high glucose.In this part,we investigate the effect of VCP979 in diabetes nephropathy.Materia Is and Methods:(1)Construction of diabetes nephropathy model in rats: Twenty-four healthy male Wistar rats were randomly divided into Sham group(n=8),diabetic nephropathy(DN + Veh,n=8)group and VCP979-treated(DN + VCP979,n=8)group.The rat model of diabetic nephropathy is built by injection of streptozotocin(STZ)intraperitoneally.Blood glucose and pathological staining were used to verified the establishment of animal model.(2)Evaluation of the effect of VCP979 on diabetic nephropathy in rat model: VCP979 was administered by gavage,and multi-parameter magnetic resonance imaging was performed.Serum creatinine(SCr)and blood urea nitrogen(BUN)of rats were analyzed to assess renal function.After treatment,renal tissues were collected to perform histopathological analysis,and evaluate the renal damage.Results:(1)After the construction of diabetes nephropathy,blood glucose of rats exceeds 16.6 mmol/L and periodate Schiff(PAS)staining shows typical pathological changes.These results verify the establishment of diabetic nephropathy.(2)VCP979 decrese SCr by 29 %,BUN by 23% and UACR by 31%,indicating VCP979 effectively improved renal function.VCP979 reduced markers of lipid peroxidation MDA by 42 %,and enhance enzyme activity of SOD by 76 %,suggesting VCP979 improves antioxidant enzyme activity,and attenuates oxidative injury.VCP979 increase the expression of GPX4 by 63 %,indicating that VCP979 mitigates the level of ferroptosis in renal tissues.(3)Magnetic resonance image of T2*WI shows VCP979 increases value of T2* by 10 % in renal tissues,which indicating that VCP979 reduces the degree of iron deposition.ASL shows VCP979 increases the renal blood flow by 102%,which indicating that VCP979 improves disorders of microcirculation.Conclusion:(1)In diabetes nephropathy,VCP979 improves renal function and attenuates renal injury significantly.(2)In diabetes nephropathy,VCP979 enchanes the antioxidant enzyme activity,improves antioxidant capacity and reduce damage induced by oxidative stress in renal tissues.VCP979 also mitigates the degree of ferroptosis.(3)In diabetes nephropathy,VCP979 reduces the degree of iron deposition and improves disorders of microcirculation.Part III Evaluation the effect of VCP979 on renal fibrosis induced by unilateral ureteral obstraction in ratsObjective: In renal fibrosis,p38 MAPK is activated through high glucose,oxidative stress,inflammatory cytokines and other stressors to promote epithelial mesenchymal transformation of renal tubular epithelial cells and mediate inflammatory response.In part I,we have verified that VCP979 could improve the degree of EMT.In this part,we investigate the effect of VCP979 on renal fibrosis,EMT and inflammation in rat model of unilateral ureteral obstruction(UUO).Materia Is and Methods:(1)Construction of diabetes nephropathy model in rats: Twenty-four healthy male Wistar rats were randomly divided into Sham group(n=8),UUO(UUO + Veh,n=8)group and VCP979-treated(UUO + VCP979,n=8)group.The model of renal fibrosis was established by ligation of the left ureter of rats.The morphology and pathological staining were used to verified the establishment of animal model.(2)Evaluation the effect of VCP979 on renal fibrosis induced by unilateral ureteral obstraction in rats: Rats were administered VCP979 by gavage.Multiparameter magnetic resonance imaging(MRI)was used to monitor renal injury.And serum creatinine(SCr),blood urea nitrogen(BUN)and urinary protein excretion were measured to evaluate renal function.In addition,renal tissues were collected after treatment and histopathological analysis was performed to assess the degree of renal fibrosis.Results:(1)After the establishment of renal fibrosis model,urine continuously accumulates in the renal pelvis,causing the appearance of the kidney to increase and the renal parenchyma is stretched to be thin.The results of Masson and Sirius red staining showed significant increase in fibrotic area,confirming the successful construction of the renal fibrosis.(2)VCP979 improves renal function significantly,and reduces the fibrotic area in Masson and Sirius red staining by 41 % and 46 %,respectly.VCP979 reduces the expression of E-cadherin by 95 % and decreases the expression of α-SMA and Vimentin by 19 % and 53 %,indicating VCP979 alleviates epithelial-mesenchymal transition in UUO rats.(3)VCP979 also decreases the expression of inflammatory cytokines and the infiltration of macrophages in renal tissue,suggesting VCP979 exerting anti-inflammatory effect.(4)Multi-parameter MRI images further showed that VCP979 decreases the value of T1 rho by 13 % in T1 rho imaging,enhances renal blood flow by 119 % in ASL,and reduces the value of R2* in BOLD.Furthermore,VCP979 may inhibit TGF-β1/Smads signaling pathway to alleviate renal fibrosis.Conclusion:(1)In renal fibrosis,VCP979 improves renal function,reduces fibrotic area in Masson and Sirius red staining,indicating VCP979 alleviates renal fibrosis.VCP979 enhances the expression of epithelial marker,and decrease the expression of mesenchymal cell marker,suggesting VCP979 could mitigate EMT.(2)In renal fibrosis,VCP979 decreases the expression of inflammatory cytokines and the infiltration of macrophages in renal tissue,suggesting VCP979 could reduce the inflammatory response in the rat model of UUO.(3)In T1 rho imaging,VCP979 could attenuate renal fibrosis.In ASL,VCP979 could improve disorders of microcirculation.In BOLD,VCP979 could mitigate hypoxia in renal fibrosis.