| Objective:FG-4592(Roxadustat),a novel HIF-proline hydroxylase inhibitor,has been shown to confer renoprotection against acute kidney injury.However,its role in the progression of chronic kidney disease is largely unknown.The purpose of this study was to evaluate whether FG-4592 alleviated renal fibrosis by interfering with necrotic inflammation of Unilateral ureteral obstruction(UUO)in rats,and had renal protective effect on chronic renal disease.Methods:Eighty SPF Sprague-Dawley male rats aged 7 weeks(body weight 240?250g)were randomly divided into 10 groups(n=8/group)after adaptive feeding for 1 week.?Before unilateral ureteral ligation,the rats were fasted for 12 hours and given normal water.Before modeling,the rats were divided into sham group and UUO model group.In the UUO model group,the left ureter of rats was separated for double ligation,while in the Sham group,only the left ureter was separated and no other operations were performed.Before surgery,rats in the UUO model group were pretreated 48 hours in advance by gavage of FG-4592(10mg/kg/d)and/or intraperitoneal injection of Nec-1(2mg/kg/d).After UUO modeling,the rats were treated for 14 days,and the rats were sacrificed at 7 and 14 days,respectively.(1)Sham7 group:the same amount of normal saline was given intragastrically for 7 days;(2)Group Sham 14 was given the same amount of normal saline intragastrically for 14 days;(3)UUO7 group:7 days after unilateral ureteral ligation;(4)UU014 group:14 days after unilateral ureteral ligation;(5)UUO7+FG group:Unilateral ureteral ligation was performed and FG-4592(10mg/kg/d)was given by gavage for 7 days;(6)UUO14+FG group:unilateral ureteral ligation was performed and FG-4592(10mg/kg/d)was given by gavage for 14 days;(7)UUO7+Nec-1 group:Unilateral ureteral ligation and intraperitoneal injection of Nec-1(2mg/kg/d)for 7 days;(8)UUO14+Nec-1 group:Unilateral ureteral ligation and intraperitoneal injection of Nec-1(2mg/kg/d)for 14 days;(9)UUO7+FG+Nec-1 group:Unilateral ureteral ligation and gavage of FG-4592(10mg/kg/d)and intraperitoneal injection of Nec-1(2mg/kg/d)for 7 days;(10)UUO14+FG+Nec-1 group:Unilateral ureteral ligation and gavage of FG-4592(10mg/kg/d)and intraperitoneal injection of Nec-1(2mg/kg/d)for 14 days.Blood samples were collected from the right internal jugular vein.Serumcreatinine(SCR)and Blood urea nitrogen(BUN)were detected by automatic biochemical analyzer.Renal tissue specimens were collected,and routine paraffin embalment and section were performed.Masson staining was used to observe the degree of Tubulo interstitial fibrosis(TIF).PAS staining was used to observe renal tubule tissue injury,and electron microscopy was used to observe the ultrastructural changes of renal tissue.At the molecular level,and western blot method to detect the RIP1 RIP3-axis MLKL signal protein expression,IL-1?,IL-18 and NLRP3 and proinflammatory medium(MCP-1?TNF-?and TLR-2)expression,detection of renal tubule interstitial fibrosis related to fiber factor TGF-?1 and CTGF expression,from necrotizing apoptosis,inflammation of NLRP3 body and proinflammatory medium Angle of FG-4592 of UUO induced necroinflammation.The expression levels of SOD,MnSOD,NOX-2,NOX-4 and 8-OHdG in serum and urine were detected.The expressions of CHOP,BIP,IRE-1? and ATF-6 were detected to clarify the effect of ER stress,and TOMM20,NDUFA10,SDHA,DRP-1 and OPA1 were detected to clarify the effect on mitochondrial dysfunction.In addition,the expression of Bcl-2/Bax ratio,activated caspase-3 protein and HIF-1?/Wnt3?/?-catenin/GSK-3? signaling pathway were detected.Results:1.At the molecular level,the expression of RIP 1-RIP3-MLKL signal axin in UUO kidney tissues increased gradually,and both FG-4592 and Nec-ltreatment could reduce the expression of RIP 1-RIP3-MLKL signal axin,and the reduction of related proteins was more significant in the FG-4592 and Nec-1 combined treatment group.Under electron microscope,the tubular epithelial cells of UUO kidney tissue were observed to be aggregated and necrotic,swelling,shedding of microvilli,and cytolysis.2.The expressions of IL-1?,IL-18,NLRP3 and pro-inflammatory mediators(MCP-1,TNF-?and TLR-2)were significantly upregulated in the UUO7 or UUO14 groups,while these expressions were reversed by both FG-4592 and Nec-1,and the decrease of related proteins was more significant in the combined treatment group of FG-4592 and Nec-1.3.TIF was significantly increased in the UUO7 or UUO 14 groups,while fibrosis score was significantly decreased in the UUO7 and UUO14 groups treated with FG-4592 or Nec-1,while it was further significantly decreased in the group treated with FG-4592 and Nec-1.Immunoimpregnation analysis showed that FG-4592 or Nec-1 significantly inhibited the expression of fibrinogenic factors TGF-1 and CTGF in a time-dependent manner compared with the UUO group.4.After FG-4592 pretreatment,the protein expressions of SOD 1 and MnSOD were up-regulated,while the protein expressions of NOX-2 and NOX-4 were down-regulated,suggesting that the oxidative stress induced by UUO could be inhibited by maintaining the balance of oxidase and antioxidant enzymes.In addition,by 8-OHdG oxidative stress markers in serum and urine concentration in UUO7 than sham group,the highest in the UUO14 group,and gives the FG-4592 UUO group 8-OHdG concentration in serum and urine were lower than control group,but FG-4592 for a joint and Nec-1 UUO group of serum and urine of a significant decline in 8-OHdG,reaffirmed UUO cause kidney damage is closely related to oxidative stress,and FG-4592 reversible UUO cause oxidative stress effect.5.Rough endoplasmic reticulum ribosomal degranulation,lumen disconnection,expansion,vesicular degeneration and peroxisomal vacuolization were observed in UUO kidney tissue under electron microscope,while smooth endoplasmic reticulum almost maintained normal structure.Western blot analysis showed that UUO significantly upregulated the expression of ER stress-related genes,including CHP,BIP,IRE-1? and ATF-6,in a time-dependent manner,consistent with the morphological results,but FG-4592 significantly reduced their expression at both time points observed.6.Electron microscopy clearly shows that the damaged mitochondrial structure in UUO kidney tissue is characterized by significant reduction in the number of mitochondria,vacuolar degeneration,cristal dilatation,mitochondrial fusion,mitophagy,and more than two or three suborganelle of mitochondrial fission.Western blotting analysis showed that the expression of TOMM20,NDUFA10 and SDHA in UUO kidney tissue was significantly inhibited compared with that in Sham group,while FG-4592 pretreatment could reverse the expression of its protein.In addition,FG-4592 significantly decreased the expression of DRP-1(fission protein),but increased the expression of OPA1(fusion protein).7.Quantitative count of TUNEL positive cells showed that the number of TUNEL positive cells was increased in the UUO group,and the number of TUNEL positive cells was significantly decreased in the FG-4592 treatment group on the 7th and 14th days of UUO.Western blotting analysis showed that FG-4592 pretreated UUO rats could significantly regulate the Bcl-2/Bax ratio and restore the expression of activated caspase-3 protein,which was beneficial to the survival of cells.8.UUO induced the activation of Wnt3?/?-catenin/GSK-3? in a time-dependent manner,while FG-4592 inhibited the expression of Wnt3?.Conclusion:1.UUO can cause the overexpression of RIP 1-RIP3-MLKL-related proteins,and simultaneously induce cell pyrotosis,necrotic inflammation and renal fibrosis in a time-dependent manner.2.Necrotizing inflammation is closely related to mitochondrial dysfunction,endoplasmic reticulum stress and cell apoptosis caused by oxidative stress.3.All the above changes were reversed by FG-4592 or the RIP1 inhibitor Nec-1,suggesting that FG-4592 improved UUO renal fibrosis by inhibiting necrotizing inflammation induced by RIP1-RIP3-MLKL.4.The anti-fibrosis effect of FG-4592 may be achieved through the intervention of HIF-1?/Wnt/catenin/GSK signaling pathway. |
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