| Objective AKI is a syndrome,characterized by a rapid raise in serum creatinine,decrease in urine output,or both.Sepsis is also a life-threatening clinical syndrome that develops as dysregulated host response to an infection,which is characterized by acute organ dysfunction and high risk of death.Kidney is one of the commonly affected organs during the sepsis,resulting in SI-AKI.AKI and sepsis are inextricably connected,AKI is one of the most common complications,about 60% sepsis patients have AKI.Besides,sepsis is the most common cause of AKI,is associated with up to 50% AKI.The patients with SI-AKI have a significantly increased mortality relative to patients without AKI and those with AKI of other etiologies.Therefore,it is necessary to explore novel treatments to improve the clinical outcomes.Nowadays,natural compounds have gained increasing attention due to their low cost,high bioavailability,and limited toxicity.Several natural compounds have been reported to protect against AKI.UA is produced by the human gut microbiota from ellagitannins and ellagic acid.UA is reported to be associated with antiinflammatory,antioxidant,mitophagy,as well as protective effect of cisplatin-induced nephrotoxicity and renal ischemia reperfusion injury.However,the role of UA in the SI-AKI is unclear,so the effect of UA on SI-AKI is explored in the present study.Materials and Methods 1.Mice were randomly divided into four groups: control group,UA group,LPS group and LPS+UA group.The model of SI-AKI was established by intraperitoneal injection of LPS.UA was injected at 50 mg/kg 30 min prior LPS injection.Blood,urine and kidney samples were collected 4 hours after LPS injection.The levels of serum creatinine and urea nitrogen were detected by automatic biochemical analyzer.The serum proinflammatory cytokines,such as IL-1β,IL-6 and TNF-α,were analyzed by ELISA kit.The level of KIM-1and NGAL in urine samples were detected by ELSIA kit.The renal tissue sections were stained with hematoxylin and eosin stain,then the morphology of renal tubules and glomeruli were observed under light microscope,and the renal injury score was calculated based on the percentage of injury.2.In vitro,mouse renal tubular epithelial cell(TCMK-1)and podocyte(MPC5)were cultured with LPS.CCK-8 kit was used to test the effect of different concentrations of LPS on the cell viability of TCMK-1 and MPC5 cells.Then the appropriate concentration of LPS was selected,and the effects of different concentrations of UA on cell viability were also observed at different time points(6,12,24 h)using CCK-8 kit.RNA next-generation sequencing was performed for LPS and/or UA treated mouse podocytes(MPC5).The differentially expressed genes were then analyzed and the possibly related genes and signaling pathways were analyzed by GO and KEGG.The effect of LPS on the ultrastructural changes of MPC5 was observed under transmission electron microscope(TEM).GFP-LC3 was transfected into MPC5,and the fluorescence spots of GFP-LC3 were observed by fluorescence microscope to detect autophagy.Western blot was used to detect the expression of podocyte-specific markers(nephrin and podocin),inflammatory factors(cleaved-caspase-1,pro-caspase-1,cleaved-IL-1β,NLRP3 and ASC)and autophagy-related markers(BNIP3、LC3-Ⅱ and SQSTM1).3.BNIP3 si RNA was transfected to interfere with BNIP3 expression,and cleaved-caspase-1,cleaved-Il-1β,NLRP3,and BNIP3 were detected by Western blot.Immunofluorescence was used to observe the colocalization of LC3 and TOM20,TOM20 and LAMP1,BNIP3、LC3 and LAMP1.The expression of BNIP3,NLRP3 and LC3-Ⅱ in differently treated mice was observed by immunohistochemical analysis.Results 1.Compared with the mice in the control group,the levels of serum urea nitrogen,creatine,IL-1β,IL-6 and TNF-α were significantly increased in LPStreated mice(P<0.01).Compared with LPS treated mice,the levels of serum urea nitrogen,creatinine,IL-1β,IL-6 and TNF-α in LPS+UA treated mice were significantly decreased(P<0.05).The concentrations of urine KIM-1 and NGAL in LPS group were also significantly higher than those in control group(P<0.01),while those in LPS+UA group were significantly lower(P<0.05).Under the light microscope,the pathological changes in the kidney tissue of LPS treated mice were observed,and the renal injury score was significantly increased.The addition of UA could significantly improve the pathological changes,and significantly reduce the renal injury score.2.CCK-8 assay showed that LPS could significantly inhibit the cell viability of TCMK-1 and MPC5,and with the increase of LPS concentration,the cell viability also decreased significantly.The cell viability was significantly affected at a concentration of 0.5 ng/ml of LPS.And MPC5 was more sensitive to LPS,and the cell viability decreased more significantly.The cell viability was significantly increased after the addition of UA,and the best therapeutic effect was achieved at a concentration of 200 ng/ml of UA.And MPC5 is more sensitive to the treatment of UA,the cell viability increased more significantly.Next-generation sequencing showed that there were 865 differentially expressed genes between LPS and LPS+UA groups.GO and KEGG analysis showed that the differential genes were significantly enriched in cellular response to hypoxia,reduction of molecular oxygen,HIF-1 signaling pathway,cytokine-cytokine receptor interaction and mitophagy.Mitochondrial damage and mitochondrial membrane destruction were observed in LPS-treated MPC5 cells under TEM.However,MPC5 cells incubated with LPS and UA induced an increase in the number of autophagic vacuoles,and damaged mitochondria was engulfed.After transfection of GFPLC3,green GFP-LC3 fluorescence spots were observed in control group.Compared with the control group,GFP-LC3 spots were significantly decreased in LPS treated podocytes.However,GFP-LC3 spots were increased and aggregated in UA treated podocytes.Western blot showed that compared with the control group,LPS significantly reduced the expression levels of nephrin and podocin(P<0.05).After adding UA,the decrease of nephrin and podocin expression induced by LPS was significantly improved(P<0.05).The protein expression levels of cleaved IL-1β,cleaved-caspase-1,NLRP3 and ASC in LPS treated podocytes were significantly higher than those in control group(P<0.05).The protein levels of cleaved IL-1β and cleaved-caspase-1 in LPS+UA group were significantly lower than those in LPS treated group(P<0.05).There was no significant difference in expression level of procaspase-1 among four groups.The expression of NLRP3 in LPS+UA group was lower than that in LPS group,but there was no significant difference.Western blot showed that LPS could significantly inhibit the expression of BNIP3 and upregulate the expression of SQSTM1(P<0.05).Compared with LPS group,LPS + UA group significantly increased the protein levels of LC3-Ⅱ and BNIP3(P<0.05),while the expression level of SQSTM1 was significantly decreased(P<0.05).3.Immunofluorescence showed compared with the LPS group,the colocalization of LC3 and TOM20 fluorescence spots was enhanced in the LPS+UA group.Meanwhile,compared with the LPS group,the fluorescence spots of LAMP1 and TOM20 were aggregated after the addition of UA,and the colocalization of LAMP1 and TOM20 was also enhanced.In the LPS+UA group,the colocalization of BNIP3、LC3 and LAMP1 was also enhanced compared with LPS group.The level of BNIP3 in LPS+si-BNIP3 group was slightly lower than that in LPS group,but there was no significant difference.The expression level of BNIP3 in LPS+US group was significantly higher than that in LPS+si-BNIP3(P<0.05).The BNIP3 protein level of LPS+UA+siBNIP3 was also slightly lower than that of LPS+UA,without significant difference.Compared with LPS,LPS+si-BNIP3 could significantly up regulate the expression of cleaved-caspase-1(P<0.05).The protein level of cleaved-caspase-1 in LPS+UA+si-BNIP3 group was also significantly higher than that in LPS+UA group(P<0.05).The level of cleaved-IL-1β in LPS+siBNIP3 treated podocyte was significantly higher than that in LPS treated podocyte(P<0.05).Compared with LPS+UA,LPS+UA+si-BNIP3 significantly increased the expression of cleaved-IL-1β(P<0.05).There was no significant difference in NLRP3 between LPS and LPS+si-BNIP3,LPS+UA and LPS+UA+si-BNIP3 groups.Immunohistochemical analysis showed that the stained density of BNIP3 in control group was significantly higher than that in LPS group(P<0.05),while the stained density in LPS+UA group was significantly higher than that in LPS group(P<0.05).Similarly,the stained density of LC3-Ⅱ in LPS treated group was significantly lower than that in control group(P<0.05).After addition of UA treatment,the stained density of LC3-Ⅱ increased significantly(P<0.05).However,the stained density of NLRP3 in LPS treatment mice was significantly higher than that in control group(P<0.05),and LPS+UA treatment significantly decrease the stained density of NLRP3(P<0.05).Conclusion 1.Intraperitoneal injection of LPS in mice could damage renal function,significantly increase the levels of kidney injury biomarkers,proinflammatory cytokines,and cause pathological changes in renal tissues.Injection of UA prior to LPS significantly improved renal injury,indicating that UA has protective effect on LPS induced AKI.2.In vitro,LPS could significantly inhibit the cell viability of TCMK-1 and MPC5 cells,and the addition of UA could improve the cell viability, which further confirmed the protective effect of UA.MPC5 is more sensitive to LPS and UA.Bioinformatics analysis showed that cytokine-cytokine receptor signaling pathway,HIF-1 signaling pathway and mitophagy may be involved in the regulation.UA may attenuate kidney injury by regulating autophagy.3.UA could regulate BNIP3-mediated mitophagy and partially reverse LPS-induced NLRP3 activation,thereby alleviating LPS induced acute kidney injury. |
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