The Role And Mechanism Of Pannexin1 In Sepsis-induced Acute Kidney Injury

Background: Sepsis-induced acute kidney injury(SI-AKI)is the fifth most common cause of hospital-acquired acute kidney injury.SI-AKI has become a medical research hotspot and a major public health issue with its characteristics of high mortality,poor prognosis and heavy economic burden.The pathogenesis of SI-AKI is complex.Apoptosis and inflammation play crucial roles in the development of SI-AKI.Pannexin1(Panx1)is a transmembrane channel glycoprotein,which plays an important role in the physiological and pathological process.Panx1 participates in the process of immune response,cell damage,cell differentiation and tumor development.Previous studies showed that Panx1 was increased in the kidney ischemia / reperfusion injury(IRI)and cisplatin-induced acute kidney injury(CIA),and inhibiting Panx1 could ameliorate IRI and CIA.However,the role of Panx1 in SI-AKI has yet to be determined.Therefore,this study is to explore the role of Panx1 in SI-AKI and its effect on apoptosis and inflammation.Methods:Part I: 1)SI-AKI mouse model was established by intraperitoneal injection of lipopolysaccharide(LPS)into C57BL/6N mouse.Mice were randomly divided into three groups: Con group,10mg/kg LPS group and 20mg/kg LPS group.Renal tissue injury was assessed by serum creatinine,blood urea nitrogen,hematoxylin and eosin(HE)and periodic acid Schiff(PAS)staining.We also measured the m RNA level of AKI biomarkers,kidney injury molecule(KIM-1)and neutrophil gelatinase associated lipocalin(NGAL),by q PCR in mouse kidney.The expression of Panx1 was detected by q PCR,Western blot and immunohistochemistry(IHC).The mice were pretreated with carbenoxolone(CBX),an inhibitor of Panx1.Then mice were randomly divided into three groups: Con group,LPS group and LPS+CBX group.2)From August 2014 to August 2019,patients who underwent renal biopsy and diagnosed as SI-AKI were retrospectively enrolled.Control group was patients underwent renal biopsy which is proven as slight pathological changes.The general and clinical data were collected,and the expression of Panx1 in renal tissue was detected by IHC.Part II: We used LPS-induced HK-2 to establish SI-AKI model in vitro.We incubated HK-2 cells with LPS at different concentrations and time to determine optimal induced condition by assessing its cell viability.Then HK-2 cells were divided into four groups: NC group,Si-Panx group,NC+LPS group and Si-Panx+LPS group.We detected the number of apoptotic cells by TUNEL as well as the expression of cleaved caspase-3,Bax and Bcl2 by western blot in mouse kidney tissue and HK-2 cells.Part III: Inflammatory cytokines IL-1β,IL-6 and TNF-α as well as antiinflammatory cytokine IL-10 were measured by q PCR and ELISA.The activation of NLRP3 inflammasome,including NLRP3,cleaved caspase-1 and active IL-1β,were evaluated by western blot.The infiltration of macrophages in the kidney were assessed by IHC.Results:Part I: 1)Compared with the control group,10mg/kg LPS group had significant changes in renal histopathology,whereas insignificant increases in serum creatinine,blood urea nitrogen as well as m RNA level of KIM-1 and NGAL in the kidney,while20mg/kg LPS group was significantly upraised the level of serum creatinine,urea nitrogen as well as KIM-1 and NGAL m RNA in the kidney and had significant renal histopathological changes.Therefore,SI-AKI model was established by intraperitoneally injected with 20 mg/kg LPS.Compared with the Con group,Panx1 m RNA and protein was significantly increased in the LPS group.Moreover,Panx1 was mainly expressed in renal tubules proven by IHC.Pretreatment of CBX alleviated the renal function and pathological damage,as well as decreased the renal tubular injury score index,KIM-1and NGAL m RNA in SI-AKI mice.2)Three SI-AKI patients and three Control patients were enrolled.Compared with the control group,Panx1 expression was upregulated in SI-AKI patients.The levels of IL-1β,IL-6,TNF-α and IL-10 were also increased in SIAKI patients.Part II: Compared with the Con group,the number of TUNEL positive cells,apoptosis index,the expression of cleaved caspase-3 and Bax were significantly increased,while Bcl2 expression was significantly decreased in the LPS group.Pretreating with CBX significantly reversed these phenomena of SI-AKI mice.The activity of HK-2 cells was the lowest when exposed to 1000 ng/ml LPS for 24 hours.Therefore,we used 1000ng/ml LPS to induce HK-2 cells for 24 hours for further investigation.Inhibiting Panx1 by si-RNA decreased the number of TUNEL positive cells,apoptosis index,the expression of cleaved caspase-3 and Bax,otherwise upregulated in LPS+NC group.Bcl2 decreased in LPS+NC group and reversed by inhibiting Panx1.Part III: Compared with the Con group,serum and kidney IL-1β,IL-6,TNF-α and IL-10 level were significantly increased in the LPS group.We also found significant elevated expressions of NLRP3,cleaved caspase-1 and activated IL-1β protein and the increased infiltration of macrophages in kidney tissue in LPS group.Pretreating with CBX significantly reduced the levels of IL-1β,IL-6 and TNF-α and downregulated the expression of NLRP3,cleaved caspase-1 and activated IL-1β.Tough IL-10 and the infiltration of macrophages slightly decreased when blocking Panx1,but we failed to find the difference between the two groups.Compared with NC group of HK-2 cells,the levels of IL-1β,IL-6,TNF-α and IL-10 m RNA as well as the expression of NLRP3,cleaved caspase-1 and activated IL-1 β were significantly increased in LPS + NC group.Inhibiting Panx1 by si-RNA significantly decreased IL-1β,IL-6,TNF-α and IL-10 m RNA as well as the expression of NLRP3,cleaved caspase-1 and activated IL-1β.Conclusion: Panx1 upregulated in SI-AKI,both in vivo and in vitro.Inhibiting of Panx1 prevented tubular cell apoptosis and decreased inflammation via decrease macrophage infiltration and NLRP3 activation,resulting in kidney protective effects in SI-AKI.These observations suggest that pharmacological inhibition of Panx1 might be a potential approach in the clinical therapy of SI-AKI.