The Role And Mechanism Of STING-activated Glycolysis In Hypoxia-related Renal Fibrosis

Objective: Renal fibrosis is characterized by deposition of extracellular matrix and excessive proliferation of fibroblasts,which is a common pathological manifestation of chronic kidney disease(CKD).Hypoxia plays a key role in the fibrosis progression of CKD,but its specific mechanism is unclear.Stimulator of interferon genes(STING)has been a hot topic of research in the fields of oncology,immunity and infection in recent years,and its role in the immune and inflammatory responses associated with kidney diseases is also gaining attention.This study focuses on exploring the role and mechanism of STING in hypoxia-related renal fibrosis and providing new ideas and targets for the treatment of renal fibrosis.Methods:(1)Two chronic hypoxia-related renal fibrosis models of ischemia-reperfusion injury(IRI)and unilateral ureteral obstruction(UUO)were established in 6～8-week-old wild type C57BL/6J male mice by unilateral renal pedicle clamping and unilateral ureteral ligation respectively.An in vitro model was constructed using hypoxic stimulation of human kidney-2(HK-2)for 48 hours.Masson staining,hematoxylin-eosin(HE)staining,immunofluorescence staining,immunohistochemical staining,western blot and quantitative real-time PCR(q PCR)were used to detect the successful establishment of renal fibrosis models and the expression levels of cyclic GMP-AMP synthase(c GAS)and STING.The renal tissues were collected from clinical CKD patients,and the expression of STING in the kidneys of patients with CKD was detected by immunohistochemical staining.(2)The expression of STING was interfered by intraperitoneal injection of STING inhibitor C-176 and targeted injection of STING lentivirus in renal parenchyma of experimental mice.The extent of renal fibrosis and the expression level of STING were assessed by immunohistochemical staining,masson staining,western blot and q PCR.In vitro,STING molecules in HK-2 cells were knocked down by STING lentivirus,and the expression of epithelial mesenchymal transition(EMT)markers and extracellular matrix proteins in HK-2 cells were evaluated by western blot and q PCR.(3)Western blot and q PCR were used to detect the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3)in IRI model and hypoxia-stimulated HK-2 cells after inhibition of STING.The activity of glycolysis rate-limiting enzymes was detected by absorption spectrum,and the level of extracellular acidification rate was detected by time-resolved fluorescence intensity measurement.PFKFB3 inhibitors 3PO and PFKFB3 si RNA were used to inhibit PFKFB3 in HK-2 cells,and the expression of EMT markers and extracellular matrix proteins in HK-2 cells were detected by western blot and q PCR.The expression of interferon regulatory factor 3(IRF3)in HK-2 cells was specifically knocked down by IRF3 si RNA,and the changes of PFKFB3 expression and glycolysis level in HK-2 cells were detected by western blot,q PCR,absorption spectroscopy and time-resolved fluorescence intensity measurement.Results:(1)The expression of c GAS and STING were low in normal mouse kidney,but significantly increased in renal fibrosis model induced by IRI and UUO.In vitro,hypoxic stimulation induced EMT and extracellular matrix accumulation in HK-2 cells,while upregulating the expression of c GAS and STING.(2)The use of specific STING inhibitors and STING lentiviral interventions attenuated IRI and UUO-induced renal fibrosis.In vitro,knocking down the expression of STING can reduce the production of EMT markers and extracellular matrix proteins in HK-2 cells induced by hypoxia.(3)The key enzyme of glycolysis,PFKFB3,and the level of glycolysis were up-regulated in hypoxia-induced renal fibrosis models,while inhibition of STING and its downstream IRF3 could reduce the expression of hypoxia-induced PFKFB3 and glycolysis level.In addition,inhibition of PFKFB3 reduced the levels of EMT markers and extracellular matrix production in hypoxia-induced HK-2 cells.Conclusions: The c GAS-STING-IRF3 signaling pathway activated in the hypoxia environment affects the glycolysis level by regulating the expression of PFKFB3,which increases the EMT of renal tubular epithelial cells and the production of extracellular matrix,thus promoting the progression of renal fibrosis.Inhibition of STING attenuates hypoxia-induced renal fibrosis by blocking PFKFB3-mediated glycolysis.