The Role Ofstimulator Of Interferon Genes (STING) In Diabetic Nephropathy

At present,the increasing incidence of diabetes worldwide has become a major global public health problem.Diabetic nephropathy(DN)is one of the most common complications of diabetes.It refers to chronic kidney injury caused by persistent heigh level of blood glucose,which affects glomeruli,renal tubules,renal mesenchyme and blood vessels.It is an important cause of chronic renal failure and end stage renal disease(ESRD).Although the pathogenesis of DN has not been fully elucidated,it’s believed that inflammation plays an important role in DN.Moreover,studies have also found that a large number of inflammatory cells infiltrating in the kidney,such as macrophages,neutrophils and the inflammatory factors secreted by them are also important reasons for the occurrence of renal fibrosis.Stimulator of interferon genes(STING)is an endoplasmic reticulum transmembrane protein,consisting of 379 amino acids(aa).STING is expressed in various endothelial and epithelial cells as well as cells such as T cells,macrophages,and plays an important role in the immune response to a variety of diseases as a major regulator of type Ⅰ interferon(IFN-Ⅰ)and the innate immune system.Studies on STING in chronic inflammation,such as autoinflammatory diseases and skin vascular lesions,have found that STING plays an important role in promoting the occurrence and development of inflammation.In addition,STING is considered as a potential protein mediator of aseptic chronic inflammation and play an important role in aseptic inflammation,such as chronic cardiovascular diseases and metabolic diseases caused by obesity and metabolic disorders.It has been found that STING plays an important role in renal fibrosis,but the role of STING in renal injury in DN,especially in podocyte and renal tubule cells,remains unclear.In this study,we found that STING aggravated the inflammatory response of DN,a metabolic related chronic disease of the kidney,and promotes the morphological and functional injury of the kidney tissue.Pharmacological inhibition of STING activity could significantly attenuate DN glomerular injury,infiltration of renal interstitial immune cells and inflammatory mediators,and inhibit renal fibrosis.DN tubular epithelial cells could promote chemokines secretion by activating STING-IFN-Ⅰ pathway and NF-κB pathway,thereby recruiting circulating immune cells to migrate into renal tissues and aggravate glomerular and tubular inflammation.ObjectivesIn order to clarify the role of STING in DN,we mainly carried out the following studies:(1)To clarify the expression changes of STING in DN.(2)To explore the effects of STING on glomeruli and renal tubules in DN.(3)To clarify the therapeutic effect and feasibility of STING inhibitors in chronic kidney disease.(4)To explore the molecular mechanisms of STING in vitro cell experiments.Methods and Results:1 Expression of STING in diabetic nephropathy1.1 The protein expression level of STING increased in kidney tissue samples of clinical diabetic nephropathy patientsImmunohistochemical staining was used to detect the expression level of STING in pathological sections of kidney biopsy of DN patients,and the results showed that STING was increased in renal tissues of DN patients,including glomeruli,mesenchyme and tubules.1.2 The protein expression level of STING increased in diabetic nephropathy miceWestern Blot was used to detect the expression of STING in STZ/HFD and db/db mice,and the results showed that STING expression was increased in both DN mice models.1.3 STING signaling was active in human podocyte and renal tubular epithelial cells under the condition of high glucoseIn vitro cells,HG was used to treat human podocytes(HPC)and renal tubular epithelial cells(HK-2),the expression and phosphorylation of TBK1 and IRF3 were detected by Western Blot.The results showed that STING signaling was active in HPC and HK-2,and more stably in HK-2.2 To detect the effect of podocyte specific knockout STING on glomerular injury in diabetic nephropathy mice2.1 Construction of podocyte specific loss of STING miceCre-loxP system was used to construct podocyte specific knockout STING mice,and the genotypes of mice were identified by agarose gel electrophoresis to verify the success of STING knockout.2.2 Podocyte specific loss of STING ameliorated glomerular injury in diabetic nephropathy mice(1)STING is transcribed by gene Tmem173.Tmem173fl/fl mice with or without Nphs2.cre were injected with STZ and fed a high-fat diet(HFD)for 3 months.(2)The urine of mice was collected to detect the ratio of urinary microalbumin to urinary creatinine(UACR),and it was found that podocyte specific loss of STING could reduce UACR in DN mice.(3)The mice glomerular basement membrane(GBM)area was observed by transmission electron microscopy(TEM),and the results showed that STING podocyte specific deletion could attenuate the STZ/HFD-induced foot process fusion,basement membrane thickening and other glomerular injuries.(4)The mice kidney was paraffin-embedded and sliced,and immunohistochemical staining of Podocin,WT1 and Desmin showed that STING podocyte specific deletion could alleviate the glomerulus injury induced by STZ/HFD.(5)By PAS staining,STING podocyte specific deletion was found to inhibit STZ/HFDinduced glomerular fibrosis.(6)Immunohistochemical staining showed that STING podocyte specific deletion could down-regulate the infiltration of macrophages in the glomerulus.3 To detect the effect of RTEC-specific loss of STING on renal tubules injury in diabetic nephropathy mice3.1 Construction of RTEC-specific loss of STING mice(1)Cre-loxP system was used to construct RTEC-specific loss of STING mice,and the genotypes of mice were identified by agarose gel electrophoresis to verify the success of STING knockout.(2)The success of STING knockout was further proved by Western Blot3.2 RTEC-specific loss of STING alleviated renal inflammation and fibrosis in diabetic nephropathy mice(1)Tmem173fl/fl mice with or without Cdh16.cre were injected with STZ and fed a highfat diet for 20 weeks.(2)mRNA expression levels of pro-inflammatory cytokines including TNF-α,IL-1β and IL-6 in renal cortex were detected by RT-qPCR,and the results showed that the RTECspecific loss of STING reduced the expression of these pro-inflammatory cytokines.Western Blot results showed that RTEC-specific loss of STING could reduce the increased expression levels of MPO and MHCI induced by STZ/HFD.It was found that RTEC-specific loss of STING could attenuate the tubular morphological injury by PAS staining.Immunohistochemical results showed that RTEC-specific loss of STING induced the secretion of IL-6 induced by STZ/HFD,and alleviated macrophages and neutrophils,as well as adaptive immune cells such as CD4+T cells and CD8+T cells in the renal interstitial.(3)Western Blot indicated that RTEC-specific loss of STING could down-regulate the expression levels of α-SMA and Vimentin induced by STZ/HFD.By Sirius red staining and COL1A1 immunohistochemistry,RTEC-specific loss of STING was found to ameliorated STZ/HFD-induced increased renal interstitial extracellular matrix proteins.In conclusion,RTEC-specific loss of STING attenuated renal interstitial fibrosis induced by STZ/HFD.4 To detect the effect of pharmacological inhibition of STING on kidney injury in diabetic nephropathy mice4.1 Injection STING inhibitor C176 in diabetic nephropathy miceWT mice were injected with STZ and fed a high-fat diet for 20 weeks.C176 was intraperitoneally injected into mice starting from the 9th week and injected once every other day until the models were obtained.4.2 Pharmacological inhibition of STING ameliorated glomerular injury in diabetic nephropathy mice(1)Mice urine was collected to detect the UACR,and it was found that the inhibition of STING activity by C176 could reduce it.(2)The mice GBM area was observed by TEM.The results showed that inhibition of STING activity by C176 could ameliorate the glomerular injury induced by STZ/HFD,such as foot process fusion and GBM thickening.(3)The mice kidney was paraffin-embedded and sliced.PAS staining and immunohistochemical staining of WT1 and Desmin showed that inhibition of STING activity by C176 ameliorated glomerular injury induced by STZ/HFD.4.3 Pharmacological inhibition of STING alleviated renal inflammation and fibrosis in diabetic nephropathy mice(1)mRNA expression levels of pro-inflammatory cytokines including TNF-α,IL-1β and IL-6 in the renal cortex were detected by RT-qPCR,and the results showed that inhibition of STING activity by C176 could reduce the expression of these pro-inflammatory cytokines.Western Blot results showed that inhibition of STING activity by C176 ameliorated the increased expression levels of MPO and MHCI induced by STZ/HFD.The mice kidney was paraffin-embedded,sliced and stained by PAS,and it was found that inhibition of STING activity by C176 attenuated the renal tubular morphological injury induced by STZ/HFD.Immunohistochemical results showed that the inhibition of STING activity by C176 could reduce the secretion of IL-6,and alleviate the infiltration of macrophages and neutrophils,as well as adaptive immune cells such as CD4+T cells and CD8+T cells in the interstitial of the kidney.In conclusion,inhibition of STING activity by C176 could attenuate the kidney inflammatory response induced by STZ/HFD.(2)Western Blot results showed that the increased expression levels of α-SMA and Vimentin induced by STZ/HFD could be reduced by C176.By FN1 and COL1A1 immunohistochemistry,inhibition of STING activity by C176 could reduce the increase of extracellular matrix protein in renal mesenchyme induced by STZ/HFD.In conclusion,inhibition of STING activity by C176 could ameliorate renal fibrosis induced by STZ/HFD.5 To investigate the effect and mechanism of STING in diabetic nephropathy renal cells5.1 Inhibition of STING signaling attenuated podocyte injury stimulated by HG(1)By silencing STING in HPC with small interfering RNA,the results showed that the silencing STING could attenuated the decrease of Nephrin and Podocin and the increase of Desmin induced by HG.(2)STING-TBK1-IRF3 signaling could be inhibited by STING silencing.The results of RT-qPCR showed that silencing STING could suppress the increase of GM-CSF and CCL5 induced by HG.5.2 Inhibition of STING signaling attenuated inflammation in HK-2(1)STING-TBK1-IRF3 pathway and NF-κB pathway up-regulation induced by HG were inhibited by silencing STING in HK-2 by small interfering RNA.The results of RT-qPCR showed that STING silencing could inhibit the increase of GM-CSF,CCL5 and CXCL10 induced by HG.ELISA showed that silencing STING could inhibit the level of IFN-α in the supernatant of HK-2 culture induced by HG.Western Blot results showed that the MHCI level was decreased after silencing STING.(2)STING was inhibited by inhibitor H-151,and the results showed that STING signaling activation induced by HG could be inhibited after the suppression of STING activity.The results of RT-qPCR showed that suppression of STING could inhibit the increase of GMCSF and the chemokines such as CCL5 and CXCL10 induced by HG.(3)The inflammatory stimulator LPS was used to stimulate HK-2,and the results showed that STING-TBK1-IRF3 pathway and NF-κB pathway activation induced by LPS could be inhibited by silencing STING by small interfering RNA.The results of reducing STING activity by H-151 is similar to the former.5.3 STING signaling was regulated by TBK1 and cGAS(1)The small molecule inhibitor GSK8612 was used to inhibit TBK1 activity.The results showed that GSK8612 could not only inhibit IRF3 phosphorylation and the activation of NF-κB pathway induced by HG,but also inhibit STING phosphorylatio(2)The silencing of cGAS by small interfering RNA showed that STING-TBK1-IRF3 and NF-κB pathways activation induced by HG or LPS could be inhibited.Conclusions and innovations:1.STING increased in DN and involved glomeruli,renal tubules and renal interstitium,and the specific loss of STING in podocytes and renal tubular epithelial cells can improve the injury in the corresponding parts of kidney in DN mice.2.Pharmacological inhibition of STING activity could improve renal function,attenuate glomerular injury,and inhibit renal interstitial inflammation and fibrosis process in DN mice.3.Inhibition of cGAS-STING pathway could improve podocyte injury and renal tubular epithelial cell inflammation,and down-regulate the expression of MHCI by reducing the secretion of IFN-I,thus affecting the role of adaptive immune cells in the kidney.4.In this study,we found that STING,as a key regulator of cGAS-STING pathway,can aggravate podocyte injury in DN and promote renal inflammation and fibrosis.We have also proved the possibility of using STING inhibitor to treat DN,which provides a new therapeutic target and experimental basis for the treatment of DN.