The Molecular Mechanism Of TNFSF14-HVEM/LTβR Pathway In The Pathogenesis Of Renal Fibrosis

BackgroundRenal fibrosis is the common outcome of all reversible kidney damage,which accelerates the transition to kidney failure and places a heavy burden on families and society.However,there isn’t an effective treatment strategy for patients with renal fibrosis.Therefore,it is urgent to find an effective treatment strategy by in-depth study of the underlying mechanisms of renal fibrosis and the specific target for renal fibrosis treatment.Tumor necrosis factor superfamily 14（TNFSF14）,one of the members of the tumor necrosis factor superfamily,is a ligand for HVEM and LTβR.As a costimulatory molecule,TNFSF14 promotes the development of inflammatory responses and inflammatory-related diseases.The inflammatory response that initiates tissue fibrosis and sustains inflammatory stimulation is an important factor leading to renal fibrosis.Therefore,we speculate that the TNFSF14 pathway is involved in the pathological process of renal fibrosis and may be a potential target for the treatment of renal fibrosis.Sphingosine kinase 1（Sphk1）,an enzyme that produces sphingosine-1-phosphate（S1P）,is closely related to various biological activities such as cell proliferation,differentiation and migration.The expression of Sphk1 is significantly increased in many inflammation-related kidney diseases,including diabetic nephropathy and polycystic kidney disease.Sphk1/S1P signaling promotes renal tubule epithelial-mesenchymal transition（EMT）and collagen deposition by up-regulating miR-21.In contrary,it is significantly inhibited high glucose-induced fibrin deposition in kidney tissue after Sphk1 gene silencing.It is indicated that Sphk1 is involved in the development of renal fibrosis.However,the correlation between the TNFSF14 pathway and the Sphk1 expression in renal fibrosis is unclear.As an important component of non-specific immunity,the complement system plays an important role in promoting inflammation and tissue damage.It has reported that C5a directly induce EMT in renal tubular epithelial cells.The deficiency of C5 gene suppresses renal inflammatory response and fibrosis.In addition,the activation of C5a/C5aR pathway is essential for Sphk1 expression.Numerous studies are suggested that there is a correlation between the TNFSF14 and complement pathway,because both of them are closely related to inflammatory responses.Objectives1.To study the expression of TNFSF14 and its receptor HVEM/LTβR during the pathogenesis of renal fibrosis.2.To investigate the role of TNFSF14-HVEM/LTβR pathway in the development of renal fibrosis.3.To explore the mechanism of TNFSF14-HVEM/LTβR pathway in the potentiating of renal fibrosis.4.To elucidate the relation of complement C5aR signaling with TNFSF14 pathway-mediated renal fibrosis.Methods1.Expression of TNFSF14 and its receptor in fibrotic renal tissue.（1）Collecting renal biopsy specimens from patients with chronic kidney disease（CKD）,and the expression of TNFSF14,HVEM and LTβR were measured by IHC.（2）Collecting peripheral blood from patients with CKD,and the level of sTNFSF14 in serum was measured by ELISA.（3）The murine renal fibrosis model of unilateral urethral obstruction（UUO）was established.The renal tissues and peripheral blood at different time points after UUO surgery were collected.Quantitative real-time PCR（RT-qPCR）,immunohistochemistry（IHC）and enzyme linked immunosorbent assay（ELISA）were used to detect the expression of TNFSF14 and its receptors.2.Effect of TNFSF14-HVEM/LTβR pathway on the pathological changes of renal fibrosis.Renal tissues were collected at 7 days after UUO and the effects of Tnfsf14 gene defect on pathological changes were detected.（1）PAS staining was used to observe the degree of renal injury and score.（2）The collagen deposition was measured by Sirius Red and Masson staining.（3）The expression ofα-SMA,E-cadherin,inflammatory related factors and the infiltration of inflammatory cells were observed by IHC、Western Blot、IF and RT-qPCR.3.The mechanism of TNFSF14-HVEM/LTβR pathway in the mediating of renal fibrosis.（1）IHC was used to measure the expression of Sphk1 in human and mouse fibrotic kidney tissues.Masson,Sirius Red and IHC were used to detect collagen deposition and expression ofα-SMA in renal tissue at 7 days after continuous intraperitoneal injection of PF543（Sphk1 inhibitor）.（2）Renal tissues were collected from Tnfsf14^+/+and Tnfsf14^-/-mice at 7 days after UUO,and the expression of Sphk1 was measured by IHC,RT-qPCR and Western Blot.（3）Primary mouse renal epithelial cells（mTECs）were isolated and cultured,and after stimulation with recombinant TNFSF14 protein,IF was used to detect the expression of Sphk1 in mTECs.4.The role of complement C5a/C5aR signaling in TNFSF14-mediated renal fibrosis.（1）Renal tissues were collected from C5aR^+/+and C5aR^-/-mice at 7 days after UUO,and the expression of Sphk1 was measured by Western Blot.To clarify that complement C5a/C5aR upregulates Sphk1 expression and promotes renal fibrosis.（2）Collection renal biopsy specimens from patients with CKD,and IF was used to detect the localization of C5aR and TNFSF14 receptor.（3）Renal tissues and peripheral blood were collected from Tnfsf14^+/+and Tnfsf14^-/-mice at 7 days after UUO,and the expression of C5aR in renal tissues was measured by IHC and RT-qPCR;The level of C5a in serum was detected by ELISA.（4）In vitro,the expression of C5aR was detected after stimulating mTECs with recombinant TNFSF14 protein;Sphk1 was detected by Western Blot after stimulating mTECs with recombinant TNFSF14 protein alone or in combination with recombinant C5a;Sphk1was detected after stimulating mTECs with recombinant TNFSF14 protein alone or in combination with C5aR antagonist（C5aRA）.Results1.Increased expression of TNFSF14 and its receptor in fibrotic renal tissue.（1）Compared with normal kidney tissue,the expression of TNFSF14 and its receptor in humanized fibrotic kidney tissue was significantly increased;compared with healthy human serum,the serum sTNFSF14 content in patients with CKD was significantly increased.（2）Compared with the sham operation group,the expression of TNFSF14 and its receptors in serum and kidney tissues of UUO injured mice were significantly up-regulated.2.TNFSF14-HVEM/LTβR pathway promotes UUO-induced renal injury,collagen deposition and inflammatory response.In the UUO model,Tnfsf14 gene deficiency significantly improved UUO-induced renal injury;inhibited deposition of collagen and expression of pro-fibrotic factorα-SMA in renal tissue;inhibited UUO-induced degradation of E-cadherin in tissues and protected the integrity of renal tubular epithelial cells;significantly reduced the expression of pro-inflammatory factors IL-6,IL-1βand TNF-αand the infiltration of inflammatory cells in renal tissues.3.TNFSF14-HVEM/LTβR pathway promotes pro-fibrotic Sphk1 expression in renal fibrosis.（1）The expression of Sphk1 was significantly increased in human or mouse fibrotic kidney tissues,and the expression level of Sphk1 was positively correlated with the severity of renal fibrosis.After treatment with PF543,the deposition of collagen in renal tissue induced by UUO was significantly reduced.（2）Tnfsf14 gene deficiency significantly inhibited Sphk1 expression in renal tissue.（3）Recombinant TNFSF14 protein directly promoted the expression of the profibrotic factor Sphk1 protein in mTECs.4.TNFSF14-HVEM/LTβR pathway promotes pro-fibrotic Sphk1 expression by activating C5aR signaling.（1）C5aR gene deficiency significantly reduced the levels of both TGF-β1,Colla1,Vim mRNA and Fibronectin,α-SMA protein.（2）C5aR gene deficiency significantly inhibited the expression of Sphk1 in renal tissue.（3）It was showed that C5aR and TNFSF14 receptors were highly co-expressed in human fibrotic kidney tissue.Moreover,after UUO surgery,the expression of C5aR in the kidney tissue and the level of C5a in serum was significantly down-regulated in Tnfsf14 gene deficient mice than that of wild-type mice.（4）In vitro experiments showed that TNFSF14 directly promoted the expression of C5aR in mTECs,and TNFSF14 synergized with C5a to up-regulate the expression of Sphk1in mTECs.In contrast,C5aRA significantly inhibited the TNFSF14-induced increase in Sphk1 expression.