Role Of TWEAK On Renal Injury And Its Related Mechanism In Lupus Nephritis

Part Ⅰ:Tumor necrosis factor-like weak inducer of apoptosis(TWEAK)activates type Ⅰ interferon signals in lupus nephritisObjectives:Numerous studies have demonstrated that type Ⅰ interferon(IFN)plays a critical role in the pathogenesis of SLE.Type Ⅰ IFN-inducible gene signature has been used to classify SLE patients,and patients with high IFN gene signature are correlated with poor prognosis.Tumor necrosis factor-like weak inducer of apoptosis(TWEAK),first described as an inducer of apoptosis,has been proved to be related to the pathogenic role in lupus nephritis(LN).Therefore,this study investigated whether TWEAK could induce the activation of type Ⅰ IFN pathway in LN.Methods:LN animal model MRL/lpr mice and peripheral blood mononuclear cells(PBMCs)derived from LN patients were studied in vivo and in vitro.shRNA was used to inhibit the expression of TWEAK gene in vivo,and TWEAK siRNA and protein were used to intervene PBMCs in vitro.The pathological changes of mouse kidney were evaluated by hematoxylin&eosin,periodic acid schiff(PAS)and masson staining,and the concentration of IFNa in renal interstitial fluid was detected by enzyme-linked immunosorbent assay.Quantitative reverse transcription PCR was used to detect type ⅠIFN-inducible gene LY6E(lymphocytic antigen 6 complex locus E),OASL(2’,5’oligoadenylate synthetase like)and ISG15(IFNα-inducible protein(clone IFI-15K)).LY6E protein was examined by western blotting.Results:MRL/lpr mice showed histological evidence of severe glomerulonephritis,characterized by glomerular hypercellularity,PAS-positive material and collagenous fiber deposition.The levels of serum creatinine,blood urea nitrogen and proteinuria were significantly higher in MRL/lpr mice than those in MRL/MpJ mice(negative control).Moreover,the concentration of IFNa of renal interstitial fluid and the gene expression levels of LY6E,OASL and ISG15 of renal tissue in MRL/lpr mice significantly increased.Western blotting showed that MRL/lpr mice exhibited higher renal LY6E protein levels than MRL/MPJ mice.The TWEAK shRNA intervention not only significantly alleviated the renal histopathological changes,decreased the levels of serum creatinine,blood urea nitrogen and proteinuria,but also reduced the concentration of IFNα in renal interstitial fluid and the expression of LY6E,OASL and ISG15 in renal tissue.In vitro,compared with control siRNA,TWEAK siRNA also significantly down-regulated the expression of LY6E,OASL and ISG15 in PBMCs,while TWEAK stimulation significantly up-regulated the gene expression of LY6E,OASL and ISG15.Conclusion:Our results illustrate a novel regulatory role of TWEAK,in which its activity positively regulates type Ⅰ IFN pathway in LN based on pre-clinical models.Our findings suggest TWEAK could act as a critical target in preventing renal damage in patients with LN.Part Ⅱ:Anti-TWEAK antibody alleviates renal interstitial fibrosis by increasing PGC-1α expression in lupus nephritisObjectives:Lipid metabolic deficiency in the renal tubular epithelium has received extensive attention in the context of kidney injury.Lipid metabolism is regulated by many factors,such as peroxisome proliferator activated receptor γ-coactivator-1α(PGC-1α).As a transcription regulator,peroxisome proliferator-activated receptor-gamma coactivator-la(PGC-1α)can activate many types of intranuclear receptors and regulate the expression of downstream target genes,such as fatty acid translocase(FAT),carnitine palmitoyl-transferase 1(CPT1),long-chain acyl dehydrogenase(LCAD),cytochrome oxidase Ⅱ(COX Ⅱ).Current studies on the mechanism of tumor necrosis factor-like weak inducer of apoptosis(TWEAK)in lupus nephritis(LN)mainly focus on the inflammatory pathway.Herein,we aimed to determine whether TWEAK could promote the progression of renal interstitial fibrosis by regulating PGC-1α expression and intervening in lipid metabolism in LN.Methods:MRL/lpr mice,an animal model of lupus,were studied in vivo,the groups are as follows:MRL/lpr mice group,anti-TWEAK antibody group,isotype control antibody group,anti-TWEAK antibody+PGC-1α shRNA group,anti-TWEAK antibody+control shRNA group,and MRL/MpJ mice were used as negative control.Human proximal tubular epithelial cells(HK2 cells)were incubated with recombinant human TWEAK protein and ammonium pyrrolidine dithiocarbamate(PDTC)in vitro.A total of 44 free fatty acids in mice kidneys were measured using a gas chromatograph system coupled with a mass spectrometer(GC-MS),the accumulation of triglycerides in renal tissue was examined by oil red O staining.The collagen fiber deposition in mouse kidney was evaluated by Masson staining and Sirius red staining.The gene and protein expression were measured by quantitative reverse transcription PCR and western blotting,repevtively.The expression of type Ⅰ collagen and fibronectin in renal interstitium was detected by immunohistochemistry,and the distribution of PGC-1α in kidney was measured by immunofluorescence.Results:The contents of free fatty acids and triglycerides in kidney of MRL/lpr mice were higher than those of MRL/MpJ mice;however,these contents were decreased by treatment with the anti-TWEAK antibody.According to immunohistochemical staining,the expression of PGC-1α in renal tubules of MRL/MpJ mice was markedly more than that in glomeruli.The expression levels of PGC-1α and its downstream target genes FAT,CPT1,LCAD,and COX Ⅱ in MRL/lpr mice were significantly lower than those in MRL/MpJ mice;after anti-TWEAK antibody intervention,the expression levels of these genes in MRL/lpr mice were significantly increased.Anti-TWEAK antibody effectively alleviated renal interstitial fibrosis,which is characterized by reducing collagen fiber deposition and inhibiting the expression of type Ⅰ collagen and fibronectin.However,PGC-1α shRNA eliminated the therapeutic effect of the anti-TWEAK antibody.In vitro,recombinant human TWEAK decreased the expression of PGC-1α in HK2 cells in a dose and time-dependent manner.PDTC,an inhibitor of NF-κB signal pathway,suppressed the decrease in the PGC-1α protein level induced by recombinant human TWEAK treatment.Conclusion:Our results show that TWEAK promotes NF-κB activation to prevent the expression of PGC-1α in renal tubules,resulting in insufficient lipid metabolism and the progress of renal interstitial fibrosis.The up-regulation of renal tubular PGC-1αexpression to improve lipid metabolism is one of the mechanisms of anti-TWEAK antibody in the treatment of renal interstitial fibrosis.