| Background Tenascin-C(TNC)is an extracellular matrix glycoprotein and plays an important role in regulating cell survival,proliferation and differentiation.TNC is abundantly expressed during embryonic development and induced in a variety of organ after injury.It is noteworthy that there is little or no expression of TNC in normal adult kidneys,but strong upregulation in chronic kidney diseases(CKD).Previous reports show its over-expression is highly related to tissue repair and renal fibrosis.Through binding to surface receptors of integrin or the toll-like receptors(TLR),TNC exerts potent effects on fibroblasts survival,inflammatory cell migration and formation of stem cell or progenitor cell niche.However,the effect of TNC after AKI has not been elucidated.In this study,we investigated the status of tenascin-C in AKI and set forth its mechanism.Methods In vivo,experiments were conducted to establish glycerol,we used rhabdomyolysis-associated acute kidney injury bilateral ischemia-reperfusion and cisplatin models to observe whether TNC is up-regulation in these three AKI models.The renal biopsy specimens of AKI patients were stained to observe the expression of TNC.In mouse models of ischemia-reperfusion injury(IRI)and cisplatin nephropathy,renal TNC was knocked down by shRNA-mediated inhibition.Serum creatinine and urea nitrogen were detected by fully automated biochemical analyzer.PAS staining was used to detect renal tubular injury.Immunohistochemistry was used to detect TNC,Cleaved caspase-3 and ?-catenin.Apoptotic cells were labeled by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)staining.The expression of 19 Wnt mRNAs was detected by RT-PCR.Western blot was used to detect the protein expression of TNC,Cleaved caspase-3,Bax,KIM-1,wnt1,wntlOb and ?-catenin.In vitro,in cultured Human proximal tubular epithelial cells(HKC,clone 8),cisplatin and staurosporine were used to induce apoptosis,and incubated with TNC recombinant protein.Apoptotic cells were determined by both FACS and TUNEL staining.The protein expression of Cleaved caspase-3,Bax,Fasl,P53,wntl,active ?-catenin,PCNA and Survivin were detected by western-blotAnd the protein expression of PCNA was also determined by immunofluorescence.The interaction between TNC and Wnt was also examined by co-stimulation of TNC recombinant protein and Wntl plasmids.The protien expression of active ?-catenin was detected by Western blot.Nuclei and cytoplasmic protein were extracted and the protein expression of ?-catenin in nuclei and cytoplasm was detected by Western-blot respectively.The interaction between Wnt1 or Wnt4 and TNC was detected by co-immunoprecipitation.To clarify the facilitating role of TNC on Wnt/?-eatenin signaling,we assessed the Wnt-mediated gene transcription in a ?-catenin responsive TOPFlash luciferase reporter assayResults In vivo,western blot analysis showed that TNC protein level significantly increased at 30 hour after glycerol intramuscular injection.Immunohistochemical staining showed that TNC was significantly upregulated in the renal interstitial area of the cortical and medulla junction in the bilateral ischemia-reperfusion model.RT-PCR results showed that TNC mRNA in IRI group up-regulated at 6 hour after operation,increased significantly at 12 hour after operation and continued to express at 24 hour after reperfusion.And the expression level of TNC mRNA in cisplatin model group increased on the first day after cisplatin intraperitoneal injection,significantly increased on the second day and continued to expresse on the third day.At the same time,immunohistochemical results showed that TNC significantly upregulated in renal biopsy specimens of AKI patients.In the bilateral ischemia-reperfusion and cisplatin model,knockdown of TNC further deteriorated serum creatinine and blood urea nitrogen compared to AKI alone.PAS staining suggested injured tubules,characterized by loss of brush border,tubular cell depletion and cast formation,were obviously aggravated after IRI injury and further exacerbated by knockdown of TNC.Western blot showed that the protein expression of Cleaved caspase-3,Bax and KIM-1 upregulated and p-AKT,wnt1,wntlOb and ?-catenin decreased.RT-PCR indicated that the expression of 12 Wnt mRNA remained unchanged.Immunohistochemical results suggest that the protein level of Cleaved caspase-3 upregulated and ?-catenin reduced.TUNEL staining suggested the number of cell apoptosis increased.This effect of TNC inhibition was associated with a reduced?-catenin upregulation,suggesting the relevance TNC to an altered Wnt signaling.In vitro,in cultured HKC-8 cells,recombinant TNC significantly inhibited cisplatin or staurosporine-induced apoptosis.Western blot showed that the expression level of Cleaved caspase-3,Bax,Fasl and P53 significantly decreased,wntl and active?-catenin significantly increased and Survivin and PCNA also significantly increased.Flow cytometry and TUNEL results suggested that apoptosis significantly reduced,and imunofluorescence indicated that PCNA increased.In addition,the expression of active ?-catenin significantly increased after co-stimulation with Wntl overexpression plasmid and recombinant TNC.Western blot showed that the expression of ?-catenin significantly increased in nuclei and significantly reduced in cytoplasm.We found that TNC physically interacted with Wnt ligand,as shown by co-immunoprecipitation.Wntl and Wnt4 can interact with TNC.Transfection of Wntl overexpression vector markedly induced luciferase reporter activity,as expected,however,co-stimulation with TNC augmented Wntl-mediated reporter gene expression.These data suggest that TNC-rich microenvironment niche could capture Wnt ligands to facilitate transcription and activation of ?-catenin signaling.Conclusion TNC expression is rapidly upregulated after AKI and this induction of TNC is renal protective.(2)The protective effect of TNC in acute renal injury is mediated by its regulation of Wnt/?-catenin signaling via physical interaction. |
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