| Part I The role of mindin in renal ischemia-reperfusion injuryObjective: To explore the expression change of mindin in renal ischemia-reperfusion injury and to verify the effect of mindin deficiency on renal ischemia-reperfusion injury.Methods: To investigate whether mindin has a pathogenic role,wild-type C57BL/6 mice were used to establish renal ischemia-reperfusion injury model.After 30 min of ischemia and 24 h of reperfusion,the expression level and distribution of mindin were detected by Western blot and immunohistochemistry.In addition,mindin knockout and wild-type mice were used to establish renal ischemia-reperfusion model,after renal ischemia-reperfusion injury,the serum creatinine and blood urea nitrogen levels of the mice in each group were measured.The pathological change of the kidney was observed by PAS staining,and the kidney damage was evaluated by acute tubular necrosis score.Results: The results showed that the expression of mindin in sham operated mice was very low.However,we observed an up-regulation of mindin expression in kidney tissues after renal ischemia-reperfusion injury and it was mainly located in renal tubules.Meanwhile,following ischemia-reperfusion,the deletion of mindin provided significant protection for mice against ischemia-reperfusion induced renal injury as manifested by attenuated the increase of serum creatinine and blood urea nitrogen.In addition,histologic examination showed extensive tubular injury characterized by tubular cell death,dilation of tubules,loss of brush border and cast formation in the outer medulla region in wild-type mice after ischemia-reperfusion.However,kidney specimens from mindin knockout mice demonstrated less tubular injury.More precisely,we assessed overall tissue injury by acute tubular necrosis score and we confirmed the mitigated tissue injury in mindin knockout mice after renal ischemia-reperfusion,as compared to wild-type miceConclusion: Mindin is significantly increased after renal ischemia-reperfusion injury,and the knockout of mindin can significantly reduce renal ischemia-reperfusion injury in mice.Part II Mindin deficiency ameliorates renal ischemiareperfusion injury by inhibiting apoptosis and inflammatory responseObjective: Extensive studies have documented that inflammatory response and cells apoptosis significantly contributed to ischemic renal dysfunction and determined the outcome of renal damage in the pathological process of renal ischemia-reperfusion injury.This part investigates the effect of mindin ablation on the inflammatory response and cells apoptosis induced by renal ischemia-reperfusion injury and the potential molecular mechanism.Methods: The upregulation of mindin in kidney prompted us to investigate the role of mindin during the progression of ischemia-reperfusion induced renal damage.To address this,wild-type and mindin knockout mice were used to established renal ischemia-reperfusion model.After 30 min of ischemia and 24 h of reperfusion,HE staining was used to observe the infiltration of inflammatory cells in the renal tissues of each group.Moreover,we examined neutrophils and macrophage accumulation by immunostaining of MPO and CD68,respectively.In response to acute insult,tubular epithelial cells and activated leukocytes release inflammatory mediators,and these cytokines and chemokines act in concert to promote inflammation in a positive feedback loop promoting further kidney damage.Therefore,we examed the production of TNF-? and MCP-1 in kidney specimens by immunohistochemistry staining following renal ischemia-reperfusion injury in wild-type and knockout mice.To determine mindin deficiency whether had an impact on apoptosis,next we performed TUNEL assay and cleaved caspase-3 staining on the renal samples.Moreover,the Western blot analysis was employed to detect the expression levels of TLR-4,phosphorylated JNK,JNK,phosphorylated p65,p65,phosphorylated I?B?,I?B?,Bcl-2,Bax and cleaved caspase-3 in kidney tissues of mice in each group.Results: The HE staning results showed that prominent inflammatory cells was noted in wild-type mice after ischemia-reperfusion insult,however,ablation of mindin suppressed inflammatory cells infiltration.Immunohistochemistry staining revealed that the accumulation of neutrophils and macrophage and the local production of proinflammatory cytokines were markedly increased after renal ischemia-reperfusion injury in wild-type mice,but this effect was reduced by mindin deletion.In addition,few TUNEL-positive cells were observed in sham operated kidneys.However,renal ischemia-reperfusion insult leads to the appearance of TUNEL-positive cells in the outer medulla region in wild-type mice,while the number of these positive cells was significantly decreased in mindin deficiency mice.Cleaved caspase-3 staining results also confirmed this effect.Furthermore,the western blot results showed that TLR-4,phosphorylated JNK,phosphorylated P65,phosphorylated I?B?,Bax,and cleaved caspase-3 expression levels were upregulated and Bcl-2 expression levels were downregulated in the kidneys with ischemic injury.However,higher Bcl-2 levels and lower TLR-4,phosphorylated JNK,phosphorylated P65,phosphorylated I?B?,Bax,and cleaved caspase-3 levels were observed in renal specimen from mindin knockout mice compared with wild-type mice.Conclusion: These observations imply that the deletion of mindin protects against renal ischemia-reperfusion injury by inhibiting both the post-ischemic inflammatory response and cells apoptosis.The mechanistic studies revealed that mindin deficiency represses TLR-4/JNK/NF-?B signalling to inhibit ischemia-reperfusion induced inflammatory responses,while the anti-apoptotic effect is linked with suppression of mitochondrial pathways.Part III Overexpression of mindin aggravates hypoxia/reoxygenation mediated apoptosis and activation of inflammatory signaling pathway in human renal tubular epithelial cellsObjective: In vitro,hypoxia/reoxygenation model was used to mimic renal ischemiareperfusion injury.We investigated the expression change of mindin in human renal tubular epithelial cells after hypoxia/reoxygenation stimulation.Moverover,mindin stably overexpression cell line were generated and used to evaluate the influence of mindin on renal tubular epithelial cells with injury induced by hypoxia/reoxygenation.Methods: Normal human renal tubular epithelial cells(HK-2)were challenged by hypoxia for 24 h,the expression of mindin was detected by Western blot and immunofluorescence at 2,4 and 8 h after reoxygenation.In addition,we established mindin stably overexpression cell line(ad Mindin)and control cells(ad GFP),after 24 h hypoxia and 8 h reoxygenation,cell apoptosis in response to hypoxia/reoxygenation stimulation were examined by TUNEL and Hoechst 33258 staining.The production of inflammatory mediators was detected by ELISA.Moreover,the Western blot analysis was employed to detect the expression levels of TLR-4,phosphorylated JNK,JNK,phosphorylated p65,p65,phosphorylated I?B?,I?B?,Bcl-2,Bax and cleaved caspase-3 in each group.Results: The Western blot results showed that mindin expression was elevated after 2 h reoxygenation and significantly increased after 8 h reoxygenation in HK-2 cells compared with untreated cells.Meanwhile,immunofluorescence analyses of cultured cells were also confirmed this upregulation.Moreover,the results revealed that apoptosis of cultured tubular epithelial cells was significantly increased following hypoxia/reoxygenation.However,compared to ad GFP group,mindin overexpression resulted in a significant increase in the number of apoptotic cells after hypoxia/reoxygenation.The results of ELISA demonstrated that overexpression of mindin significantly promoted the secretion of TNF-? and MCP-1.The Western blot results showed that higher TLR-4,phosphorylated JNK,phosphorylated P65,phosphorylated I?B?,Bax,and cleaved caspase-3 expression levels and lower Bcl-2 levels were found in hypoxia/reoxygenation induced cells compared with untreated cells.Moreover,mindin overexpression cells showed substantially lower Bcl-2 expression and higher TLR-4,phosphorylated JNK,phosphorylated P65,phosphorylated I?B?,Bax,and cleaved caspase-3 levels when compared with the ad GFP cells after hypoxia/reoxygenation.Conclusion: Our results suggest that overexpression of mindin can upregulate the hypoxia/reoxygenation mediated apoptosis level,secretion of inflammatory mediators,and the activation of TLR-4/JNK/NF-?B/signaling in vitro.Meanwhile,this proapoptotic effect is linked with the promotion of mitochondrial pathways. |
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