| Background Current epidemiological studies report that there are about 260,000 to 300,000 patients with chronic kidney disease(CKD)caused by various causes,of which 11%of patients with CKD eventually develop into end-stage renal disease,which seriously affects people’s quality of life has developed into a major public health problem that seriously endangers human health.At this stage,treatment methods to prevent and alleviate the progression of CKD disease mainly including drug therapy,nutritional therapy,and artificial kidney replacement therapy to alleviate various symptoms of toxin accumulation and metabolic disorders in the body.Despite these therapies,their efficacy is limited and needs to be addressed urgently.According to current literature reports,tubulointerstitial fibrosis is one of the main causes of CKD,which is mainly due to long-term chronic inflammatory response,which stimulates tubulointerstitial cells to release a large number of profibrotic-related cytokines,resulting in a large amount of extracellular matrix deposition.To.Nuclear factor(NF-κB)signaling pathway plays an important role in regulating the body’s inflammatory response.In addition,a large number of literature have reported that the transforming growth factor TGF-β/Smad signaling pathway is one of the important signaling pathways in regulating renal interstitial cell fibrosis.Mindin belongs to the extracellular matrix protein family,which can recruit a large number of immune response cells in the group,activate the immune-inflammatory response in the body,and play an important role in maintaining the stability of disease metabolism in the body.At present,relevant kinds of literature have reported that the Mindin plays an important role in the tumor,cardiovascular,diabetic nephropathy,and liver fibrosis,but its specific mechanism in the progression of renal interstitial fibrosis has not yet been elucidated.Therefore,combined with the current literature reports and the results of our previous research,we hypothesized that the Mindin may induce long-term inflammation of renal tubular epithelial cells by regulating the NF-κB signaling pathway and TGF-β/Smad signaling pathway,and further promote extracellular matrix proteins.Deposition causes fibrosis of renal tubulointerstitial cells.This project aims to explore the role of the Mindin in the development of renal fibrosis to provide a new theoretical basis for the clinical treatment of patients with renal fibrosis.Method Part I: We used C57BL/6 wild-type mice to establish a renal ischemia-reperfusion-induced renal fibrosis model.We used HE staining and Masson staining to determine the model construction.Real-time quantitative PCR(RT-q PCR),immunofluorescence,and Immunohistochemical technique were used to detect the expression and distribution of Mindin in kidney tissues of the sham-operated group and model group.At the cellular level,we used TGF-β reagent to stimulate renal tubular epithelial cells to construct a renal fibrosis model at different time periods to explore the relationship between the Mindin and renal fibrosis.Part II: Using renal tubular epithelial cells to construct a stably transfected cell line overexpressing the Mindin,explore the effect of Mindin on extracellular matrix proteins during renal interstitial fibrosis.Subsequently,C57BL/6 wild-type mice and Mindin knockout mice were used to construct a renal fibrosis model.The effects of Mindin on mouse kidney tissue structure and expression were detected by HE staining and western blotting.Masson staining,Suez Red staining,immunohistochemistry,immunofluorescence,and western blotting techniques were used to detect the effects of Mindin on extracellular matrix proteins,namely,fibronectin,cadherin,and type 1collagen during interstitial fibrosis in mice.Part III: Establish a renal fibrosis model,use immunofluorescence technology to detect the expression of core subunit p65 in the NF-κB signaling pathway,and use immunoblotting to detect phosphorylation of p65(P-p65)in NF-κB signaling pathway-related proteins And p65,P-IκBα and IκBα expression,followed by immunoblot analysis of blank control group,transfection control group and transfection group HK2 cells with and without TGF-β drug stimulation,TGF-β/Smad signaling pathway is related The expression of proteins TGF-β,P-smad2,Smad2,P-smad3,Smad3 and Smad7.C57BL/6 wild-type mice and Mindin knockout mice were used to construct a renal fibrosis model.Immunohistochemistry was used to detect the expression of p65 in mouse kidney tissue.NF-κB signal was detected by immunoblot technology Expression of pathway-related proteins.RT-q PCR was used to analyze wild type mice in the sham operation group,Mindin knockout mice in the sham operation group,and wild type mice in the model group and IL-1β mice in the model group,IL-6 and TNF-α transcription levels were expressed.Then,immunohistochemical and western blot techniques were used to detect the expression of TGF-β/Smad signaling pathway-related proteins in mouse kidney tissues in different groups.Result Part I: Compared with the kidney tissue of the sham operation group,the kidney tissue of the model group has significantly enlarged renal tubules,and the renal tubules and glomeruli are infiltrated with blood cells and inflammatory cells.The kidney tissue scores of the mice in the surgery group increased significantly.Masson staining results showed that collagen was mainly concentrated around the dilated tubules in the kidney tissue of the model group,while there was almost no collagen deposition in the kidney tissue of the sham operation group.In addition,the expression of Mindin in the kidney tissue of the model group was significantly higher than that of the sham operation group,and the Mindin was mainly expressed in the dilated renal tubular cytoplasm,and the results of RT-q PCR and immunoblot detection were consistent with the immunofluorescence results.At the cell level,the expression of Mindin gradually increased at 6 hours,12 hours,and 24 hours after stimulation with 20 ng/ml TGF-β drug,and the expression was highest at 24 hours.Part II: We used lentivirus to interfere with renal tubular epithelial cells HK2,and constructed a stable cell line expressing Mindin.Fluorescence results suggest that the cells in the transfection group(p LV-Mindin)group have significantly increased fluorescence expression under the microscope compared to the cells in the control group(p LV-Vector).The expression level increased significantly.When stimulated with TGF-β,the expression level of Mindin increased further,and the expression level of Mindin in the cells of p LV-Mindin group was significantly higher than that of control group.Moreover,the immunoblot results showed that no TGF-β drug was given.When stimulated,the expression of fibronectin and type I collagen of p LV-Mindin was higher than that of p LV-Vector and the blank control group(Control),while the expression of cadherin of p LV-Mindin was lower than that of p LV-Vector,giving 20 ng/ml This trend was further increased when HK2 cells were stimulated to establish a renal fibrosis model under ml 24-hour TGF-β conditions.The results of HE staining showed that compared with the normal group,the kidney tissue of wild-type mice in the model group had significantly expanded renal tubules,a large number of blood cells appeared between the renal tubules,and renal tubular epithelial cells were necrotic and inflammatory.Cell infiltration was obvious.Compared with wild-type mice,Mindin knockout mice had less tubular dilatation,blood cell and inflammatory cell infiltration range,and necrosis degree;Suez red and Masson staining results suggested that the kidneys of wild-type mice in the model group A large amount of collagen fibrous protein deposits appeared in the dilated tubules of tissues.However,after the Mindin was knocked out,collagen deposition in the renal tubules of mice decreased.Furthermore,immunoblotting results showed that compared with the normal group,the expression of type 1 collagen in the kidney tissue of wild-type mice in the model group was deeper,and the renal tubulointerstitial was mainly concentrated.However,after the Mindin was knocked out,the expression of type 1 collagen was weakened,and the scope has narrowed.Immunofluorescence results showed that the expression of fibronectin in the kidney tissue of wild-type mice in the model group increased compared with wild-type mice in the sham operation group.After the Mindin was knocked out,the expression of fibronectin was weakened.Immunoblotting results suggest that renal fibrosis promotes the expression of type 1 collagen and fibronectin and inhibits the expression of cadherin.However,after Mindin knockout,the expression of type 1 collagen and fibronectin were decreased and the expression of cadherin was increased.Part III: Immunofluorescence results suggest that p LV-Mindin NF-κB signaling pathway core protein p65 cytoplasmic expression is higher than that of p LV-Vector and Control groups when TGF-β drug is not given The renal fibrosis model p65 protein was translocated from the cytoplasm of renal tubular epithelial cells to the nucleus,and the expression of p LV-Mindin p65 was higher than that of the control group.Renal tubular cell nuclear protein immunoblotting results showed that p LV-Mindin and control cells hardly expressed p65 when TGF-β was not given,and p65-mindin p65 expression was increased when TGF-β was given.Higher than the control group.In addition,without TGF-β drug stimulation,the expression levels of p LV-Mindin phosphorylated p65(P-p65)and P-IκBα were higher than those in the control group,while the expression levels of p LV-Mindin IκBα were lower than those in the control group.TGF-After beta drug stimulation,this trend has further expanded.TGF-β/Smad signaling pathway-associated Western blot results showed that p LV-Mindin TGF-β,P-smad2 and P-smad3 were higher than those in the control group without TGF-β drug stimulation,while p LV-Mindin Smad7 The expression level was lower than that in the control group,and the change was further increased after stimulation with TGF-β.Immunohistochemical results suggest that renal fibrosis-induced cytoplasmic p65 expression in mouse kidney tissues is significantly up-regulated,and p65 is expressed in most nuclei of dilated renal tubular epithelial cells.However,after Mindin knockout,the cytoplasmic p65 expression level decreased.And the expression data of nuclear p65 was reduced;the immunoblotting results of mouse kidney tissues showed that compared with wild type mice in sham operation group,the expression of total protein P-p65 and P-IκBα in kidney tissue of wild type mice in renal fibrosis model group The expression levels of P-p65 and P-IκBα were higher than those of the control group.The expression level of IκBα in Mindin stable transgenic cell lines in the knockout group was lower than that in the control group.However,the Mindin knockout mice in the model group and the model group Compared with wild-type mice,the expression of cytoplasmic P-p65 and P-IκBα decreased,and the expression of IκBα increased.RT-q PCR results showed that the expression levels of IL-1β,IL-6 and TNF-α in the kidney tissue of wild-type mice in the renal fibrosis model group were higher than those in the control group.Levels,however,knocking out Mindin reduced the transcription levels of these transcription factors.In addition,the results of Smad2 immunohistochemistry showed that compared with wild-type mice in the sham operation group,wild-type mice in the renal fibrosis model group had significant up-regulation of Smad2 expression in the cytoplasm and nucleus of the kidney tissue,and mainly concentrated in the expansion of renal tubular epithelial cells However,after the Mindin knockout,the expression of Smad2 in cytoplasm of mouse kidney tissue decreased.Subsequently,the results of immunoblotting indicated that renal fibrosis promotes the expression of TGF-β/Smad signaling pathway-related proteins such as TGF-β,P-smad2 and P-smad3,inhibits the expression of Smad7 protein,and inactivates TGF-β/Smad signaling pathway.Conclusion Part I: The expression of Mindin was significantly up-regulated in vivo and in vitro,indicating the existence of Mindin was closely linked with renal interstitial fibrosis.Part II: It was found Mindin induces the expression of profibrotic-related factors,promotes the deposition of extracellular matrix proteins,accelerates the occurrence and development of renal interstitial fibrosis,and prevents the Mindin from protecting the structure and function of mouse kidney tissue in vivo and in vitro.It shows that Mindin plays an important role in the process of renal interstitial fibrosis.Part III: The Mindin may induce renal tubular epithelial cells to undergo an inflammatory response by activating the NF-κB signaling pathway and the TGF-β/Smad signaling pathway,thereby promoting the deposition of extracellular matrix proteins and causing fibrosis of renal tubular interstitial cells.Inhibit Mindin inactivation of NF-κB and TGF-β/Smad signaling pathways,reduce mouse kidney tissue damage.It is confirmed that Mindin may be a new target for treating patients with renal fibrosis. |
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