| BackgroundAs a major risk factor for cardiovascular and cerebrovascular diseases,hypertension is an important cause of renal injury.Hypertensive Renal Disease(HRD)is a kind of Renal structure and function damage caused by essential hypertension.It is one of the common diseases in internal medicine,but the pathogenesis has not been fully explained.The main manifestations of hypertensive renal damage are increased proteinuria,benign glomerulosclerosis,renal interstitial fibrosis and inflammatory cell infiltration.Fibrosis,inflammation and immune activation of renal tubules and interstitium are important factors leading to the continuous progression of HRD.Excess matrix deposition is a characteristic feature of the fibrotic process,resulting from impaired balance between extracellular matrix protein synthesis and its degradation.The secretion of inflammatory cytokines and infiltration of inflammatory cells are the initiators of renal fibrosis.There is currently no effective method to prevent the progression of fibrosis,so it is essential to improve the understanding of the cellular and molecular mechanisms involved in the progression of HRD fibrosis and inflammation.The common pathogenesis of HRD in current studies includes genetic factors,salt sensitivity,oxidative stress,endothelial cell dysfunction,and activation of the renin-angiotensin-aldosterone System(RAAS).Inhibition of RAAS overactivation is one of the most effective methods to slow down the progression of renal disease.Angiotensin ?(Ang?),as the main effector of RAAS,is involved in the regulation of blood pressure and target organ damage through both hemodynamic and non-hemodynamic aspects.It can act on kidney vascular smooth muscle cells,resulting in vasoconstriction.It can also be used as a proinflammatory agent to activate the intracellular signal transduction system and promote the inflammatory response of the kidney.Moreover,it can accelerate the process of renal fibrosis by up-regulating the expression of TGF-?1,increasing the proliferation of fibroblasts,promoting the synthesis of extracellular matrix,inhibiting the degradation of extracellular matrix,and accelerating the process of renal fibrosis.Therefore,Ang? is a key regulator in the progression of HRD.Ubiquitin system is a common post-translational modification system of proteins.Ubiquitin modification is not only a marker for proteasomal degradation of target proteins,but also a regulatory factor for protein-protein interactions and enzyme activation.A variety of E3 ubiquitin ligases have been found to regulate hypertension and damage to target organs,and drugs targeting ubiquitin proteasome have been developed for the treatment of hypertension,but there are few reports on the ubiquitination modificatioin of renal injury in hypertension.The discovery of a novel E3 ubiquitin ligase regulating HRD and the exploration of its regulatory mechanism will provide a good theoretical basis for the elaboration of the pathogenesis of HRD and the research and development of HRD drugs,which has become a hot issue in this field.As a member of the TRIM family,TRIM31 also is a very important E3 ubiquitin ligase.Whether TRIM31 also plays an important role in hypertension and HRD has not been reported.Previous studies have shown that TRIM31 plays a key regulatory role in the development of a variety of diseases,especially in the process of tumor proliferation and migration.The mechanism is mainly involved in the regulation of NF-?B activation by TRIM31 and its involvement in inflammatory response.The activation of NF-?B plays a crucial role in the pathological progression of HRD,suggesting that TRIM31 may also be involved in the occurrence and progression of HRD.In this study,we proposed the following scientific hypothesis:E3 ubiquitin ligase TRIM31 improves renal function,fibrosis,and inflammatory response in an HRD mouse model constructed by Ang?.To test the above hypothesis,we designed a series of in vivo and in vitro experiments.Objectives1.To investigate the correlation between TRIM31 and the pathological progress of human and mouse HRD.2.To investigate whether TRIM31 is involved in regulating renal function damage in HRD mice.3.To investigate whether TRIM31 is involved in the regulation of renal fibrosis in HRD mice.4.To investigate whether TRIM31 is involved in the regulation of renal inflammation in HRD mice.Methods1.AnimalsIn the background of C57BL/6J mice,TRIM31 knockout(TRIM31-/-)mice were constructed by Transcription activator-like effectors Nucleases(TALEN)technique.C57BL/6J male mice were purchased fromg Vital River Laboratory Animal(Beijing).All animal experiments are conducted in accordance with the requirements and regulations of the Animal Control Regulations of the Ministry of Health(Documentation No 55,2001)and the Ethics Committee of Shandong University Qilu Hospital for animal experiments.Animal experiments are grouped as follows.2.Animal experiments grouping(1)Eight--week-old male wildtype(WT)C57BL/6J mice were randomly divided into 2 groups:saline group and Ang? group.(n=15 per group)(2)Eight-week-old male TRIMS1-/-mice and WT littermates(TRIM31+/+)mice were selected and randomly divided into four groups:TRIM31+/++saline group,TRIM31-/-+ saline group,TRIM31+/+ +Ang? group,TRIM31-/-+saline group.(n=15 per group)3.Mouse model of HRDOsmotic pumps were pre-infused with an appropriate dose of Ang? and buried in the dorsal subcutaneous area of 8-week-old male mice,respectively.Ang? was continuously pumped at a speed of 1000ng/Kg/min for 42 days to establish a HRD mouse model.The control group was pumped with the sterile saline(n=15 per group).Blood pressure and body weight of mice were measured at regular intervals weekly before and after modeling,and urine of mice was collected for renal function index detection.The mice were euthanized at the end of the experiment,body weight and kidney weights were measured,and the blood,kidney,heart,liver,small intestine and other tissue samples of the mice were collected for further tissue and molecular studies4.Renal biopsy specimens collection of patients with clinical HRDThe normal adjacent tumor tissues(Control),glomerular minor lesion biopsy tissues(GML)without hypertension,and renal biopsy samples of HRD were obtained from the department of pathology of Shandong University.IHC was used to detect the expression of TRIM31 protein,Hematoxylin and eosin(H&E)and Masson's Trichrome staining were used to investigate the correlation between TRIM31 protein expression and renal injury and fibrosis.5.Mouse genotype identificationThe mouse tail DNA was extracted by the mouse tail extraction kit,and the genotypes were identified by PCR amplification and sequencing.At the same time,the protein of mouse kidney tissue was extracted,and the protein expression level of TRIM31 was detected by Western blot analysis to evaluate the gene knockout efficiency of TRIM31.6.Cell culture and treatment Human proximal renal tubular epithelial cell-2(HK2)was used in the experiment,which was cultured in RPMI1640 medium with 10%FBS and incubated at 37?incubator containing 5%CO2.The main treatments are as follows:(1)Ang? time gradient test:HK2 cell were cultured in vitro and treated with different stimulation times(0,4h,8h,12h,24h,36h).(2)Ang? concentration gradient test:HK2 were cultured in vitro and treated with different concentrations(0,10-8 M,10-7 M,1 0-6 M,10-5 M,10-4 M)of Ang?.7.Blood pressure measurement of miceRadiotelemetry(HD-X11 Transmitter,DataSciences International)was applied to detect mouse(TRIM31+/++saline group,TRIM31-/-+saline group,TRIM31+/++Ang? group,TRIM31-/-+saline group)systolic(SBP)and diastolic(DBP)blood pressure once a week before and after modeling,respectively.8.Tissue sampling of miceBlood pressure and body weight of mice were measured at regular intervals every week before and after modeling,and urine of mice was collected for detection of renal function indexes.Euthanasia,at the end of the experiment,the mice,and to return the mice blood,kidneys,heart,liver,small intestine tissue samples,to weigh around the heart and kidneys,removing renal capsular,in accordance with the experimental requirements will be late organizations put in liquid nitrogen preservation or put in 4%paraformaldehyde fixed 24-48 h or fixed in 2%glutaraldehyde,used for follow-up study.9.Detection of kidney function and 24h urinary protein in miceThe serum creatinine(Cr),blood urea nitrogen(BUN),uric acid(UA)and 24-hour urinary protein of TRIM31-/-and TRIM31+/+mice were detected by Automatic Biochemical Analyzer and ELISA.10.Transmission electron microscope(TEM)was used to observe the ultrastructure of the mice kidneyThe renal cortex of mice in each group were collected and fixed with 2.5%glutaraldehyde.The ultrastructure of glomerular podocytes and basement membrane was observed by TEM11.Meso Scale Discovery(MSD)was used to detect inflammation-related indexes in serumBased on the principle of electrochemiluminescence,the contents of TNF-?,IL-6,IL-1? and MCP-1 in serum of mice were detected by using pre-embedded MSD multi-factor detection plate and MSD detector.12.Histological and IHC staining of mouse kidneyMouse kidneys were fixed overnight in 4%paraformaldehyde.Tissues were dehydrated and embedded into corresponding paraffin blocks,and paraffin sections(thickness:4?m)were made for subsequent experiments.Masson's Trichrome and Picrosirius Red staining were used to detect renal interstitial fibrosis of mice in each group.Morphological differences of renal glomeruli in paraffin sections were observed by Perioacid-Schiffstain(PAS).IHC was used to detect the expression of fibrosis related molecules(TRIM31,KIM-1,Nephrin,Collagen ?,Collagen ?,Collagen ?,Fibronectin,?-SMA,CD68,TNF-?,IL-6 and IL-1?)in kidney tissues of mice in each group.13.Tissue RNA extraction,reverse transcription and quantitative real-time PCR(RT-PCR)RNA was extracted from mice kidney tissues and HK2 cells,The TAKARA reverse transcription kit was used for mRNA reverse transcription,and RT-PCR were applied to get Ct values of trim31,collagen ?,collagen ?,collagen ?,fibronectin,?-sma,tnf-?,il-6 and il-1?.Using ?-actin as internal parameter,the Ct values were calculated by 2-??CT formula.14.Western blot analysisKidney tissue protein of mice in each group were extracted by protein extraction kit,and protein of HK2 cells was extracted by cell lysate.After the protein concentration was adjusted by BCA,SDS-PAGE gel electrophoresis was performed to detect the protein contents of TRIM31,KIM-1,Nepharin,Collagen?,?,?,Fibronectin,?-SMA,TNF-?,IL-6 and IL-1?.15.Statistics Analysis All data were presented as mean ± SEM.Normality distributionassumption of the data was assessed using Shapiro-Wilk test.For normally distribution,data were analyzed by unpaired two-tailed Student's t-tests to determine the statistical difference between two groups.And one-way ANOVA followed by Dunnett or Tukey post hoc tests were performed to determine the statistical difference between multiple groups with one variable and normally distribution.To compare multiple groups with more than one variable,two-way ANOVA followed by Tukey or Sidak post hoc tests was used.For data with a non-Normally distribution,we performed a nonparametric statistical analysis using the Kruskal-Wallis test followed by the Dunn post-hot test for multiple comparisons with one variable.In all statistical comparisons,a p value of<0.05 was considered statistically significant.Results1.TRIM31 expression was significantly decreased in the renal tissues of the mouse model of HRD induced by Ang?We successfully established a mouse model of HRD by subcutaneous implantation of Ang? pump.IHC,Western blot and RT-PCR were used to detect the expression of TRIM31.Compared with normal saline group,the expression of TRIM31 in Ang? group was significantly down-regulated.2.TRIM31 expression was significantly down-regulated in renal tubular epithelial cells stimulated by Ang?The protein and mRNA levels of TRIM31 in HK2 cultured in vitro were observed by Western blot and RT-PCR using the time gradient(0,4h,8h,12h,24h,36h)and concentration gradient(0,10-8 M,10-7 M,10-6 M,10-5 M,10-4 M)of Ang?stimulation,which is a pathogenic factor of HRD.Results showed that the expression of TRIM31 decreased in protein and mRNA levels with gradient stimulation of Ang?.The results of in vivo and in vivo experiments showed that TRIM31 expression was related to HRD model,suggesting that TRIM31 might be involved in the progression of HRD.3.Successful construction of HRD mice model by TRIM31 knockout mice(1)In the background of C57BL/6J mice,TRIM31 knockout mice(TRIM31-/-)were successfully constructed by TALEN technique and confirmed by mouse tail Genomic DNA sequencing.At the same time,the protein of mouse kidney tissue was extracted,and Western blot was used to further confirm that the expression of TRIM31 protein was significantly reduced in TRIM31 knockout mice.(2)The body weight before and after modeling and weight of mice in each group were measured,and it was found that TRIM31 knockout did not affect the body weight and kidney weight of mice.4.TRIM31 deficiency did not affect the blood pressure changes induced by Ang? in miceThe systolic and diastolic blood pressure of mice in each group were measured by radiotelemetry method.The results showed that compared with the saline group,the blood pressure of mice in the Ang? group increased significantly from the first week after the infusion,and remained at a higher level in the following weeks.The results of DSI measurement showed that the SBP in the last week before the end of modeling in the Ang? group was more than 140mmHg,reaching the level of hypertension,which confirmed the successful construction of the hypertension model in the mice.After the injection of normal saline or Ang?,mice in the TRIM31-/-group showed no significant changes in blood pressure compared with mice in the TRIM31+/+ group.These results indicate that TRIM31 gene deletion does not affect the baseline level of blood pressure in mice,nor does it affect the changes of blood pressure induced by Ang?.5.TRIM31 deficiency aggravated renal function injury in HRD mice induced by Ang?BUN,Cr and 24h urinary protein datas showed that TRIM31 deficiency aggravated the renal function impairment and renal filtration barrier impairment in mice induced by hypertension.Western blot and IHC staining of renal Nephrin and KIM-1 indicated that TRIM31 gene knockout increased the damage of renal tubule and renal podocyte caused by hypertension.Meanwhile,PAS staining indicated that TRIM31 gene knockout aggravated glomerulosclerosis induced by hypertension in mice.Meanwhile,transmission electron microscopy showed that TRIM31 gene knockout significantly aggravate the damage of the foot process and basement membrane of the kidney induced by hypertension.These results indicate that TRIM31 gene deletion significantly aggravates the renal function damage of HRD mice.6.TRIM31 deficiency aggravated renal fibrosis in HRD mice induced by Ang?Masson's Trichrome and Sirius staining showed significant renal fibrosis in HRD mice induced by Ang?,and it was more severe in TRIM31 knockout mice.IHC,Western blot,and RT-PCR analysis of Collagen ?,?,?,Fibronectin,and?-smooth muscle actin(?-SMA)showed that the expression in TRIM31-/-HRD mice were significantly increased after Ang? infusion compared with TRIM31+/+mice.These results suggest that the expression deficiency of TRIM31 may aggravate the renal fibrosis induced by Ang?.7.TRIM31 deficiency aggravated renal inflammation in HRD mice induced by Ang?Serum MSD measurament showed that the expressions of TNF-?,IL-6 and IL-1?in TRIM31-/-HRD mice were significantly aggravated after Ang? infusion compared with TRIM31+/+ mice.CD68 IHC staining showed significant infiltration of macrophages in renal tissues after TRIM31 knockout compared with WT mice after Ang? infusion.Meanwhile,IHC,Western blot and RT-PCR also indicated that the expressions of inflammation-related molecules TNF-?,IL-6 and IL-1? in the kidney tissues of mice were significantly enhanced after TRIM31 gene knockout.8.The expression of TRIM31 was significantly decreased in renal biopsy tissues of patients with HRDIHC analysis showed that TRIM31 expression was significantly down-regulated in renal tissues of patients with HRD compared with normal paracancerous renal tissues of patients without hypertension and with glomerular biopsy with minor lesions without hypertension.Conclusions1.The expression level of TRIM31 was decreased in renal pathological tissues of HRD.2.TRIM31 deficiency significantly increased renal function impairment,fibrosis and inflammation in HRD mice.Background Inflammation and fibrosis are the two main patiiological features of hyperisolated Renal Disease(HRD).Angiotensin ?(Ang?)plays a key role in inducing renal tissue inflammation and fibrosis.Transforming growth factor ?1(TGF-?1)was recognized as the key mediator of renal fibrosis,and was the main regulatory factor in the pathogenesis of Renal inflammation and fibrosis in HRD.It can induce the formation and deposition of Extracellular Matrix(ECM),promote the proliferation and differentiation of fibroblasts and the transdifferentiation of epithelial cells.In terms of mechanism,TGF-?1 mainly regulates renal fibrosis and inflammation by mediating the activation and transduction of Smad-dependent and non-Smad-dependent signaling pathways(mainly MAPKs and NF-kB signaling pathways).The targeted inhibition of the activation of TGF-?1 downstream signaling pathway is of great significance for the treatment of HRD.It has been shown that Ang? induces renal oversecretion of cytokines such as TGF-?1,which fiirther aggravates hypertensive renal injury through TGF-?1.The study of the regulation mechanism of TGF-?1 signaling pathway is beneficial to further elucidate the pathogenesis of hypertensive nephropathy and provide theoretical basis for the research and development of drugs for hypertensive nephropathy.TGF-P-activated kinase 1(TAK1)is a mitogen-activated protein kinase,kinase,kinase(MAP3K).The kinase activity of TAK1 is regulated by various cytokines such as TGF-?1.Studies have shown that activated TAK1 further phosphorylates key adaptor proteins downstream of the TGF-?1 signaling pathway,which plays a key role in the activation of non-Smad-dependent signaling pathways(mainly MAPKs and NF-kB)and the production of inflammatory factors.However,it remains to be further explored whether TAK1 is involved in regulating TGF-?1-mediated classical Smad signaling pathway and renal fibrosis during the occurrence and development of HRD.Ubiquitin modification is an important post-translational modification of proteins,in which ubiquitin itself forms a polyubiquitin chain through seven internal lysine(K6,K11,K27,K29,K33,K48,and K64).E3 ubiquitin ligase determines the specificity of ubiquitin molecules binding to target proteins,and is therefore the most critical molecule in protein ubiquitin process.Protein ubiquitination plays a key role in the development of many diseases.It is of great scientific significance and clinical value to study the molecular mechanism of protein ubiquitination modification regulating the occurrence of HRD and to search for new therapeutic targets.It has been reported that protein ubiquitin modification plays an important role in TGF-?1 downstream signal transduction and the biological function of TAKL However,the related E3 ubiquitin ligase needs to be further discovered.In The first part of our study,it has been confirmed that the tripartite motif 31(TRIM31)is involved in and regulates injury,fibrosis and inflammatory response of HRD renal fimction.Whether TRIM31 plays a role in HRD through the regulation of TGF-pi signaling pathway,and the specific molecular mechanism of the regulation of HRD remains to be further studied.Therefore,in this study,we proposed the following scientific hypothesis: TRIM31 can modify the target protein in TGF-pi signaling pathway through ubiquitination,thus participating in the activation of TGF-?1-related signaling pathway in HRD,and thus playing a protective role in the pathological process of HRD.In this study,we will further explore the specific molecular mechanism of TRJM31 in HRD through in vitro and in vivo experimental studies,so as to find a new theoretical basis for the search of target gene therapy for renal damage in hypertension.Objectives1.To investigate the effect of TRIM31 on the activation of TGF-?1 signaling pathway in the pathological progression of HRD.2.To investigate the target molecules of TGF-?1 signaling pathway TRIM31.3.To investigate the ubiquitination modification of target molecules by TRIM31.4.To investigate the role of TAK1 in TGF-?1 signaling pathway.Methods1.Animal experiments grouping Eight-week-old male TRIM31-/- mice and WT littermates(TRIM31+/+)mice were selected and randomly divided into four groups: TRIM31+/+ + saline group,TRIM31-/-+saline group,TRIM31+/+ + Ang? group,TRIM31-/-+ saline group.(n= 15 per group)2.Mouse model of HRD Osmotic pumps were pre-infused with an appropriate dose of Ang? and buried in the dorsal subcutaneous area of 8-week-old male mice,respectively.Ang? was continuously pumped at a speed of 1000ng/Kg/min for 42 days to establish HRD mouse model.The control group was pumped with the sterile saline(n=15 per group).At the end of the experiment,the mice were euthanized,and the kidney of the mice were collected for further tissue and molecular studies.3.Renal biopsy specimens collection of patients with clinical HRD The normal adjacent tumor tissues(Control),glomerular minor lesion biopsy tissues(GML)without hypertension,and renal biopsy samples of HRD were obtained from the department of pathology of Shandong University.IHC was used to detect the expression of TRIM31,TGF-?1,Collagen I,pSmad3,TAK1,TNF-?,and pP65.4.Cell culture and treatment Human proximal renal tubular epithelial cell-2(HK2),Mouse Primary renal tubular epithelial cells(MRPTEpiC)and HEK-293 T were used in the experiment,which were cultured respectively in RPMI1640 medixxm,DMEM and EpiCM-A with 10% FBS and incubated at 37°C incubator containing 5%C02.The main treatments are as follows:HK2 ceUs:(1)HK2 cells were cultured in vitro and treated with different concentrations(0*2ng/mL,4ng/mL,6ng/mL,8ng/mL,10ng/mL)of hTGF-?1 or stimulation for different times(0,4h,8h,12 h,24h,36h).(2)HK2 cells were cultured in vitro and pretreated with TGF-?1 neutralizing antibody.The cells were stimulated with 10-5 M Ang? at a time gradient(0,4h,8h,12 h,24h,36h)to extract the cell proteins.(3)HK2 cells were cultured in vitro,and si-RNA of TRIM31 was transfected into HK2 to down-regulate TRIM31 protein by RNAiMAX.And the cells were stimulated with 10ng/mL hTGF-?1 for 0,12 h or 24 h to extract the cell proteins.(4)We constructed TRIM31-WT overexpression vector,TRIM31-ARing mutant and E3 ubiquitin ligase function point mutation TRIM31-C53A/C56 A overexpression vector to transfect HK2 cells and stimulated by 10ng/mL hTGF-?1 for 24 h respectively.(5)HK2 cells were cultured in vitro,md stimulated by 10ng/mL hTGF-?1 for0.5hami Ih respectively.Western blot and Co-imimmoprecipitation(Co-IP)were applied for ftirther study.(6)HK2 cells were cultured in vitro,and the 5z-7-oxozeaenol(5z7)inhibitor and si-RNA of TAK1 were used to intervene the kinase activity or expression of TAK1 in these two cells,respectively.After 0.5h and lh stimulation by 10ng/mL TGF-?1,cells were lysed to extract the cell protein.MRPTEpiC cells:MRPTEpiC cells were cultured in vitro,and the 5z-7-oxozeaenol(5z7)inhibitor and si-RNA of TAK1 were used to intervene the kinase activity or expression of TAK1 in these two cells,respectively.After 0.5h and lh stimulation by 10ng/mL TGF-?1,cells were lysed to extract the cell protein.HEK-293T:(1)Overexpression vectors of TGF-(31 signaling pathway related molecules TRAF6,TAK1,Smad2,Smad3 and Smad4 were constructed and co-transfected with TRJM31 overexpression vector into HEK293 T cell line.Co-immimoprecipitation(Co-IP)was used to detect the bindings.(2)In vitro, GFP-labeled TRIM31 and Myc-labeled TAK1 overexpression vectors were co-transfected into HEK293 T cell,and cells were collected for immunofluorescence staining.(3)TRM31 mutants(TRIM31-?Ring,TRIM31-AB,TRIM31-AC-C,TRIM31-AC)and TAK1 mutants(1-300aa,1-480aa,301-579aa)were constructed and co-transfected into HEK293 T cell for further Co-IP.(4)Different concentrations of Flag-TRIM31 and Myc-TAK1 were co-transfected to HEK293 T to extract the cell protein.(5)Flag-TRIM31 and Myc-TAK1 were co-transfected to JIEK293 T and culture for24 h culture.Then,proteasomal pathway inhibitor Bortezomib and lysosomal pathway inhibitor Chloroquine were added respectively for 4h before protein extraction.(6)Flag-TRIM31-WT,Flag-TRIM31-ARing,Flag-TRIM31-C53A/C56 A co-transfected HEK293 T with Myc-TAK1 to extract the cell protein.(7)Different types(WT,K48 or K63)HA-ubiquitin expression vector was built and co-transfected with Flag-TRJM31,Myc-TAK1 expression vector for 24 hours.(8)A series of Myc label TAK1 lysine point mutations overexpression plasmid were constructed(Myc-TAK 1-K72 R,Myc-TAK 1-K15 8R)respectively.And cotransfection HEK293 T cell lines with Flag-TRIM31 and HA-ubiquitin plasmid for fiirther Co-IP.5.Sampling of mouse tissue samples At the end of modeling,mice were euthanized,kidney specimens were retained,kidney capsule was removed,and each tissue was stored in liquid nitrogen or fixed in 4% paraformaldehyde for 24-48 h for subsequent experiments according to the requirements of later experiments.6.Histological and IHC staining of mouse kidney Mouse kidneys were fixed ovemi^it in 4% paraformaldehyde.Tissues were dehydrated and embedded into corresponding paraffin blocks,and paraffin sections(thickness: 4?m)were made for subsequent experiments.IHC was used to detect the expression of TGF-?1 in kidney tissues of mice in each group.7.Tissue RNA extraction,reverse transcription and quantitative real-time PCR(RT-PCR)RNA was extracted from mice kidney tissues and HK2 cells,The TAKARA reverse transcription kit was used for mRNA reverse transcription,and RT-PCR were applied to get Ct values of trim3 J,tgfpif collagen?,collagen ?,collagen ?,fibromectin,?-sma,tnf-? and il-1? Using P-actin as internal parameter,the Ct values were calculated by 2-AACT formula.8.Western blot analysis Kidney tissue protein of mice in each group were extracted by protein extraction kit,and protein of HK2 cells was extracted by cell lysate.After the protein concentration was adjusted by BCA,SDS-PAGE gel electrophoresis was performed to detect the target protein contents.9.Laser confocal detection of TRIM31 and TAK1 co-localization In vitro,GFP-labeled TRIM31 and Myc-labeled TAK1 overexpression vectors were co-transfected into HEK293 T cell,and the co-localization of TRIM31 and TAK1 in cells was detected by laser confocal staining.10.Construction of expression vectors and point mutations The recombinant plasmid and point mutant plasmid were constructed and extracted by priming design,amplification,digestion and ligation of target gene for further overexpression and in vitro transcriptional translation experiments.11.Co-immunoprecipitation(Co-IP)HK2 was cultured in vitro and Co-IP was used to detect the binding of endogenous target molecules.HEK293 T cells were cultured in vitro and transfected with overexpressed plasmids of various target molecules.Co-IP was used to detect endogenous binding of target molecules.The target protein was expressed by in vitro translation system,and Co-IP was used to detect the exogenous binding of the target molecule.12.Cell transfection HK2,HEK293 T or MRPTEpic cells were cultured in vitro,and the small interfering RNA of TRIM31 or TAK1 was transfected into the cells with RNAiMAX to knock down the expression of the target gene.The overexpressed plasmid was transfected with Lipo3000 to overexpress the target gene.13.In vitro protein expression test was carried out by in vitro translation system PCDNA3.1 expression plasmid with T7 promoter were build and TNT Quick Coupled Transcription/translation System kit from Promega company was used for Reticulocyte Transcription in vitro translation.The Mini Expression Module kit was used for e.coli in vitro translation of TAK1 and TRIM31 expression plasmid.14.In vitro ubiquitination modification experiment The proteins obtained from the in vitro translation system were added to the in vitro ubiquitination modification system(including El,UBCH5 A,K48,K63,etc.)of BostonBiochem.After the reaction at room temperature for 30 minutes,the in vitro ubiquitination modification and the types of ubiquitination modifications were detected by Western blot.15.Statistics Analysis All data were presented as mean ± SEM Normality assumption of the data distribution was assessed using Shapiro-Wilk test.For normally distribution,data were analyzed by unpaired two-tailed Student's t-tests to determine the statistical difference between two groups.And one-way ANOVA followed by Dunnett or Tukey post hoc tests were performed to determine the statistical difference between multiple groups with one variable and normally distribution.To compare multiple groups with more than one variable,two-way ANOVA followed by Tukey or Sidak post hoc tests was used.For data with a non-Normally distribution,we performed a nonparametric statistical analysis using the Kruskal-Wallis test followed by the Dunn post-hot test for multiple comparisons with one variable.In all statistical comparisons,a p value of < 0.05 was considered statistically significant.Results1.TGF-?1 was involved in AagH-induced hypertensive renal damage in mice TGF-?1 is a key fibrogenic cytokine in hypertensive renal injury and renal tubulointerstitial fibrosis.Western blot,JHC and RT-PCR showed that TGF-?1 expression was significantly increased in the renal tissues of mice with Ang? induced hypertensive nephropathy,but there was no significant difference in TGF-?1 expression between TRIM31+/+ and TRIM31-/- mice.These results indicate tibiat TRIM31 knockout does not affect the expression of TGF-?1 in HRD kidney.2.TGF-?1 affects the expression of TRIM31 Western blot and RT-PCR showed that TGF-?1 stimulated renal tubular epithelial cells with time and concentration gradients,and the expression of TRIM31 decreased gradients.These results suggest that TGF-?1 inhibits the transcription and translation of TRIM31 gene,and TRJM31 may be involved in TGF-?1 mediated signaling pathway.TGF-?1 neutralizing antibody was pretreated,and TRIM31 protein level did not decrease under Ang? time gradient stimulation.These results suggest that TGF-?1 negatively regulates the protein expression of TRIM31.and Ang? may regulate TRIM31 protein expression through TGF-?1.3.Protective effect of TRIM31 on renal tubular epithelial cells stimulated by hTGF-?1 Western blot and RT-PCR showed that the decreased expression of TRIM31 significantly increased the levels of fibrosis and inflammation-related index proteins and mRNA in renal tubular epithelial cells stimulated by hTGF-?1.Overexpression of TRIM31 significantly improved the expression of fibrosis and inflammation-related indexes in renal tubular epithelial cells stimulated by hTGF-?1.And TRIM31-?Ring and TRIM31-C53A/C56 A overexpression did not have the effect,which proved that The E3 ubiquitin ligase function of TRIM31 plays an important role.4.Effect of TRIM31 on TGF-?1 signaling pathway activation Classical Smad signaling pathways and non-Smad signaling pathways,especially NF-?B and MAPK signaling pathways,play an important role in renal fibrosis.The phosphorylation of Smad2 and Smad3 represents the activation of classical Smad signaling pathway,and the phosphorylation of ERK,JNK,p38 mid NF-?B represents the activation of non Smad signaling pathway.Western blot analysis showed that the kidneys of hypertensive mice induced by Ang? showed significant activation of classical Smad signaling pathway and non-Smad signaling pathway,and it was more serious in TRJM31 knockout mice.In HK2 cultured in vitro,si-RNA down-regulation of TRJM31 protein also significantly promoted the activation of TGF-?1-induced classical Smad signaling pathways and non-Smad signaling pathways.These results indicate that TRIM31 expression deficiency can promote the activation of TGF-?1 signaling pathway.5.Binding experiment of TRIM31 with target molecule in TGF-?1... |
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