| BackgroundObstructive nephropathy(ON)refers to the disorder of urine discharge caused by urinary tract obstruction,which leads to the increase of pressure in renal pelvis and renal tubules,and finally leads to the damage of renal parenchyma and renal function.ON is the main cause of Chronic Kidney Disease(CKD).In addition,the invisibility of ON makes its diagnosis difficult,and patients often can’t get timely treatment,which will aggravate the progress of CKD.ON is also a common cause of Acute Kidney Injury(AKI),accounting for about 17%of all AKI cases,and AKI will increase the risk of CKD.Renal fibrosis plays a key role in the process of CKD induced by ON,and it is the ultimate way for CKD to develop into End Stage Renal Disease(ESRD).Therefore,inhibiting fibrosis can delay or prevent ESRD.There is no ideal treatment for renal fibrosis at present,and the reason may be related to the fact that the mechanism of renal fibrosis has not been fully understood.ON fibrosis is characterized by the loss of epithelial cells in glomerulus and tubulointerstitium and the increase of extracellular matrix protein(ECM)deposition,which will gradually replace the normal tissue structure and prevent the normal function of kidney cells.At present,the cellular mechanism driving renal fibrosis is still not completely clear.It is reported that inflammatory factors and inflammatory cells are the main causes of fibrosis in the literature,but the mechanism of inflammation affecting fibrogenesis still needs to be further explored.Thus understanding the relationship among them may be the key to formulate treatment strategies that can prevent the progression of fibrosis.Many studies have shown that the inflammatory characteristics of ON are excessive innate and acquired immune response,infiltration of inflammatory cells and release of cytokines.Generally,inflammation is a protective response to potentially harmful endogenous or exogenous damage,aiming at eliminating the threat of diseased cells and promoting tissue repair through fibrosis.However,persistent inflammation will cause damage to organism tissues,and if not corrected in time,it will cause persistent fibrosis,which will lead to organ failure.Therefore,targeting inflammation is a reasonable strategy in the treatment of ON.Unfortunately,the underlying pathophysiological mechanism of long-term inflammation and fibrosis on,including the role of immune system,little is known at present.Complete unilateral ureteral obstruction(CUUO)has become an important model to study the mechanism of renal fibrosis and evaluate the influence of potential treatment methods to improve chronic renal diseases.The pathological features of CUUO kidney are tubular dilatation,interstitial dilatation,proximal tubular tissue loss,hydronephrosis,leukocyte infiltration,tubular epithelial cell death and fibroblast proliferation.Experimental CUUO of rodents is considered to be able to simulate human CKD in an accelerated way.Due to oxidative stress,activation of RAS system and stimulation of cytokines,nuclear factor κB(NF-κB)will transfer to the nucleus,which will start transcription of target genes leading to inflammation and fibrosis.The increase of inflammation will also accelerate the process of fibrosis,because tumor necrosis factor α(TNFα)can stimulate fibroblasts to produce transforming growth factor β1(TGF-β1)by activating NF-κB pathway,and TGF-β1 can promote the process of fibrosis.TRIM protein is a member of the RING family in E3 ubiquitin ligase,which is characterized by the existence of three conserved domains,namely RING,B-Box and coiled-coil.Recently,as E3 ubiquitin ligase,TRIM protein plays a key role in regulating NF-κB signaling pathway,which has attracted the attention of many researchers around the world.Mitsugumin 53(MG53),also known as TRIM72,is a member of TRIM family protein mainly expressed in skeletal muscle and myocardium.It is an indispensable part of repairing cell membrane damage.When the cell membrane is damaged,MG53 can quickly locate the damaged part and seal the damaged cell membrane,thus playing its protective role.Therefore,MG53 is also known as a molecular bandage for cell injury.Renal dysfunction can be divided into AKI and CKD.The incidence of CKD in the elderly population is increasing.The main reason is that the kidney’s ability to repair damage decreases with age in the elderly population,while the animal model research related to this is less.Whether knocking out MG53 gene will accelerate the process of renal fiber has not been reported in the literature.It has been reported that MG53 can regulate TLR/NF-κB signaling pathway to exert its anti-inflammatory effect,thereby protecting the mouse hippocampal neuron cell line(HT22 cell line)and the mouse hippocampal nerve tissue from Lipopolysaccharides(LPS)-induced neurotoxicity.Macrophages used to be considered as the key factor to promote renal fibrosis in CUUO.However,the latest research shows that infiltrating macrophages have anti-fibrosis effect,It can protect the renal structure in the process of progressive renal scar formation caused by ON.Therefore,we constructed a kind of RAW 264.7 monocyte macrophage which could deliver MG53 induced by DOX to the kidney damage of CUUO mice,and studied whether it colud play a biological role in alleviating renal fibrosis after overexpression of MG53.When endogenous MG53 expressed and secreted by tissues can’t meet the requirements of injury repair,exogenous rhMG53(recombinant human mg53,rhMG53)has become a means of supplementing mg53 at this time.Studies have confirmed that exogenous rhmg53 can play a very good protective role in Ischemia/Reperfusion Injury(IRI)of various organs including heart,skeletal muscle,lung,liver,brain and kidney.However,there is no literature report on whether rhMG53 can play its anti-inflammatory role in CKD process and then control renal fibrosis,and the study of this problem plays an important role in promoting the clinical transformation and application of rhMG53 protein.Therefore,this study is divided into the following four parts,as follows:Part 1 Study on the effect CUUO on renal fibrosis in MG53KO mice ObjectiveTo investigate the effects of CUUO on renal inflammation and fibrosis in MG53KO mice.Materials and methods1.Select 8-12 weeks years WT and MG53KO mice of 16 each,weighing(23.51.2)g,and randomly divide WT and MG53KO mice into Sham group and CUUO group,namely Sham WT,Sham KO,CUUO WT and CUUO KO,n=8;Isoflurane(1.5-2.0%)was used to anesthetize mice,and laparotomy was performed on the midline of abdomen to separate the left ureter.The left ureter was double ligated with 5-0 surgical silk thread as CUUO group,and only abdominal incision and suture was used as Sham group.On the 1st,3rd and 7th day after operation,Sham group and CUUO group mice were killed by cervical dislocation after CO2 asphyxia,and bilateral kidney specimens were collected.2.HE staining was used to evaluate the renal morphological changes of WT and MG53KO mice on the 1st,3rd and 7th day after operation.Masson staining was used to compare the renal fibrosis changes of WT and MG53KO mice on the 1st,3rd and 7th day after operation.Immunohistochemical staining was used(Immumohistochemical,IHC)and Western Blot were used to detect and compare the changes of collagen,leukocyte antigen marker CD 11b,macrophage infiltration antigen markers F4/80 and MG53 in WT and MG53KO mice on the 1st,3rd and 7th day.3.Statistical methods:All experimental data are represented by mean standard error((x±S)),and the data are analyzed and processed by SPSS22.0 statistical software.The mean of two samples is compared by T-test,and the mean of multiple samples is compared by one-way ANOVA.The difference is statistically significant(P<0.05).Results1.At the 1st,3rd and 7th day after CUUO operation,HE staining showed that the degree of renal lesions in MG53KO group and WT group increased with the increase of days,and the degree of renal lesions in MG53KO group were all more severe than that in WT group respectively on the 1st,3rd and 7th day after CUUO operation.2.Masson staining showed that the degree of renal fibrosis in MG53KO group and WT group increased with the number of days,and the degree of renal fibrosis in MG53KO group were all more severe than that in WT group(0.85±0.05)vs(0.56±0.02)respectively on the 1st,3rd and 7th day after CUUO operation.3.On the 1st,3rd and 7th day after CUUO operation,IHC staining and WB detection showed that the expression levels of Collagen 1,Collagen 3 and FN in the kidneys of MG53KO group and WT group increased with the increase of days,and the expression levels of Collagen 1,Collagen 3 and FN in MG53KO group were all higher than those of WT group respectively on the 1st,3rd and 7th day after CUUO operation.4.On the 1st,3rd and 7th day after CUUO operation,IHC staining and WB detection showed that the expression levels of leukocyte antigen marker CD11b and macrophage infiltration antigen marker F4/80 in MG53KO group and WT group increased with the increase of days,and the expression levels of CD11b and F4/80 in MG53KO group were higher than those in WT group respectively on the 1st,3rd and 7th day after CUUO operation.5.On the 1st,3rd and 7th day after CUUO operation,IHC staining and WB detection showed that the expression of MG53 in kidney of WT group increased with the increase of days,while on the 1st,3rd and 7th day after CUUO operation,no MG53 expression was detected in MG53KO group.ConclusionOn the 1st,3rd and 7th day after CUUO operation,CUUO aggravated the renal inflammation and fibrosis of MG53KO mice compared with WT mice.Part 2 Study of the mechanism of MG53 inhibiting NF-κB activation ObjectiveTo investigate whether MG53 can regulate the activity of NF-κB in proximal renal tubular epithelial cell(PTEC)of mice and its mechanism.Materials and methods1.Primary mouse PTEC was isolated from WT and MG53KO kidneys aged 2-3 months respectively.PTEC was starved in serum-free medium for 8 hours and then treated with LPS(Escherichia coli O111:B4,Sigma L2630,USA).Cells were collected for WB,cell fragments were removed after centrifugation and frozen in-80 refrigerator for cytokine detection,and PTEC was fixed with 4%paraformaldehyde for cellular immunofluorescence analysis.Collect the serum of animals in different treatment groups and supernatant of culture medium in all groups of primary mouse PTEC,and keep it at-80 C until use.The cytokine levels of IL-6,TNFα,IL-10 and IL-1β released by PTEC in WT group and MG53KO group after LPS stimulation were measured and compared by ELISA.2.Immunofluorescence staining was used to analyze the position of p65 and IκBα in the cells of PTEC in WT and MG53KO groups under hypoxia and control.3.PCMS-MG53 and p65 with MYC tag were transfected into HEK293 cells for 24 hours,then the transfected cells were cultured in the medium containing proteasome inhibitor MG132 for 16 hours,and then the cells were collected for CO-IP experiment.4.Equal amount(200 ng/well)of PCMS tag expressing MG53 construct(as described above)and IκBα promoter plasmid were co-transfected into HEK293FT cells.Wait another 18 hours after transfection,and stimulate the cells with 20 ng/ml TNFa within the specified time.5.Methods:All experimental data were expressed by mean standard error(x±s),and the data were analyzed by SPSS22.0 statistical software.The mean of two samples was compared by T-test,and the mean of multiple samples was compared by one-way ANOVA.The difference was statistically significant(P<0.05).Results1.Western Blot results showed that the ratios of p-IκBα/IκBaand p-p65/p65 in WT+LPS and MG53KOLPS groups were significantly higher than those in WT Control group and MG53KOControl group.Compared with WT+LPS group,the ratio of p-IκBα/IκBα and p-p65/p65 of MG53KO PTEC increased significantly after LPS stimulation.ELISA results showed that compared with WT Control group,WT+LPS group obviously secreted more TNFα,IL-6 and IL-1β.Compared with inflammatory factors of TNFα,IL-6 and IL-1β secreted by WT+LPS group,MG53KO PTEC group secreted more TNFα,IL-6 and IL-1β into the culture medium supernatant after LPS stimulation.2.Immunofluorescence results showed that in WT and MG53KO control groups,the fluorescence intensity expression of p-IκBα protein was weak.The fluorescence intensity expression of p65 protein was also low,mainly in cytoplasm.In WT+LPS and MG53KO+LPS groups,the expression of fluorescence intensity of p-IαBa protein increased,and the expression of fluorescence intensity of p65 protein also increased,mainly in the nucleus.Under hypoxia stress,p65 in WT and MG53KO groups can enter the nucleus of PTEC.However,the fluorescence intensity of p-p65 and p-IκBa protein in MG53KO+LPS group was higher.3.CO-IP results show that MG53 and IκBa can be directly combined with each other.4.The luciferase test showed that after 4 hours of TNF-α stimulation,the relative fluorescence intensity of luciferase in pcms-MG53 group was significantly higher than that in empty vector group(PCDNA),and the difference was statistically significant(P<0.05).ConclusionMG53 can combine with the transcription region of IKBA promoter to promote the expression of IκBα.Part 3 Construction of Tet-On system to regulate the secretion of MG53 macrophages to reduce obstructive fibrosisObjectiveConstruct macrophages based on the Tet-On system that can regulate the secretion of MG53 and explore whether overexpression of MG53 has the biological effect of improving renal fibrosis in CUUO MG53KO mice.Materials and methods1.Cloned the gene sequence expressing tPA to the front of the full-length sequence of MG53 cDNA,then design primers to amplify the tPA-MG53 sequence by PCR method,and clone the PCR product into the empty vector of adenovirus modified by pShuttle-TetON-CMV-GFP,which contains tetracycline response element(Tre)in the promoter region to generate Ad-tPA-MG53-GFP which is induced by DOX.Then transformed and extract this plasmid.The Ad-tPAMG53-GFP adenovirus was packaged,amplified and purified with a specific kit.RAW264.7 cells were infected with Ad-tPA-MG53-GFP and adenovirus in the control group.After DOX induction for 24 hours,4%paraformaldehyde fixed cells were used for immunofluorescence staining.Lysate of RAW264.7 cells transfected with adenovirus and protein secreted from concentrated medium were collected for Western Blot.2.24 MG53KO mice aged 8-12 weeks,weighing(20.7±1.9)g,were randomly divided into 4 groups,with 6 mice in each group.Among them,three groups were performed with CUUO as the experimental group,and one group in the experimental group was only given drinking water containing DOX after operation,without cell therapy,which was called CUUO none group;In one group,106 RAW264.7 cells infected with control adenovirus were injected via tail vein after operation,and drinking water containing DOX was given,which called Ad-tPA-GFP group;In the third group,106 RAW264.7 cells infected with Ad-tPA-MG53-GFP adenovirus were injected via tail vein after operation,and drinking water containing DOX was given,which called Ad-tPA-MG53-GFP group.Sham group only underwent abdominal incision and suture,and drinking water containing DOX was given as a blank control group.Every other day,the mice in the experimental group were injected with RAW 264.7 cells infected with Ad-tPA-MG53-GFP adenovirus and control adenovirus for 24 hours,with a total of 4 injections.Seven days after operation,all mice were killed,and kidney tissue samples were collected for Western Blot,Masson and immunofluorescence staining.3.Statistical methods:All experimental data are represented by mean standard error(x±s)),and the data are analyzed and processed by SPSS22.0 statistical software.The mean of two samples is compared by T-test,and the mean of multiple samples is compared by one-way ANOVA.The difference is statistically significant(P<0.05).Results1.confocal immunofluorescence staining was performed on RAW264.7 macrophages with anti-MG53 antibody after adtpa-vector-GFP adenovirus infected RAW26.7 cells for 12 hours,and DOX induction culture was given for 24 hours,and fixed cells were immunofluorescence.The results showed that there was no MG53 expression.The Ad-tPA-MG53-GFP adenovirus infected RAW26.7 cells for 12 hours,and was induced by DOX for 24 hours.After 24 hours,there was red fluorescence expression(MG53).The results indicated that Ad-tPA-MG53-GFP-RAW cells could induce the expression of MG53 by DOX.2.Masson staining showed that compared with the CUUO mice injected with Ad-tPA-MG53-GFP-RAW264.7 cells via tail vein and without cell therapy.The area of renal fibrosis in mice injected with Ad-tPA-Vector-GFP-RAW264.7 cells via tail vein was significantly reduced,which were(42.78±5.31)vs(64.67±7.80)、(60.62±8.03).3.Compared with sham group,CUUO+DOX group and CUUO Ad-Vector+DOX group,only CUUO+Ad-MG53+DOX group could detect the expression of MG53,which indicated that MG53 was presented to the damaged kidney tissue by RAW264.7 cells.Compared with sham group,the expressions of COL-1,COL-3 and FN in CUUO+DOX group and CUUO Ad-Vector+DOX group were significantly higher,and there was a significant difference in statistics,which indicated that the model was established effectively.Compared with CUUO+DOX group and CUUO Ad-Vector+DOX group,The protein expression of p-IκBa and p-p65 in CUUO+AdMG53+DOX group decreased significantly,and there was a statistically significant difference.This indicates that MG53 presented by RAW264.7 cells can alleviate the activation and fibrosis of NF-κB signaling pathway in damaged kidney tissues.ConclusionMacrophages secreting MG53 based on the Tet-On system can alleviate the inflammation and fibrosis of obstructive kidneys in mice.Part 4 Study on rhMG53 in the treatment of obstructive fibrosisObjectiveTo explore whether systemic administration of rhMG53 can alleviate NF-κB activation and fibrosis in CUUO mice,and provide a theoretical basis for further promoting the application of rhMG53 protein in the treatment of ON.Materials and methods1.Eighteen 8-12 week years old WT mice weighing(21.5±1.3)g were randomly divided into Sham group(n=6)and experimental group(n=12).The experimental mice in group were treated with CUUO to induce renal fibrosis into CUUO group.The mice in CUUO group were randomly divided into CUUO+saline group and CUUO+rhMG53 group,with n=6 mice.The mice in the two groups were treated with saline or rhMG53(2mg/Kg)every day for the first seven days,and then treated every other day.Sham group was used as the control group.Only incision and suture were performed,and no normal saline or rhMG53 treatment was given after operation.The mice were killed on the 12th day.The changes of renal fibrosis were detected by Western Blot,HE staining,Masson staining and IHC staining.2.Statistical methods:All experimental data are expressed by mean standard error(x±s),and the data are analyzed and processed by SPSS22.0 statistical software.The mean of two samples is compared by T-test,and the mean of multiple samples is compared by one-way ANOVA.The difference is statistically significant(P<0.05).Results1.HE staining showed that compared with Sham group,the renal tissue morphology of CUUO side in saline treatment group was disordered and the renal tubules were swollen,while after rhMG53 treatment,the renal tissue morphology of obstructive side recovered and the renal tubule swelling was alleviated.Masson staining showed that the renal tissue of obstructive side in saline treatment group was significantly more fibrotic than that in Sham group.However,the degree of renal fibrosis on the obstructive side was reduced after rhMG53 treatment.2.IHC staining showed that compared with Sham group,the expression of MG53 in kidney tissue on the obstructive side of the group treated with normal saline slightly increased,and the swelling of renal tubules became more serious.However,after rhMG53 treatment,immunofluorescence staining showed that MG53 in the injured part increased significantly.After rhMG53 treatment,MG53 was found in the kidney on the obstructive side,and the degree of tubule swelling was reduced.ConclusionrhMG53 can reduce kidney inflammation and fibrosis in CUUO mice. |
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