| 1.BackgroundAcute kidney injury(AKI)occurs in approximately 50%of patients in the intensive care unit(ICU).It is an abrupt decrease in kidney function that occurs over 7 days or less according to the 2012 Kidney Disease:Improving Global Outcomes(KDIGO)criteria.Renal early reversal and recovery from AKI are closely associated with better long-term prognosis,while a longer AKI duration is associated with not only increased mortality and morbidity,but also the development of acute kidney disease(AKD;persisting between 7 and 90 days)and chronic kidney disease(CKD,persisting more than 90 days).As there is no effective therapy for AKI,early differentiation of patients at a high risk for AKD or CKD is of great significance in clinical assessment.However,there is no validated method to date to predict renal prognosis.While having been extensively explored for early detection of AKI,biomarkers for renal outcomes are of new concern and the same important,as biomarker-guided treatment adjustmentmay modify resolution pattern to better outcomes.Hence,identifying biomarkers related to renal outcomes are highly demanded.The primary reasons for the difficulty in renal outcome prediction are that renal biopsy is often unavailable in AKI and thus renal repair process is unrevealed.As a form of extracellular vesicles(EV)loaded with abundant biomarkers and poorly filtered,exosomes from the urine provide an intriguing alternative approach as’liquid biopsy’.Importantly,these extracellular vesicles deliver their content to downstream cells and act as intercellular messengers.In experimentalresearch,they have been reported to play a role in the reversal of kidney ischemia-reperfusion(IR)injury.Hence,urinary exosomes and some exosomal molecules may be also involved in renal repair and thus may be utilized as biomarkers for AKI prognosis.Among various urinary exosomal proteins,CD26,also known as dipeptidyl peptidase-Ⅳ,is a widely distributed multifunctional membrane protein.It acts as proteolytic enzyme,receptor and co-stimulator,that are widely involved in inflammation and immune response.Specifically,previous studies have shown that CD26 exerts protection against apoptosis in IR.As IR is an important cause and apoptosis is an essential mechanism in AKI,it is highly probable thatCD26 is involved in AKI repair and thus indicates renal sequelae,which was to our knowledge never studied before.There are two forms of CD26 in urine-free CD26 and microvesicle-bound CD26,with the latter having been reported to be the main form in urine and correlate with diabetic kidney disease.Hence,urinary exosoma1CD26 seems to be a potential biomarker in renal prognosis and future intervention for AKI.2.Objectives(1)To verify whether urine exosmalCD26 was in involved in AKI(2)To verify whether urine exosmalCD26 was associated with AKI stage(3)To examine the possible association between urinary exosomalCD26 and major adverse kidney events(MAKE)(4)To examine the possible association between urinary exosomalCD26 and renal improvement in AKI(5)To explore the predictive potential of urinary exosomalCD26 as a novel biomarker for AKI prognosis.3.Method3.1 Study designThis was a prospective cohort study which enrolled AKI patients as a cohort.To clinically verify whether urinary exosomalCD26 is involved in AKI,a non-AKI control cohort was also needed to compare urinary exosomalCD26 between the AKI and non-AKI patients.For AKI patients,bi-directional comparisons were performed.Urinary exosomalCD26 was firstly compared as a continuous variable between the opposite values of each outcome.Vice versa,we divided the AKI cohort into two groups with a low-and a high-CD26 level,and the outcomes were then compared between these two CD26 levels.Besides,baseline clinical characteristics were also compared to check the comparability and define covariates in multiple analyses.The association between CD26 level and AKI stage was first analyzed via bi-directional.Within the AKI cohort,MAKE 90 was compared between high-and low-CD26 levels,via KM analyses,and reversal within 28 days was compared between high-and low-CD26 levels,via KM and Cox analyses.The association between urinary exosomalCD26 and secondary endpoints were analyzed within the AKI survivors via bi-directional comparisons,after comparation for those endpoints between CD26 levels in AKI patients.Based on the results,sensitivity analyses were performed to optimize the predictive capacity of urinary exosomalCD26 for clinical application.3.2 Study populationThis single-center prospective cohort study was conducted ICU in Qilu Hospital,Shandong University from January 2017 to January 2018.The study protocol was approved by the Ethics Committee of Qilu Hospital,Shandong University(IERB:2017074).Signed informed consent was obtained from patients or legally authorized representatives.For theAKI cohort,consecutive adult patients admitted to the ICU were screened within 24 h after admission,among whom patients with AKI were enrolled in this study.AKI diagnose and stage judgment were based on the 2012 KDIGOcriteria.Exclusion criteria included anuria,severe urinary tract infection,urological neoplasms,obstructive nephropathy and previous use of CD26 inhibitors.For the control cohort,consecutive adult patients without AKI at ICU admission were enrolled from January to March 2017.The two cohorts were matched in terms of the overall severity of illness other than kidney by the constraint of the non-renal Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ)score,which was calculated as the score of the APACHE Ⅱcomponents other than serum creatinine(SCr).As AKI is usually secondary to severe primary disease,the overall severity of illness,even apart from kidney,is significantly lower for the patients without AKI than the AKI patients.Hence,patients with the non-renal APACHE Ⅱscore<8 were excluded from the control cohort based on a pilot study in the ICU.Other exclusion criteria were similar to those for the AKI cohort.CKD was defined as abnormalities of kidney structure or function(eGFR<60ml/min/1.73m2)present for≥3 months.AKI patients were treated following the KDIGO guidelines.3.3 Clinical data collectionThe urine output was recorded(usually every 8 h,hourly during a relapse)andSCr was measured(every 48 h to 72 h)continuously until discharge.Clinical parameters were noted at ICU admission,such as demographics,severe comorbidities,reasons for ICU admission,use of nephrotoxic drugs/vasopressors/ventilator and AKI stage.APACHE II/non-renal APACHE Ⅱscore were calculated at 24h after ICU admission.Length of ICU/hospital stay was also recorded as a clinical parameter.Patients were followed up for(non-)survival,SCr and receipt of RRTvia calls to their delegator or doctors until 90 days from ICU admission.3.4 Outcomes of AKI and follow-upThe primary outcomes were major adverse kidney events(MAKE),including death,new receipt of renal-replacement therapy(RRT)and persistent renal dysfunction(a creatinine value≥200%of the enrollment value).Previous studies have shown that AKI follows several patterns for resolution,including early or late reversal,sustained reversal or relapse,and ultimate recovery or non-recovery.On this basis,the second outcomeswere renal improvement-early reversal,recovery and reversal.The value of deathwas recorded as survival/non-survival at hospital discharge.To compare survival over time between the low/high CD26 levels,thetime of death was also noted within 90 days after ICU admission.Similarly,new receipt of renal-replacement therapy(RRT)and persistent renal dysfunction were noted within 90 days after ICU admission.Concerning the renal improvement,reversal was defined as the patient no longer meeting the criteria for any AKI stages.Similar to death,to capture when AKI began to resolve,the time of reversal was also noted within 28 days after ICU admission.For a small number of patients who reversed more than once due to another relapse,only the first occurrence was considered.It was regarded as early reversal if it occurred within 7 days after ICU admission.Recovery was defined as reversal being present at discharge from the hospital.AKI Patients were monitored until hospital discharge to record thevalues of each outcome,namely,early reversal/no early reversal,recovery/non-recovery and reversal(which means that the patient reversed at least once)/non-reversal.3.5 Sampling scheduleAt enrolment,15 ml of urine was collected for exosomes isolation.After sample collection,the samples were centrifuged at 300 x g for 10 min and the sediments were discarded.Urine samples were then frozen at-80℃.3.6 Exosomes isolation and purificationAfter thawed,urine samples were vortexed immediately.For depleting the abundant Tamm-Horsfall protein(THP)contamination,the urine samples mixed with reducing agent dithiothreitol(DTT,200 mg/ml)were incubated at 95℃ for 2 min.Then urine exosomes were isolated by differential ultracentrifugation:17000 ×g 15min to remove microvesicles and cell debris,filtered through 0.22μm membrane filter,200000 × g 1h for pellet,resuspended pellet with phosphate buffered solution(PBS)and 200000 × g 1h for exosomes pellet.Then they were diluted to 100ul with PBS and frozen at-80℃.3.7 Electron microscopyExosomes isolated from urine were fixed in 2%paraformaldehyde-PBS for 10min.The 10μL samples were then deposit on Formvar-carbon-coated grid and air dried for 20 min in a dry environment.The grids were contrasted and embedded with a mixture of 4%uranyl acetate and 2%methylcellulose(1:9 ratio).Grids were air dried and observed with a JEOL electron microscope(JEOL1200,Japan).3.8 Detection of exosomal biomarkerThe well-established markers were assessed in the urine pellets using western blot analyses and beads-based flow cytometry analysis.3.9 Flow cytometry analysis of exosomes-bound beadsExosomes-coated latex beads prepared by incubating exosomes with 4μ diameter aldehyde/sulfate latex beads(Invitrogen)by mixing 30 mg exosomes in a 1 ml volume of beads for 15 min at room temperature and 4℃ overnight with continuous rotation,as previously described.The reaction was stopped with 100mM glycine and 2%BSA in PBS for 30 min at room temperature.Exosomes-bound beads were washed with 2%BSA in PBS and centrifuged for 5 min at 14,800 × g.Then the beads were blocked with 2%BSA in PBS with rotation at room temperature for 30 min,and incubated with PE Mouse anti-Human CD26(555437,BD Pharmingen,1:200 diluted in 2%BSA)for 30min at room temperature.Later,beads were washed in 2%BSA and centrifuged for 1 min at 14,800 × g.After washing twice,beads were analyzed by flow cytometry.All buffer used above was filter through 0.22μm PVDF-membrane filter twice.Negative control was obtained by incubating the beads coupled with the exosomes,with an isotype control antibody,or in the absence of antibody.Gating of beads was performed based on FCS/SSC parameters,so antibody and protein aggregates are excluded from the analysis.The percentage of positive beads was calculated relative to the total number of beads analyzed per sample(100,000 events).This percentage was therein referred to as the percentage of CD26 positive exosomes.Fluorescein tagged anti-CD61,CD81 and TSG101 antibodies were used the same protocol for analyzing positive percentage.3.10 Statistical analysesThe obtained data were expressed as either percentages for categorical variables or medians with interquartile ranges(IQRs)for continuous variables.Variables were compared via Student’s t-test,Mann-Whitney test,Kolmogorov-Smirnov test or Pearson’s chi-squared test,as appropriate.SPSS 17.0 was used for the above statistical tests.GraphPad Prism version 5 was used for Receiving Operator Characteristic(ROC)analyses.To verify the involvement of urinary exosomalCD26 in AKI,CD26 and clinical variables were firstly compared between the AKI cohort and the non-AKI control cohort.As not all comorbidities/confounders were matched between these two cohorts such as hypertension and cardiovascular events,multivariate linear regression analysis was performed after bivariate Spearman correlation analyses to adjust for the possible impact of covariates on CD26 percentage,including demographics,severe comorbidities,ICU admission reasons and illness severities.Within the AKI cohort,bidirectional comparisons were conducted to examine the association between urinary exosomalCD26 and outcomes.Clinical characteristics were also compared between AKI patients with low/high-CD26 levels.To examine the correlation between urinary exosomalCD26 and AKI severity,CD26 percentage was compared among the three AKI stages,then the three AKI stages were compared between the low/high-CD26 levels.Similarly,for the outcomedeath,CD26 percentage was firstly compared between survivors and non-survivors,and then the mortalities of patients with low/high-CD26 levels were compared.In addition,the cumulative morality for each level was calculated for each day until 90 days after ICU admission and compared via Kaplan-Meier analysis and log-rank test.The cumulative incidence of RRT and persistent renal dysfunction for each level were analyzed via the same methods.In the same way,to examine the association between the reversal time and CD26 level,we used Kaplan-Meier analysis with log-rank testing to analyze the cumulative incidence of reversal within 28 days after ICU admission.Reversal was defined as an event.Patients without a reversal were censored at death.Hazard ratio(HR,which is the relative likelihood of reversal in high-CD26 level vs.low-CD26 level)and 95%confidence interval(CI)were estimated with multivariable Cox proportional hazards regression.Covariates in the models were selected on the basis of Kaplan-Meier analyses with p<0.10,including demographics,acute critical illnesses,severe comorbidities,severity of illness and AKI stages.For the three renal improvement outcomes-early reversal,recovery and reversal,the correlation between CD26 and the three renal outcomes was investigated only in AKI survivors,to eliminate the effect of death.Bidirectional comparisons were performed.CD26 percentage was firstly compared between the two opposite values of each renal outcome,and then the rates of the two opposite values of each renal outcome were compared between the low/high-CD26 levels via univariable Chi-squared tests and multivariable logistic regression.Continuous variables,such as age and non-renal APACHE Ⅱ score,were transformed to categorical variables.Chi-squared tests were performed between CD26 level and each baseline clinical variable.On the basis of Chi-squared tests,covariates with p<0.10 were selected to be adjusted in multivariate logistic regression analyses.As sepsis is the main cause for AKI in ICU,post hoc ROC analyses were further performed for all AKI survivors and then for septic/non-septic AKI survivors to evaluate the prediction performance of urinary exosomalCD26 on renal outcomes for these groups.4.Results4.1 Study populationAn AKI cohort of 133 patients and a non-AKI control cohort of 68 patients were enrolled in this study.4.2 Morphology and size of the pelletsThe morphology of these isolated pellets and the size distribution evidenced by electron microscopy and dynamic light scattering confirmed the constituent of exosomes.4.3 Biomarker of exosomes Western blot analyses evidenced that CD63,calnexin,GRP94 and TSG101 were enriched.Furthermore,the bead-based flow cytometry analyses revealed high percentages of CD61,CD81 and TSG101 expression.4.4 Comparison of the CD26 percentage and clinical characteristic between the AKI cohort and the non-AKI control cohortThe results showed that the AKI and control cohorts were matched in terms of age,gender and non-renal APACHE II score.CD26 percentage was significantly lower in the AKI cohort than in the control cohort with a skewed distribution[median(IQR):6.4%(1.2%-22.0%)vs.23.9%(6.6%-64.5%);p<0.001]4.5 Correlation between the CD26 percentage and clinical covariatesMultiple linear regression analysis showed that CD26 percentage was negatively correlated with AKI in all the patients(β=-15.95,p<0.001)after adjustment for covariates.No significant correlations were found between CD26 percentage and the covariates.4.6 Comparison of clinical characteristics within the AKI cohortWithin the AKI cohort,comparisons of clinical characteristics between patients with a low-CD26 level(CD26 percentage<6.4%)and a high-CD26 level(CD26 percentage≥6.4%)showed no significant difference in terms of age,gender,APACHE Ⅱ score and non-renal APACHE Ⅱ score.4.7 Correlation between the CD26 percentage and AKI stageThe comparison of the CD26 percentage among the three AKI stages showed no significant difference,and the AKI severity at baseline showed no significantdifference between patients with low/high CD26 levels.4.8 Correlation between the urinary exosomalCD26 and endpoints4.8.1 Correlation between urinary exosomalCD26 and MAKE 90 within the AKI cohort For the outcome death,the AKI cohort had 81(60.9%)survivors and 52(39.1%)non-survivors.No significant difference was found for CD26 percentage between survivors and non-survivors,and no significant difference was found for death between the low-and the high-CD26 levels.For the cumulative morality,Kaplan-Meier curves showed no significant difference of death between the low-and the high-CD26 level within 90 days after ICU admission,either.For the cumulative incidence of RRT,patients with a high-CD26 level were significantly lower than a low-CD26 level within 90 days(p=0.013).For the cumulative incidence of persistent renal dysfunction,no significant difference was found between the high-and low-CD26 levels.MAKE90-the composite of death,RRT and persistent renal dysfunction-was significantly lower in patients with a high-CD26 level than those with a low-CD26 level,mainly due to receipt of RRT(p=0.018).4.8.2 Correlation between urinary exosomalCD26 and renal reversal within the AKI Cohort4.8.2.1 Correlation between urinary exosomalCD26 and renal reversal within 28 days in the AKI CohortFor the cumulative incidence of the outcome reversal within 28 days after ICU admission,Kaplan-Meier curves showed that it was significantly higher for the high-CD26 level than the low-CD26 level.After adjustment for covariates selected on the basis of Kaplan-Meier analyses,multivariable Cox regression analysis showed that the high-CD26 level was associated with increased adjusted rates of reversal(HR,1.90;95%Cl,1.15-3.14;p=0.012).4.8.2.2 Correlation between urinary exosomalCD26 and renal improvement endpoints within the AKI survivorsFor the three renal improvement outcomes-early reversal,recovery and reversal,we have showed that AKI patients with a high-CD26 level had a significantly higher rate of early reversal,recovery and reversal in unadjusted analyses.Their associations with urinary exosomalCD26 were further analyzed in AKI survivors.The results revealed that the CD26 percentage for a preferable value(early reversal,recovery and reversal)was significantly higher than a corresponding adverse value(no early reversal,non-recovery and non-reversal)in AKI survivors.Within the 81 AKI survivors,44 showed early reversal(high-CD26 level:29(72.5%)vs.low-CD26 level:15(36.6%),p=0.001),57 recovered(ligh-CD26 level:34(85.0%)vs.low-CD26 level:23(56.1%);p=0.004),58 reversed at least once during hospitalization(high-CD26 level:35(87.5%)vs.low-CD26 level:23(56.1%);p=0.002).After adjustment for covariates,the association between the low/high-CD26 level and renal outcomes still persisted.4.8,3 Sensitivity AnalysesWhile the ROC analyses for recovery and reversal were not significant at 0.05 level,the ROC analysis revealed a significant effect for early reversal[area under the curve 0.65;95%CI,0.53-0.77;p=0.021].According to the ROC curve,we propose a CD26 percentage of 6.9%as the optimal cut-off value to differentiate early reversal and no early reversal in AKI survivors.Patients with CD26 percentage greater than or equal to 6.8%were classified as at high probability to early reversal.After applying this optimal cut-off(6.8%),the ORs were 4.73(1.77-11.48),5.23(1.72-13.95)and 6.73(2.00-19.67)for early reversal,recovery and reversal,respectively,ROC analyses also revealed that CD26 level had a higher prediction value in non-septic AKI survivors,with(AUC,0.71;95%Cl,0.52-0.89;p=0.046)for early reversal,(AUC,0.79;95%CI,0.63-0.94;p=0.009)for recovery and(AUC,0.83;95%CI,0.70-0.97;p=0.003)for reversal.5.ConclusionsWe report that urinary exosomalCD26 is negatively associated with AKI.For AKI,high-CD26 level is associated with lower rate of major adverse kidney events.Furthermore,high-CD26 level is associated with higher rate of renal reversal within 28 days and with preferable endpoints defined in this study-reversal,early reversaland recovery.Moreover,urinary exosomalCD26has predictive value for early reversal,and its predictive capability can be greatly improved for reversal,early reversaland recovery in non-septic AKI.1 BackgroundAcute kidney injury(AKI)is a serious complication of hospitalized patients,especially critically ill patients.In addition to a significant increase in hospital mortality,it can also lead to increased long-term mortality,chronic kidney disease(chronickidneydisease,CKD)and end-stage renal disease.Occurs,causing a heavy burden of disease.At present,there are no effective preventive or treatment measures for AKI,and it is still supportive treatment.Exosomes are extracellular vesicles that are widely present in various body fluids.The structure of its lipid bilayer carrying the protein and nucleic acid of the source cell makes it a kind of intercellular messenger.By binding to the recipient cell,it is widely involved in the regulation of various physiological and pathological processes.In AKI,it is more and more used as a disease marker.It has received more and more attention.Several recent studies have found that exogenous exosomes can reduce AKI kidney damage and promote the recovery of renal function,but its mechanism and key molecules are still unclear.The study of Paper I found that compared with the control group,the expression rate of CD26 in urinary exosomes of AKI patients was significantly lower;and within the cohort of AKI patients,the group with high urinary exosomes CD26 had higher discharge renal function than the low group Recovery rate:After adjusting for gender,age,chronic comorbidities,acute etiology,criticality,and AKI classification,urinary exosomalCD26 is still independently associated with renal prognosis such as 7-day renal function recovery and discharge renal function recovery.Due to the presence of glomerular filtration membrane,urinary exosomes seldom come from the circulation,and the exosomes produced by kidney cells are the main source of urinary exosomes.From this we inferred that exosomes derived from kidney cells expressing CD26 may have the function of reducing kidney damage and promoting kidney repair.CD26 is a transmembrane glycoprotein on the cell surface.CD26 is also commonly called Dipeptidyl peptidase-4(DPP4).It has serine protease activity and is widely expressed in many types of cells.In the kidney,it is expressed in glomerular podocytes.The basement membrane is highly expressed in the brush border microvilli of distal tubular epithelial cells(TEC).Studies have found that CD26 is related to processes such as cell proliferation and inflammation regulation.The cell regeneration of TEC is the main part of AKI repair.Studies have found that CD26 can promote cell proliferation.And CD26 is involved in the regulation of inflammatory response,especially its substrate contains a series of chemotactic cytokines.The main pathological process of AKI occurs in the TEC,especially the damage,necrosis or apoptosis of the distal TEC,and then the infiltration of inflammatory cells and the occurrence of interstitial inflammation.The main process of kidney repair is the proliferation of TEC,which replaces the lost TEC and repairs renal tubular epithelial tissue.Inflammation is involved in the regulation of this process.Good proliferation and regulation lead to complete repair and recovery of renal function;otherwise,it leads to aggravation of fibrosis and ultimately leads to CKD.Therefore,we hypothesize that CD26+exosomes may promote TEC proliferation,inhibit inflammation,reduce fibrosis,reduce kidney damage,and promote kidney repair.Therefore,we plan to cultivate the mouse renal tubular epithelial cell line TCMK1 in vitro,and obtain the physiological exosomes in the culture supernatant by simulating ischemia/reperfusion injury(IR),the main pathogenic process of AKI in vitro.ExoNormal,IR program obtains ExoIR to explore the expression of CD26 in urine under physiological and IR conditions.TCMK1 was transfected with overexpressing CD26 adenovirus to construct TEC-derived CD26 overexpressing exosomes(ExoCD26);an IR-related AKI mouse model was constructed by surgery,and exosomes were further used for intervention.Comparison of renal function,histopathology,hyperplasia,inflammation and other indicators between IR-AKI and controls,to explore the effect of CD26+exosomes on AKI kidney repair and its possible mechanism.2 Purpose(1)In vitro experiments to explore whether hypoxia culture can affect the expression of exosomes CD26;(2)Use renal tubular epithelial cell line to construct CD26 overexpression exosomes in vitro;(3)In the AKI animal model,trace whether the exosomes derived from the renal tubular epithelial cell line can be taken up by kidney cells;(4)Through in vivo experiments,explore whether CD26 overexpression exosomes can reduce kidney damage and promote kidney repair,and explore its possible mechanism.3 Methods3.1 Cellculture and virus transfection:TCMK1 cells are routinely cultured and completed the ischemia and hypoxia procedures,and the standard procedures are transfected with CD26 overexpressing adenovirus and empty vectors.Retain the cell culture supernatant.3.2 Isolation,purification and expression identification of exosomesThe exosomes in the supernatant of TCMK-1 mouse renal tubular cells were extracted by ultra-speed differential centrifugation,ExoNormal released from cells cultured under normal conditions,ExoIR released from cells cultured under hypoxia,and CD26?The cells released exosomes ExoCD26 after transfection,and ExoIR+CD26 released from cells cultured and transfected under hypoxia,respectively,were detected for CD26 expression.After the exosomes were fully reacted with the anti-CD26 antibody and the fluorescein-conjugated secondary antibody,they were added to flow cytometry analysis.3.3 Establishment of AKI animal model(IR)C57B1/6 male adult mice(8 weeks)were anesthetized with pentobarbital,and the right renal pedicle was tightly ligated under a microscope.The right kidney was removed;the left renal artery was clamped with an arterial clip to observe the discoloration of the kidney.The ischemia changes,covered with moist gauze,and clamped for 30 minutes.3.4 In vivo tracing of exosomesOperate in thedark,label exosomes with PKH26,and inject them into IR mice through the tail vein;take the kidney tissue under anesthesia at 15 minutes,2 hours,and 12 hours after injection;OCT embeds the tissue blocks and prepares frozen sections;After the sections were hydrated with double distilled water and washed with PBS,the reaction was terminated with BSA;AQP1 primary antibody was added and incubated overnight in a humidified box at 4 degrees,then washed with PBS,fluorescein-conjugated AQP1 secondary antibody was added,and incubated at 37℃ for 30 minutes.Wash with PBS,stain with DAPI,mount the slide,and observe immediately under a fluorescence microscope3.5 IR mice intervention measures and groupingDivided into control group,IR+BSA group,IR+ExoNormal group,IR+ExoIR group,IR+ExoCD26 group,each group 10 mice;the control group did not undergo kidney treatment,the rest of the operations were the same as the IR operation groups;the IR groups were injected with equal protein quality BSA,ExoNormal,ExoIR and ExoCD26 through the tail vein 12 hours after the operation.3.6 Renal function index detection72 hours after the intervention,blood samples were taken.After centrifugation of each blood sample,the serum was preserved.The urea nitrogen and creatinine test kits were used to detect the concentration of urea nitrogen and creatinine.3.7 Histopath ological examination of kidney72 hours after the intervention,kidney samples were taken.Each kidney tissue was stained with HE and PAS to show the damage of renal tubular epithelium,and a semi-quantitative method was used to compare between each group.3.8 Detection of kidney proliferation indicatorsImmunofluorescence technology was used to detect the expression of proliferating cell nuclear antigen(PCNA)in the kidneys of each group;the Western blot method was used to detect the expression of p21 and p53 in the kidneys.3.9 Detection of kidney inflammation indicatorsImmunofluorescence technique used anti-MOMA2 antibody,anti-neutrophil antibody and anti-CXCR4 antibody to show the infiltration of macrophages and neutrophils in the kidney and the expression of chemotactic cell receptor CXCR4 in the kidney.And the Western blot method was used to detect the expression of the chemotactic cytokine SDF-1(the ligand of CXCR4)in the kidney.3.10 Detection of kidney fibrosis indicatorsWestern blot method was used to determine the expression of type 1 collagen in the kidneys of each group.4 Results4.1 The effect of hypoxia on the expression of exosomes CD26Exosomes derived from the TCMK-1 renal tubular epithelial cell line hardly express CD26,and the hypoxic cell culture environment increases the expression of exosomes CD26.4.2 Identification of CD26 overexpression exosomes The CD26 promoter was transfected into cells with adenovirus as a vector.After successful transfection,the expression of exosomalCD26 was detected to increase significantly,with a positive rate of over 70%.4.3 Identification of AKI animal modelsAfter one renal artery was clamped,the contralateral kidney was removed.After 72 hours,the mouse urea nitrogen and creatinine increased significantly,and the renal tissue showed IR pathological changes,which confirmed the success of AKI modeling.4.4 Tracing of Exosomes in AnimalsPKH26-labeled exosomes were injected into IR mice through the tail vein,and samples were taken within 2 hours,frozen sections were made,and fluorescently labeled with AQP1,which showed that fluorescent markers of exosomes were visible in renal tubular epithelial cells,and there was no fluorescence of exosomes in other types of kidney cells.Marking indicates that exosomes derived from renal tubular epithelial cells are selectively taken up by renal tubular epithelial cells.4.5 The influence of exosomes on kidney function indexesSerum urea nitrogen and creatinine levels in IR mice were significantly increased;compared with BSA,ExoNormal and ExoIR had no effect on renal function indexes,while ExoCD26 significantly reduced serum urea nitrogen and creatinine levels.4.6 The effect of exosomes on kidney tissue damageHE and PAS staining showed that the kidneys of IR mice showed obvious damage,including renal tubule dilation,brush border loss,necrosis of renal tubular epithelial cells,and cast formation.Compared with BSA,ExoNormal and ExoIR failed to relieve the kidney.Damage,and ExoCD26 significantly reduced the score of kidney damage and the score of damaged renal tubules,and reduced the damage of kidney tissue.4.7 The effect of exosomes on the proliferation of TECs Immunofluorescence staining showed that Proliferating Cell Nuclear Antigen(PCNA)in IR mice was significantly reduced.Compared with BSA,ExoNormal and ExoIR failed to increase the expression of PCNA,while ExoCD26 significantly increased the expression of PCNA.In addition,the expression of p21 and p53 in the kidney of IR mice was significantly increased,suggesting cell division and proliferation.Compared with BSA,ExoNormal and ExoIR failed to reduce the expression of p21 and p53,but ExoCD26 significantly reduced the expression of both.The above results suggest that ExoCD26 promotes the proliferation of TECs cells.4.8 The influence of exosomes on renal inflammationImmunofluorescence showed that the infiltration of neutrophils and macrophages in IR increased significantly;compared with BSA,ExoNormal and ExoIR failed to significantly reduce the infiltration of neutrophils and macrophages,while ExoCD26 significantly reduced the two types of inflammatory cells of infiltration.At the same time,western blot and immunofluorescence results showed that the expression of SDF-1... |
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