Cd80 And CTLA-4 As Antidiastole Markers In Adult Minimal Change Disease

BackgroundMinimal change disease(MCD)is a common form of idiopathic nephrotic syndrome(INS)that accounts for 10 to 15%of nephrotic diseases in adults.MCD is a serious and challenging disease for adult-onset patients,of which around 25%are steroid-resistant.Compared to children,adult-onset MCD patients were reported to delay responses to glucocorticoids treatment.It has been reported that 73%of MCD patients experienced at least one relapse,of which 28%suffered frequent relapses.The mechanism of steroid-resistant MCD is unknown.The characteristics of patients(e.g.,age or medication compliance)and pathologic misdiagnosis,which may be due to similar imaging features of MCD,focal segmental glomerulosclerosis(FSGS),and stage I of idiopathic membranous nephropathy(IMN)under light microscopy are possible causes.Long-term prognosis may not be favorable,as indicated in a study in which a large number of patients with adult-onset MCD were found to have FSGS on a second kidney biopsy and experienced progression to end-stage renal disease(ESRD).According to the North American Pediatric Renal Trials and Collaborative Studies,steroid-resistant nephrotic syndrome constitutes the second most frequent cause of ESRD in the first 2 decades of life.Therefore,early-stage identification of steroid-resistant nephritic syndrome is needed in MCD patients.The pathogenesis of MCD remains unclear;however,several hypotheses have been proposed.For several decades,MCD has been considered a T-cell disorder,and increased levels of several cytokines were also suggested.Recently,a proposed"two-hit" theory proposed the induction of CD80(B7-1)and regulatory T-cell dysfunction,with or without impaired autoregulatory function of podocytes.Several studies of children suggested detecting urinary CD80 level to distinguish MCD from FSGS,but there have been few studies of adultMCD.Abatacept(cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein[CTLA-4-Ig]),a costimulatory inhibitor that targets CD80,has been used in CD80-accociated nephropathy.However,its effectiveness is controversial.Here,we aimed to evaluate whether CD80 and CTLA-4 could be diagnostic markers in adult MCD,and whether these markers could be useful for predicting the effectiveness of single-glucocorticoid treatment in adult-onset MCD patients.Materials and methods1.PatientsAll patients were followed at Shandong Provincial Hospital.Patients over 14 years old are recommended by specialist physicians instead of pediatricians in China and receive diagnosis and treatment following standards for adults.Before participation in this study,written informed consent was obtained from all patients and their parents/guardians.2.Inclusion and exclusion criteria:Our research subjects were first selected from the hospitalized patients in the nephrology department of Shandong Provincial Hospital,from March 2014 to December 2016,with a mean follow-up visit time of 14.9 months per case.Our inclusion criteria were:(1)serum albumin<30 g/1 and 24 hours urinary protein>3.5g;(2)age no less than 14 years old;(3)renal pathological diagnoses as MCD or IMN;renal pathologic diagnoses of patients were established using light and electron microscopy;(4)eGFR calculated by Creatinine Equation higher than 60 ml/min per 1.73 m2.The exclusion criteria were:(1)pregnancy,malignant tumor,or urinary system lithiasis;(2)seconary nephrotic syndrome,for example,hepatitis B associated glomerulonephritisand lupus nephritisand so on.(3)combined with other autoimmune diseases,such as rheumatoid arthritis.The IMN patients were the control group.3.Therapeutic effect assessment indexComplete Remission(CR):urinary protein excretion<0.3 g/d or urine protein:creatinine ratio(uPCR)<30 mg/mmol.Partial Remission:urinary protein excretion<3.5 g/d or uPCR<350 mg/mmol and a 50%or greater reduction from peak values,accompanied by an improvement or normalization of the serum albumin concentration.Relapse:proteinuria>3.5 g/d or uPCR>350 mg/mmol.Steroid resistance:failure to achieve remission after 16 weeks of corticosteroid therapy.Steroid sensitivity:achieved CR during16 weeks of corticosteroid therapy.Glucocorticoid treatment:prednisone 1 mg/kg per day for 8 weeks,and then reduce the 10%of total dosage every two weeks.4.CD80 and CTLA-4 measurementsSerum and urinary CD80 and CTLA-4 were detected when patients were in relapse or complete/partial remission,and 24-hour urinary protein,uPCR and serum albumin were measured in the same day.CD80 and CTLA-4 levels were measured using a commercially available ELISA kit(Bender MedSystems,eBioscience,Vienna,Austria),and results were adjusted for urinary creatinine excretion.Urinary creatinine level and protein and serum albumin levels were measured using an autoanalyzer.5.ImmunohistochemistryKidney samples were obtained from excess tissue corresponding to kidney nephrectomy specimens donated to the biobank of Shandong University after diagnostic evaluation.The primary antibodies were mouse anti-human monoclonal CD80(1:150)and CTLA-4(1:100,both Santa Cruz Biotechnology),and the secondary antibodies were a rabbit anti-mouseBiotin-Streptavidin HRP Detection Systems(Zhongshanjinqiao Biotechnology company,Beijing,China).Sections were counterstained with Carazzi's hematoxylin.6.Statistical analysisData were described as means±SEor means±SD(only for age).Means were compared by one-way Anova and t-test and comparions of 95%CI.Statistical analyses were performed with SPSS 17.0.Figures were performed with GraphPad.Prism.v5.0 and Photoshop CS6.Receiver operating characteristic(ROC)curve analyses were performed with Medcalc 17.0.Results were considered significant at p<0.05.ResultsWe detected serum and urinary CD80 and CTLA-4 levels by ELISA in 55 patients with biopsy-proven MCD and 26 patients with biopsy-proven idiopathic membranous nepliropathy(IMN).1.Comparison of CD80 and CTLA-4 levels ofsteroid-sensitive MCD patients in relapse and remissionWe detected all the laboratory data(levels of serum albumin,24-hour urinary protein,serum CD80 and CTLA-4,urinary CD80 and CTLA-4)for all our steroid-sensitive MCD patients when they were in relapse and remission.Urinary CD80 excretion was lower for MCD patients in remission than relapse(156.65 ±24.62vs1066.40±176.76 ng/g creatinine;p<0.0001).However,mean urinary CTLA-4 levels were greater for MCD patients in remission than relapse(728.73± 89.93vs 151.70± 27.01 ng/g creatinine;p<0.0001).The area under the receiver operating characteristic curve(AUC)comparing MCD patients in relapse versus remission was 0.957 for urinary CD80 and 0.928 for urinary CTLA-4,with no significant difference between these two AUCs.The serum CD80 and CTLA-4 levels of steroid-sensitive MCD patients in relapse were not statistically different from these patients when they were in remissionA limited number of biopsies were available for study byimmunohistochemistry,including 12 cases of MCD in relapse,4 cases of MCD in remission.The remission in the 4 cases of MCD was partial(proteinuria<1 g/24 h)at the time of renal biopsy,and several days after biopsy,all 4 cases showed CR.The results showed that CD80 was present in the glomeruli of patients with steroid-sensitive MCD in relapse,but was minimal or absent for those with steroid-sensitive MCD in remission.CTLA-4 was minimal or absent in the glomeruli of patients with steroid-sensitive MCD in relapse,but was present in the glomeruli of those with steroid-sensitive MCD in remission.2.Comparison of urinary CD80 and CTLA-4 excretion of steroid-sensitive MCD patients in relapse,steroid-resistant MCD patients in relapse and IMN in relapseWe compared the CD80 and CTLA-4 levels of 32 steroid-sensitive MCD patients in relapse,23 steroid-resistant MCD patients in relapse,and 26 IMN patients in relapse.The urinary CD80 level of steroid-sensitive MCD patients in relapse was significantly higher than that of steroid-sensitive MCD patients in relapse(1066.40±176.76 vs 203.78 ± 30.65 ng/g creatinine;p<0.0001),and it was also significantly higher than that of IMN patients in relapse(1066.40± 176.76 vs 294.95± 34.08 ng/g creatinine;p<0.0001).Urinary CTLA-4 levels of steroid-sensitive MCD patients in relapse were significantly lower than those of steroid-sensitive MCD patients in relapse(151.70±27.01 vs 457.83 ± 99.45 ng/g creatinine;p = 0.006),and they were also significantly lower than those of IMN patients in relapse(151.70±27.01 vs 299.53±47.46 ng/g creatinine;p = 0.006).The urinary CD80 levels of steroid-resistant MCD patients in relapse was not different statistically from IMN patients in relapse,as were the urinary CTLA-4 levels of steroid-resistant MCD patients in relapse.Serum CD80 or CTLA-4 levels did not differ among groups.In comparing steroid-sensitive and steroid-resistant MCD patients in relapse,the AUC for urinary CD80 level was 0.937,and for urinary CTLA-4,it was 0.736.In comparing steroid-sensitive MCD patients and IMN patients in relapse,the AUC for urinary CD80 was 0.867,and for urinary CTLA-4,it was 0.721.A limited number of biopsies were available for study,including 12 cases of steroid-sensitive MCD in relapse,5 cases of steroid-resistant MCD in relapse,and 6 cases of IMN in relapse.CD80 was present in the glomeruli of patients with steroid-sensitive MCD in relapse,meanwhile,CTLA-4 was minimal or absent in the glomeruli of patients with steroid-sensitive MCD in relapse.Both CD80 and CTLA-4 were present in the glomeruli of patients with steroid-resistant MCD and IMN in relapse,however,cpmpared with steroid-sensitive MCD in relapse,the levels were minimal.ConclusionsIn conclusion,for patients with MCD,strongly positive CD80 expression and simultaneous negative CTLA-4 expressionin glomeruli,or higher urinary CD80 level and lower urinary CTLA-4 level,glucocorticoids therapy may achieve complete remission.Urinary CD80 and CTLA-4 levels may play a role in diagnosis and steroid therapy effectivity as non-invasive biomarkers.Further studies investigating the precise mechanisms of the interaction of CD80 and CTLA-4 during the whole course ofMCD are needed.