Detecting Serum Biomarkers For Chronic Renal Allograft Dysfunction And Initial Study Of Galectin-7, A Candidate Biomarker

Objective:Most late renal allograft failure is attributed to chronic allograft dysfunction(CGD),but its precise diagnosis,pathogenesis and treatment are largely unknown.Renalbiopsy remains the most reliable method for detecting CGD post-transplantation,but it isassociated with significant patient morbidity and mortality.The purpose of this study was toidentify biomarkers of chronic renal allograft dysfunction in human serum usingtwo-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phasehigh-performance liquid chromatography (RP-HPLC) followed by electrospray ionizationmass spectrometry (ESI-MS).The expression of Galectin-7,a candidate biomarker,inallograft tissue and the effect of Galectin-7 on immunological function were further studied.Methods:Serum samples were divided into four groups:chronic allograft dysfunction(CGD),long-term stable renal function (SRF),acute rejection (AR) and normal volunteer (N).Firstly,the highest abundance proteins in serum samples were selectively removed usingMultiple Affinity Removal Column.Differential protein analysis was performed using 2-DDIGE.These spot features were excised,digested by trypsin,and analyzed byRP-HPLC-ESI/MS.Galectin-7 and Clusterin,two of these identified proteins was confirmedby ELISA analysis in the independent set of serum samples.The expression of Galectin-7 inrenal biopsy specimen from CGD,SRF and AR groups was detected using immunohistochemistry technology.Recombined Galectin-7 at different concentrations(0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL) and T lymphocytes of mouse had been coculturedwith or without anti-CD3 and anti-CD28 antibody for 5 days in vitro.The proliferation of Tlymphocytes and subpopulations was analyzed using FACS.Recombined Galectin-7 atanother concentration gradient (2ng/mL,4ng/mL,8ng/mL,20ng/mL,200ng/mL) had beencocultured with T lymphocytes of mouse for 4 hours and 24 hours in vitro.The earlyapoptosis and late apoptosis of T lymphocytes were analyzed using FACS.Results:A total of 39 protein spots were found to be significantly up regulated or downregulated in serum from CGD group compared to other three groups.22 differentiallyexpressed proteins were identified in CGD serum,including apolipoprotein A-I precursor,complement C4-A precursor,complement factor H-related protein 1 precursor,component C9precursor,galectin-7,clusterin precursor,homerin,Beta-2-glycoprotein 1 precursor,et al.ELISA result showed that Galetin-7 was significantly up regulated in serum from CGD groupcompared to SRF group and to N group and Clusterin was significantly up regulated in serumfrom CGD group compared to SRF group.Galectin-7 was high expressed in endochylema ofatrophied tubular epithelial cells of renal biopsy specimen from CGD patients.The count ofGalectin-7 positive cells per high power field was significantly greater in renal biopsyspecimen from CGD group compared to SRF group and to AR group.In vitro recombinedGalectin-7(0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL) could significantly induce theproliferation of T lymphocytes of mouse.Recombined Galectin-7 could synergy to promotethe proliferation of T lymphocytes when it was used with anti-CD3 and anti-CD28 Antibody.CD8+T lymphocytes were the major proliferated T lymphocytes.RecombinedGalectin-7(2ng/mL,4ng/mL,8ng/mL,20ng/mL,200ng/mL) had no effect on the earlyapoptosis and late apoptosis of T lymphocytes of mouse after 4 hours or 24 hours' cultivationwith T lymphocytes.Conclusion:There are set of differentially expressed proteins from CGD groupcompared with AR,SRF and N groups.These differentially expressed proteins were involvedin different physiologic functions and this research provided the basis for further validation oflarger sample from multiple centers.Our data also showed that confirmed Galectin-7 andClusterin may be serum biomarkers of CGD patients.Galectin-7 was significantly up regulated in serum and renal biopsy specimen from CGD group.Recombined Galectin-7promoted the proliferation of T lymphocyte but not apoptosis in vitro which suggestsGalectin-7 may play an important role in mechanisms of CGD.Galectin-7 may become a newtarget for treatment of CGD.