Application Of CD248 Protein As A Urine Marker For Chronic Kidney Disease

BackgroundChronic kidney disease is a chronic renal structural and dysfunction caused by various causes.Renal interstitial fibrosis is one of the major pathological changes of chronic kidney disease.Pathological findings are the most direct diagnostic evidence in fibrotic diagnosis and treatment,but as invasive examinations,clinical applications are limited.Serological indicators of type ? collagen,GFR,albumin creatinine ratio,imaging ultrasound elastography,shear wave elastography and other methods due to repeatable,non-invasive researchers are concerned.The fibrosis process is complex and involves many cells and molecules.Although the current indicators directly or indirectly reflect the level of fibrosis in patients through different angles,the specificity is insufficient.The study suggests that irreversible activation of myofibroblasts is a crucial factor in fibrosis.Node,but lacks corresponding detection indicators.Screening for specific markers remains a major challenge in fibrosis research and treatment processes.CD248(endo-sialic acid protein),a type ? transmembrane glycoprotein.Recent studies have demonstrated a significant up-regulation of CD248 expression in pathological fibrotic diseases.And the degree of expression is positively correlated with the degree of fibrosis of the tissue.Studies have shown that it is closely related to myofibroblast activation,and our study also determined that interference with CD248 molecules can affect fibrosis progression.Whether CD248 molecule can be used as a new diagnostic marker for the progression of fibrosis in chronic kidney disease is of concern.ObjectivesIn this study,urine samples from patients with urnnary protein positive were used as the research object.By detecting CD248 protein in urine,combining with other indicators of renal injury and disease characteristics,we can explore whether CD248 protein can be used as a new urine marker for chronic kidney disease.Methods1.CD248-specific monoclonal antibody preparation:mice immunized with prokaryotic expression CD248 recombinant protein,high-valent mouse spleen PEG cell fusion method for fusion to obtain hybridoma cells,indirect ELISA screening for CD248-positive hybridoma cell lines;hybridoma cells After intraperitoneal injection of mice,the aspergic acid was purnfied by caprylic acid-ammonium sulfate method.The purification effect of the antibody was confirmed by SDS-PAGE.The immunoblot analysis of antibody and CD248 recombinant protein,SJSA-1 cell CD248 protein reaction,flow cytometry detection antibody and SJSA-1 cell membrane protein CD248 reaction;ELISA detection of antibody affinity.2.Clinical specimen verification:5 samples of proteinuria were randomly selected and irmmunoblotting was used to detect the content of urine CD248 protein.137 urine samples were collected(healthy human urine:10 cases;non-CKD proteinuria:22 cases;CKD proteinuria:105 cases).Among them,34 cases of diabetic nephropathy;13 cases of renal transplantation;9 cases of IgA nephropathy;49 cases of chronic glomerulonephritis.Blood creatinine,cystatin C and disease course data were collected on the day of sample collection.Immunoblotting was used to detect the expression of CD248 in urine samples of patients with chronic kidney disease.Nonparametic test(÷2 test of row x list data)analysis and CKD staging Correlation between CRF serum creatinine stage,cystatin C content,and disease course.Results1.Six CD248-specific monoclonal antibodies(M3F,M3H,M4A,M4B,M4C,M5)were screened.Western blot results showed that all 6 antibodies could bind to MBP-CD248 recombinant protein and SJSA-1 expressed CD248 protein.Three antibodies in flow cytometry could bind to CD248 protein expressed by SJSA-1:M3F,M4C The affinity of the 6 strains of antibodies was(unit:mM):M3F:0.07,M3H:0.07,M4A:0.08,M4B:0.105 M4C:0.05,M5:0.32.2.1.Western blot analysis showed that CD248 was expressed in proteinuria;2.CD248 positive rate in non-CKD:13.6%,CD248 positive rate in CKD urine:53.3%;P<0.05;3.CKD1-5 The positive rates of CD248 in urine were 33.3%,53.3%,55.5%,51.7%,and 61.3%,respectively.P<0.05;4.In the CRF creatinine stage,the positive rates of CD248 in the 5 groups were 36.6%and 61.5%,respectively.50%,62.5%,66.7%;P<0.055;5.Cystatin C normal reference value group CD248 positive rate:33.3%,greater than the reference value of the upper line group positive rate:61.2%;P<0.056;6.diabetic nephropathy course 1-5 The positive rates of CD248 in the groups of 5,1-10,and 10.1 years were:50%,52%,and 66.7%,respectively;P<0.05;the distribution of CKD in each group was P>0.05;6After 7.years of transplantation and more than 5 years of transplantation,the positive rates of CD248 were 14%and 50%,respectively.The distribution of CKD in the two groups was P>0.05.Conclusion1.All the 6 antibodies could bind to the denatured epitope of CD248 protein;3 antibodies could bind to the CD248 spatial conformation site;the affinity of M6 was the highest in the affinity detection of 6 antibodies.2.CD248 may have potential as an early detection marker for chronic kidney disease,and the expression of CD248 in urine may be more clinically valuable than CKD in the progression of the disease.