Association of the presence and quantity of C4D with the severity of the kidney injury and the kidney longevity in patients with primary membranous glomerulonephritis

Object and hypothesis: The aim of this study is to evaluate the correlation between the glomerular C4d staining and demographic, clinic, histopathologic findings and kidney longevity of patients with idiopathic membranous glomerulonephritis. Lectin pathway of the complement system is thought to be activated in patients with C4d positive glomerular staining, thus, in those patients severe renal injury and decreased renal survival rate is expected. Methodology: Of the 50 patients who applied to Dokuz Eylül University School of Medicine (DEUTF) Department of Nephrology between September 2007 and September 2014 and on whom performed biopsy that diagnosed as membranous nephropathy by DEUTF Department of Pathology and evaluated as idiopathic membranous nephropathy by (DEUTF) Department of Nephrology , 23 patients are included. After the 23 patients with age of 18 and older and which were followed properly as primary (idiopathic) membranous glomerulonephritis by DEUTF Department of Nephrology choosen, they were invited to the department and discussed face to face, written informed volunteer consent was obtained for staining with C4d on previus kidney biopsy material and for the usage of history of disease data. Patients' files in the archive of hospital of DEUTF researched retrospectively. İn all of cases, biopsy specimens were obteined by Department of Nephrology, and evaluated both at the time of biopsy and reassesment for C4d staining by Department of Pathology. Kidney tissue specimens in the Department of Pathology archival were analysed for glomerular C4d staining with C4d kit (Cell Marque R; C4d SP91 monoclonal antibody). Glomerular C4d staining was performed via immunohistochemistry method. Glomerular C4d staining degrees are determined as negative (staining less than %25 in one glomerulus), mild degree (fine granular staining), intermediate degree (thichened granular) and severe degree (coarse deposites). According to these degrees of C4d, at first patients grouped as negative and mild degree stained and intermediate-severe stained. Those groups were compared according to their demographical (age, sex, follow up period) , clinical and laboratory follow up variables (proteinuria of 24 hours, serum creatinine level, creatinine clerence in terms of MDRD formula, serum albumine level at initial, in term of 3th month, in term of 6th month and last visit; existance of hypertension, conservative treatment and/or immunosupressive treatment, doubling time of serume creatinine level, %50 decrease in glomerular filtration range and existance of renal replacement) and also histopathologic (glomerular basal membrane properties, percentages of global and segmental sclerotic glomerulus, interstitial fibrosis and tubular atrophy, thickening in arteries and arteriols, existance of mesangial hypercelluarity and interstitial inflamation at light microscopy; examination of IgG, C3, Ig kappa and lambda stainings at immonofluorescence microscopy) findings. After determining the groups according to C4d staining degrees; groups which based on C4d staining pattern was composed as segmental pattern group (C4d staining less than %50 of a glomerulus) and global pattern group (C4d staining in almost full of glomerulus) were composed. Segmental and global staining pattern groups were also compared according to the same demographic, clinical, laboratory follow up variables and histopathologic variables. For statistical analysis SPSS 15.0 was used; findings were evaluated as statistically significant if p value is to be found less than 0.05. For categorical data analysis Chi-square and Fisher's exact test; for analysis of variance Mann-Whitney U and correlation analyses tests are used. Kaplan-Meier and Cox proportional hazard analyses were planned to be performed to evaluate the effect of glomerular C4d staining on renal survival. Results: Of the patients who were between ages of 34 and 72, %60 were (n=12, mean 48.83 years) male and %40 were (n=8, mean 51.75 years) female. Of 20 patients, %90 were identified as glomerular C4d-positive (n=18), and %10 (n=2) as negative. During mean follow up of 43.7 months (minimum 6; maximum 130 months), as neither any of the patients needed renal replacement theraphy, nor creatinine levels increased two-fold and GFR decreased half during the periods; we could not perform renal survival analysis. When we compared the negative-mild degreed C4d staining group with the intermediate-severe degreed C4d staining group; we did not find correlation between severity of C4d staining degrees and between demografical, clinical findings statistically. Among histopathogical variables, percentage of segmental sclerosis, deposites of immunoglobulin A, kappa and lambda staining degrees were associated with negative and mild degreed C4d staining group (p=0.017, p=0.029, p=0.049 respectively).in the same group, global glomerulosclerosis was also dominant according to intermediate-severe degreed C4d staining group, but was not statistically significant. Mean value of IgG staining degrees accompanied to global sclerosis dominancy in the same group as we compared to the intermediate-severe C4d staining group. However, yet not statistically significant; further thickening in basal membranes and arteries and interstitial inflamation in more patients attracted notice. When we evaluated gruops based on C4d staining of pattern; we determined 6 biopsies that C4d stained in segmental pattern and 12 biopsies that C4d stained as global. Staining in global pattern was statistically associated with further degrees (intermediate and severe) of C4d staining (p=0.001). After this finding, groups based on global and segmental pattern compared to groups based on C4d staining degrees in the name of segmental/global pattern factor scoring. Group with the global pattern showed statistically significant correlation with the further degrees of C4d stainig in the extent of segmental/global pattern factor scoring too (p<0.01). Also about the clinical and laboratory follow up variables, in the groups based on C4d staining pattern it was found that group of global C4d staining was associated with higher levels of proteinuria according to segmental groups when the findings analysed via proteinuria classification (p=0.027). Conclusions:. C4d staining can be confirmed usually by the immunohistochemically methods retrospectively. But it could not be shown statistically if C4d is a marker of poor prognosis in idiopathic membranous nephropathy. İn our study we found that both C4d staining degree and pattern was not correlated with traditional prognostic factors and renal survival markers. Among histopathologic findings, the significant finding of the segmental sclerosis percentage which correlated with the C4d negative and mild degreed staining statistically; and accompanied global sclerosis of glomeruli correlated with the same group without statistical meaning was remarkable. This finding is considered to focal glomerulosclerosis was a independent worse renal outcome predictor that affected by many factors and could exist from variable pathologies. The assocation between negative-mild degreed C4d staining and IgA, kappa and lambda is considered to be associated with secondary factors like post-infectious situation except primary membranous nephropathy. Yet it is not significant statistically; there were some other histopathological findings like interstitial inflamation, also thickening of basale membrane and arteries correlated with further degreed C4d staining, new studies should be performed with more patients to evaluate the correlation between thickening in arteries and degrees of C4d staining. The finding in which C4d with global pattern was highly correlated with further degrees of C4d is found to be concerned with the prevalance of global pattern of immune deposites in primary membranous nephropathy in adults. Here, C4d is considered to be a stigmate of the previous or ongoing execarbations or relapses in illness. Our study is the first report involving if C4d is effective on prognosis of idiopathic membranous glomerulonephritis in adults extensively. C4d is a split product of classical and lectin pathway of complement, so with our negatively correlated findings of C4d with prognostic factors; it comes to mind that alternative pathway is also affective on pathogenesis as a different mechanism. To research that if C4d is efficient on prognosis and renal survival in idiopathic membranous nephropathy; it is suggested that more investigations which membrane phospholipase A2 antibody and factor-B the component of alternative pathway are concluded should be performed with more patient series and longer follow-up periods. Keywords: C4d, membranous nephropathy, renal injury