Evaluation of the rate of progression of chronic renal transplant rejection using the proliferating cell nuclear antigen

Abstract

Since the introduction of kidney transplantation, the short-term patient and graft survival have been progressively improving, but the long-term graft survival and half-life of transplants have not. Beyond doubt, chronic rejection (CR) remains the major cause of chronic graft failure and is responsible for the loss over 10 years of 50% of grafts. (1-4). CR is characterized clinically by proteinuria, hypertension and declining renal function, and pathologically by arteriosclerosis, glomerulosclerosis and interstitial fibrosis. The progressive morphological changes of glomerular, vascular and tubulointerstitial (TIN) compartments in renal allografts experiencing chronic rejection correlate directly with declining renal function and the eventual graft loss [5]. In consideration of this fact and using proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation, a retrospective analysis of renal allograft biopsies was carried out with the aim to determine whether quantification of the cell proliferation activity in three tissue compartments may be used as a prognostic index of CR progression. Clinical course and biopsy specimens of 27 patients with clinically and morphologically confirmed (Banff schema) diagnosis of CR were retrospectively evaluated. Patients were transplanted between 1988 and 1994 at our Institute and regularly followed-up for at least 2 years after transplantation (14 patients), or till haemodialysis has been resumed (11 patients) or till death (2 patients). The progression rate of renal graft function deterioration was estimated by slopes of regression lines obtained by plotting 1/sCr vs time. According to the progression rate after biopsy patients were divided into two groups: fast progression group (10 patients) and slow progression group (17 patients). Immunosuppressive regimens included induction with anti-lymphocyte globulin followed with cyclosporine, steroids and azathioprine. Immunohistochemistry. Sections of formalin-fixed, paraffinembedded tissue were stained for PCNA with monoclonal mouse anti-proliferating cell nuclear antigen (clone PC 10, Dako) as follows in ref. [6]. PCNA immunoreactivity of smooth muscle cells and lymphocytes in vascular intima, lymphocytes in TIN and glomerular mesangium proliferation was assessed by semiquantitative scoring (scale 0-3). Normal tonsil was used as a positive control. Differences between groups were evaluated using Student's t-test. Pearson's correlation test was used to study the relationship between variables. P values < 0.05 were considered as significant. Data on patients are presented in Table 1. The only statistical difference was in progression of chronic graft failure presented by regression lines obtained by plotting 1/sCr vs time. PCNA protein expressions in vascular, glomerular and TIN compartments for both groups are presented in Figs. 1-3.. PC 10 immunoreactivity was confined to the nucleus. The pattern of staining was granular or diffuse. Individual values of the scores of PCNA immunoreactivity in different tissue compartments for the groups examined are presented in Tables 2 and 3. Comparison of the means of PCNA immunoreactivity scores in different tissue compartments of fast and slow progression group showed that cells proliferation in glomerular, TIN and vascular compartments are significantly higher in the fast than in the slow progression group (Table 4). Besides, significant positive correlation was found between the slopes of the regression lines plotting 1/sCr vs. time and cells proliferation scores for all compartments (Table 5). Chronic graft failure may occur due to CR, cyclosporine nephrotoxicity, repeated acute rejection, recurrent glomerular diseases, surgical and urological complications, but CR was recognized as the most common cause of chronic graft failure and renal graft loss [7]. Progression of renal disease reflects the interactive effects of changes in the structure and function. This notion has been examined many times in the native kidneys by studies correlating glomerular filtration rate with architectural deterioration in the glomerular or interstitial compartment [8-10]. Meanwhile, the similar studies in human renal allograft are scarce. Increased PCNA immunodetection in glomerular cells as well as in interstitial infiltrates was described in acute and chronic renal graft rejection and diagnostic value of this finding was analyzed [12, 15]. Similarly, PCNA was often examined immunohistochemically in lymphoma and solid tumours as a marker of cell proliferation, but its reliability as a prognostic factor was also evaluated [18-20]. In the present study the prognostic value of PCNA expression in different tissue compartments of renal grafts experiencing CR was examined for the first time. The present study revealed that two groups examined with the different chronic graft failure progression rate as the main difference had significantly different proliferation of cells in glomerular, TIN and vascular compartments. PCNA immunoreactivity scores in these three compartments were significantly higher in the fast than in slow progression group. The significant positive correlation between the slope of the regression line plotting 1/sCr vs. time and cell proliferation in glomerular, TIN and vascular compartments was also found. Therefore, it was suggested that the measurement of PCNA expression in renal graft tissue might be used as a prognostic index.