Increased content of fibronectin and laminin in glomeruli isolated from chronically rejected human renal allografts.

Abstract

Chronic rejection is frequently associated with the development of glomerular lesions consisting of mesangial cell proliferation, increase in mesangial extracellular matrix (ECM)* along with thickening of glomerular basement membrane (GBM). The GBM and mesangial matrix are composed of a mixture of collagens, glycoproteins, proteoglycans, and glycosaminoglycans that not only create a structural support for glomerular cells but also influence their activity (1). Fibronectin (FN), a 200-kD glycoprotein, is found in plasma as well as in the GBM and mesangial ECM. The FN is a chemotactic factor for monocytes, macrophages, and fibroblasts, and provides a mitogenic signal for fibroblasts and mesangial cells (2). The FN seems to play a role in the initiation of in situ immune complexes in some forms of glomerulonephritides (3). Laminin (LN) is the major adhesive glycoprotein of GBM and appears as a key molecule in mediating cell interactions with basement membranes (1); LN may also possess growth-promoting activity (4). Interactions between cells and ECM proteins are mediated through receptors belonging toβ1 and β3 integrin family that are present on glomerular as well as circulating cells (5). These cell-matrix interactions may affect migration of lymphocytes as well as provide a costimulatory signal to T cells (6). We measured the content of FN and LN in glomeruli isolated from renal allografts with chronic rejection. Samples of renal tissue were obtained during graftectomy from 13 patients (6 male, 7 female patients) of 33.9±1.6 years (mean ±SEM; range, 36-76 years). All recipients had terminal failure of kidney allograft due to chronic rejection after 32±19 months (mean ±SEM; range, 14-155 months) posttransplant. In all specimens from removed grafts, severe obstructive vascular changes, typical for chronic rejection, were observed as well as mesangial matrix expansion with thickening of basement membrane representing transplant glomerulopathy. According to Banff schema (7), mild chronic transplant glomerulopathy was presented in 38%, while moderate and severe changes were found in 38% and 23% of chronically rejected kidneys, respectively. The glomeruli also exhibited a progression of ischemic changes with sclerosis. The percentage of completely fibrosed glomeruli varied from 0% to 35% (mean 9%) of total number of glomeruli present in specimens. By immunofluorescence, in most cases there were nonspecific segmental deposits of IgM and fibrin, sometimes associated with trace amounts of C3. Normal renal tissue was obtained during nephrectomy performed due to limited kidney carcinoma. Samples from 13 patients matched according to age and gender were studied. The differential sieving method was employed (8) yielding a highly purified (>95%) fraction of intact glomeruli. Isolated glomeruli were sonicated, and the content of FN in the homogenate was measured by means of enzyme-linked assay as previously described(9). The LN concentration in homogenates was estimated with a laminin EIA kit (Biomedical, Kyoto, Japan), and protein content using BCA-Protein Assay (Pierce, Beijerland, the Netherlands). Glomeruli isolated from renal tissue obtained during graftectomy contained significantly higher amounts of FN and LN as compared with normal kidney(Table 1). We calculated the amounts of intraglomerular FN and LN according to protein content. Thus our results were not affected by the number of glomeruli in preparation and/or differences in the diameter of glomeruli, and they reflect actual increases in ECM proteins studied in glomeruli during chronic rejection. It has been previously shown that in the course of various kidney diseases LN and FN accumulate in glomeruli (1). We analyzed kidneys with histopathologic changes of chronic rejection; cases of de novo or recurrent glomerulopathy were excluded. Therefore, our findings reflect changes in ECM occurring during chronic rejection. In normal kidney FN is found in the mesangial area, stroma, and endothelium while laminin has been detected only within basement membranes(1). In dysfunctional renal allografts, Bukovsky et al.(10) did not find increased reactivity with anti-FN antibody. However, also using immunohistochemistry, Gould et al.(11) observed that during chronic rejection there was a strong expression of isoforms of cellular FN in most glomerular mesangial area and in vascular walls in human allografts. In the model of accelerated rejection of cardiac allograft (Kupiec-Weglinski JW, personal communication), there was an early increase in intragraft FN synthesis. Using immunohistochemistry, increased expression of LN has been described in vascular lesions developed during acute and chronic rejection(12). By employing a quantitative method, a 2.5-fold increase in LN in the glomerulus was found. In all specimens studied, the histopathologic changes typical for allograft glomerulopathy were observed. Allograft glomerulopathy represents a distinct entity that should be differentiated from glomerular ischemic lesions induced by vascular and interstitial changes of chronic rejection(13). Immune mechanisms are probably involved in the development of allograft glomerulopathy. Increased content of FN and LN changes the architecture of glomerulus but also affects immune response and cell-matrix interactions (1). Accumulation of FN has been implicated in the development of glomerular changes in the course of chronic glomerulonephritides (14). Also, LN influences lymphocyte migration (15), and quantitative changes in the content of this ECM glycoprotein may contribute to the pathomechanism of chronic rejection.