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C4d complement split product expression in chronic rejection of renal allograft
C4d Complement Split Product Expression in Chronic Rejection of Renal Allograft A. Mro´z, M. Durlik, T. Cieciura, J. Pazik, T. Ba˛czkowska, A. Chmura, S. Nazarewski, and M. Lao ABSTRACT Chronic allograft rejection remains the major cause of late renal graft loss. Its pathogenesis is complex, depending on both immunological and nonimmunological factors. An important role in development of chronic rejection is ascribed to an ongoing immunological reaction mainly of the humoral type. C4d complement split product, as a stable fragment of complement degradation activated by antigen-antibody complexes, is considered to be an indicator of humoral activity in allografts. The aim of the present study was to establish a correlation between C4d expression and morphological findings specific for chronic rejection among biopsy specimens from patients with deteriorating graft function versus protocol biopsy specimens versus biopsy specimens of native kidneys with glomerular diseases. C4d deposits in peritubular capillaries and glomeruli were observed in 83% of patients with morphological changes of chronic rejection. No C4d expression was found in the protocol biopsy group. C4d deposits in glomeruli localizations were found in kidneys from patients with glomerulopathies; the pattern of distribution was similar to that for antibodies characteristic for glomerulonephritis. There was a positive correlation between C4d expression and morphological features of chronic rejection. In our opinion, only peritubular capillary localization is specific for a rejection process; glomerular localization is nonspecific and probably secondary to antigen-antibody complex deposition in course of some types of glomerulopathies.
C
HRONIC REJECTION still remains a major cause of late graft loss. It is by no means a homogeneous process; a number of immunological and nonimmunological factors contribute to its development.1 It is a clinicopathological diagnosis. From the clinical point of view, the main features of chronic rejection include slow, but continuous, deteriorating graft function often accompanied by proteinuria and worsening hypertension. Graft biopsies, which must be performed to exclude other causes such as glomerular disease recurrence or drug toxicity, show changes affecting all compartments of the kidney. The interstitial changes of fibrosis and tubular atrophy are regarded to be nonspecific, whereas arterial intimal thickening with mononuclear infiltration and chronic transplant glomerulopathy, as evidenced by mesangial matrix widening with duplication of glomerular basement membrane, are believed to be distinctive for chronic rejection. However, transplant glomerulopathy also may be seen in thrombotic microangiopathy and certain chronic immune complex diseases. The pathogenesis of chronic rejection remains obscure.
According to one of the recent theories, its development is connected to an ongoing vascular reaction of mainly the humoral type, which results in endothelial damage and a subsequent cascade of events leading to obliteration of vessels.1 This view is supported by the fact that many patients with morphological features of chronic rejection display anti-donor HLA antibodies in the serum as well as by experimental studies showing an important role of humoral alloreactivity in the development of arteriosclerosis in mice.2 C4d is a stable complement split product covalently bound to vascular endothelium. The complement degradaFrom the Department of Transplantation Medicine and Nephrology (A.M., M.D., T.C., J.P., J.B., M.L.), Department of General and Transplantation Surgery (A.C.) and Department of General, Vascular and Transplant Surgery (S.N.), Transplantation Institute Warsaw, Warsaw, Poland. Address reprint requests to A. Mro´z, Department of Transplantation Medicine and Nephrology, Transplantation Institute Warsaw, Nowogrodzka 59 str. Warsaw PL02-006, Poland.
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00799-1
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2190
Transplantation Proceedings, 35, 2190 –2192 (2003)
C4d COMPLEMENT SPLIT PRODUCT
tion cascade is initiated by antibodies, hence the presence of C4d deposits in tissues is considered to be proof of a preceding humoral reaction. Renal biopsy specimens display 2 possible sites of C4d deposition; those in the cortical peritubular capillaries are considered specific for an alloantigen-dependent reaction while localization in glomeruli is nonspecific. The role of peritubular capillary C4d deposition for the diagnosis of acute humoral rejection is established,3 its usefulness as an indicator of immunological activity in chronic renal allograft rejection is still under investigation.4,5 The aim of this study was to determine the usefulness of C4d expression to diagnose patients with immunologically active chronic rejection. MATERIAL AND METHODS Nineteen patients, who were at least 1 year after renal transplantation and showed deteriorating graft function, were included in the study. Control groups included 10 protocol biopsy specimens from patients with stable graft function at a year after transplantation and 10 biopsy specimens with glomerular disease. All renal biopsy specimens were assessed histologically and immunomorphologically. In addition, C4d expression was localized and graded on frozen tissue (monoclonal antibodies from Quidel Company, USA) by the intensity of staining from 0 to 3.
RESULTS
C4d deposits were present in 8 study patients, 6 of whom showed C4d deposits in peritubular capillaries and glomerular tufts; 2 only had deposits in glomeruli. Specific histopathological changes for chronic rejection namely, chronic transplant glomerulopathy and/or arterial intimal thickening with scant lymphocytes infiltrates, were observed in 6 patients, 5 of whom (83%) were C4d-positive with deposits in peritubular capillaries and glomeruli. Another 3 C4d-positive patients (C4d expression in both localizations in 1 case and only glomerular localization in 2 others) displayed nonspecific interstitial changes on biopsy. C4d deposition in protocol biopsies group was not detected. Immunomorphological findings in both groups were not useful, consisting mainly of scant C3 and IgM in deposits in collapsed segments of glomerular tufts and in the walls of hyalinized vessels. C4d deposits were present in 6 of 10 biopsy specimens from the glomerulopathy group. The pattern of staining was that associated with antibodies responsible for glomerular disease: membranous in 4 cases of membranous glomerulonephritis, and mesangial in 2 cases of IgA nephropathy. The other 4 biopsy specimens represented focal, segmental glomerulosclerosis (n ⫽ 3) and minimal change disease (n ⫽ 1). DISCUSSION
One of the most important factors in the pathogenesis of chronic rejection seems to be ongoing immunological response of the humoral type.1,4,6 This theory is based on the
2191
observation of the de novo appearance of antibodies against donor HLA among recipients with deteriorating graft function.4,5 These antibodies activate the classical pathway of complement degradation, which results in the synthesis of several minute peptides. C4d complement split product is the only fragment showing a covalent binding to tissue and thereby remaining at the site of complement activation and not being degraded.6 The most vulnerable structures to antibody action are peritubular capillaries, which have been proved to remain of donor origin even 25 years after transplantation6,7 and be the site of immunological activation. In contrast C4d deposits are frequently found in the native kidneys,8 but the localization is mainly glomerular. Interestingly, in our study, the deposition pattern in the group of glomerulopathies followed the pattern of dominant causative antibody, ie, it was membranous in membranous glomerulonephritis and mesangial in mesangio-proliferative IgA nephropathy. Four other biopsy specimens from this group with focal segmental sclerosis and minimal change disease, which showed scant antibody deposition, were C4d-negative. Morphological lesions distinctive for chronic rejection were found in 6 of 19 patients from the study group. In all but 1 of these patients (5 of 6, 83%) C4d-positive deposits localized in peritubular capillaries and glomeruli were present. The correlation between C4d expression and other morphological findings (interstitial fibrosis, tubular atrophy, and glomerulosclerosis) was not studied. The immunomorphological findings in the study group were irrelevant (only scant IgM and C3 deposits) showing no recurrence of glomerulopathy. The frequency of C4d expression in peritubular capillaries among patients with morphological features of chronic rejection in our study was even greater than that in most other articles (83% vs approximately 60%),6 but the number of patients in the present series is small and the matter needs further investigation. Additionally, in the present study we did not assess the presence of antidonor HLA antibodies in serum nor did we perform ultrastructural studies to detect multilayering of peritubular capillary basement membranes, which is also believed to be a marker of immunological activity in chronic allograft rejection. The patients from the protocol biopsy control group were all C4d-negative, and their graft function remained stable. No significant global and/or segmental glomerulosclerosis was found in this group, nor were there specific immunological findings pointing to recurrent glomerulopathy.
CONCLUSIONS
Our study showed a positive correlation between C4d expression and morphological features widely accepted as specific for chronic rejection. Only peritubular capillary localization of the deposits seemed specific for immunologically active chronic rejection; glomerular localization appeared to be probably nonspecific and secondary to immune complex entrapment in the course of glomerular
2192
diseases. This observation requires further analysis in a larger study. REFERENCES 1. Paul LC: Kidney Int 56:183, 1999 2. Russel PS, Chase CM, Colvin RB: Transplantation 21:235, 1997 3. Collins AB, Schneeberger EE, Pascual MA, et al: J Am Soc
MRO´ Z, DURLIK, CIECIURA ET AL Nephrol 10:2208, 1997 4. Regele H, Bohmig GA, Habicht A, et al: J Am Soc Nephrol 13:2371, 2002 5. Watschinger B, Pascual M: J Am Soc Nephrol 13:2420, 2002 6. Mauiyyedi S, Della Pelle P, Saidman S, et al: J Am Soc Nephrol 12:574, 2001 7. Randhawa PS, Starzl T, Ramos HC, et al: Am J Kidney Dis 24:72, 1994 8. Zwirner J, Felber E, Herzog V, et al: Kidney Int 36:1069, 1989
C
HRONIC REJECTION still remains a major cause of late graft loss. It is by no means a homogeneous process; a number of immunological and nonimmunological factors contribute to its development.1 It is a clinicopathological diagnosis. From the clinical point of view, the main features of chronic rejection include slow, but continuous, deteriorating graft function often accompanied by proteinuria and worsening hypertension. Graft biopsies, which must be performed to exclude other causes such as glomerular disease recurrence or drug toxicity, show changes affecting all compartments of the kidney. The interstitial changes of fibrosis and tubular atrophy are regarded to be nonspecific, whereas arterial intimal thickening with mononuclear infiltration and chronic transplant glomerulopathy, as evidenced by mesangial matrix widening with duplication of glomerular basement membrane, are believed to be distinctive for chronic rejection. However, transplant glomerulopathy also may be seen in thrombotic microangiopathy and certain chronic immune complex diseases. The pathogenesis of chronic rejection remains obscure.
According to one of the recent theories, its development is connected to an ongoing vascular reaction of mainly the humoral type, which results in endothelial damage and a subsequent cascade of events leading to obliteration of vessels.1 This view is supported by the fact that many patients with morphological features of chronic rejection display anti-donor HLA antibodies in the serum as well as by experimental studies showing an important role of humoral alloreactivity in the development of arteriosclerosis in mice.2 C4d is a stable complement split product covalently bound to vascular endothelium. The complement degradaFrom the Department of Transplantation Medicine and Nephrology (A.M., M.D., T.C., J.P., J.B., M.L.), Department of General and Transplantation Surgery (A.C.) and Department of General, Vascular and Transplant Surgery (S.N.), Transplantation Institute Warsaw, Warsaw, Poland. Address reprint requests to A. Mro´z, Department of Transplantation Medicine and Nephrology, Transplantation Institute Warsaw, Nowogrodzka 59 str. Warsaw PL02-006, Poland.
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00799-1
© 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2190
Transplantation Proceedings, 35, 2190 –2192 (2003)
C4d COMPLEMENT SPLIT PRODUCT
tion cascade is initiated by antibodies, hence the presence of C4d deposits in tissues is considered to be proof of a preceding humoral reaction. Renal biopsy specimens display 2 possible sites of C4d deposition; those in the cortical peritubular capillaries are considered specific for an alloantigen-dependent reaction while localization in glomeruli is nonspecific. The role of peritubular capillary C4d deposition for the diagnosis of acute humoral rejection is established,3 its usefulness as an indicator of immunological activity in chronic renal allograft rejection is still under investigation.4,5 The aim of this study was to determine the usefulness of C4d expression to diagnose patients with immunologically active chronic rejection. MATERIAL AND METHODS Nineteen patients, who were at least 1 year after renal transplantation and showed deteriorating graft function, were included in the study. Control groups included 10 protocol biopsy specimens from patients with stable graft function at a year after transplantation and 10 biopsy specimens with glomerular disease. All renal biopsy specimens were assessed histologically and immunomorphologically. In addition, C4d expression was localized and graded on frozen tissue (monoclonal antibodies from Quidel Company, USA) by the intensity of staining from 0 to 3.
RESULTS
C4d deposits were present in 8 study patients, 6 of whom showed C4d deposits in peritubular capillaries and glomerular tufts; 2 only had deposits in glomeruli. Specific histopathological changes for chronic rejection namely, chronic transplant glomerulopathy and/or arterial intimal thickening with scant lymphocytes infiltrates, were observed in 6 patients, 5 of whom (83%) were C4d-positive with deposits in peritubular capillaries and glomeruli. Another 3 C4d-positive patients (C4d expression in both localizations in 1 case and only glomerular localization in 2 others) displayed nonspecific interstitial changes on biopsy. C4d deposition in protocol biopsies group was not detected. Immunomorphological findings in both groups were not useful, consisting mainly of scant C3 and IgM in deposits in collapsed segments of glomerular tufts and in the walls of hyalinized vessels. C4d deposits were present in 6 of 10 biopsy specimens from the glomerulopathy group. The pattern of staining was that associated with antibodies responsible for glomerular disease: membranous in 4 cases of membranous glomerulonephritis, and mesangial in 2 cases of IgA nephropathy. The other 4 biopsy specimens represented focal, segmental glomerulosclerosis (n ⫽ 3) and minimal change disease (n ⫽ 1). DISCUSSION
One of the most important factors in the pathogenesis of chronic rejection seems to be ongoing immunological response of the humoral type.1,4,6 This theory is based on the
2191
observation of the de novo appearance of antibodies against donor HLA among recipients with deteriorating graft function.4,5 These antibodies activate the classical pathway of complement degradation, which results in the synthesis of several minute peptides. C4d complement split product is the only fragment showing a covalent binding to tissue and thereby remaining at the site of complement activation and not being degraded.6 The most vulnerable structures to antibody action are peritubular capillaries, which have been proved to remain of donor origin even 25 years after transplantation6,7 and be the site of immunological activation. In contrast C4d deposits are frequently found in the native kidneys,8 but the localization is mainly glomerular. Interestingly, in our study, the deposition pattern in the group of glomerulopathies followed the pattern of dominant causative antibody, ie, it was membranous in membranous glomerulonephritis and mesangial in mesangio-proliferative IgA nephropathy. Four other biopsy specimens from this group with focal segmental sclerosis and minimal change disease, which showed scant antibody deposition, were C4d-negative. Morphological lesions distinctive for chronic rejection were found in 6 of 19 patients from the study group. In all but 1 of these patients (5 of 6, 83%) C4d-positive deposits localized in peritubular capillaries and glomeruli were present. The correlation between C4d expression and other morphological findings (interstitial fibrosis, tubular atrophy, and glomerulosclerosis) was not studied. The immunomorphological findings in the study group were irrelevant (only scant IgM and C3 deposits) showing no recurrence of glomerulopathy. The frequency of C4d expression in peritubular capillaries among patients with morphological features of chronic rejection in our study was even greater than that in most other articles (83% vs approximately 60%),6 but the number of patients in the present series is small and the matter needs further investigation. Additionally, in the present study we did not assess the presence of antidonor HLA antibodies in serum nor did we perform ultrastructural studies to detect multilayering of peritubular capillary basement membranes, which is also believed to be a marker of immunological activity in chronic allograft rejection. The patients from the protocol biopsy control group were all C4d-negative, and their graft function remained stable. No significant global and/or segmental glomerulosclerosis was found in this group, nor were there specific immunological findings pointing to recurrent glomerulopathy.
CONCLUSIONS
Our study showed a positive correlation between C4d expression and morphological features widely accepted as specific for chronic rejection. Only peritubular capillary localization of the deposits seemed specific for immunologically active chronic rejection; glomerular localization appeared to be probably nonspecific and secondary to immune complex entrapment in the course of glomerular
2192
diseases. This observation requires further analysis in a larger study. REFERENCES 1. Paul LC: Kidney Int 56:183, 1999 2. Russel PS, Chase CM, Colvin RB: Transplantation 21:235, 1997 3. Collins AB, Schneeberger EE, Pascual MA, et al: J Am Soc
MRO´ Z, DURLIK, CIECIURA ET AL Nephrol 10:2208, 1997 4. Regele H, Bohmig GA, Habicht A, et al: J Am Soc Nephrol 13:2371, 2002 5. Watschinger B, Pascual M: J Am Soc Nephrol 13:2420, 2002 6. Mauiyyedi S, Della Pelle P, Saidman S, et al: J Am Soc Nephrol 12:574, 2001 7. Randhawa PS, Starzl T, Ramos HC, et al: Am J Kidney Dis 24:72, 1994 8. Zwirner J, Felber E, Herzog V, et al: Kidney Int 36:1069, 1989