C4d AND KIDNEY TRANSPLANTATION

1. C4d AND KIDNEY TRANSPLANTATION

2. C4d AND KIDNEY TRANSPLANTATION. Dr.Abhijit Kishore Korane.Department of Nephrology,Apollo Hospitals , Chennai.

3. Acute humoral rejection and some forms of chronic rejection are initiated by the binding of anti-donor antibodies to the endothelial lining of blood vessels in the graft.

4. The gold standard for the diagnosis of rejection and for guiding patient management is the histological evaluation of a renal allograft biopsy. • Detection of C4d is regarded as an indirect sign, a ‘footprint’ of an antibody response.

5. During the post-transplantational period, C4d is detected in 30% of all diagnostic graft biopsies. It is typically seen early after transplantation. • C4d can also be detected years after grafting (as late as 15 years).

6. C4d is ‘transplant specific’, As it is practically never detected along peritubular capillaries in the native diseased kidney, such as active lupus nephritis, antineutrophil cytoplasmic antibody (ANCA) disease or anti-glomerular basement membrane (GBM) disease its detection seems ‘transplant specific’. (Nickeleit V, et al. Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol2002; 13: 242–251)

7. HISTORY OF C4d

8. As early as 1960, the detrimental effect of preformed donor-specific antibodies became apparent. • 1970 by Jeannetet al. who suggested that de novo production of DSA against a transplanted donor organ can lead to severe graft injury. • Halloran et al. in the early 1990s established that acute rejection associated with the development of de novo anti-HLA DSA in recipient’s serum is a defined clinicopathologic entity carrying a poor prognosis. • They subsequently postulated that the “complement-neutrophil pathway is probably the major mechanism of antibody injury”

9. However, a specific diagnostic marker of humoral immunity was not identified, and deposits of immunoglobulins or C3 in biopsies were not found to correlate with alloantibody-positive acute rejections. • Same time Feucht et al. detected C4d, in capillaries of biopsies from patients at “high immunological risk,” and they suggested that capillary C4d deposition was evidence for humoral alloreactivity against the graft. • Basel was the first transplant centre in the world which considered C4d to be a very valuable diagnostic tool and incorporated it into the diagnostic decision-making process.

10. Acute antibody-mediated rejection is only recently recognized as a distinctive clinicopathologic entity. It was not listed in both the original (1993) and revised (1997) Banff schemes for renal transplant diseases but was officially added to this scheme in 2003.

11. COMPLEMENT SYSTEM AND THE C4d

12. Binding of complement fixing antibodies to a cell surface recruits C1qrs complexes. C1qrs cleaves and activates C4 and C2. C4b formed in this way may form covalent bonds with the cell surface and associate with C2a to form C4b2a, the classical complement pathway C3 convertase. C4b2a catalyzes cleavage of C3 and C5, amplifying complement activation. C3 convertases are controlled by various mechanisms. One mechanism involves cleavage of C4b by factor I plus MCP or C4-binding proteins as cofactors to yield C4d, which is catalytically inactive.

14. Following activation and degradation of the C4 molecule, thioester groups are exposed which allow transient, covalent binding of the degradation product C4d to endothelial cell surfaces and extracellular matrix components of vascular basement membranes near the sites of C4 activation.

15. C4d is also found in intracytoplasmic vacuoles of endothelial cells. (Regele H, et al. Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 2002; 13: 2371–2380)

16. Apart from the classical antibody-mediated route of complement activation, C4 can also be activated via an alternative, antibody-independent mechanism, the ‘mannan-binding lectin’ pathway. Thus, C4d may also be potentially deposited without prior antibody binding. (Petersen SV et al. An assay for the mannan binding lectin pathway of complement activation. J Immunol Methods 2001; 257: 107–116)

17. Based on the location of the C4d deposits along peritubular capillaries, antibodies are probably directed against peritubular capillary endothelial antigens.

18. METHODS OF DETECTING C4d IN TRANSPLANT KIDNEY BIOPSY.

19. Two methods for the detection of C4d in allograf biopsies are available. • A three-step immunofluorescence is performed on unfixed frozen tissue with a monoclonal anti-C4d antibody, • Immunohistochemistry with a polyclonal anti-C4d antibody can be done when only paraffin sections of biopsies are available. • Both methods allow identical endothelial staining of peritubular capillaries for C4d. • Differences between frozen and paraffin sections exist in the results of glomerular or mesangial C4d staining.

20. The detection of C4d by paraffin-embedded tissue samples demonstrated a substantially lower prevalence and extent of C4d expression. • On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by paraffin-embedded tissue samples than by immunofluorescence for frozen sample.

21. Staining patterns for C4d and there significance. • Of diagnostic relevance is the focal or diffuse, strong accumulation of C4d along peritubular capillaries in the renal cortex and/or medulla. • Only non-fibrotic and non-necrotic parenchymal regions should be evaluated.

22. Graph comparing two staining techniques(IF,IHC)

23. Relation of C4d and demonstration of Donar specific antibodies.

24. Depending on the testing method used e.g. crude panel-reactive antibody (PRA) titre testing vs more sensitive flow cytometry analyses, antibodies can be detected in 43–90% of C4d-positive vs 0–50% of C4d-negative patients.

25. Acute Humoral Rejection in Kidney Transplantation: II. Morphology, Immunopathology, and Pathologic Classification. J Am Soc Nephrol 13: 779–787, 2002

27. Impact of humoralalloreactivity early after transplantation onthe long-term survival of renal allograftsKidney International, Vol. 59 (2001), pp. 334–341

28. Capillary C4d Deposition in Kidney Allografts: A Specific Marker of Alloantibody-Dependent Graft Injury J Am Soc Nephrol 13: 1091–1099, 2002

30. HISTOPATHOLOGICAL FEATURES AND DETECTION OF C4d.

31. Detection of the Complement Degradation Product C4d in Renal Allografts: Diagnostic and Therapeutic ImplicationsJ Am Soc Nephrol 13: 242–251, 2002

33. Impact of humoralalloreactivity early after transplantation onthe long-term survival of renal allograftsKidney International, Vol. 59 (2001), pp. 334–341

34. Acute Humoral Rejection in Kidney Transplantation: II. Morphology, Immunopathology, and Pathologic Classification. J Am Soc Nephrol 13: 779–787, 2002

36. Expression of C4d of a normal biopsy and a case withhumoral rejection

37. Pure acute humoral rejection Peritubular capillaries contain numerous polymorphonuclear leukocytes

39. TRANSPLANT GOLMERULITIS WITH C4d STANING.

40. The minimal threshold level to call a biopsy ‘positive’ is the detection of C4d in at least 10 capillaries surrounding adjacent tubules. (Nickeleit V et al. Detection of the complement degradation product C4d in renal allografts:diagnostic and therapeutic implications. J Am Soc Nephrol 2002; 13: 242–251)

41. C4d is found in, type I rejection episodes(i.e. tubulo-interstitial):- 24-43% . type II rejection (transplant endarteritis):- 45%. type III rejection (i.e. vascular rejection with fibrinoid vascular wall necrosis or thrombosis):- 50%. Glomerular rejection (i.e. Transplant glomerulitis or glomerulopathy):- 50%. (Nickeleit V et al. Detection of the complement degradation product C4d in renal allografts:diagnostic and therapeutic implications. J Am Soc Nephrol 2002; 13: 242–251)

42. C4d and Acute Humoral Rejection

43. Acute Humoral Rejection in Kidney Transplantation: II. Morphology, Immunopathology, and Pathologic Classification. J Am Soc Nephrol 13: 779–787, 2002

47. C4d and Chronic Humoral Rejection

48. Chronic Humoral Rejection: Identification of Antibody- Mediated Chronic Renal Allograft Rejection by C4d Deposits in Peritubular Capillaries. J Am Soc Nephrol 12: 574–582, 2001

49. Some authors have suggested a specific association between C4d and chronic rejection: sclerosing transplant vasculopathy, multilayering of peri-tubular capillary basement membranes and splitting of glomerular basement membranes.

50. Most of the C4d-positive patients, • CR tested had antidonor HLA antibody (88%). • None of the C4d-negative CR tested had antidonor antibody. • 1-yr graft survival rates were C4d positive was 62% compared to C4d negative patients(25%).