| Abstract|| |
The chronic dysfunction stands as the most common cause of renal allograft loss. During the nineties of the past century, this condition was referred to as chronic allograft nephropathy (CAN). Since 2005, CAN has been assigned by the eighth Banff schema to four main categories via histopathological and immunohistochemical findings including chronic antibodymediated rejection (CAMR), chronic T-cell-mediated rejection (CTMR), chronic cyclosporine toxicity (CNITOX), and "interstitial fibrosis (IF)/tubular atrophy; not otherwise specified (NOS)" to eliminate the term CAN. We conducted a retrospective study of renal allograft cases with biopsy-proven chronic damage diagnosed at our nephropathology units, between January 2007 and September 2013, to assign them to the defined categories. Differences between groups were tested using one-way analysis of variance. The frequencies of the diagnostic categories were as follow: CNITOX (43.1%), CAMR (27.5%), CTMR (17.6%), and NOS (11.8%). The serum creatinine level, time posttransplant, and global sclerosis frequency were insignificant among the categories. Nine categorized cases showed transplant glomerulopathy; five of them were seen in association with CAMR. There was a positive relationship between the number of interstitial CD8 + T cells and the degree of IF in CTMR cases. Two cases showed combined features of CAMR and CTMR. Protocol renal allograft biopsy starting 3 months after transplantation with proper monitoring and adjustment of the calcineurin inhibitors level may reduce the potential risk of chronic damage in renal allograft.
|How to cite this article:|
Hamza WM, Ali HH, Gabal SM, Fadda SA. Chronic renal allograft dysfunction in Egyptian population: Histopathological and immunohistochemical study. Saudi J Kidney Dis Transpl 2016;27:921-8
|How to cite this URL:|
Hamza WM, Ali HH, Gabal SM, Fadda SA. Chronic renal allograft dysfunction in Egyptian population: Histopathological and immunohistochemical study. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2020 May 31];27:921-8. Available from: http://www.sjkdt.org/text.asp?2016/27/5/921/190818
| Introduction|| |
Revising the treatment options of chronic renal failure, the renal allograft transplant is favored with longer survival, better life quality, and lower budget than the dialysis.  The chronic dysfunction of the allograft is the most predominant cause of renal graft failure through the first 10 years following transplantation with a mysterious etiopathogenesis.  Many terms were used to signify such condition;  some cornered the condition as alloimmune-mediated while most lack signifying the condition etiology.
Although the term chronic allograft nephropathy was introduced by the first Banff working classification group to encourage the investigators to define more precisely the lesions underlying the chronic allograft damage;  it was misused as a wastebasket diagnosis. Thus, the eighth Banff meeting eliminated this term and replaced it with etiology-based diagnostic categories that included: the chronic antibody-mediated rejection (CAMR), the chronic T-cell-mediated rejection (CTMR), and the chronic toxicity of the calcineurin inhibitors immunosuppressive drugs [chronic cyclosporine toxicity (CNITOX)], together with the still enigmatic chronic damage termed "interstitial fibrosis and tubular atrophy (IF/TA); not otherwise specified (NOS)." 
Intuitively, the determination of the incriminated factors underlies the chronic injury is of utmost value as it may suggest changes in patients' treatment strategies and can recognize categories of allograft injury that are at higher risk of progression. 
This study was designed to categorize cases of biopsy-proven chronic renal allograft damage presented to our nephropathology units according to the eighth Banff classification schema, and to determine the most prevalent underlying cause of chronic damage among these cases. Correlation was done with the available clinical data as well as other associated histopathologic parameters.
| Materials and Methods|| |
Between January 2007 and September 2013, a total of 418 renal allograft biopsies were received at our nephropathology units. All cases diagnosed as "IF/TA" (101) were collected as paraffin blocks; those with recurrent/de novo glomerulonephritis diagnosed on clinical and histopathological bases were excluded from the study. Local Ethics Committee approved the study.
Patients' data: age, sex, duration of the allograft, laboratory findings notably the serum creatinine level, and the current therapeutic regimen were recorded.
Material preparation: for histopathological evaluation, the available archived slides were collected. For immunohistochemical evaluation, additional sections (3 μ thick) were prepared from nonexhausted paraffin blocks on charged glass slides, treated for antigen retrieval, and then treated with antibodies using avidinbiotin-peroxidase technique. 3,3'-Diaminobenzidine was used as a substrate and chromogen. Hematoxylin was used as a counterstain. Sections were treated with: (1) CD8 "monoclonal antibody, ready to use" (Thermo scientific, "SP16," Fremont, CA, US): a marker of cytotoxic/suppressor T lymphocytes.  (2) C4d "polyclonal antibody, ready to use" (Spring bioscience, Pleasanton, CA, US): an immunological marker for the humoral alloresponse.  Sections from a lymph node with florid reactive follicular hyperplasia were used as a control material for both makers.
Histopathological examination: All cases were examined using light microscope by two observers, the percent of glomerular sclerosis was estimated. The following items were scored (0-3) as defined in the Banff schema:  Glomerulitis (g), transplant glomerulopathy (cg), tubulitis (t), tubular atrophy (ct), interstitial inflammation (i), interstitial fibrosis (ci), vasculitis (v), chronic vascular changes (cv), arteriolar hyalinosis (ah), and peritubular capillaritis (ptc). Both acute and chronic sum scores were calculated, then the type of acute rejection (if present) and grade of chronicity were determined.
Immunohistochemical examination: The number of CD8 + interstitial Tcells was counted at a high power field (×400) throughout the entire cortical region of the biopsies in a single section, combined with a subjective assessment of the percentage of CD8 + cells relative to the whole interstitial infiltrate, and detection of CD8 + cells incriminated in tubulitis and chronic vascular changes. A cutoff limit for a predominant CD8 + T-cell infiltrate was postulated to dominate ≥60% of the entire interstitial infiltrate. C4d staining was scored (0-3); positive staining was considered by the presence of circumferential C4d deposition in more than 50% of ptc cross-sections (score 3). 
Cases were assigned to four diagnostic groups according to eighth Banff schema:  (1) CAMR: in presence of diffuse C4d positivity in the ptcs and associated chronic lesions (i.e., transplant glomerulopathy (TG) and/or chronic vasculopathy). (2) CTMR: in the presence of evidence of ongoing T-cell activity at the tubulointerstitial level including tubulitis in nonatrophic tubules and interstitial infiltrate in noncompletely scarred areas (below the threshold of acute T-cell-mediated rejection) and associated chronic lesions (i.e., chronic vasculopathy and TG). (3) CNITOX: in presence of both nodular arteriolar hyalinosis and striped interstitial fibrosis, and absence of sings of both CAMR and CTMR. (4) IF/TA; NOS: considered in the absence of speciﬁ c histopathologic and immunohistochemical features.
To assign cases showing concomitant features of more than one category, a diagnostic hierarchy was adopted in this study categorizing cases showing C4d positivity as CAMR; nevertheless, the percentage of associated CD8 + interstitial T cells. Cases showed a predominant CD8 + interstitial cellular infiltrate were categorized as CTMR. The distinction between CNITOX and IF/TA; NOS cases were based on the presence/absence of drug toxicity features.
| Statistical Analysis|| |
Data were handled using the GraphPad Instat computer software version 3.10 (GraphPad Software, Inc., La Jolla, CA, USA) and summarized using the mean and standard deviation for quantitative variables, whereas the frequency and percentages were used for the qualitative ones. Differences between groups were tested using one-way analysis of variance.
| Results|| |
In this survey of renal allograft biopsies (418 cases), about one-fourth (101 cases) were histologically documented to have "IF/TA." These cases included 76 males (75.2%) and 25 females (24.8%), with M:F ratio (3.04:1) and wide age range (7-66 years); their biopsies obtained between 3 and 252 posttransplant months. Only 51 out of 101 cases were feasible for further categorization as the rest of the cases had insufficient material for further sectioning of the paraffin blocks to perform immunohistochemical staining. Near half of the categorized cases were attributable to drug toxicity. The categorized cases were CAMR (14 cases, 27.5%), CTMR (9 cases, 17.6%), CNITOX (22 cases, 43.14%), and IF/TA; NOS (6 cases, 11.8%). The chronic biopsy changes presented as early as three months posttransplantation. The main demographic and clinical data are presented in [Table 1].
About one-fifth of all cases showed TG as well as chronic vascular changes [Table 2]. The IF was the backbone in all cases. Twenty cases were accompanied by an active rejection process. The borderline changes were encountered in 21 categorized cases (excluding the CTMR cases).
Although 55.5% of cases with TG were encountered with CAMR and 51.7% of those with tubular injury were seen with CNITOX, both lesions were encountered in other categories [Table 3] . Only one categorized case showed marked TA, on the same line; marked IF was encountered in three categorized cases; two of them were CAMR. More than half of cases with chronic vascular changes were seen with CNITOX. There was no direct relationship between the degree of arteriolar hyalinosis and the grade of IF. Marked arteriolar hyalinosis was encountered only in two cases of CNITOX and CTMR groups; one case per each. Marked chronic allograft changes were encountered only in CAMR and CNITOX. There was a highly significant difference in the mean number of CD8 + T cells, and their percentage relative to the whole interstitial infiltrate in CTMR versus the other categories [Table 4]. Only two CAMR cases showed about 60% interstitial CD8 + T-cell infiltrate; they were categorized as CAMR based on the presence of diffuse C4d positivity.
|Table 3: Frequency of various encountered histopathological findings in categorized cases.|
Click here to view
|Table 4: Number and percentage of interstitial CD8+ T lymphocytes (relative to the entire interstitial infiltrate) in categorized cases.|
Click here to view
We encountered a positive relationship between the number of interstitial CD8 + T cells and the grade of IF [Figure 1]. No CTMR cases with Grade (3) interstitial fibrosis were detected in our study. There was no direct relationship between the number of CD8 + T cells and the degree of chronic vascular changes in CTMR cases.
|Figure 1: Relation between the number of interstitial CD8+ T cells and the degree of interstitial fibrosis (ci) in CTMR cases.|
Click here to view
| Discussion|| |
In the present study, all cases of renal allograft biopsy presented to our units were reviewed and those diagnosed as IF/TA were subjected for further evaluation to be categorized according to the eighth Banff schema.  The current study highlighted the magnitude of IF/TA in biopsied renal allograft patients in our hospital over the past six years, where chronic changes were detected in 24% of them. Serón et al  and Ali et al  reported higher frequencies (53% and 38% respectively). These considerable percentages alarm the necessity for better planning of posttransplant monitoring with more clinicopathological correlation to prevent or at least minimize the frequency of such graft problems. The lack of protocol biopsy application in our hospital with reliance on indication biopsy might also have hindered application of possible preventive measures.
By a personal contact with the authors of a similar work in Italy,  we ascertained that there is no dominant percentage of active Tcell component in IF/TA cases reported in the literature, however; to categorize cases enrolled in this study in a hierarchical manner, we postulated a percentage cutoff limit for interstitial CD8 + T cells dominance relative to the whole interstitial infiltrate (≥60%).
Drug toxicity (viz.; cyclosporine) was the most frequent cause of IF/TA in this cohort (43.14%) that is consistent with findings reported by Divella et al  where the frequency of CNITOX was also the highest (35.44%) among their cases; on the other hand, they reported higher frequencies of CTMR and IF/TA; NOS (25.3% and 22.8%), respectively, whereas the frequency of CAMR was lower (16.45%). These differences can be partially attributed to different ethnic groups with the subsequent varied genetic basis of immunologic responses as well as possibly dissimilar immunosuppressive regimens. This dominance of cyclosporine toxicity points to a possible role of omitting regular monitoring in our patients.
Although the posttransplant durations and the serum creatinine levels at the time of graft biopsy were not significantly different among the categories; earlier presentation and lower mean level of serum creatinine were observed in CNITOX cases compared to CTMR and CAMR cases. These data are also concordant with the results of Divella et al. 
In the current study, TG was encountered in 18% of all IF/TA cases; an incidence very close to that reported by Kieran et al  (20%). Al Aly et al  encountered higher frequency of TG (37%). On the other hand, Vongwiwatana et al  and Sis et al  reports were much lesser (2.7% and 5.1%), respectively.
TG was reported being C4d positive ,,,, and negative. ,,,, Many possible causes of such variation were explained with hypotheses. ,, In our work, however, five cases (55.5%) were C4d-positive, TG was detected in all categories.
Furthermore, in the current study, the diagnostic challenge of TG was intensified by the unavailability of electron microscope examination of histologically suspicious cases as well as the morphological overlapping between TG, thrombotic microangiopathy, and recurrent membranoproliferative glomerulonephritis of varied etiologies that is in most of such cases lack proper clinical information aiding in solving the dilemma.
Among all categorizes, CAMR monopolized marked TA (one case) and marked IF (two cases) which in line with the observation that CAMR group showed the highest serum creatinine level points out that CAMR is likely associated with poorer graft prognosis.
In harmony with previous reports, ,, a direct relationship between the number of CD8 + interstitial cells and the degree of IF was observed in CTMR cases, a finding that threw light on the direct relationship between the degree of interstitial infiltrate and the associated tubulointerstitial histologic parameters suggestive of chronicity.
Chen et al  supported that acute interstitial cellular rejection can lead to IF but not chronic vasculopathy. In the current study, six CTMR cases were not associated with chronic vasculopathy.
Acute active rejection (Type IA) was encountered in 17.8% of all IF/TA cases in the present work. The same type of rejection was reported by Shishido et al  in 34% of their IF/TA cases. It worth noting that our cases were maintained on posttransplant triple therapy (cyclosporine, mycophenolate mofetil, and steroids) without routine induction protocol.
A much higher proportion of CD8 + T cells was observed in all groups relative to that reported by Divella et al;  notably in CTMR group, this could be explained by the association of acute active rejection Type IA in five CTMR cases in the current study.
We also encountered acute active rejection (Type IA) in cases categorized as CAMR and CNITOX. This could be explained by being either an acute exacerbation of a smoldering T-cell-mediated rejection process or a superadded acute rejection on top of a chronic one with antibody-mediated or drug-induced etiopathogenesis, respectively.
Although a diagnostic hierarchy to assort the cases in different categories was adopted in the current work, it worth noting that two cases showing combined features CTMR and CAMR were encountered, both were enrolled under CAMR group based on the hierarchical sequence. Such mixed cases reflect the complexity of the underlying etiopathogenesis of IF/TA as well as the problematic magnitude for prophylaxis and managing IF/TA cases.
| Conclusion|| |
From this study, we concluded that the over dosage of CNI immunosuppressive regimens is the most common cause of chronic graft dysfunction in the studied cases. Therefore, a thorough proper adjustment and monitoring of the CNI level may reduce the potential risk of chronic graft dysfunction. Compulsory renal biopsy should be applied for all renal allograft cases three months posttransplant for early detection of IF/TA. C4d and CD8 immunostaining should be applied routinely for all biopsied cases with impaired graft functions. Further studies with case follow-up are recommended for better evaluation of the prognosis and outcome of categorized cases, and also to bare the underlying etiologies of the NOS cases.
| Source of Support|| |
This study is partially funded by the Faculty of Medicine, Cairo University, Egypt.
Conflict of interest: None declared.
| References|| |
Paul LC. Chronic allograft nephropathy: An update. Kidney Int 1999;56:783-93.
Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 1993; 44:411-22.
Solez K, Colvin RB, Racusen LC, et al. Banff ′05 Meeting Report: Differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (′CAN′). Am J Transplant 2007;7:518-26.
Divella C, Rossini M, Loverre A, et al. Immunohistochemical characterization of glomerular and tubulointerstitial infiltrates in renal transplant patients with chronic allograft dysfunction. Nephrol Dial Transplant 2010;25: 4071-7.
Mason DY, Cordell JL, Gaulard P, Tse AG, Brown MH. Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence. J Clin Pathol 1992;45:1084-8.
Feucht HE, Schneeberger H, Hillebrand G, et al. Capillary deposition of C4d complement frag-ment and early renal graft loss. Kidney Int 1993; 43:1333-8.
Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999;55:713-23.
Seemayer CA, Gaspert A, Nickeleit V, Mihatsch MJ. C4d staining of renal allograft biopsies: A comparative analysis of different staining tec-hniques. Nephrol Dial Transplant 2007;22:568-76.
Serón D, Moreso F, Fulladosa X, Hueso M, Carrera M, Grinyó JM. Reliability of chronic allograft nephropathy diagnosis in sequential protocol biopsies. Kidney Int 2002;61:727-33.
Ali AA, Al-Mudhaffer AJ, Al-Taee Q, AlWindawi S. Allograft biopsy in kidney transplant recipients in the medical city of Baghdad. Saudi J Kidney Dis Transpl 2013;24:1039-43.
Kieran N, Wang X, Perkins J, et al. Combination of peritubular c4d and transplant glomerulopathy predicts late renal allograft failure. J Am Soc Nephrol 2009;20:2260-8.
Al Aly Z, Yalamanchili P, Cortese C, SalinasMadrigal L, Bastani B. C4d peritubular capillary staining in chronic allograft nephropathy and transplant glomerulopathy: An uncommon finding. Transpl Int 2005;18:800-5.
Vongwiwatana A, Gourishankar S, Campbell PM, Solez K, Halloran PF. Peritubular capillary changes and C4d deposits are associated with transplant glomerulopathy but not IgA nephropathy. Am J Transplant 2004;4: 124-9.
Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007;7:1743-52.
Nickeleit V, Zeiler M, Gudat F, Thiel G, Mihatsch MJ. Detection of the complement degradation product C4d in renal allografts: Diagnostic and therapeutic implications. J Am Soc Nephrol 2002;13:242-51.
Rotman S, Collins AB, Colvin RB. C4d deposition in allografts: Current concepts and interpretation. Transplant Rev 2005;19:65-77.
Horita S, Nitta K, Kawashima M, et al. C4d deposition in the glomeruli and peritubular capillaries associated with transplant glomerulopathy. Clin Transplant 2003;17:325-30.
Akalin E, Dinavahi R, Dikman S, et al. Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining. Clin J Am Soc Nephrol 2007;2:1261-7.
Hayde N, Bao Y, Pullman J, et al. The clinical and genomic significance of donor-specific antibody-positive/C4d-negative and donor-specific antibody-negative/C4d-negative transplant glomerulopathy. Clin J Am Soc Nephrol 2013;8:2141-8.
Smith RN, Colvin RB. Chronic alloantibody mediated rejection. Semin Immunol 2012;24: 115-21.
Bishop GA, Hall BM, Duggin GG, Horvath JS, Sheil AG, Tiller DJ. Immunopathology of renal allograft rejection analyzed with monoclonal antibodies to mononuclear cell markers. Kidney Int 1986;29:708-17.
Halloran PF, Melk A, Barth C. Rethinking chronic allograft nephropathy: The concept of accelerated senescence. J Am Soc Nephrol 1999;10:167-81.
Chen Y, Baltzan M, George D, Bohm C, Okasha K, Shoker A. Fate of recurrent acute interstitial cellular rejection in an HLA identical kidney transplant recipient: Impact of donor microchimerism. Clin Nephrol 1997;48: 300-6.
Shishido S, Asanuma H, Nakai H, et al. The impact of repeated subclinical acute rejection on the progression of chronic allograft nephropathy. J Am Soc Nephrol 2003;14:1046-52.
Wael M Hamza
Department of Pathology, Faculty of Medicine, Cairo University, Cairo
[Table 1], [Table 2], [Table 3], [Table 4]