Chronic Allograft Dysfunction - Is There a Treatment?

BACKGROUND: The major causes of renal transplant loss are death and chronic allograft dysfunction (CAD). The aims of this study were to determine the incidence of CAD in our population and the relation between allograft survival and immunosuppressive regimens. METHODS: We studied retrospectively 473 patients who received deceased donor kidney transplants with at least 1 allograft biopsy between January 1990 and May 2007. Clinical data included age, gender, biopsy data, and immunosuppression before and after kidney biopsy. Mean age was 45.4 +/- 12.7 years including 65% males with a mean follow-up of 6.7 +/- 4.5 years. CAD was observed in 177 of 473 biopsies: 48 patients showed interstitial fibrosis (IF); 101 chronic rejection (CR); 16 transplant glomerulopathy (TG); and 12, CR and TG. Mean follow-up since the discovery of the histologic feature was 60.5 +/- 50.5 months for IF; 38.3 +/- 40.8 for CR, and 18.2 +/- 19.2 for TG. RESULTS: CAD, which was more common in younger patients (P = .03), correlated upon univariate and multivariate analysis with CKD stage 5d development (P < .001). Deposition of C4d in peritubular capillaries was more frequent among CAD patients (P = .004), an association with particular relevance to recipients with CR (P = .02) and TG (P < .001). When we analyzed CAD subpopulation, we observed a positive correlation between allograft survival and immunosuppression modification after biopsy. Substitution of sirolimus (40/177) was shown in univariate, multivariate and Cox regression analyses to be a renal protector (P < .002). Allograft survival was also correlated with initial mycophenolate mofetil versus azathioprine, (62/177) immunosuppression (P < .001). CONCLUSION: CAD, a frequent histologic feature, may benefit from sirolimus conversion.

Specificity and Sensitivity of Screening for Anti-HLA Antibodies in Kidney Allograft Dysfunction

BACKGROUND: Prospective testing for posttransplant circulating anti-HLA antibodies seems to be a critical noninvasive tool, but confirmatory data are lacking. MATERIALS AND METHODS: Over the last 3 years, peritubular capillary (PTC) C4d deposition was prospectively sought by an immunofluorescence technique applied to frozen tissue in biopsies obtained for allograft dysfunction. Screening for circulating anti-HLA class I/II alloantibodies (AlloAb) by the flow cytometric test was performed simultaneously. RESULTS: We evaluated 132 sets of biopsies and simultaneous serum samples. PTC C4d deposition was demonstrated in 15.9% (21/132) of biopsies. Circulating anti-HLA I/II AlloAb were detected in 25% (33/132) of serum samples. Employing receiver-operator characteristic (ROC) curves for all C4d-positive biopsies, screening for AlloAb showed a global specificity of 82% and sensitivity of 61.9%. When this analysis was restricted to biopsies obtained in the first month posttransplantation, the sensitivity increased to 81.8%, but the specificity decreased to 76.9%. After the first month posttransplantation, we observed sensitivity of 40.0% and a specificity of 86.4%. In the first month posttransplantation, all patients with a diagnosis of acute antibody-mediated rejection displayed circulating anti-HLA class I/II, but not always at the same time as the C4d-positive biopsy. CONCLUSIONS: In the first month posttransplantation, prospective monitoring of anti-HLA antibodies may be useful. The high sensitivity allows the identification of patients at risk, affording an earlier diagnosis of antibody-mediated rejection. After the first month, the test can be used to evaluate allograft dysfunction episodes, since positivity is highly suggestive of an antibody-mediated process.

Impact of Donor and Recipient Cytokine Genotypes on Renal Allograft Outcome

Allelic differences in gene promoter or codifying regions have been described to affect regulation of gene expression, consequently increasing or decreasing cytokine production and signal transduction responses to a given stimulus. This observation has been reported for interleukin (IL)-10 (-1082 A/G; -819/-592 CT/CA), transforming growth factor (TGF)-beta (codon 10 C/T, codon 25 G/C), tumor necrosis factor (TNF)-alpha (-308 G/A), TNF-beta (+252 A/G), interferon (IFN)-gamma (+874 T/A), IL-6 (-174 G/C), and IL-4R alpha (+1902 G/A). To evaluate the influence of these cytokine genotypes on the development of acute or chronic rejection, we correlated the genotypes of both kidney graft recipients and cadaver donors with the clinical outcome. Kidney recipients had 5 years follow-up, at least 2 HLA-DRB compatibilities, and a maximum of 25% anti-HLA pretransplantation sensitization. The clinical outcomes were grouped as follows: stable functioning graft (NR, n = 35); acute rejection episodes (AR, n = 31); and chronic rejection (CR, n = 31). The cytokine genotype polymorphisms were defined using PCR-SSP typing. A statistical analysis showed a significant prevalence of recipient IL-10 -819/-592 genotype among CR individuals; whereas among donors, the TGF-beta codon 10 CT genotype was significantly associated with the AR cohort and the IL-6 -174 CC genotype with CR. Other albeit not significant observations included a strong predisposition of recipient TGF-beta codon 10 CT genotype with CR, and TNF-beta 252 AA with AR. A low frequency of TNF-alpha -308 AA genotype also was observed among recipients and donors who showed poor allograft outcomes.

C4d Presence in Kidney Allograft Biopsy: Sensitivity and Specifity of Immunoperoxidase vs Immunofluorescence

OBJECTIVES: Evaluate the sensitivity/specificity of immunoperoxidase method in comparison with the standard immunofluorescence. MATERIAL AND METHODS: Retrospective review of 87 biopsies made for allograft dysfunction. Immunofluorescence (IF) was performed in frozen allograft biopsies using monoclonal antibody anti-C4d from Quidel®. The indirect immunoperoxidase (IP) technique was performed in paraffin-embebbed tissue with polyclonal antiserum from Serotec®. Biopsies were independently evaluated by two nephropathologist according Banff 2007 classification. RESULTS: By IF, peritubular C4d deposition were detected in 60 biopsies and absent in 27 biopsies. The evaluation of biopsy by IP was less precise due to the presence of background and unspecific staining. We find 13.8% (12/87) of false negative and Banff classification concordance in 79.3% (69/87) of cases (table1). The ROC curve study reveal a specificity of 100% and sensitivity of 80.0 % of IP method in relation to the gold standard (area under curve:0.900; 95% Confidence interval :0.817-0.954; p=0.0001). Banff Classification C4d Cases Immunofluorescence Immunoperoxidase n =87 Diffuse Negative 3 (3.4%) Focal Negative 9 (10.3%) Negative Negative 27 (31.0%) Diffuse Diffuse 33 (37.9%) Focal Focal 9 (10.3%) Diffuse Focal 6 (6.9%) CONCLUSION: The IP method presents a good specificity, but lesser sensitivity to C4d detection in allograft dysfunction. The evaluation is more difficult, requiring more experience of the observer than IF method. If frozen tissue is unavailable, the use of IP for C4d detection is acceptable.

Peritubular Capillaries C4d Deposits in Renal Allograft Biopsies and Anti HLA I/II Alloantibodies Screening. Incidence and Clinical Importance

Aim: To characterise clinically the patients with C4d in peritubular capillaries deposits (C4dPTCD) and/or circulating anti-HLA class I/II alloantibodies. To determine the correlation between positive C4dPTCD and circulating anti-HLA class I/II alloantibodies during episodes of graft dysfunction. Subjects and Methods: C4d staining was performed in biopsies with available frozen tissue obtained between January 2004 and December 2006. The study was prospective from March 2005, when a serum sample was obtained at the time of biopsy to detect circulating anti-HLA class I/II alloantibodies. Results: We studied 109 biopsies in 86 cadaver renal transplant patients. Sixteen of these (14.7%) presented diffuse positive C4dPTCD. There was a 13.5% rate of +C4dPTCD incidence within the first six months of transplantation and 16% after six months (p>0.05). Half of the +C4dPTCD in the first six months was associated with acute humoral rejection. After six months, the majority of +C4dPTCD (n=7/8) was present in biopsies with evidence of interstitial fibrosis/tubular atrophy and/or transplant glomerulopathy. The C4dPTCD was more frequent in patients with positive anti-HCV antibodies(p<0.0001), a previous renal transplant (p=0.007), and with a panel reactivity antibody (PRA) ≥ 50%(p=0.0098). The anti-HCV+ patients had longer time on dialysis (p=0.0019) and higher PRA(p=0.005). Circulating anti-HLA I/II alloantibodies were screened in 46 serum samples. They were positive in 10.9% of samples, all obtained after six months post transplant. Circulating alloantibodies were absent in 92.5% of the C4d negative biopsies. Conclusion: We found an association between the presence of C4dPTCD and 2nd transplant recipients,higher PRA and the presence of anti-HCV antibodies. The presence of HCV antibodies is not a risk factor for C4dPTCD per se, but appears to reflect longer time on dialysis and presensitisation. In renal dysfunction a negative alloantibody screening is associated with a reduced risk of C4dPTCD (<10%).

C4d Detection in Renal Allograft Biopsies: Immunohistochemistry vs. Immunofluorescence

Introduction. Peritubular capillary complement 4d staining is one of the criteria for the diagnosis of antibody-mediated rejection, and research into this is essential to kidney allograft evaluation. The immunofluorescence technique applied to frozen sections is the present gold-standard method for complement 4d staining and is used routinely in our laboratory. The immunohistochemistry technique applied to paraffin-embedded tissue may be used when no frozen tissue is available. Material and Methods. The aim of this study is to evaluate the sensitivity and specificity of immunohistochemistry compared with immunofluorescence. We describe the advantages and disadvantages of the immunohistochemistry vs. the immunofluorescence technique. For this purpose complement 4d staining was performed retrospectively by the two methods in indication biopsies (n=143) and graded using the Banff 07 classification. Results. There was total classification agreement between methods in 87.4% (125/143) of cases. However, immunohistochemistry staining caused more difficulties in interpretation, due to nonspecific staining in tubular cells and surrounding interstitium. All cases negative by immunofluorescence were also negative by immunohistochemistry. The biopsies were classified as positive in 44.7% (64/143) of cases performed by immunofluorescence vs. 36.4% (52/143) performed by immunohistochemistry. Fewer biopsies were classified as positive diffuse in the immunohistochemistry group(25.1% vs. 31.4%) and more as positive focal (13.2% vs. 11.1%). More cases were classified as negative by immunohistochemistry (63.6% vs. 55.2%). Study by ROC curve showed immunohistochemistry has a specificity of 100% and a sensitivity of 81.2% in relation to immunofluorescence (AUC: 0.906; 95% confidence interval: 0.846-0.949; p=0.0001). Conclusions. The immunohistochemistry method presents an excellent specificity but lower sensitivity to C4d detection in allograft dysfunction. The evaluation is more difficult, requiring a more experienced observer than the immunofluorescence method. Based on these results, we conclude that the immunohistochemistry technique can safely be used when immunofluorescence is not available.

Atheroembolic Renal Disease As a Cause of Allograft Primary Non-Function

Atheroembolic renal disease, also referred to as cholesterol crystal embolization, is a rare cause of renal failure, secondary to occlusion of renal arteries, renal arterioles and glomerular capillaries with cholesterol crystals, originating from atheromatous plaques of the aorta and other major arteries. This disease can occur very rarely in kidney allografts in an early or a late clinical form. Renal biopsy seems to be a reliable diagnostic test and cholesterol clefts are the pathognomonic finding. However, the renal biopsy has some limitations as the typical lesion is focal and can be easily missed in a biopsy fragment. The clinical course of these patients varies from complete recovery of the renal function to permanent graft loss. Statins, acetylsalicyclic acid, and corticosteroids have been used to improve the prognosis. We report a case of primary allograft dysfunction caused by an early and massive atheroembolic renal disease. Distinctive histology is presented in several consecutive biopsies. We evaluated all the cases of our Unit and briefly reviewed the literature. Atheroembolic renal disease is a rare cause of allograft primary non -function but may become more prevalent as acceptance of aged donors and recipients for transplantation has become more frequent.

Treatment of Hepatitis C Virus Infection in Kidney Transplant Recipients: Case Report

Chronic hepatitis C virus (HCV) infection exists in a large proportion of patients undergoing renal transplantation. Nowadays it is not considered to be an absolute contraindication to transplantation; however, it is associated with an increased risk for the patient and accounts for a shorter half-life of the renal allograft. We present three transplant recipients who displayed serious hepatic dysfunction after renal transplantation due to an HCV infection. In two of these cases, the liver biopsies established the diagnosis of FCH. In the third case, the liver biopsy was compatible with the early stages of FCH. All patients were started on peg-interferon alfa 2-b and ribavirin with subsequent normalization of hepatic function and early complete viral responses.

Zero-Time Kidney Histology Predicts Early Graft Function

Our purposes are to determine the impact of histological factors observed in zero-time biopsies on early post transplant kidney allograft function. We specifically want to compare the semi-quantitative Banff Classification of zero time biopsies with quantification of % cortical area fibrosis. Sixty three zero-time deceased donor allograft biopsies were retrospectively semiquantitatively scored using Banff classification. By adding the individual chronic parameters a Banff Chronic Sum (BCS) Score was generated. Percentage of cortical area Picro Sirius Red (%PSR) staining was assessed and calculated with a computer program. A negative linear regression between %PSR/ GFR at 3 year post-transplantation was established (Y=62.08 +-4.6412X; p=0.022). A significant negative correlation between arteriolar hyalinosis (rho=-0.375; p=0.005), chronic interstitial (rho=0.296; p=0.02) , chronic tubular ( rho=0.276; p=0.04) , chronic vascular (rho= -0.360;P=0.007), BCS (rho=-0.413; p=0.002) and GFR at 3 years were found. However, no correlation was found between % PSR, Ci, Ct or BCS. In multivariate linear regression the negative predictive factors of 3 years GFR were: BCS in histological model; donor kidney age, recipient age and black race in clinical model. The BCS seems a good and easy to perform tool, available to every pathologist, with significant predictive short-term value. The %PSR predicts short term kidney function in univariate study and involves extra-routine and expensive-time work. We think that %PSR must be regarded as a research instrument.

Decreased Vascular Endothelial Growth Factor (VEGF) Expression in Focal Segmental Glomeruloesclerosis Lesions of Patients Under Sirolimus

Background: Proteinuria (PT) with SRL appears not only after conversion from a calcineurin inhibitor (CI), but also in de novo patients. The PT may be related to a hemodynamic effect of CI withdrawal or to a direct effect of SRL in glomerulus (GL). Recently an association between PT in SRL patients and FSGS lesions has been described. It is also known that SRL decrease VEGF synthesis and experimental data suggest that VEGF is essential to podocyte survival and differentiation. Aim: To determine if glomerular lesions and PT in SRL patients could be related to altered glomerular VEGF expression. Material and methods: We evaluated glomerular VEGF expression in 10 biopsies: A-allograft kidney in backtable (n=3); B-native normal kidney (n=1); C-native kidney with FSGS lesions (n=2); D-allograft kidney with FSGS lesions from proteinuric patients under SRL after conversion from CI (n=3); E-allograft kidney in proteinuric patient under SRL with a membranous glomerulonephritis (n=1). We employed indirect immunohistochemistry in paraffin-embedded sections using a mouse monoclonal antibody against human VEGF-C1 (Santa Cruz). Results: The controls biopsies (A; B) showed normal global VEGF expression, with strong podocyte staining. The VEGF expression in the group C was similar to the controls, although no FSGS lesions were observed in the stained GL. The group D showed normal VEGF expression in the apparently normal GL, hypertrophied podocytes with reduction of VEGF in anomalous GL, and no staining in slcerotic lesions. We observed a gradual reduction of VEGF expression with progressive dedifferentiation of podocytes. In the group E the VEGF was globally reduced, with some hypertrophied podocytes expressing decreased VEGF. Conclusion: We confirmed the diminished VEGF expression in injured podocytes of SRL patients.This decreased expression may result from a direct effect of SRL and precede the appearance of FSGS lesions and PT. Further studies are needed with greater number of cases and controls, including early biopsies of patients under SRL.

Terapêutica Endovascular de Pseudoaneurisma e Fístula Arteriovenosa em Enxerto Renal

A 31 year old male Caucasian received a renal cadaveric allograft. Reconstruction of an inferior polar artery was corrected pre-implantation. Delayed graft function occurred leading to dialysis support for one month. Graft biopsies(days 7, 15) showed acute tubular necrosis(ATN) and no rejection. Serial ultrasound (US), performed on average weekly, were compatible with ATN. On day 31, Doppler US and a CAT scan suggested for the first time a pseudoaneurysm adjacent to the implantation of the graft artery on the external iliac artery. For clinical and technical reasons, arteriography was only performed on day 67, when serum creatinine was 3.3 mg/dl. It showed a large pseudoaneurysm with an arteriovenous fistula to the right common iliac vein. Compression of the right external iliac artery was clear. In an attempt to close the arteriovenous fistula, the communication with the pseudoaneurysm was embolised with gelfoam and metallic coils with partial success. One week later, by right femoral approach a covered wallstent was placed immediately below the origin of the graft artery.Subsequent Doppler US and arteriography con-firmed closure of the communication with thepseudoaneurysm and of the arteriovenous fistula. The calibre of the right external iliac artery was then normal. By month 18, serum creatinine is stable at 2.1 mg/dl. We can only speculate on the origin of thepseudoaneurysm and of the AV fistula, whichwere not evident until one month post-transplantation. Backtable surgery was performed on thepolar not the main graft artery. Invasive angiography was irreplaceable in this unusual clinical situation.

Transplantation in Highly Sensitised Patients Treated with Intravenous Immunoglobulin and Rituximab

Renal transplant in highly sensitised patients is associated with increased morbidity. The aim of this retrospective study was to evaluate the clinical evolution of 30 highly sensitised deceased donor kidney transplants and the influence of different timing of B cell directed treatment and its importance in the outcome of these patients. All recipients had negative complement dependent lymphocytotoxicity cytotoxic T cell crossmatch and no identified anti human leucocyte antigen class I donor specific antibodies. T cell flow crossmatch was performed within 24h of transplantation with serum obtained pretransplant (historic, recent or baseline). Posttransplant flow crossmatch were performed prospectively starting on the 3rd posttransplantation day. The immunosuppressive regime included thymoglobulin, tacrolimus, mycofenolate mofetil and steroids. Positive flow crossmatch occurred in 20/29 patients by the 3rd posttransplantation day, and in 17/27 patients after the 3rd posttransplantation day. All patients were started on intravenous immunoglobulin before transplantation: in nine patients (group A) at 400mg/kg/day for five days; in the remaining 21 patients (group B), as a continued infusion of 2g/kg during 48h. In group A, Rituximab was added only in the presence of antibody mediated rejection; in group B, introduced on the 3rd posttransplantation day whenever a positive flow crossmatch (with serum obtained pre or posttransplant) was reported. Antibody mediated rejection was observed in 44.4% of patients in group A, and 19% of those in group B. Mean follow-up was 12.2±5.5 months. Overall allograft survival was 76.6%, 81% in group B, and 66.6% in group A. At last follow up, mean serum creatinine was 1.3±0.6 mg/dl. Renal transplantation with pretransplant positive flow crossmatch is highly associated with antibody mediated rejection, despite introduction of intravenous immunoglobulin pretransplantation. However high dose intravenous immunoglobulin for 48h plus Rituximab by the 3rd posttransplantation day reduce the incidence of antibody mediated rejection by more than 50% and allowed for allograft survival of 81% at one year, with an excellent renal function.

Leflunomide and Polyomavirus-Associated Nephropathy in Renal Transplant

Polyomavirus nephropathy is a major complication in renal transplantation, associated with renal allograft loss in 14 to 80% of cases. There is no established treatment, although improvement has been reported with a variety of approaches. The authors report two cases of polyomavirus infection in renal allograft recipients. In the first case, a stable patient presented with deterioration of renal function, worsening hypertension and weight gain following removal of ureteral stent placed routinely at the time of surgery. Ultrasound examination and radiology studies revealed hydronephrosis due to ureteral stenosis. A new ureteral stent was placed, but renal function did not improve. Urinary cytology revealed the presence of decoy cells and polyomavirus was detected in blood and urine by qualitative polymerase chain reaction. Renal biopsy findings were consistent with polyomavirus -associated nephropathy. In the second case, leucopaenia was detected in an asymptomatic patient 6 months after transplantation. Mycophenolate mophetil dosage was reduced but renal allograft function deteriorated, and a kidney biopsy revealed polyomavirus -associated nephropathy, also with SV40 positive cells. In both patients immunosuppression with tacrolimus was reduced, mycophenolate mophetil stopped and intravenous immune globulin plus ciprofloxacin started. As renal function continued to deteriorate, therapy with leflunomide (40 mg/day) was associated and maintained during 5 and 3 months respectively. In the first patient, renal function stabilised within one month of starting leflunomide and polymerase chain reaction was negative for polyomavirus after 5 months. A repeated allograft biopsy 6 months later showed no evidence of polyomavirus nephropathy. In the second patient, polyomavirus was undetectable in blood and urine by polymerase chain reaction after 3 months of leflunomide treatment, with no evidence of polyomavirus infection in a repeated biopsy 6 months after beginning treatment.

Transplant Glomerulopathy: Clinico-Pathologic Features

Transplant glomerulopathy is a sign of chronic kidney allograft damage. It has a distinct morphology and is associated with poor allograft survival. We aimed to assess the prevalence and clinic-pathologic features of transplant glomerulopathy, as well as determine the functional and histological implications of its severity. We performed a single-centre retrospective observational study during an eight-year period. Kidney allograft biopsies were diagnosed and scored according to the Banff classification, coupled with immunofluorescence studies. The epidemiology, clinical presentation, outcomes (patient and graft survival) and anti-HLA alloantibodies were evaluated. Transplant glomerulopathy was diagnosed in 60 kidney transplant biopsies performed for clinical reasons in 49 patients with ABO compatible renal transplant and a negative T-cell complement dependent cytotoxicity crossmatch at transplantation. The estimated prevalence of transplant glomerulopathy was 7.4% and its cumulative prevalence increased over time. C4d staining in peritubular capillaries (27.6%) was lower than the frequency of anti-HLA antibodies (72.5%), the majority against both classes I and II. Transplant glomerulopathy was associated with both acute (mainly glomerulitis and peritubular capillaritis) and chronic histologic abnormalities. At diagnosis, 30% had mild, 23.3% moderate and 46.7% severe transplant glomerulopathy. The severity of transplant glomerulopathy was associated with the severity of interstitial fibrosis. Other histological features, as well as clinical manifestations and graft survival, were unrelated to transplant glomerulopathy severity.