Long-term Allograft Failure Research Articles

The Impact of Obesity on Allograft Failure After Kidney Transplantation: A Competing Risks Analysis.

The impact of pretransplant body mass index (BMI) on long-term allograft outcomes after kidney transplantation remains controversial. The conventional approach of using Kaplan-Meier method to calculate the cumulative risk of death-censored allograft failure may overestimate the risk of failure especially when competing failure risks are present. A retrospective cohort of adult first-time kidney transplant recipients was drawn from the Organ Procurement and Transplantation Network database (2001 to 2009). Based on World Health Organization obesity classification, BMI was categorized as: less than 18.5, 18.5 to <25, 25 to < 30, 30 to < 35, 35 to <40 and ≥40 kg/m. Both unadjusted and adjusted risk models were used to assess for risk of allograft failure in the presence of death as a competing event. A total of 108 654 recipients were studied. In both unadjusted and adjusted models, increasing BMI level was associated with increased risk of long-term allograft failure. In the adjusted model with BMI 18.5 to less than 25 as the reference, the subhazards ratios (SHRs) for BMI were: less than 18.5: SHR, 0.96; P = 0.41; 25 to less than 30: SHR, 1.05; P = 0.01; 30 to less than 35: SHR, 1.15; P = <0.001; 35 to less than 40: SHR, 1.21; P < 0.001; and greater than 40: SHR, 1.13; P = 0.002. Handling of death as a competing event demonstrates a graded, detrimental impact of increasing pretransplant BMI on the risk of graft failure after kidney transplantation in both unadjusted and adjusted models. Compared with previous studies, a lower BMI was not associated with an increased risk of graft loss in a competing risk model.

Pre-transplant immune state defined by serum markers and alloreactivity predicts acute rejection after living donor kidney transplantation.

Acute rejection (AR) remains a major cause for long-term kidney allograft failure. Reliable immunological parameters suitable to define the pre-transplant immune state and hence the individual risk of graft rejection are highly desired to preferably adapt the immunosuppressive regimen in advance. Donor and third party alloreactivities were determined by mixed lymphocyte cultures. Soluble forms of CD25, CD30, and CD44 were detected in patients' serum by ELISA. Various lymphocyte subpopulations were measured using flow cytometry. All patients received triple immunosuppression (tacrolimus/mycophenolate mofetil/steroids) and were grouped according to biopsy results within the first year: rejection-free (RF, n=13), borderline (BL, n=5), or acute rejection (AR, n=7). Patients with AR showed the highest pre-transplant alloreactivities and serum levels (sCD25/sCD30/sCD44) according to the pattern RF<BL<AR. Relying on serum analysis only, multivariate logistic regression (logit link function) yielded a prognostic score for prediction of rejection with 75.0% sensitivity and 69.2% specificity. Patients with rejection showed markedly higher pre-transplant frequencies of CD4(+) /CD8(+) T cells lacking CD28, but lower numbers of CD8(+) CD161(bright) T cells and NK cells than RF individuals. Pre-transplant immune state defined by alloreactivity, serum markers, and particular lymphocyte subsets seems to correlate with occurrence of graft rejection after kidney transplantation. A prognostic score based on pre-transplant serum levels has shown great potential for prediction of rejection episodes and should be further evaluated.

Colonisation with methicillin-resistantStaphylococcus aureusprior to renal transplantation is associated with long-term renal allograft failure

Renal transplant recipients are at an increased risk of developing Methicillin-resistant Staphylococcus aureus due to their immunosuppressed status. Herein, we investigate the incidence of MRSA infection in patients undergoing renal transplantation and determine the effect of MRSA colonisation on renal allograft function and overall mortality. Between January 1st 2007 and December 31st 2012, 1499 consecutive kidney transplants performed in our transplant unit and a retrospective 1:2 matched case-control study was performed on this patient cohort. The 1-, 3- and 5-year overall graft survival rates were 100%, 86% and 78%, respectively, in MRSA positive recipients compared with 100%, 100% and 93%, respectively, in the control group (P<0.05). The 1-, 3- and 5-year overall patient survival rates were 100%, 97% and 79%, respectively, in MRSA positive recipients compared with 100%, 100% and 95%, respectively, in the control group (P=0.1). In a multiple logistic regression analysis, colonisation with MRSA pre-operatively was an independent predictor for renal allograft failure at 5 years (hazard ratio: 4.6, 95% confidence interval: 1-30.7, P=0.048). These findings demonstrate that the incidence of long-term renal allograft failure is significantly greater in this patient cohort compared with a matched control population.

Predictors of 10-Year Pancreas Allograft Survival After Simultaneous Pancreas and Kidney Transplantation

This study aimed to identify the preoperative, perioperative, and postoperative factors affecting 10-year pancreas allograft survival after simultaneous pancreas and kidney (SPK) transplantation. Analysis was performed on a prospectively maintained database of 56 SPK transplants consecutively performed between January 1992 and October 2002. The definition cutoff points of specific variables were obtained by the receiver operating characteristic curve and multiple logistic regression analyses that were performed to determine the predictors of pancreas allograft survival after 10 years. In total, 44 (79%) patients had an overall survival of more than 10 years, and the overall 10-year pancreas allograft survival rate was 57% (n = 32/56). The significant predictors for pancreas allograft failure in 10 years and above were kidney allograft failure (P = 0.04), serum creatinine 1 year postoperatively (P = 0.002), and serum hemoglobin A1c (HbA1c) level 2 years postoperatively (P = 0.003). A serum creatinine cutoff value of more than 129 μmol/L at 1 year was 87.5% sensitive and 70% specific for predicting pancreas allograft failure at 10 years. Serum HbA1c of more than 5.6% at 2 years was 85.7% sensitive and 62.5% specific for predicting pancreas allograft failure at 10 years. On multivariate analysis, 129 μmol/L and above of serum creatinine and more than 5.6% of serum HbA1c were the independent predictors of pancreas allograft failure at 10 years. These findings may provide important information for identifying patients at risk for long-term pancreas allograft failure after SPK transplantation.

The impact of CTLA4 and PTPN22 genes polymorphisms on long-term renal allograft function and transplant outcomes

Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) downregulates the immune system. Lymphoid tyrosine phosphatase (Lyp)—PTPN22 protein—is suggested to be negative regulator of T-cell reaction. There are several polymorphisms in the CTLA4 and PTPN22 genes, which can influence the immune response and allograft function after kidney transplantation. The aim of this study was to examine the impact of CTLA4 and PTPN22 genes polymorphisms on the long-term renal transplant function and recipients’ outcomes during a 5-year follow-up observation. The study enrolled 268 Caucasian renal transplant recipients. Genotyping of the rs231775 (+49AG) CTLA4 gene polymorphism was performed using real-time PCR and rs2476601 (C1858T) PTPN22 gene polymorphism using PCR-RFLP method. The 5-year graft survival rate was 81.7%. Dialysis was necessary in 22 (8%) patients, 7 (2.6%) patients died and 20 (7.4%) switched to another transplantation center. We found no association between studied polymorphisms and graft loss/dialysis. Comparison of the distribution of the +49AG CTLA4 and C1858T PTPN22 genes polymorphisms genotypes among dead and living patients showed no statistically significant differences. Above results suggest that the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 genes polymorphisms are not associated with long-term allograft failure, graft loss and mortality after transplantation.

Renal Ischemia-Reperfusion Injury Amplifies the Humoral Immune Response

Renal transplant recipients who experience delayed graft function have increased risks of rejection and long-term graft failure. Ischemic damage is the most common cause of delayed graft function, and although it is known that tissue inflammation accompanies renal ischemia, it is unknown whether renal ischemia affects the production of antibodies by B lymphocytes, which may lead to chronic humoral rejection and allograft failure. Here, mice immunized with a foreign antigen 24-96 hours after renal ischemia-reperfusion injury developed increased levels of antigen-specific IgG1 compared with sham-treated controls. This amplified IgG1 response did not follow unilateral ischemia, and it did not occur in response to a T-independent antigen. To test whether innate immune activation in the kidney after ischemia affects the systemic immune response to antigen, we repeated the immunization experiment using mice deficient in factor B that lack a functional alternative pathway of complement. Renal ischemia-reperfusion injury did not cause amplification of the antigen-specific antibodies in these mice, suggesting that the increased immune response requires a functional alternative pathway of complement. Taken together, these data suggest that ischemic renal injury leads to a rise in antibody production, which may be harmful to renal allografts, possibly explaining a mechanism underlying the link between delayed graft function and long-term allograft failure.

The impact of ICAM1 and VCAM1 gene polymorphisms on long-term renal transplant function and recipient outcomes

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are implicated in endothelial cell-leukocyte adhesion. Several functional polymorphisms that affect the expression of ICAM-1 and VCAM-1, and therefore post-transplant immunological response, have been found in genes of these molecules. The aim of this study was to examine the impact of ICAM1 and VCAM1 gene polymorphisms on long-term renal transplant function and recipient outcome during a 5-year follow-up period. The study enrolled 269 Caucasian renal transplant recipients (165 males, 104 females, mean age 47.58 ± 12.96 years) remaining under observation for 5 years. Genotyping of the c.1405A>G (Lys469Glu, rs5498) ICAM1, T(-1592)C (rs1041163), and T(-833)C (rs3170794) VCAM1 genes polymorphisms was performed using real-time PCR. The rs5498 ICAM1 GG genotype was an independent risk factor associated with increased creatinine concentration at 12, 24, 36, 48, and 60 months after transplantation (p=0.0004, p=0.02, p=0.01, p=0.01 and p=0.04, respectively). Creatinine concentrations 36 and 48 months after transplantation were higher among CC homozygotes of the rs1041163 VCAM1 (TT+CT vs. CC, p=0.049 and p=0.04, respectively). The C allele of the rs1041163 VCAM1 gene polymorphism was identified as a risk factor for dialysis after transplantation (HR=3.323, 95%CI=1.44-7.67, p=0.005). The C allele of the rs1041163 VCAM1 gene was at the border of statistical significance as a risk factor for death after transplantation (p=0.06). These results suggest that the rs5498 ICAM1 GG genotype is correlated with early and long-term allograft failure. The C allele of the rs1041163 VCAM1 gene may be associated with long-term allograft failure and graft loss, as well as increased morbidity after transplantation.

Genetic or Pharmaceutical Blockade of Phosphoinositide 3-Kinase p110δ Prevents Chronic Rejection of Heart Allografts

Chronic rejection is the major cause of long-term heart allograft failure. A prominent feature of chronic graft rejection is tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signalling, regulating both T cell activation and, importantly, migration of primed T cells to non-lymphoid antigen-rich tissue. The objective of this study was to investigate the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model of minor histocompatibility antigens-mismatched heart allografts. HY-mismatched hearts were transplanted into recipient mice with either genetic or pharmacological targeting of PI3K p110δ signalling. We show that suppression of p110d activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment, while not inducing specific T cell tolerance. PI3K p110d pharmacologic inactivation is effective when initiated after transplantation. We further show that PI3K p110d inhibition acts by impairing the localization of antigen-specific T cells to the heart grafts. We propose that targeting of PI3K p110δ activity is a viable strategy for the treatment of heart allograft chronic rejection in humans.

Genetic or Pharmaceutical Blockade of Phosphoinositide 3-Kinase p110δ Prevents Chronic Rejection of Heart Allografts

Chronic rejection is the major cause of long-term heart allograft failure. A prominent feature of chronic graft rejection is tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signalling, regulating both T cell activation and, importantly, migration of primed T cells to non-lymphoid antigen-rich tissue. The objective of this study was to investigate the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model of minor histocompatibility antigens-mismatched heart allografts. HY-mismatched hearts were transplanted into recipient mice with either genetic or pharmacological targeting of PI3K p110δ signalling. We show that suppression of p110d activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment, while not inducing specific T cell tolerance. PI3K p110d pharmacologic inactivation is effective when initiated after transplantation. We further show that PI3K p110d inhibition acts by impairing the localization of antigen-specific T cells to the heart grafts. We propose that targeting of PI3K p110δ activity is a viable strategy for the treatment of heart allograft chronic rejection in humans.

Genetic or Pharmaceutical Blockade of Phosphoinositide 3-Kinase P110δ Prevents Chronic Rejection of Heart Allografts

Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.

Limiting Hepatitis C Virus Progression in Liver Transplant Recipients Using Sirolimus-Based Immunosuppression

Hepatitis C virus (HCV) causes progressive liver fibrosis in liver transplant recipients and is the principal cause of long-term allograft failure. The antifibrotic effects of sirolimus are seen in animal models but have not been described in liver transplant recipients. We reviewed 1274 liver recipients from 2002 to 2010 and identified a cohort of HCV recipients exposed to sirolimus as primary immunosuppression (SRL Cohort) and an HCV Control Group of recipients who had never received sirolimus. Yearly protocol biopsies were done recording fibrosis stage (METAVIR score) with biopsy compliance of >80% at both year one and two. In an intent-to-treat analysis, the SRL Cohort had significantly less advanced fibrosis (stage ≥2) compared to the HCV Control Group at year one (15.3% vs. 36.2%, p < 0.0001) and year two (30.1% vs. 50.5%, p = 0.001). Because sirolimus is sometimes discontinued for side effects, the SRL Cohort was subgroup stratified for sirolimus duration, showing progressively less fibrosis with longer sirolimus duration. Multivariate analysis demonstrated sirolimus as an independent predictor of minimal fibrosis at year one, and year two. This is the first study among liver transplant recipients with recurrent HCV to describe the positive impact of sirolimus in respect of reduced fibrosis extent and rate of progression.

Lymphoid neogenesis in chronic rejection

Although chronic rejection is currently one of the main causes of long-term allograft failure, its pathogenesis remains elusive, thereby preventing the development of effective therapy. Recent advances in the comprehension of the pathophysiology of chronic inflammatory diseases could shed new light on the pathogenesis of chronic rejection. Lymphoid neogenesis is a mechanism responsible for the progressive organization of chronic inflammatory infiltrates into functional ectopic germinal centers, which has been recently evidenced in various pathological situations sharing a common feature: the failure of the immune response to eradicate the targeted antigens. Chronic rejection is such a situation as it results from a sustained alloimmune response against the donor's antigens that are constantly replenished by the grafted tissue. Accordingly, functional ectopic germinal centers develop within chronically rejected organs. During chronic rejection, the graft is simultaneously the target and the site of elicitation of the alloimmune response.

Can mTOR inhibitors reduce the risk of late kidney allograft failure?

The most frequent causes of late kidney allograft failure are chronic rejection, nonalloimmune injury and death, all of which may depend on the characteristics of the donor and recipient, but may also be influenced by the type of immunosuppression. Combining calcineurin inhibitors (CNIs) and corticosteroids offers potent immunosuppression, but may also cause side effects leading to progressive graft dysfunction or an increased risk of death. New immunosuppressive strategies may come from the availability of inhibitors of mTOR, a downstream effector of phosphatidylinositol-3 kinase that provides the signal for cell proliferation by phosphorylating a cascade of kinases. Recent trials have shown that it is possible to minimize the dose or withdraw CNIs a few weeks after transplantation when they are combined with mTOR inhibitors and their combination may also make it possible to minimize or avoid the use of corticosteroids. Moreover, by inhibiting the signal for cell proliferation, mTOR inhibitors may reduce the replication of cytomegalovirus inside host cells, prevent transplant vasculopathy, and exert anti-oncogenic activity. All of these characteristics offer a ray of hope for reducing the risk of long-term allograft failure.

The Impact of Daily Sodium Intake on Posttransplant Hypertension in Kidney Allograft Recipients

Introduction Posttransplant hypertension is a well-known risk factor for long-term allograft failure and mortality in kidney recipients. Although dietary sodium restriction is a widely recommended nonpharmacological measure for control of blood pressure (BP), no detailed investigation has been conducted regarding the impact of dietary sodium restriction on this condition. Methods Thirty-two patients on antihypertensive treatment completed the study. They were randomly divided into two groups: controls (group 1) versus strict sodium diet (group 2; 80 to 100 mmol sodium daily). After randomization, 24-hour urine for sodium measurement, BP, and allograft functions were recorded at baseline and after 3 months. BP treatment was reevaluated at each visit throughout the study. Results At baseline, there was no significant difference in age, sex, serum creatinine, systolic and diastolic BP, antihypertensive drugs, or 24-hour urinary sodium levels between the groups. After 3 months, daily urinary sodium excretion (from 190 ± 75 to 106 ± 48 mEq/d, P < .0001), systolic BP (from 146 ± 21 to 116 ± 11 mm Hg), and diastolic BP (from 89 ± 8 to 72 ± 10 mm Hg) had significantly decreased in group 2, while no significant changes were observed in group 1. Conclusion Low sodium intake in combination with antihypertensive treatment appears to efficiently control BP in kidney allograft recipients with hypertension. Twenty-four-hour urinary sodium excretion should be checked regularly in these patients as a useful marker to indicate whether the patient complies with low sodium intake.

Frequency and Clinical Implications of Development of Donor-Specific and Non–Donor-Specific HLA Antibodies after Kidney Transplantation

The involvement of immunologic and nonimmunologic events in long-term kidney allograft failure is difficult to assess. The development of HLA antibodies after transplantation is the witness of ongoing reactivity against the transplant, and several studies have suggested that the presence of HLA antibodies correlates with poor graft survival. However, they have not discriminated between donor-specific (DS) and non-specific (NDS) antibodies. A total of 1229 recipients of a kidney graft, transplanted between 1972 and 2002, who had over a 5-yr period a prospective annual screening for HLA antibodies with a combination of ELISA, complement-dependent cytotoxicity, and flow cytometry tests were investigated; in 543 of them, the screening was complete from transplantation to the fifth year postgrafting. Correlations were established between the presence and the specificity of the antibodies and clinical parameters. A total of 5.5% of the patients had DS, 11.3% had NDS, and 83% had no HLA antibodies after transplantation. NDS antibodies appeared earlier (1 to 5 yr posttransplantation) than DS antibodies (5 to 10 yr). In multivariate analysis, HLA-DR matching, pretransplantation immunization, and acute rejection were significantly associated with the development of both DS and NDS antibodies and also of DS versus NDS antibodies. The presence of either DS or NDS antibodies significantly correlated with lower graft survival, poor transplant function, and proteinuria. Screening of HLA antibodies posttransplantation could be a good tool for the follow-up of patients who receive a kidney transplant and allow immunosuppression to be tailored.

Inhibition of Matrix Metalloproteinases During Chronic Allograft Nephropathy in Rats

Chronic allograft nephropathy (CAN) belongs to the major causes of long-term kidney allograft failure. One of the histologic hallmarks of CAN is interstitial fibrosis, influenced by matrix metalloproteinases (MMPs) that are controlling extracellular matrix (ECM) degradation. Whether MMPs affect the development and progression of CAN is not clear so far. To analyze the role of MMPs in CAN, we investigated the effects of an early and a late application of BAY 12-9566, an inhibitor of MMP-2, -3, and -9 on the development and progression of CAN in a rat kidney-transplantation model. Fisher kidneys were orthotopically transplanted into Lewis recipients that were treated with BAY 12-9566 (15 mg/kg per day) or vehicle either for the first 10 days after transplantation (early treatment) or from week 12 to week 20 after transplantation (late treatment). Proteinuria was analyzed every 4 weeks up to week 20 after transplantation when kidney grafts were removed for further analysis. Early MMP-inhibition resulted in a significantly reduced 24-hour protein excretion that was paralleled by a lower grade of CAN after 20 weeks. However, late MMP inhibition starting at week 12 after transplantation resulted in significantly higher proteinuria and a higher grade of CAN as compared with controls. Furthermore, transforming growth factor-beta and platelet-derived growth factor-B chain mRNA levels were significantly increased in these animals. Inhibition of MMPs early after transplantation reduced the development and progression of CAN but promoted CAN if initiated at later stages. Thus, MMPs are involved in the development and progression of CAN.

Protocol biopsies after kidney transplantation

Numerous studies have investigated features of allograft injury in renal biopsies obtained in stable kidney transplants. Evaluation of protocol biopsies has revealed a considerably high prevalence of subclinical acute rejection (SAR) and chronic allograft nephropathy (CAN) already in early phases after transplantation. The meanwhile well-established association of SAR and CAN in protocol biopsy with long-term allograft failure and the finding of superior allograft outcome after treatment of SAR in a randomized prospective study may point to clinical relevance of this procedure. In this review, potential benefits and risks associated with kidney allograft biopsy in stable renal transplant recipients are discussed.

THE SPECTRUM OF COMPLICATIONS OF IMMUNOSUPPRESSION

Brenner, Michael J. MD; Tung, Thomas H. MD; Jensen, John N. MD; Mackinnon, Susan E. MD Author Information

Highly altered V beta repertoire of T cells infiltrating long-term rejected kidney allografts.

Chronic rejection represents a major cause of long-term kidney graft loss. T cells that are predominant in long-term rejected kidney allografts (35 +/- 10% of area infiltrate) may thus be instrumental in this phenomenon, which is likely to be dependent on the indirect pathway of allorecognition only. We have analyzed the variations in T cell repertoire usage of the V beta chain at the complementary determining region 3 (CDR3) level in 18 human kidney grafts lost due to chronic rejection. We observed a strongly biased intragraft TCR V beta usage for the majority of V beta families and also a very high percentage (55%) of V beta families exhibiting common and oligoclonal V beta-C beta rearrangements in the grafts of patients with chronic rejection associated with superimposed histologically acute lesions. Furthermore, V beta 8 and V beta 23 families exhibited common and oligoclonal V beta-J beta rearrangements in 4 of 18 patients (22%). Several CDR3 amino acid sequences were found for the common and oligoclonal V beta 8-J beta 1.4 rearrangement. Quantitative PCR showed that biased V beta transcripts were also overexpressed in chronically rejected kidneys with superimposed acute lesions. In contrast, T lymphocytes infiltrating rejected allografts with chronic rejection only showed an unaltered Gaussian-type CDR3 length distribution. This pattern suggests that late graft failure associated with histological lesions restricted to Banff-defined chronic rejection does not involve T cell-mediated injury. Thus, our observation suggests that a limited number of determinants stimulates the recipient immune system in long-term allograft failure. The possibility of a local response against viral or parenchymatous cell-derived determinants is discussed.

Computerized histomorphometric assessment of protocol renal transplant biopsy specimens for surrogate markers of chronic rejection.

Chronic transplant rejection has emerged as the commonest cause of long-term renal allograft failure, and early identification of those grafts at risk could allow the targeting of specific therapies aimed at delaying this process. This study explores the usefulness of quantitative immunohistochemistry in defining biopsy-based surrogate markers of allograft damage. A consecutive series of 52 renal transplant recipients immunosuppressed with cyclosporine were studied. Needle core transplant biopsies were performed at 1, 3, and 6 months postoperatively. Immunostaining for collagen III, and smooth muscle actin, tenascin, and infiltrating leukocytes was performed using an indirect immunoperoxidase technique. The interstitial area stained (%) was measured using a semiautomatic image analysis system. The results were related to glomerular filtration rates (GFR) measured at 6, 12, and 24 months after transplantation using rank correlation coefficients. The area fraction of immunostained collagen III correlated with 6-month GFR (r=-0.42, P=0.005) and was predictive of 12-month GFR (r=-0.32, P=0.03). An area fraction of immunostained collagen III of >40% at 6 months was associated with a significantly lower GFR at 24 months, compared with a percentage area of < or =40% (31+/-4 versus 45+/-4 ml/min/1.73 m2, P=0.01). Furthermore, a collagen III of >40% at 6 months identified patients who were at risk of progressive deterioration in graft function. Grafts with poorer long-term function can be predicted using 6-month protocol biopsy specimens immunostained for collagen III. This should prove to be a useful ad interim surrogate marker of allograft damage in studies addressing the effects of new immunosuppressive agents on the development of chronic rejection.