Evaluation of C1q Status and Titer of De Novo Donor-Specific Antibodies as Predictors of Allograft Survival.

De novo donor-specific antibodies (dnDSAs) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dnDSA C1q status or titer at the time of first detection can independently predict allograft loss. In a consecutive cohort of 508 renal transplant recipients, 70 developed dnDSAs. Histologic and clinical outcomes were correlated with the C1q assay or dnDSA titer. C1q positivity correlated with dnDSA titer (p < 0.01) and mean fluorescence intensity (p < 0.01) and was more common in class II versus class I dnDSAs (p < 0.01). C1q status correlated with tubulitis (p = 0.02) and C4d status (p = 0.03) in biopsies at the time of dnDSA development, but not T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR). De novo DSA titer correlated with Banff g, i, t, ptc, C4d scores, TCMR (p < 0.01) and ABMR (p < 0.01). Post-dnDSA graft loss was observed more frequently in recipients with C1q-positve dnDSA (p < 0.01) or dnDSA titer ≥ 1:1024 (p ≤ 0.01). However, after adjustment for clinical phenotype and nonadherence in multivariate models, neither C1q status nor dnDSA titer were independently associated with allograft loss, questioning the utility of these assays at the time of dnDSA development.

Trabecular bone score in kidney transplant recipients.

SUMMARY: It is uncertain whether bone mineral density (BMD) can accurately predict fracture in kidney transplant recipients. Trabecular bone score (TBS) provides information independent of BMD. Kidney transplant recipients had abnormal bone texture as measured by lumbar spine TBS, and a lower TBS was associated with incident fractures in recipients. INTRODUCTION: Trabecular bone score (TBS) is a texture measure derived from dual energy X-ray absorptiometry (DXA) lumbar spine images, providing information independent of bone mineral density. We assessed characteristics associated with TBS and fracture outcomes in kidney transplant recipients. METHODS: We included 327 kidney transplant recipients from Manitoba, Canada, who received a post-transplant DXA (median 106 days post-transplant). We matched each kidney transplant recipient (mean age 45 years, 39% men) to three controls from the general population (matched on age, sex, and DXA date). Lumbar spine (L1-L4) DXA images were used to derive TBS. Non-traumatic incident fracture (excluding hand, foot, and craniofacial) (n = 31) was assessed during a mean follow-up of 6.6 years. We used multivariable linear regression models to test predictors of TBS, and multivariable Cox proportional hazard regression was used to estimate hazard ratios (HRs) per standard deviation decrease in TBS to express the gradient of risk. RESULTS: Compared to the general population, kidney transplant recipients had a significantly lower lumbar spine TBS (1.365 ± 0.129 versus 1.406 ± 0.125, P < 0.001). Multivariable linear regression revealed that receipt of a kidney transplant was associated with a significantly lower mean TBS compared to controls (-0.0369, 95% confidence interval [95% CI] -0.0537 to -0.0202). TBS was associated with fractures independent of the Fracture Risk Assessment score including BMD (adjusted HR per standard deviation decrease in TBS 1.64, 95% CI 1.15-2.36). CONCLUSION: Kidney transplant recipients had abnormal bone texture as assessed by TBS and a lower lumbar spine TBS was associated with fractures in recipients.

Interferon Gamma ELISPOT Testing as a Risk-Stratifying Biomarker for Kidney Transplant Injury: Results From the CTOT-01 Multicenter Study.

Previous studies suggest that quantifying donor-reactive memory T cells prior to kidney transplantation by interferon gamma enzyme-linked immunosorbent spot assay (IFNγELISPOT) can assist in assessing risk of posttransplant allograft injury. Herein, we report an analysis of IFNγELISPOT results from the multicenter, Clinical Trials in Organ Transplantation-01 observational study of primary kidney transplant recipients treated with heterogeneous immunosuppression. Within the subset of 176 subjects with available IFNγELISPOT results, pretransplant IFNγELISPOT positivity surprisingly did not correlate with either the incidence of acute rejection (AR) or estimated glomerular filtration rate (eGFR) at 6- or 12-month. These unanticipated results prompted us to examine potential effect modifiers, including the use of T cell-depleting, rabbit anti-thymocyte globulin (ATG). Within the no-ATG subset, IFNγELISPOT(neg) subjects had higher 6- and 12-month eGFRs than IFNγELISPOT(pos) subjects, independent of biopsy-proven AR, peak PRA, human leukocyte antigen mismatches, African-American race, donor source, and recipient age or gender. In contrast, IFNγELISPOT status did not correlate with posttransplant eGFR in subjects given ATG. Our data confirm an association between pretransplant IFNγELISPOT positivity and lower posttransplant eGFR, but only in patients who do not receive ATG induction. Controlled studies are needed to test the hypothesis that ATG induction is preferentially beneficial to transplant candidates with high frequencies of donor-reactive memory T cells.

Rates and determinants of progression to graft failure in kidney allograft recipients with de novo donor-specific antibody.

Understanding rates and determinants of clinical pathologic progression for recipients with de novo donor-specific antibody (dnDSA), especially subclinical dnDSA, may identify surrogate endpoints and inform clinical trial design. A consecutive cohort of 508 renal transplant recipients (n = 64 with dnDSA) was studied. Recipients (n = 388) without dnDSA or dysfunction had an eGFR decline of -0.65 mL/min/1.73 m(2) /year. In recipients with dnDSA, the rate eGFR decline was significantly increased prior to dnDSA onset (-2.89 vs. -0.65 mL/min/1.73 m(2) /year, p < 0.0001) and accelerated post-dnDSA (-3.63 vs. -2.89 mL/min/1.73 m(2) /year, p < 0.0001), suggesting that dnDSA is both a marker and contributor to ongoing alloimmunity. Time to 50% post-dnDSA graft loss was longer in recipients with subclinical versus a clinical dnDSA phenotype (8.3 vs. 3.3 years, p < 0.0001). Analysis of 1091 allograft biopsies found that dnDSA and time independently predicted chronic glomerulopathy (cg), but not interstitial fibrosis and tubular atrophy (IFTA). Early T cell-mediated rejection, nonadherence, and time were multivariate predictors of IFTA. Independent risk factors for post-dnDSA graft survival available prior to, or at the time of, dnDSA detection were delayed graft function, nonadherence, dnDSA mean fluorescence intensity sum score, tubulitis, and cg. Ultimately, dnDSA is part of a continuum of mixed alloimmune-mediated injury, which requires solutions targeting T and B cells.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Class II HLA epitope matching-A strategy to minimize de novo donor-specific antibody development and improve outcomes.

De novo donor-specific antibody (dnDSA) develops in 15-25% of renal transplant recipients within 5 years of transplantation and is associated with 40% lower graft survival at 10 years. HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for 286 donor-recipient pairs. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DR dnDSA alone (21.4 vs. 13.2, p < 0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p < 0.001). An optimal threshold for epitope mismatches (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA. Applying these thresholds, zero and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome.

Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury.

Noninvasive biomarkers are needed to assess immune risk and ultimately guide therapeutic decision-making following kidney transplantation. A requisite step toward these goals is validation of markers that diagnose and/or predict relevant transplant endpoints. The Clinical Trials in Organ Transplantation-01 protocol is a multicenter observational study of biomarkers in 280 adult and pediatric first kidney transplant recipients. We compared and validated urinary mRNAs and proteins as biomarkers to diagnose biopsy-proven acute rejection (AR) and stratify patients into groups based on risk for developing AR or progressive renal dysfunction. Among markers tested for diagnosing AR, urinary CXCL9 mRNA (odds ratio [OR] 2.77, positive predictive value [PPV] 61.5%, negative predictive value [NPV] 83%) and CXCL9 protein (OR 3.40, PPV 67.6%, NPV 92%) were the most robust. Low urinary CXCL9 protein in 6-month posttransplant urines obtained from stable allograft recipients classified individuals least likely to develop future AR or a decrement in estimated glomerular filtration rate between 6 and 24 months (92.5-99.3% NPV). Our results support using urinary CXCL9 for clinical decision-making following kidney transplantation. In the context of acute dysfunction, low values can rule out infectious/immunological causes of injury. Absent urinary CXCL9 at 6 months posttransplant defines a subgroup at low risk for incipient immune injury.

Detection of clinical and subclinical tubulo-interstitial inflammation by the urinary CXCL10 chemokine in a real-life setting.

Urinary CXCL10 is a promising noninvasive biomarker for tubulo-interstitial allograft inflammation, but its diagnostic characteristics have not been assessed in a real-life setting. We investigated urinary CXCL10 in 213 consecutive renal allograft recipients having 362 surveillance biopsies at 3/6 months and 80 indication biopsies within the first year posttransplant. Allograft histology results were classified as (i) acute Banff score zero, (ii) interstitial infiltrates only, (iii) tubulitis t1, (iv) tubulitis t2-3 and (v) isolated vascular compartment inflammation. For clinical and subclinical pathologies, urinary CXCL10 correlated well with the extent of tubulo-interstitial inflammation. To determine diagnostic characteristics of urinary CXCL10, histological groups were separated into two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (i.e. tubulitis t1-3 and isolated vascular compartment inflammation). For subclinical pathologies, AUC was 0.69 (sensitivity 61%, specificity 72%); for clinical pathologies, AUC was 0.74 (sensitivity 63%, specificity 80%). A urinary CXCL10-guided biopsy strategy would have reduced performance of surveillance and indication biopsies by 61% and 64%, respectively. Missed (sub)clinical pathologies were mostly tubulitis t1 and isolated vascular compartment lesions. In real life, urinary CXCL10 had clinically useful diagnostic properties making it a candidate biomarker to guide allograft biopsies.

Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant.

The natural history for patients with de novo donor-specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pretransplant DSA, with a mean follow-up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA posttransplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years posttransplant. Independent predictors of dnDSA were HLA-DRß1 MM > 0 (OR 5.66, p < 0.006); and nonadherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p = 0.061). The median 10-year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody-mediated injury can occur and progress in patients with dnDSA in the absence of graft dysfunction and furthermore, nonadherence and cellular rejection contribute to dnDSA development and progression to graft loss.

Inflammation in areas of tubular atrophy in kidney allograft biopsies: a potent predictor of allograft failure.

The Banff scoring schema provides a common ground to analyze kidney transplant biopsies. Interstitial inflammation (i) and tubulitis (t) in areas of viable tissue are features in scoring acute rejection, but are excluded in areas of tubular atrophy (TA). We studied inflammation and tubulitis in a cohort of kidney transplant recipients undergoing allograft biopsy for new-onset late graft dysfunction (N = 337). We found inflammation ('iatr') and tubulitis ('tatr') in regions of fibrosis and atrophy to be strongly correlated with each other (p < 0.0001). Moreover, iatr was strongly associated with death-censored graft failure when compared to recipients whose biopsies had no inflammation, even after adjusting for the presence of interstitial fibrosis (Hazard Ratio = 2.31, [1.10-4.83]; p = 0.0262) or TA (hazard ratio = 2.42, [1.16-5.08]; p = 0.191), serum creatinine at the time of biopsy, time to biopsy and i score. Further, these results did not qualitatively change after additional adjustments for C4d staining or donor specific antibody. Stepwise regression identified the most significant markers of graft failure which include iatr score. We propose that a more global assessment of inflammation in kidney allograft biopsies to include inflammation in atrophic areas may provide better prognostic information. Phenotypic characterization of these inflammatory cells and appropriate treatment may ameliorate late allograft failure.

Histopathologic clusters differentiate subgroups within the nonspecific diagnoses of CAN or CR: preliminary data from the DeKAF study.

The nonspecific diagnoses 'chronic rejection''CAN', or 'IF/TA' suggest neither identifiable pathophysiologic mechanisms nor possible treatments. As a first step to developing a more useful taxonomy for causes of new-onset late kidney allograft dysfunction, we used cluster analysis of individual Banff score components to define subgroups. In this multicenter study, eligibility included being transplanted prior to October 1, 2005, having a 'baseline' serum creatinine < or =2.0 mg/dL before January 1, 2006, and subsequently developing deterioration of graft function leading to a biopsy. Mean time from transplant to biopsy was 7.5 +/- 6.1 years. Of the 265 biopsies (all with blinded central pathology interpretation), 240 grouped into six large (n > 13) clusters. There were no major differences between clusters in recipient demographics. The actuarial postbiopsy graft survival varied by cluster (p = 0.002). CAN and CNI toxicity were common diagnoses in each cluster (and did not differentiate clusters). Similarly, C4d and presence of donor specific antibody were frequently observed across clusters. We conclude that for recipients with new-onset late graft dysfunction, cluster analysis of Banff scores distinguishes meaningful subgroups with differing outcomes.

Pathological and clinical characterization of the 'troubled transplant': data from the DeKAF study.

We are studying two cohorts of kidney transplant recipients, with the goal of defining specific clinicopathologic entities that cause late graft dysfunction: (1) prevalent patients with new onset late graft dysfunction (cross-sectional cohort); and (2) newly transplanted patients (prospective cohort). For the cross-sectional cohort (n = 440), mean time from transplant to biopsy was 7.5 +/- 6.1 years. Local pathology diagnoses included CAN (48%), CNI toxicity (30%), and perhaps surprisingly, acute rejection (cellular- or Ab-mediated) (23%). Actuarial rate of death-censored graft loss at 1 year postbiopsy was 17.7%; at 2 years, 29.8%. There was no difference in postbiopsy graft survival for recipients with versus without CAN (p = 0.9). Prospective cohort patients (n = 2427) developing graft dysfunction >3 months posttransplant undergo 'index' biopsy. The rate of index biopsy was 8.8% between 3 and 12 months, and 18.2% by 2 years. Mean time from transplant to index biopsy was 1.0 +/- 0.6 years. Local pathology diagnoses included CAN (27%), and acute rejection (39%). Intervention to halt late graft deterioration cannot be developed in the absence of meaningful diagnostic entities. We found CAN in late posttransplant biopsies to be of no prognostic value. The DeKAF study will provide broadly applicable diagnostic information to serve as the basis for future trials.

Can protocol biopsy better inform our choices in renal transplantation?

The use of protocol biopsies has provided insights into the pathogenesis of many renal allograft diseases. It is now widely accepted that acute and chronic immune injury, as well as other pathologies associated with eventual graft loss, may occur initially in the absence of graft dysfunction. Indeed, renal transplant biopsies performed at the time of graft dysfunction may disclose advanced stages of renal injury that may not be amenable to treatment. However, protocol biopsies have several limitations that include their morbidity, cost, and potential for sampling error. Furthermore, the prevalence of early subclinical rejection is decreasing in the modern era of immunosuppression, which argues against their use in patients of low immunological risk. Conversely, the transplantation community has, perhaps unadvisedly, embarked upon protocols of immunosuppressive drug minimization. The consequences of these practices may result in late inflammation in the graft that could prove deleterious to its function in the long term. It is hoped that in the future "systems biology" techniques (eg, genomics, proteomics, and metabolomics) may guide clinicians regarding the safety of drug minimization protocols, inform them as to when a renal transplant biopsy should be procured, and perhaps one day replace the renal transplant biopsy altogether. Until such time, however, the renal biopsy remains an indispensable tool in the management of renal transplant recipients.

Use of cardioprotective medications in kidney transplant recipients.

Death with function causes half of late kidney transplant failures, and cardiovascular disease (CVD) is the most common cause of death in these patients. We examined the use of potentially cardioprotective medications in a prospective observational study at seven transplant centers in the United States and Canada. Among 935 patients, 87% received antihypertensive medications at both 1 and 6 months after transplantation. Similar antihypertensive regimens were used for patients with and without diabetes and CVD, but with wide variability among centers. In contrast, while 44% of patients were on angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) at the time of transplantation, the proportion taking these agents dropped to 12% at month 1, then increased to 24% at 6 months. Fewer than 30% with CVD or diabetes received ACEI/ARB therapy 6 months posttransplant. Aspirin use was uncommon (<40% of patients). Even among those with diabetes and/or CVD, fewer than 60% received aspirin and only half received a statin at 1 and 6 months. This study demonstrates marked variability in the use of cardioprotective medications in kidney transplant recipients, a finding that may reflect, among several possible explanations, clinical uncertainty due the lack of randomized trials for these medications in this population.

Preparation, structural characterization, and thermomechanical properties of poly(methyl methacrylate)/organoclay nanocomposites by melt intercalation.

Poly(methyl methacrylate) (PMMA) based nanocomposites were synthesized by melt intercalation technique using organoclays (Cloisite 30B and Cloisite 20A) as fillers. X-ray diffraction (XRD) and transmission electron microscopy (TEM) were used to determine the dispersion and the morphology of the nanocomposites obtained. Thermomechanical tests including tensile test and dynamic mechanical analysis (DMA) were used to evaluate the Young's modulus, storage modulus and the glass transition temperature. Thermogravimetric analysis (TGA) is conducted on the poly(methyl methacrylate) based nanocomposites to determine their thermal stability. The effect of filler content is studied by considering samples with filler contents varying from 1 to 5 wt%. The mechanical properties obtained from the tensile tests show an increase in the Young's modulus and a decrease in the strain to failure as function of the nanoclays concentration. Relative to the pure poly(methyl methacrylate), the dynamic mechanical analyses show an increase in the storage modulus and the glass transition temperature of both nanocomposites. The thermogravimetric analysis shows an increase of the thermal stability of both nanocomposites.