The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology.

The XIII Banff meeting, held in conjunction the Canadian Society of Transplantation in Vancouver, Canada, reviewed the clinical impact of updates of C4d-negative antibody-mediated rejection (ABMR) from the 2013 meeting, reports from active Banff Working Groups, the relationships of donor-specific antibody tests (anti-HLA and non-HLA) with transplant histopathology, and questions of molecular transplant diagnostics. The use of transcriptome gene sets, their resultant diagnostic classifiers, or common key genes to supplement the diagnosis and classification of rejection requires further consensus agreement and validation in biopsies. Newly introduced concepts include the i-IFTA score, comprising inflammation within areas of fibrosis and atrophy and acceptance of transplant arteriolopathy within the descriptions of chronic active T cell-mediated rejection (TCMR) or chronic ABMR. The pattern of mixed TCMR and ABMR was increasingly recognized. This report also includes improved definitions of TCMR and ABMR in pancreas transplants with specification of vascular lesions and prospects for defining a vascularized composite allograft rejection classification. The goal of the Banff process is ongoing integration of advances in histologic, serologic, and molecular diagnostic techniques to produce a consensus-based reporting system that offers precise composite scores, accurate routine diagnostics, and applicability to next-generation clinical trials.

The XIIIth Banff Conference on Allograft Pathology: The Banff 2015 Heart Meeting Report: Improving Antibody-Mediated Rejection Diagnostics: Strengths, Unmet Needs, and Future Directions.

The 13th Banff Conference on Allograft Pathology was held in Vancouver, British Columbia, Canada from October 5 to 10, 2015. The cardiac session was devoted to current diagnostic issues in heart transplantation with a focus on antibody-mediated rejection (AMR) and small vessel arteriopathy. Specific topics included the strengths and limitations of the current rejection grading system, the central role of microvascular injury in AMR and approaches to semiquantitative assessment of histopathologic and immunophenotypic indicators, the role of AMR in the development of cardiac allograft vasculopathy, the important role of serologic antibody detection in the management of transplant recipients, and the potential application of new molecular approaches to the elucidation of the pathophysiology of AMR and potential for improving the current diagnostic system. Herein we summarize the key points from the presentations, the comprehensive, open and wide-ranging multidisciplinary discussion that was generated, and considerations for future endeavors.

Anti-ETAR and anti-AT1R autoantibodies are elevated in patients with endstage cystic fibrosis.

Autoantibodies against endothelin-1 type A receptor (ETAR) are present in systemic sclerosis complicated by lung fibrosis and pulmonary hypertension. As increased serum levels and local overproduction of endothelin-1 in the airways are reported in cystic fibrosis (CF) patients, we reasoned that anti-ETAR antibodies could be prevalent in endstage CF patients prior to lung transplantation (LTx). Also, ETAR autoantibodies are frequently associated with autoantibodies against the angiotensin II type 1 receptor (AT1R). We analyzed the presence of anti-ETAR and anti-AT1R autoantibodies in 43 LTx patients (chronic obstructive pulmonary disease (COPD), n=20; CF, n=13; interstitial lung disease (ILD), n=1). We observed overall higher anti-ETAR and anti-AT1R autoantibody titers in sera taken prior to LTx in the CF patient group as compared to COPD. No difference was found in autoantibody levels between patients with CF versus ILD. In sera taken post-LTx we found the same difference in anti-ETAR and anti-AT1R autoantibody titers between patients with CF versus COPD. No difference was found in antibody titers between sera taken prior to or 6 months after LTx. There was no association between autoantibody levels and other relevant demographic parameters, and we found no association between autoantibody titers and the development of the bronchiolitis obliterans syndrome. Both autoantibody titers were strongly correlated. We hypothesize that due to prolonged exposure to bacterial infection, increased levels of AT1R and ETAR result in a deregulated immune response causing autoantibody formation. Further research is expedient to elucidate the occurrence of autoantibodies against ETAR and AT1R and their role in disease progression.

Serum levels for midkine, a heparin-binding growth factor, inversely correlate with angiotensin and endothelin receptor autoantibody titers in patients with macroangiopathy.

AIM: Peripheral artery disease results in impaired blood flow to the extremities, most often as a consequence of atherosclerotic disease. The hallmark of atherosclerosis is chronic inflammation in the vessel wall. The renin-angiotensin and endothelin systems are considered important pathophysiological effectors. Midkine, a multifunctional cytokine, fulfils different roles in inflammation and promotion of neoangiogenesis. The aim of this study was to assess whether circulating midkine serum levels in patients with peripheral artery disease correlate with established atherosclerosis risk factors, as well as titers of functional autoantibodies directed against receptors of the renin-angiotensin and endothelin system. METHODS: Clinical data, laboratory values and serum samples from 118 patients operated on for severe peripheral artery disease, and from 100 healthy blood donors were collected. Serum samples were analysed for midkine concentrations as well as autoantibody titers against angiotensin II type 1 and endothelin-1 type A receptors. RESULTS: Midkine values were significantly higher in the study population than in healthy controls (P<0.001). Circulating midkine levels did not correlate with neither of the traditional risk factors age, sex, obesity, smoking, hypertension, high cholesterol levels, or diabetes mellitus. An unexpected inverse correlation was found with the autoantibodies against angiotensin II type 1 receptor (P<0.05) and endothelin-1 type A receptor (P<0.01). CONCLUSION: The high levels of midkine in severe peripheral artery disease patients introduce this cytokine as a possible novel effector in the advanced atherosclerotic process. These results also suggest a functional link between vascular receptor autoantibody formation and down-regulated midkine serum levels, that may be relevant in the pathogenesis of clinically relevant peripheral artery occlusive disease.

Pretransplant sensitization against angiotensin II type 1 receptor is a risk factor for acute rejection and graft loss.

The angiotensin II type 1 receptor (AT1R) is an emerging target of functional non-HLA antibodies (Ab). We examined the potential of determining the degree of presensitization against AT1R as a risk factor for graft survival and acute rejection (AR). The study included 599 kidney recipients between 1998 and 2007. Serum samples were analyzed in a blinded fashion for anti-AT1R antibodies (AT1R-Abs) using a quantitative solid-phase assay. A threshold of AT1R-Ab levels was statistically determined at 10 U based on the time to graft failure. An extended Cox model determined risk factors for occurrence of graft failure and a first AR episode. AT1R-Abs >10 U were detected in 283 patients (47.2%) before transplantation. Patients who had a level of AT1R-Abs >10 U had a 2.6-fold higher risk of graft failure from 3 years posttransplantation onwards (p = 0.0005) and a 1.9-fold higher risk of experiencing an AR episode within the first 4 months of transplantation (p = 0.0393). Antibody-mediated rejection (AMR) accounted for 1/3 of AR, whereby 71.4% of them were associated with >10 U of pretransplant AT1R-Abs. Pretransplant anti-AT1R-Abs are an independent risk factor for long-term graft loss in association with a higher risk of early AR episodes.

Neutrophil gelatinase-associated lipocalin: pathophysiology and clinical applications.

Neutrophil gelatinase-associated lipocalin (NGAL), a 25 kDa protein produced by injured nephron epithelia, is one of the most promising new markers of renal epithelial injury. In contrast to serum creatinine and urinary output, which are the measures of kidney function, NGAL is specifically induced in the damaged nephron and then released into blood and urine, where it can be readily measured. Careful proof-of-concept studies using defined animal models have uncovered the sources and trafficking of NGAL in acute kidney injury (AKI) and have addressed the contributions of renal and non-renal sources. Clinical studies indicate that NGAL, unlike creatinine, is a marker responsive to tissue stress and nephron injury, but less so to adaptive hemodynamic responses. In certain clinical settings, NGAL is an earlier marker compared with serum creatinine. In addition, clinical studies have shown that NGAL is a powerful predictor of poor clinical outcomes, which can be used to risk stratify patients when combined with serum creatinine. NGAL has important limitations, including its responsiveness in systemic inflammation, which is partially uncoupled from its response to kidney injury and which needs to be considered when interpreting NGAL results clinically. This review covers the biology and pathophysiology of NGAL and summarizes the results of the growing body of clinical studies that have addressed the utility of NGAL in the early diagnosis of AKI, in the distinction of intrinsic AKI and in the prognostic assessment of broad patient populations.

Novel signalling mechanisms and targets in renal ischaemia and reperfusion injury.

Acute kidney injury (AKI) induced by ischaemia and reperfusion (I/R) injury is a common and severe clinical problem. Vascular dysfunction, immune system activation and tubular epithelial cell injury contribute to functional and structural deterioration. The search for novel therapeutic interventions for I/R-induced AKI is a dynamic area of experimental research. Pharmacological targeting of injury mediators and corresponding intracellular signalling in endothelial cells, inflammatory cells and the injured tubular epithelium could provide new opportunities yet may also pose great translational challenge. Here, we focus on signalling mediators, their receptors and intracellular signalling pathways which bear potential to abrogate cellular processes involved in the pathogenesis of I/R-induced AKI. Sphingosine 1 phosphate (S1P) and its respective receptors, cytochrome P450 (CYP450)-dependent vasoactive eicosanoids, NF-κB- and protein kinase-C (PKC)-related pathways are representatives of such 'druggable' pleiotropic targets. For example, pharmacological agents targeting S1P and PKC isoforms are already in clinical use for treatment for autoimmune diseases and were previously subject of clinical trials in kidney transplantation where I/R-induced AKI occurs as a common complication. We summarize recent in vitro and in vivo experimental studies using pharmacological and genomic targeting and highlight some of the challenges to clinical application of these advances.

Calprotectin and neutrophil gelatinase-associated lipocalin in the differentiation of pre-renal and intrinsic acute kidney injury.

BACKGROUND: Urinary calprotectin has recently been identified as a promising biomarker for the differentiation of pre-renal and intrinsic acute kidney injury (AKI). This study compares the diagnostic performance of calprotectin and neutrophil gelatinase-associated lipocalin (NGAL) in this differential diagnosis. METHODS: Urinary calprotectin and NGAL concentrations were assessed in a study population of 87 subjects including 38 cases of intrinsic AKI, 24 cases of pre-renal AKI and 25 healthy controls. Urinary tract obstruction, renal transplantation and metastatic cancer were defined as exclusion criteria. RESULTS: Mean calprotectin concentrations were significantly lower in pre-renal (190.2 ± 205.7 ng mL(-1) ) than in intrinsic AKI (6250.1 ± 7167.2 ng mL(-1) , P < 0.001). Receiver-operating characteristic (ROC) analysis provided an AUC of 0.99. Mean NGAL concentrations were significantly higher in intrinsic than in pre-renal AKI as well (458.1 ± 695.3 vs. 64.8 ± 62.1 ng mL(-1) , P = 0.001) providing an AUC of 0.82. A combination of the present study population with the cohort of the proof of concept study led to a population of 188 subjects (58 pre-renal AKI, 90 intrinsic AKI, 40 healthy controls). ROC analyses provided an AUC of 0.97 for calprotectin and 0.76 for NGAL yielding sensitivity and specificity values of 93.3 and 94.8% (calprotectin) vs. 75.3 and 72.4% (NGAL). Optimal cut-off values were 440 ng mL(-1) (calprotectin) and 52 ng mL(-1) (NGAL). Pyuria increased calprotectin concentrations independent of renal failure. CONCLUSION: This study shows that both calprotectin and NGAL are able to differentiate between pre-renal and intrinsic AKI after exclusion of pyuria. In the present population, calprotectin presents a higher sensitivity and specificity than NGAL.

[Clinical risk-adapted therapies in systemic sclerosis]. / Risikoadaptierte Therapien bei der systemischen Sklerose.

Systemic sclerosis is a challenging disease in terms of early risk assessment and the implications for therapy. The current article draws on a literature analysis and on personal experience of over 450 patients with systemic sclerosis and related illnesses, describing an attempt at a risk-adapted treatment strategy for patients with systemic sclerosis, and is intended to be used as a discussion paper. A major point of emphasis is on novel therapeutic options for patients who are refractory to current treatment strategies and who are at a high risk of serious complications or a limited prognosis. The paper also highlights possible future treatment options, including vasoactive, cellular and immune suppressant treatments.

Value of posttransplant antibody tests in the evaluation of patients with renal graft dysfunction.

Posttransplant HLA antibodies correlate with C4d positive rejection and decreased graft survival. However, the diagnostic value of various antibody tests in the management of patients presenting with graft dysfunction is uncertain. Whether all or some patients should be tested, how often, what antibodies to test for and how to interpret results in presensitized or transfused patients, are issues still unresolved. We tested for HLA and non-HLA antibodies by flow cytometry assays in 103 consecutive patients with graft dysfunction. The results show that: (1) C4d positive rejection was diagnosed in 75% of patients who developed posttransplant HLA antibodies, but only in 2% in antibody negative patients. (2) The correlation existed for donor specific IgG antibodies but not for IgM or nondonor specific IgG antibodies. (3) Weak antibody reactivity required confirmation by alternative testing as there were false positive results. (4) Posttransplant transfusions did not induce de novo HLA antibodies. (5) Negative antibody results were unlikely to turn positive after several months of follow-up. (6) Antibodies to the angiotensin II type 1 receptor, HLA-DP and MICA did not correlate with C4d+ rejection. We conclude that testing for posttransplant HLA antibodies is critical in narrowing the diagnostic alternatives in patients with graft dysfunction.

Effect of mycophenolate mofetil on rat kidney grafts with prolonged cold preservation.

The impact of mycophenolate mofetil (MMF) on initial renal transplant function is not well characterized. We tested how MMF may modulate graft function and survival in a syngeneic rat kidney transplantation model after prolonged cold preservation. Donor kidneys were preserved in University of Wisconsin for either 24 or 39 h prior to transplantation into nephrectomized rats. Recipients received MMF (20 mg/kg/day) or vehicle. Mycophenolic acid (MPA) blood concentrations were measured by high-performance liquid chromatography. The inflammatory response, tubular epithelial proliferation, and histologic damage 3 days post-transplantation were assessed microscopically. In the 24 h cold storage (c.s.) group serum-creatinine was measured. In the 39 h c.s. group 1-week recipient survival was determined. After 24 h of c.s., recipient survival was 100%. The number of T-cell infiltrates was low and not influenced by MMF, whereas renal ED1+ cell infiltration was significantly suppressed by MMF. Tubular cell proliferation was enhanced by MMF. Serum-creatinine levels and renal histology were comparable between MMF and vehicle-treated animals. In the 39 h c.s. group, recipient survival was 20% in MMF-treated vs 90% in vehicle-treated animals (P=0.001). MMF effectively suppressed inflammatory cell infiltration and inhibited tubular cell proliferation. MMF-induced structural damage was most striking in the renal papilla. In rat kidney grafts with moderate preservation injury (24 h c.s.), MMF, given at an immunosuppressive dose, showed predominantly antiinflammatory effects without compromising graft function. In grafts with severe preservation injury (39 h c.s.), MMF caused irreversible structural damage and inhibited tubular cell regeneration resulting in renal failure.

[Urological evaluation and follow-up of the kidney transplant patient]. / Urologische Betreuung von Patienten vor und nach Nierentransplantation.

Patients with end-stage renal disease awaiting kidney transplantation require regular urological evaluation. The urologist's main task is early diagnosis and treatment of genitourinary malignancies and evaluation of the lower urinary tract. Furthermore, urologists are often confronted with the question of whether or not to perform pretransplant urological surgery, i.e., native nephrectomy for polycystic kidney disease. Urological care after kidney transplantation involves diagnosis and treatment of ureteral complications, malignancies, lower urinary tract symptoms, and last but not least erectile dysfunction, which has a prevalence of 20-50% among kidney transplant recipients. For the evaluation and follow-up of the living kidney donor, international guidelines have been developed in recent years to also help the urologist to perform a correct evaluation and follow-up of the kidney donor.

Laparoscopic versus open donor nephrectomy in Germany: impact on donor health-related quality of life and willingness to donate.

INTRODUCTION: Laparoscopic living donor nephrectomy (LDN) offers multiple advantages to the donor. Since 1999 LDN has become the only surgical approach for living kidney donation in our department. To our knowledge a donor health-related quality of life (QoL) has not yet been performed with standardized and validated questionnaires to compare laparoscopic with open nephrectomy. We therefore performed a study with two questionnaires (SF-36/GBB-24) and one set of open questions for all donors in our department. METHODS: Questionnaires were sent out to all donors between 1983 and 2001 with at least a 1-year follow-up. To exclude a bias a maximum response rate was sought; donors who did not answer were recontacted as well as their recipients or their physicians to motivate them for participation. RESULTS: The response rate was (89.8%). Except for less limb pain in the laparoscopy group, no difference could be detected for donors QoL with respect to the surgical method. Willingness to donate again was not affected by the surgical method. Nevertheless if asked again today, most donors want laparoscopic kidney retrieval. CONCLUSIONS: Donors health-related QoL is not affected by the surgical method when queried retrospectively. Nevertheless, most donors today would favor laparoscopy, if they could chose again. How laparoscopy affects a reluctant donor to step forward must be determined in a prospective study.

Differences in reporting of acute rejections between American and European publications of large immunosuppressive trials impair comparability of study results.

This study examined the use of different definitions for acute rejection in recent large multicenter trials performed in America and Europe in order to assess whether systematic differences exist between both scientific cultures. We systematically selected recent publications on multicenter randomized controlled trials, investigating immunosuppressive regimens in de novo kidney transplant recipients. Publications included were classified according to the type of acute rejection reported: group 1 reported no or only one type of rejection rate (biopsy-proven or treated); group 2 reported information on both treated and biopsy-proven rates. Other potential factors (journal's impact-factor, study size) were compared within the subgroups. To determine the rates of treated but not biopsy-proven acute rejections, additional analyses were performed within subgroup 2. The reviewed publications were 24/44 (54.5%) European (E) and 20/44 (45.5%) American (A) origin. Eighteen of 44 publications reported no or only one type of rejection rate (group 1); 26 publications reported treated as well as biopsy-proven rates (group 2). Significantly more European publications reported both treated and biopsy-proven rates (E: 18/24 [75.0%] vs A: 8/20 [40.0%]; P = .019). Group 1 American papers were published in higher-ranked journals than European ones. The rate of blindly treated rejections did not differ significantly (A: 6.13% [range 0% to 12.8%] vs E: 8.43% [range 0% to 16.9%]) and the proportion of blindly treated rejections was slightly lower in American studies (A: 18.5% vs E: 26.5%). Our systematic review showed large discrepancies with a trend to report biopsy-proven rejection rates only in recent years.

Impact of cyclosporine on the development of immunosuppressive therapy.

With the advent of cyclosporine (CsA) 20 years ago, graft survival increased considerably to more than 80% at 2 years posttransplant. The early formulation of CsA, Sandimmun, is effective in preventing organ rejection, although its absorption profile means it is subject to a high degree of variability. The development of a microemulsion formulation, Neoral, provided a therapy with superior efficacy in kidney, liver, and heart transplantation with an improved pharmacokinetic profile. Calcineurin inhibitors (CNIs), including CsA, have a narrow therapeutic range, so frequent blood measurements to control drug levels are required. Recent research has demonstrated that the measurement of blood CsA concentration at 2 hours postdosing--C2 monitoring--has the potential to optimize efficacy and reduce the side effects associated with CNI use. In heart and de novo kidney transplantation, C2 monitoring may help to further reduce the incidence of acute rejection, while in maintenance renal transplant recipients, C2 monitoring can help to detect overexposure and thus allows safe dose reduction, which may improve blood pressure and renal function. C2 monitoring thus facilitates a better balance between effective Neoral immunosuppression and unwanted side effects. Today, CsA remains the cornerstone of immunosuppression, and ongoing studies aim to further optimize patient management strategies with Neoral. With other trials evaluating the impact of Neoral in combination with newer therapies such as Certican, myfortic, and FTY720, the use of CsA in transplant recipients looks set to continue.

Enteric-coated mycophenolate sodium: safe conversion from mycophenolate mofetil in maintenance renal transplant recipients.

Mycophenolate mofetil (MMF), in combination with cyclosporine and corticosteroids, improves long-term graft function and survival in renal transplant recipients. However, optimal MMF therapy may be limited by gastrointestinal (GI) intolerance, which may result in the need for MMF dose reduction, interruption, or discontinuation, leading to increased risk of acute rejection. Enteric-coated mycophenolate sodium (EC-MPS) is an advanced formulation delivering mycophenolic acid (MPA), developed with the objective of improving MPA-related upper GI adverse events. A pivotal, 12-month, phase III, randomized, multicenter, double-blind, double-dummy, parallel group study investigated whether stable renal transplant patients can be converted from MMF to EC-MPS therapy without compromising tolerability or efficacy. Stable renal transplant recipients received either MMF, 1000 mg b.i.d. (n=159), or EC-MPS, 720 mg b.i.d. (n=163), for 12 months. The incidence of GI adverse events was comparable between both treatment groups at 3 and 6 months, but there was a trend toward reduced severity of GI side effects in the EC-MPS group. There were fewer serious adverse events with EC-MPS and significantly fewer serious infections (P<.05). This comparable safety profile for EC-MPS and MMF also extended to elderly patients and patients with diabetes at baseline. For the composite efficacy variable of biopsy-proven acute rejection, graft loss, death, or loss to follow-up, EC-MPS had a lower 12-month efficacy failure rate (EC-MPS: 7.5% vs MMF: 12.3%; P=ns). These data demonstrate that stable renal transplant recipients receiving MMF can be converted to EC-MPS with no efficacy or tolerability compromise.

FTY720: early clinical experience.

FTY720 is the first in a new class of immunomodulators--sphingosine 1-phosphate receptor (S1P-R) agonists. It is highly effective in prolonging allograft survival in preclinical models of transplantation. Furthermore, FTY720 acts synergistically with calcineurin inhibitors and proliferation inhibitors in these models, suggesting that use of FTY720 in combination with classical immunosuppressants may be a promising new option for transplant patients. Phase I studies conducted in stable renal transplant patients maintained on a cyclosporine (CsA)-based regimen have revealed a tolerable profile of FTY720 for transplant pharmacotherapy. The pharmacokinetics of FTY720 is characterized by linear dose-proportional exposure over a wide range of doses, only moderate interpatient variability, and a prolonged elimination half-life (t(1/2) 89 to 157 hours). These factors suggest that FTY720 can be administered according to a simple once-daily schedule, without the need for blood-level monitoring or dose titration. The pharmacodynamics of FTY720 in humans are characterized by a significant reduction in peripheral blood count by up to 85%. In contrast to the nonspecific myelosuppressive effects of other immunosuppressants, this effect of FTY720 is specific for lymphocytes, with no effect observed on monocytes or granulocytes. In combination with CsA, FTY720 was well tolerated following single or multiple dosing, without any evidence of additional toxicities, indicating that FTY720 may be useful in the future design of more effective and less toxic regimens for prevention of graft rejection.