David N. Rush

Banff 2013 meeting report

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.

Proteomic-Based Detection of Urine Proteins Associated with Acute Renal Allograft Rejection

At present, the diagnosis of renal allograft rejection requires a renal biopsy. Clinical management of renal transplant patients would be improved by the development of non-invasive markers of rejection that can be measured frequently. This study sought to determine whether such candidate proteins can be detected in urine using mass spectrometry. Four patient groups were rigidly defined on the basis of allograft function, clinical course, and allograft biopsy result: acute clinical rejection group (n = 18), stable transplant group (n = 22), acute tubular necrosis group (n = 5), and recurrent (or de novo) glomerulopathy group (n = 5). Urines collected the day of the allograft biopsy were analyzed by mass spectrometry. As a normal control group, 28 urines from healthy individuals were analyzed the identical manner, as well as 5 urines from non-transplanted patients with lower urinary tract infection. Furthermore, sequential urine analysis was performed in patients in the acute clinical rejection and the stable transplant group. Three prominent peak clusters were found in 17 of 18 patients (94%) with acute rejection episodes, but only in 4 of 22 patients (18%) without clinical and histologic evidence for rejection and in 0 of 28 normal controls (P < 0.001). In addition, the presence or absence of these peak clusters correlated with the clinicopathologic course in most patients. Acute tubular necrosis, glomerulopathies, lower urinary tract infection, and cytomegalovirus viremia were not confounding variables. In conclusion, proteomic technology together with stringent definition of patient groups can detect urine proteins associated with acute renal allograft rejection. Identification of these proteins may prove useful as non-invasive diagnostic markers for rejection and the development of novel therapeutic agents.

A randomized, prospective multicenter pharmacoepidemiologic study of cyclosporine microemulsion in stable renal graft recipients. Report of the Canadian Neoral Renal Transplantation Study Group.

BACKGROUND The safety, tolerability, and pharmacokinetics of conventional cyclosporine (ConCsA) and cyclosporine microemulsion (MeCsA) were compared under conditions of normal clinical practice in a prospective, randomized, concentration-controlled, pharmacoepidemiologic study. METHODS Between September 1994 and March 1995, 1097 stable renal transplant recipients in 14 Canadian centers were randomized 2:1 to treatment with MeCsA or ConCsA. Patients were commenced on each study drug at a dose equal to their previous therapy with ConCsA, and the dose was adjusted to maintain predose whole blood cyclosporine concentrations within the therapeutic range established for each center. Prednisone and azathioprine were continued unless dose adjustment was required for clinical reasons. RESULTS The mean cyclosporine concentration was comparable in both treatment groups at all time points throughout the 6 months of follow-up. The mean dose of cyclosporine was 3.6 mg/kg/day in both treatment groups at entry to the study, and declined by 0.3% and by 2.8% in patients receiving ConCsA and MeCsA, respectively. The nature and severity of adverse events were similar in both treatment groups, but there was a transient increase in neurological and gastrointestinal complications in the group receiving MeCsA within the first month after conversion (P<0.05). Serum creatinine and creatinine clearance did not change in either treatment group throughout the study. Biopsy-proven acute rejection occurred in three patients (0.8%) receiving ConCsA and in seven patients (0.9%) receiving MeCsA, with non-histologically proven acute rejection in an additional three patients (0.8%) receiving ConCsA and five patients (0.6%) receiving MeCsA (P=NS). Serum creatinine rose transiently in 35 patients (9.8%) receiving ConCsA and 138 patients (18.7%) receiving MeCsA (P<0.05) and resolved either spontaneously or after a reduction in the cyclosporine dose. One graft was lost in the MeCsA group due to irreversible rejection, and seven patients died, three in the group receiving ConCsA and four of those receiving MeCsA (P=NS). Absorption of cyclosporine was more rapid and complete from MeCsA than from ConCsA during the first 4 hr of the dosing interval, resulting in almost 40% greater exposure to the drug (P<0.001). There was close correlation between area under the time-concentration curve (AUC) over the first 4 hr of the 12-hr dosage interval and AUC over the entire 12-hr dosage interval for both formulations, making AUC over the first 4 hr a good predictor of total cyclosporine exposure. Using this parameter, patients with low absorption randomized to receive MeCsA showed a marked increase in drug exposure by months 3 and 6, whereas there was no change in those who continued on ConCsA. A limited sampling strategy utilizing samples at the predose and postdose trough levels provided an excellent correlation with drug exposure, particularly for patients receiving MeCsA (r2=0.94 MeCsA vs. r2=0.89 ConCsA). CONCLUSIONS MeCsA appears to be a safe and effective therapy in stable renal transplant patients and provides superior and more consistent absorption of cyclosporine when compared with ConCsA. Transient toxicity after conversion to MeCsA occurs in some patients, and may reflect the increased exposure to cyclosporine. Use of a limited sampling approach combining trough and 2-hr postdose concentrations may provide an effective way to monitor this exposure.

Computerized Image Analysis of Sirius Red–Stained Renal Allograft Biopsies as a Surrogate Marker to Predict Long-Term Allograft Function

Chronic allograft nephropathy (CAN) is a major problem in posttransplant management. The lack of a reliable and early surrogate marker of CAN has hampered patient care and research. In this study, the Cortical Fractional Interstitial Fibrosis Volume (V(IntFib)), quantitated with computerized image analysis of Sirius Red-stained protocol biopsies, was examined as a potential surrogate for time to graft failure (TTGF) in 68 renal allograft recipients. At 6 mo posttransplant, V(IntFib) was highly correlated with TTGF (r = 0.64, P < 0.001). Both the Banff Chronic Sum and the Acute Sum Scores were also correlated with TTGF, but less strongly (r = 0.28, P < 0.02; r = 0.35, P < 0.003, respectively). As V(IntFib) was not correlated with the Banff Chronic Score, a multivariate model was created that incorporated V(IntFib) and both Acute and Chronic Banff pathology. This model was highly correlated with TTGF (r = 0.7, P < 0.0001). These findings suggest that V(IntFib) determined by computerized image analysis of Sirius Red-stained protocol biopsies at 6 mo posttransplant, with or without incorporation of Banff acute and chronic scoring, may provide an early surrogate for time to graft failure in renal allograft recipients.

Lack of Benefit of Early Protocol Biopsies in Renal Transplant Patients Receiving TAC and MMF: A Randomized Study

We conducted a randomized, multicenter study to determine whether treatment of subclinical rejection with increased corticosteroids resulted in beneficial outcomes in renal transplant patients receiving tacrolimus (TAC), mycophenolate mofetil (MMF) and prednisone. One hundred and twenty‐one patients were randomized to biopsies at 0,1,2,3 and 6 months (Biopsy arm), and 119 to biopsies at 0 and 6 months only (Control arm). The primary endpoint of the study was the prevalence of the sum of the interstitial and tubular scores (ci + ct)> 2 (Banff) at 6 months. Secondary endpoints included clinical and subclinical rejection and renal function. At 6 months, 34.8% of the Biopsy and 20.5% of the Control arm patients had a ci + ct score ≥ 2 (p = 0.07). Between months 0 and 6, clinical rejection episodes were 12 in 10 Biopsy arm patients and 8 in 8 Control arm patients (p = 0.44). Overall prevalence of subclinical rejection in the Biopsy arm was 4.6%. Creatinine clearance at 6 months was 72.9 ± 21.7 in the Biopsy and 68.90 mL/min ± 18.35 mL/min in the Control arm patients (p = 0.18). In conclusion, we found no benefit to the procurement of early protocol biopsies in renal transplant patients receiving TAC, MMF and prednisone, at least in the short term. This is likely due to their low prevalence of subclinical rejection.

Urinary CXCL9 and CXCL10 Levels Correlate with the Extent of Subclinical Tubulitis

Subclinical tubulitis has been associated with the later development of interstitial fibrosis and tubular atrophy (IF/TA), leading to diminished allograft survival. The aim of this study was to investigate how concentrations of urinary CXC‐receptor 3 (CXCR3) chemokines (i.e. CXCL4/9/10/11) and CCL2 relate to the extent of subclinical tubulitis. Using ELISA, urinary CXCR3 chemokines, CCL2 and tubular injury markers (i.e. urinary NGAL and α1‐microglobulin [α1 m]) were measured in patients with stable estimated GFR ≥40 mL/min exhibiting normal tubular histology (n = 24), subclinical borderline tubulitis (n = 18) or subclinical tubulitis Ia/Ib (n = 22), as well as in patients with clinical tubulitis Ia/Ib (n = 17) or IF/TA (n = 10). CXCL9 and CXCL10 were significantly higher in subclinical tubulitis Ia/Ib than in subclinical borderline tubulitis (p ≤ 0.03) and normal tubular histology (p ≤ 0.0002). By contrast, NGAL, α1‐m, CXCL4, CXCL11 and CCL2 were not or only marginally distinctive across these patient groups. All urinary chemokines and tubular injury markers were higher in clinical tubulitis Ia/Ib than in normal tubular histology (p ≤ 0.002), but only tubular injury markers were elevated in IF/TA. These results demonstrate a correlation of urinary CXCL9 and CXCL10 levels with the extent of subclinical tubulitis suggesting potential as noninvasive screening biomarkers.

Prevalence and treatment of decreased bone density in renal transplant recipients: a randomized prospective trial of calcitriol versus alendronate.

Background. Reduced bone mineral density (BMD) is common in long-term renal transplant recipients and results in a high incidence of fractures. The optimal therapy for these patients is not known. Methods. Baseline BMD determinations were obtained in 211 long-term adult renal transplant recipients. One hundred and seventeen patients with a reduced BMD (T score ≤ −1) were randomly assigned to treatment with alendronate and calcium (n=60) versus calcitriol and calcium (n=57). Of these, 46 and 51 patients, respectively, completed 1 year of treatment. Forty-nine patients who were not eligible or did not consent to the trial were followed prospectively. Results. Reduced baseline BMD (T score ≤ −1) was present in 159 (78.7%) of patients at the lumbar spine or femur. There was no significant loss of BMD in the prospectively followed patients during 2.7 years. The average lumbar BMD increased from 0.984±0.149 to 1.025±0.143 g/cm2 (P <0.001) with alendronate and from 1.014±0.15 to 1.034±0.146 g/cm2 (P =0.002) with calcitriol. BMD at the femur increased from 0.809±0.092 to 0.836±0.107 g/cm2 (P <0.001) with alendronate and from 0.830±0.144 to 0.857±0.125 g/cm2 (P =0.023) with calcitriol. Conclusions. One year of treatment with alendronate or calcitriol, both with calcium supplementation, resulted in significant increases in BMD at the lumbar spine and femur, with a trend toward alendronate being more effective at the spine (P =0.082). Further studies are needed to determine whether BMDs continue to increase after 1 year and whether there is any additional benefit to combining vitamin D and alendronate. Larger studies are needed to determine whether treatment decreases fracture rates.

Detection of subclinical tubular injury after renal transplantation: comparison of urine protein analysis with allograft histopathology.

Background. Tubulointerstitial injury due to rejection leads to tubular atrophy (TA)/interstitial fibrosis (IF) followed by deterioration of allograft function. This study investigated whether urinary tubular injury biomarkers can detect subclinical tubulitis found in protocol biopsies allowing for a noninvasive screening procedure. Methods. Four rigidly defined groups (stable transplants with normal tubular histology [n=24], stable transplants with subclinical tubulitis [n=38], patients with clinical tubulitis Ia/Ib [n=18], and patients with other clinical tubular pathologies [n=20]) were compared for differences in urinary intact/cleaved β2-microglobulin (i/cβ2m), retinol-binding protein (RBP), neutrophil-gelatinase-associated lipocalin (NGAL), and α1-microglobulin (α1m). Results. Tubular proteinuria was present in 38% (RBP) to 79% (α1m) of patients in the stable transplant with normal tubular histology group. The stable transplant with subclinical tubulitis group had slightly higher levels of i/cβ2m (P=0.11), RBP (P=0.17), α1m (P=0.09), and NGAL (P=0.06) than the stable transplant with normal tubular histology group with a substantial overlap. The clinical tubulitis Ia/Ib and the other clinical tubular pathology groups had significantly higher levels of RBP, NGAL, and α1m than stable transplants with normal tubular histology or stable transplants with subclinical tubulitis (P<0.002). Conclusions. None of the investigated biomarkers allow for clear differentiation between stable transplants with normal tubular histology and stable transplants with subclinical tubulitis. Therefore, the protocol allograft biopsy currently remains the preferred tool to screen for subclinical tubulitis. Further longitudinal studies should determine whether tubular proteinuria in stable transplants with normal tubular histology indicates a clear risk for early development of TA/IF.

Safety and tolerability of cyclosporine and cyclosporine microemulsion during 18 months of follow-up in stable renal transplant recipients: a report of the Canadian Neoral Renal Study Group.

BACKGROUND There has been concern that the increased drug exposure associated with treatment with cyclosporine microemulsion (CsA-ME) would lead to an increase in adverse events. METHODS The long-term safety and tolerability of conventional cyclosporine (CsA) and CsA-ME were compared in a randomized, multicenter, pharmacoepidemiologic study involving 1097 stable renal transplant patients after 18 months of follow-up. RESULTS No significant difference was seen in change in serum creatinine or calculated creatinine clearance between the two groups. Episodes of deterioration in renal function (change in serum creatinine > or = 20%) were categorized with the following results for CsA-ME versus CsA, respectively: acute rejection, 4.5% vs. 4.5%; chronic rejection, 8% vs. 11%; CsA nephrotoxicity, 12% vs. 7% (P=0.008); transient changes, 17% vs. 12%; other causes, 4% vs. 6%. During the first 6 months of the study, a transient increase in the incidence of gastrointestinal and neurological adverse events was seen in the CsA-ME group compared with the CsA group. Up to 18 months, patients in the CsA group reported significantly fewer hearing and vestibular disorders, but more cardiovascular problems than those in the CsA-ME group (P=0.035). CONCLUSIONS Tolerance to CsA and CsA-ME was similar. Renal function over 18 months was not adversely affected by the increased drug exposure with CsA-ME, although there was a transient increase in nephrotoxicity. The frequency of acute and chronic rejection did not change.

The urine protein to creatinine ratio (P/C) as a predictor of 24-hour urine protein excretion in renal transplant patients.

BACKGROUND The purpose of this study was to examine the utility of the random urine protein to creatinine ratio (P/C) in evaluation and longitudinal management of proteinuria in adult renal transplant recipients with or without overt nephropathy in an outpatient clinic. METHODS A total of 289 adult renal transplant recipients provided 24-hr urine collections for total protein and creatinine, followed by a random urine for protein and creatinine. For longitudinal analysis, 192 of these patients provided two 24-hr urine collections with concomitant random urine specimens separated on average by 6.8 months. As well, 134 patients provided a total of 851 multiple-paired spot and 24-hr urine samples (range 2 to 12) over a 2-year period. RESULTS The log random urine P/C ratio correlated significantly to the log 24 UP (r=0.749, P<0.0001) with or without nephrotic range proteinuria. High sensitivity (74.4-90%) and specificity values (93-98%) were found for estimating proteinuria from 0.5 to 2 g/day. However, the precision of estimation decreased as the level of urinary protein excretion increased to >3 g/day. The positive predictive value decreased as proteinuria became >3 g/day, perhaps because of the low prevalence of patients with high level proteinuria in our sample. The direction of change in P/C ratio longitudinally was accompanied by a similar direction of change in 24 UP, which was highly significant (r=0.7555, P<0.0001). CONCLUSION We conclude that the urine P/C ratio is a useful and convenient screening and longitudinal test for proteinuria.