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.

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.

Clinical predictors of proteinuria after conversion to sirolimus in kidney transplant recipients.

Proteinuria is an increasingly recognized effect of sirolimus (SRL) therapy in kidney transplant recipients. Predictors of proteinuria after conversion to SRL are not well described, and in particular the risk in African-American (AA) kidney recipients is unknown. We sought to analyze risk factors for proteinuria with SRL therapy in a cohort of 39 patients (44% AA) converted from tacrolimus to SRL at a mean time of 4 months posttransplantation. Patients were maintained on therapy with mycophenolate mofetil while most patients underwent early steroid withdrawal. Urinary protein to creatinine ratio (Up/cr) at a mean of 14 months postconversion increased to > or =500 mg/g in 65% of AAs versus 14% of non-AAs (p = 0.001). Mean arterial blood pressure at the time of conversion and pretransplant proteinuric kidney disease were also predictors of proteinuria after SRL conversion. In conclusion, AAs appear to be at high risk for proteinuria and should be monitored closely after conversion to SRL in calcineurin inhibitor sparing protocols.

Differential pharmacokinetic interaction of tacrolimus and cyclosporine on everolimus.

OBJECTIVE: We characterized the pharmacokinetics of tacrolimus and everolimus in a combined immunosuppressive regimen. METHODS: This was an open-label exploratory trial in eight maintenance renal transplant patients with calcineurin inhibitor intolerance initially receiving mycophenolate mofetil (MMF) and tacrolimus. At enrollment, MMF was discontinued and replaced with everolimus 1.5 mg twice a day in study period 1 (days 1 to 10). In period 2 (day 11 to month 3), tacrolimus dose was reduced by half. RESULTS: At study entry tacrolimus trough level (C0) was 7.9 +/- 3.9 ng/mL and area under the curve over a dosing interval (AUC) was 132 +/- 56 ng x h/mL. The addition of everolimus in period 1 did not change tacrolimus exposure: C0 8.4 +/- 4.0 ng/mL, AUC 134 +/- 70 ng x h/mL. Everolimus pharmacokinetics in the presence of tacrolimus in period 1 were: C0 3.3 +/- 1.2 ng/mL, Cmax 10.4 +/- 5.1 ng/mL, AUC 58 +/- 20 ng x h/mL. When compared to pharmacokinetic data from a previous study in 47 renal transplant patients receiving everolimus at the same fixed dose (1.5 mg twice a day) with cyclosporine, everolimus exposure was 2.5-fold higher with cyclosporine relative to the data in this study with tacrolimus. After tacrolimus dose reduction in period 2, there was no clinically relevant change in everolimus exposure: C0 3.0 +/- 1.1 ng/mL, Cmax 8.2 +/- 1.3 ng/mL, AUC 49 +/- 10 ng x h/mL. CONCLUSIONS: Tacrolimus appears to have a minimal effect on everolimus blood levels compared with the influence of cyclosporine. The dose of everolimus when combined with tacrolimus needs to be higher than when combined with cyclosporine in order to reach a given everolimus blood level.

Experience with everolimus.

Everolimus is a novel macrolide immunosuppressant that acts as a T-lymphocyte proliferation signal inhibitor. Its actions are complementary to and synergistic with those of the calcineurin inhibitors. Compared with sirolimus, everolimus has unique pharmacokinetic characteristics including greater bioavailability and a shorter half-life, allowing more rapid achievement of a steady state. Clinical experience to date, largely limited to use in kidney transplant patients receiving cyclosporine-based immunosuppression, indicates that administration of everolimus is associated with low rates of acute rejection and a tolerable safety profile. Recent observations in heart transplant patients suggest that the antiproliferative effects of everolimus may prevent allograft vasculopathy.

Use of sirolimus to facilitate cyclosporine avoidance or steroid withdrawal in kidney transplant recipients.

Sirolimus is a non-nephrotoxic cell-cycle progression antagonist that has proven to be highly effective in preventing renal allograft rejection when used in combination with cyclosporine and corticosteroids. On the basis of its immunosuppressive potency alone, sirolimus has emerged as an agent with the potential for facilitating the elimination of calcineurin inhibitors or corticosteroids from the maintenance regimens administered to kidney transplant recipients. The results of three randomized trials suggest that use of sirolimus as a substitute for cyclosporine in patients receiving steroids and either azathioprine or mycophenolate mofetil results in comparable rates of acute rejection and better renal function than in patients maintained on cyclosporine. Preliminary results from uncontrolled trials indicate that use of sirolimus in combination with either cyclosporine or tacrolimus allows withdrawal of steroid therapy with low rates of subsequent acute rejection compared to historical controls. Larger, controlled studies with longer durations of follow-up are warranted to verify the favorable results of these early experiences with the use of sirolimus as a calcineurin inhibitor or steroid-sparing agent.

Safety and efficacy of TOR inhibitors and other immunosuppressive regimens in African-American renal transplant recipients.

African-American renal transplant recipients have higher rates of acute allograft rejection and lower rates of allograft survival compared with Caucasian patients, and these differences have not been eliminated by a new generation of potent immunosuppressive drugs. In particular, African-Americans tend to exhibit higher rejection rates after withdrawal of corticosteroid therapy. Based on promising early results using the combination of sirolimus and low-dose tacrolimus in liver, kidney-pancreas, and islet-cell transplant recipients, our center is conducting a pilot study of this immunosuppression regimen in African-American patients. As of April 2001, there has been only 1 acute rejection episode (2.8%) in this cohort. Long-term follow-up of these patients will be necessary to assess the benefits and risks of this regimen.

Life satisfaction in renal transplant recipients: preliminary results from the transplant learning center.

The Transplant Learning Center (TLC) was designed to improve quality of life (QOL) and preserve graft function in solid-organ transplant recipients. To meet the specific goals of the program, the Life Satisfaction Index and Transplant Care Index were designed to serve as composite measures for measuring transplant-specific QOL and the ability to care for a transplant, respectively. In this study, we analyzed self-reported health information to examine relationships between comorbidities and individual posttransplantation side effects, life satisfaction, and transplant care, defined by renal transplant recipients. Patients entered the TLC through self-referral or referral by a health professional. Included in the analysis were 3,676 TLC enrollees with a mean time since transplantation of 4.8 years. Comorbidities and adverse effects were common, with high blood pressure reported by 89% of respondents and unusual hair growth reported by 70%. Sexual dysfunction and headache had a greater impact on QOL than more common adverse effects, such as changes in body and facial shape, hirsutism, and tremor. Regression modeling was used to identify the most significant associations between QOL indices and structural (nonmedical), medical, and psychosocial factors. Greater life satisfaction was most strongly associated with being in control of one's health and living a normally active life with satisfying emotional relationships. Management of such clinical problems as adverse effects of medication and nonadherence should be informed by the patient's perspective. Clinicians should actively solicit information about physical activity, appearance concerns, side effects of medications, nonadherence, and sexual and relationship issues when evaluating renal transplant recipients.

Immunomodulatory effect of extracorporeal photopheresis after successful treatment of resistant renal allograft rejection.

BACKGROUND: Acute renal allograft rejection contributes to patient morbidity. Standard immunosuppressives are only partially effective and have significant side effects. Extracorporeal photopheresis (ECP) has been effective in reversing the acute rejection process. T cell cytokine expression is implicated in rejection and tolerance but actual changes in the cytokine profile of ECP-treated individuals have not been documented. METHODS: ECP was administered to a patient with acute renal allograft rejection resistant to other immunosuppressives. Enzyme-linked immunosorbent spot (ELISPOT) assay was performed to determine the frequency of mitogen-induced cytokine-producing cells before and after ECP. RESULTS: ECP resulted in resolution of rejection; serum creatinine concentration fell from 7.1 to 2.2 mg/dl; ELISPOT revealed a three-fold increase in the frequency of IL-5 producing cells; IFN-gamma:IL-5 ratio shifted from 2.73 pre-treatment to 1.01 post-treatment. CONCLUSION: Effective therapy of acute allograft rejection with ECP alters the peripheral blood cytokine profile towards "type 2" cytokines, suggesting that alteration of T cell cytokine profiles may contribute to the resolution of the process.

Elevated endothelin-1 in tubular epithelium is associated with renal allograft rejection.

Vascular endothelin-1 (ET-1) levels are elevated in patients with renal allograft rejection, and the mitogenic and pressor actions of ET-1 might contribute to transplant vasculopathy, posttransplantation hypertension, and ischemia-reperfusion injury. In contrast, relatively little is known about tubular expression of ET-1 in acute or chronic rejection of renal allografts. We sought to determine whether tubular ET-1 levels were altered in patients with acute or chronic renal allograft rejection. Immunohistochemical analysis of tubular ET-1 was performed in renal biopsy specimens from 18 patients with acute rejection, 7 patients with chronic rejection, and 5 normal kidneys excised for localized neoplasm. The diagnosis of acute or chronic rejection in each patient was verified and graded using the Banff schema. Renal tubular epithelium from patients with allograft rejection had markedly elevated staining for ET-1 compared with normal kidneys. Tubular ET-1 levels were elevated in 18 of 18 patients with acute rejection and 5 of 7 patients with chronic rejection. Tubular ET-1 staining was graded from 0 to +3 as follows: normal kidneys, 1.2 +/- 0.2; acute rejection, 2.3 +/- 0.4 (P < 0.01); and chronic rejection, 2.2 +/- 0.5 (P < 0.01). ET-1 staining was prominent in both proximal and distal tubules, and we observed abundant ET-1 secretion from proximal tubular epithelium in culture. Moreover, ET-1 activated the c-fos immediate early gene promoter in proximal tubular cells transfected with a c-fos luciferase reporter. We conclude that elevated tubular ET-1 levels are associated with acute and chronic rejection of renal allografts. Our results also suggest distinct pathophysiological roles for the tubular and vascular ET-1 systems in renal allograft rejection.

Hypertension after pancreas-kidney transplantation: role of bladder versus enteric pancreatic drainage.

BACKGROUND: Recent reports suggest that hypertension may be less common after simultaneous pancreas-kidney transplantation than after kidney transplantation alone. However, the mechanisms for this beneficial effect have not been delineated. We hypothesize that lower blood pressures may result from chronic volume depletion in patients with bladder-drained pancreatic allografts. METHODS: We compared the incidence and severity of hypertension 12 months after transplantation in 79 bladder-drained pancreas-kidney recipients and 46 diabetic kidney-only recipients. These two groups were compared with a smaller group of enterically drained pancreas-kidney recipients. Blood pressure was also compared before and after surgical conversion from bladder to enteric drainage in 10 patients. RESULTS: Hypertension was significantly less common and less severe after pancreas-kidney transplantation than after kidney transplantation alone, but the benefit of the pancreas transplant was evident only in bladder-drained patients. Logistic regression analysis of the bladder-drained pancreas-kidney patients confirmed the independent impact of the pancreatic allograft on the presence of hypertension, indicated an independent association with serum creatinine concentration and donor age, but suggested no correlation with recipient age, race, or number of rejection episodes. A comparison of blood pressures before and after pancreatic conversion from bladder to enteric drainage indicated no significant change in the prevalence or severity of hypertension. CONCLUSIONS: We conclude that the beneficial effect of a pancreas transplant on the prevalence and severity of hypertension after simultaneous pancreas-kidney transplantation is limited to bladder-drained patients. Although it is possible that the effect is mediated by chronic volume depletion, the observation that blood pressure does not increase after conversion from bladder to enteric drainage suggests that other factors may be involved.

Kidney-pancreas transplantation for diabetic nephropathy.

Pancreas transplantation is being performed with increasing frequency and increasing technical success. The availability of new immunosuppressant agents has been associated with a reduction in the previously high rates of allograft rejection in recipients of simultaneous pancreas-kidney transplants. These lower rejection rates have, in turn, led to changes in surgical techniques and a resurgence of interest in isolated pancreas transplantation--either in nonuremic patients or, more commonly, in patients who have already received a prior kidney transplant. Pancreas transplantation has emerged as an important option for the management of patients with type I diabetes mellitus and diabetic nephropathy.

Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-gamma only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-gamma-producing PBLs. Studies of donor-specific IFN-gamma-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.