M. Boratyńska

The influence of non‐HLA antibodies directed against angiotensin II type 1 receptor (AT1R) on early renal transplant outcomes

Non‐HLA antibodies (Abs) targeting vascular receptors are thought to have an impact on renal transplant injury. Anti‐angiotensin II type 1‐receptor‐activating antibodies (anti‐AT1R) have been mentioned to stimulate a severe vascular rejection, but the pretransplant screening has not been introduced yet. The aim of our study was to assess the incidence and importance of anti‐AT1R antibodies and their influence on renal transplant in the 1st year of observation. We prospectively evaluated the presence of anti‐AT1R antibodies in 117 consecutive renal transplant recipients in pre‐ and post‐transplant screening. Anti‐AT1R antibodies were observed in 27/117 (23%) of the analyzed recipients already before transplantation. The function of renal transplant was considerably worse in anti‐AT1R(+) group. The patients with anti‐AT1R Abs >9 U/ml lost their graft more often. Biopsy‐proven AR was described in 4/27 (15%) pts in the anti‐AT1R(+) group and 13/90 (14.4%) in the anti‐AT1R(−) group, but more severe cases of Banff IIB or antibody‐mediated rejection (AMR) were more often observed in anti‐AT1R (+) 4/27 (15%) vs. 1/90 (1.1%) in anti‐AT1R(+) (P = 0.009). Patients with anti‐AT1R Abs level >9 U/ml run a higher risk of graft failure independently of classical immunological risk factors. The recipients with anti‐AT1R Abs developed more severe acute rejections described as IIB or AMR in Banff classification. More recipients among the anti‐AT1R‐positive ones lost the graft. Our study suggests monitoring of anti‐AT1R Abs before renal transplantation for assessment of immunologic risk profiles and the identification of patients highly susceptible to immunologic events, graft failure, and graft loss.

The influence of calcineurin inhibitors on mycophenolic acid pharmacokinetics

Despite the fact that concentrations of mycophenolic acid (MPA) are not routinely measured, accumulating data suggest the usefulness of this monitoring to optimize therapy. The aim of this study was to assess the influence of CsA and tacrolimus on MPA pharmacokinetics. Concentrations of MPA were measured using HPLC. An assay was performed before dose (the C(0)), as well as at 40 minutes and 1, 2, 4, 6, 8, 10, 12 hours after administration of mycophenolate mofetil (MMF). MPA profiles were assessed in 51 patients receiving tacrolimus at a dose of 1.0 g/d and prednisone as well as in 97 patients receiving CsA (2.0 g/d) and prednisone. Significant correlations of MPA levels with serum albumin and GFR were observed in both groups. Women presented with higher levels of MPA than men. C(0) MPA level among the tacrolimus group were significantly higher than those in CsA group: 3.18 +/- 2.21 microg/mL versus 1.68 +/- 1.03 microg/mL (P </=.001). The level of MPA AUC((0-12)) in the tacrolimus group was nonsignificantly higher than that in the CsA group. There was no second peak of MPA level in a group of patients receiving CsA. We developed a limited sampling strategy to estimate MPA AUC((0-12)) in both tacrolimus and CsA groups. We observed a correlation between C(0) MPA and C(0) CsA (r =.35; P </=.001) as well as, between tacrolimus dose and MPA C(40) and MPA C(max) (r =.24; P </=.05; r =.27; P </= 0.05, respectively). No relationship between MPA pharmacokinetics and tacrolimus blood concentrations was noticed. Tacrolimus and CsA both affect the pharmacokinetics of MPA; high MPA concentrations in patients treated with tacrolimus justify MMF dose reduction in this group. Alterations of CsA concentrations must be used to guide MMF dose adjustments.

The impact of non-HLA antibodies directed against endothelin-1 type A receptors (ETAR) on early renal transplant outcomes.

BACKGROUND Non-HLA antibodies (Abs) targeting vascular receptors are considered to have an influence on renal transplant injury. Anti-endothelin-1 type A receptor (anti-ETAR) antibodies were associated with cellular and antibody-mediated rejection and early onset of vasculopathy in heart transplant patients but their role in renal transplantation remains unclear. The aim of our study was to assess the incidence and importance of anti-ETAR antibodies and their impact on renal transplant during the first year observation. METHODS We evaluated the presence of anti-ETAR antibodies in 116 consecutive renal transplant recipients in pre- and post-transplant screening (before and in 1st, 3rd, 6th, 12th month after transplantation). Additionally, we assessed the presence of anti-HLA antibodies. Anti-ETAR antibodies were assayed by ELISA. The diagnosis of acute rejection was based on the Banff criteria. RESULTS Anti-ETAR antibodies were observed in 55 (47.4%) of the analyzed recipients before transplantation. The function of renal transplant was significantly worse in the anti-ETAR(+) group compared to the anti-ETAR(-) group during the first post-transplant year. One month after transplantation the serum creatinine in anti-ETAR (+) patients (pts) was 1.86±0.8mg/dl and 1.51±0.5 in anti-ETAR(-) pts (p=0.009). Twelve months after transplantation the difference between the groups was still observed 1.70±0.7 vs. 1.40±0.4 (p=0.04). Biopsy proven acute rejection was recognized in 8/55 (14.5%) in ETAR(+) and 9/61 (14.8%) in ETAR(-) patients but cases with mild to severe intimal arteritis (v1-v3) were more often observed in patients with the presence of anti-ETAR Abs 4/55 (7.2%) comparing with 1/61 (1.6%) in anti-ETAR(-) patients. The anti-ETAR antibody levels varied at different measurement intervals during the one-year follow-up. CONCLUSIONS The presence of anti-ETAR antibodies is associated with a worse renal transplant function during the first 12months after transplantation. Including anti-ETAR antibodies in the diagnostics of renal transplant recipient immune status should be considered to provide comprehensive assessment of humoral alloimmunity.

Long-term Follow-up of Non-HLA and Anti-HLA Antibodies: Incidence and Importance in Renal Transplantation

BACKGROUND Detection of antibody-mediated injury is becoming increasingly important in post-transplant patient care. The role of donor-specific anti-human leukocyte antigen (HLA) antibodies in kidney transplant damage is known, whereas the significance of non-HLA antibodies remains an unresolved concern. The aim of the study was to determine the presence and influence on renal function of non-HLA and anti-HLA antibodies in stable patients at 5 years after kidney transplantation. METHODS We evaluated the antibodies in 35 consecutive patients with stable renal function at 5 years after transplantation. RESULTS Pretransplant screening for donor-specific antibodies by CDC cross-matches was negative in all patients. Anti-endothelial cell antibodies (AECA), anti-angiotensin II type 1 receptor antibodies (anti-AT1R), and anti-endothelin receptor antibodies (anti-ETAR) were assayed as non-HLA antibodies. Non-HLA antibodies were observed in 12 (34%) patients, including AECA (n = 5; 14%), anti- AT1R (n = 6; 17%), anti-ETAR (n = 4; 11%), and both anti-AT1R and anti-ETAR (n = 3). Among 13 (37%) patients with anti-HLA antibodies, 7 also had both non-HLA antibodies: AECA (n = 1), anti-AT1R (n = 3), and anti-ETAR (n = 3). The antibody-negative group (n = 13) showed significantly better renal function than the antibody-positive group (non-HLA and/or anti-HLA; n = 22). Biopsy-proven acute rejection had occurred in 2 of 13 (15%) antibody-negative versus 8 of 22 (36%) antibody-positive patients. These preliminary data revealed an high prevalence of autoantibody and alloantibody production among stable patients at 5 years after kidney transplantation. CONCLUSION Simultaneous production of these antibodies and their association with reduced renal function suggests that active humoral immune responses are poorly controlled by immunosuppression.

Hyperuricemia is a mediator of endothelial dysfunction and inflammation in renal allograft recipients.

BACKGROUND Hyperuricemia is common in renal transplant recipients treated with calcineurin inhibitors. Uric acid induces glomerular hypertension, microvascular disease, and renal interstitial fibrosis and is an independent risk factor for cardiovascular complications. The mechanisms by which uric acid injures renal allografts and the cardiovascular system remain unclear. OBJECTIVE To assess the influence of uric acid on biomarkers of endothelial dysfunction and inflammation in renal allograft recipients. PATIENTS AND METHODS The study included 78 allograft recipients with normal allograft function. Exclusion criteria were abnormal renal function, proteinuria, diabetes mellitus, obesity, and inflammation. Participants were divided into 2 groups: 48 patients with hyperuricemia (mean [SD] uric acid concentration, 7.72 [1.33] mg/dL) and 30 patients with normouricemia (5.48 [0.92] mg/dL; control group). Concentrations of plasma resistin, CD146, and soluble vascular cell adhesion molecule-1 (sVCAM-1), which are markers of endothelial dysfunction and inflammation, were assessed in both groups. No significant differences were noted for patient demographic data including age, sex, cause of renal failure, number of HLA mismatches, delayed graft function, and number of acute rejection episodes. RESULTS Concentrations of the examined biomarkers were increased in the group with hyperuricemia compared with the control group: plasma resistin, 7.15 (2.42) ng/mL vs 6.29 (2.76) ng/mL; CD146, 389.7 (150.0) microg/mL vs 330 (117) microg/mL; and sVCAM-1, 1126 (371) ng/mL vs 955 (269) ng/mL (P < .03). In addition, resistin correlated significantly with sVCAM-1 (P < .01). CONCLUSIONS Hyperuricemia mediates endothelial dysfunction and inflammation and via this pathway, possibly contributes to chronic allograft injury and cardiovascular events in renal allograft recipients.

The Impact of De Novo Donor-specific Anti-Human Leukocyte Antigen Antibodies on 5-Year Renal Transplant Outcome

Numerous studies have shown that circulating donor-specific antibodies targeting human leukocyte antigen (HLA) are associated with accelerated renal transplant failure, but many patients with these antibodies have good graft function. The aim of our study was to investigate the long-term graft function and survival in patients with de novo post-transplant donor-specific anti-HLA antibodies (DSA). Our prospective study included 78 consecutive recipients with a negative crossmatch before transplantation. Recipient serum samples were assayed for DSA in week 2 and 1, 3, 6, 9, 12 months after transplantation using a complement-dependent lymphocytotoxic technique with donor lymphocytes. Additionally, patients with DSA and stable renal function in the first year were tested with a more sensitive flow-panel-reactive antibody. DSA were present in 34 (44%) of our patients during the first 12 months after transplantation. Biopsy-proved acute rejection occurred in 11 DSA-positive and 10 DSA-negative patients. Seven DSA-positive patients had antibody-mediated rejection and no DSA-negative ones developed humoral rejection. The serum creatinine level in DSA-positive patients was significantly higher (2.48 vs 1.43 mg/dL) in year 5. The 13 (38%) DSA-positive patients with good graft function in month 12 were stable during a 5-year follow-up: their serum creatinine was 1.46 ± 0.4 in year 1 and 1.56 ± 0.4 mg/dL in year 5 and nobody lost their allograft. One- and 5- year graft survivals were appropriately 85% and 59% in DSA-positive patients compared to 93% and 93% in DSA-negative patients. To sum up, post-transplant DSA had a significant influence on kidney function and graft survival but in 38% of patients the presence of DSA did not decrease a 5-year renal function. A good renal allograft function in the presence of DSA in the first year after transplantation and cessation of their production in the subsequent years may be a good prognostic marker for a long-term allograft function and survival.

Mycophenolate mofetil severely depresses antibody response to CMV infection in early posttransplant period

Estimation of anti-CMV-IgG and anti-CMV-IgM is considered a relatively inexpensive screening tool of CMV status. The aim of study was to estimate how the immunosuppressive protocol influence serum anti-CMV IgG and IgM concentration in renal graft recipients and to estimate the adequacy of anti-CMV-IgG concentration and anti-CMV-IgM index as screening parameters of active CMV disease in patients receiving different immunosuppression. The study group consisted of 33 patients with clinical signs of CMV disease who received one of three types of immunosuppression: (1) azathioprine (Aza) + cyclosporine (CyA) + prednisone (Pr), 20 patients; (2) mycophenolate mofetil (MMF) + CyA + Pr, eight patients; tacrolimus (Tac) + MMF, five patients. Patients were enrolled when the pp65-antigen (pp65) of PBL was positive within 1 to 5 months after transplant (75 patients tested). The IgM-i in the Aza + CyA + Pr group was higher than in MMF + CyA + Pr group (2.73 + 1.8 vs 1.08 +/- 1.07, P =.021). The IgM-i in the Aza + CyA + Pr group was higher than in Tac + MMF (2.73 +/- 1.8 vs 0.78 +/- 0.69; P =.014). There was no difference in IgM-i between MMF + CyA + Pr and Tac + MMF. There was no difference in relative increase of IgG-c among all groups but there was a difference in relative increase of IgM-i between Aza + CyA + Pr and MMF + CyA + Pr groups (6.7 +/- 9.4 vs 2.3 +/- 5.9; P =.007) and between Aza + CyA + Pr and MMF + Tac groups (6.7 +/- 9.4 vs 0.6 +/- 0.54; P =.003). Immunosuppressive protocols including MMF exert an inhibitory influence on B-cell response and synthesis of anti-CMV-IgM. It makes the anti-CMV-IgM index an inadequate rough screening diagnostic parameter of active CMV disease.

Non-HLA antibodies: angiotensin II type 1 receptor (anti-AT1R) and endothelin-1 type A receptor (anti-ETAR) are associated with renal allograft injury and graft loss.

INTRODUCTION Non-HLA antibodies specific for angiotensin II type 1 receptor (anti-AT1R) and endothelin-1 type A receptor (anti-ETAR) of vascular cells activate signaling pathways leading to cell proliferation and vascular injury. The aim of this study was to evaluate the impact of non-HLA antibodies on kidney allograft morphology and function in patients who underwent a kidney biopsy due to renal function impairment. PATIENTS AND METHODS The study included 65 consecutive renal transplant patients who were evaluated for the presence of non-HLA and anti-HLA antibodies at the time of transplant biopsy. Results of pre-transplant CDC cross-match were negative. A kidney allograft biopsy was performed between 6 days and 13 years (42 ± 49 months) after transplantation, and the diagnosis was made on the basis of the Banff criteria. The level >9 U/L of anti-AT1R and anti-ETAR antibodies was considered high. RESULTS A high level of non-HLA antibodies (anti-AT1R and/or anti-ETAR) was found in 7 (10.7%) of 65 patients at the time of biopsy. Graft loss in the non-HLA-positive patients was significantly higher (71% in non-HLA-positive cases after 7.8 ± 2.6 months vs 11% after 6 months in non-HLA-negative cases [P = .00099]). In these non-HLA-positive patients, the mean anti-AT1R level was 15.3 ± 9.4 U/L and the mean anti-ETAR level was 13.8 ± 8.6 U/L. In only 2 of these patients were anti-HLA antibodies additionally detected: anti-class I in 1 and anti-class II in both patients. The mean serum creatinine level was 2.34 ± 0.6 mg/dL at the time of biopsy. Results of an early biopsy revealed acute vascular rejection (Banff grade IIB). Chronic allograft injury was found (grading cg1-3, cv1-2, ci1-2, ct1-2) in the remaining 6 patients. C4d was present in 3 of 7 patients. CONCLUSIONS High levels of anti-AT1R and/or anti-ETAR antibodies were associated with morphological and functional allograft injury and graft loss in these study patients. Non-HLA antibodies can be helpful in assessing the risk of graft failure.

1,25-Dihydroxyvitamin D deficiency predicts poorer outcome after renal transplantation.

OBJECTIVE To assess 1,25-dihydroxyvitamin D status and the effect of vitamin concentration on transplantation outcome in renal allograft recipients. PATIENTS AND METHODS Ninety patients underwent renal transplantation between 2002 and 2005. All received alfacalcidol supplementation before surgery. 1,25-Dihydroxyvitamin D concentration was determined on day 3 posttransplantation and at 1-, 6-, 12-, 18-, and 24-month follow-up. RESULTS Severe 1,25-dihydroxyvitamin D deficiency was noted in 83% of patients immediately posttransplantation. From 1 to 12 months thereafter, concentrations increased almost 3-fold, and remained constant to 24 months. In 50% of patients, the 1,25-dihydroxyvitamin D concentration reached a concentration of more than 30 pg/mL, similar to that in healthy volunteers; in the other 50%, the concentration reached 17.2 pg/mL. A high incidence of delayed graft function was observed in patients with 1,25-dihydroxyvitamin D deficiency (44% vs 6%). There was a negative correlation between the initial 1,25-dihydroxyvitamin D and serum creatinine concentrations at day 3 and month 6 (P < .03). Similarly, the 1,25-dihydroxyvitamin D concentration at 1 month was negatively correlated with creatinine concentration at months 1 through 24 (P < .01). Poor outcome was observed primarily in patients with 1,25-dihydroxyvitamin D deficiency; 2 patients developed cancer, 5 grafts were lost, and 4 patients died of cardiovascular events. CONCLUSIONS 1,25-Dihydroxyvitamin D deficiency is highly prevalent in renal allograft recipients. Patients with 1,25-dihydroxyvitamin D deficiency are at greater risk of delayed graft function, and the graft is more likely to be lost. These findings suggest the necessity of adequate vitamin D supplementation both before and after transplantation.

Inhibition of mTOR by sirolimus induces remission of post-transplant lymphoproliferative disorders

A recent study showed ubiquitous activation of the mammalian Target of Rapamycin (mTOR) signaling pathway in cells in post-transplant lymphoproliferative disorders (PTLD), regardless of their Epstein-Barr virus (EBV) genome expression status [1]. The inhibition of mTOR kinase activity by sirolimus and its analogs impairs cell growth and proliferation and may be effective in PTLD treatment. Sirolimus and its water-soluble ester, temsirolimus, are currently undergoing phase I and phase II clinical trials in several hematological malignancies and solid tumors [2]. We present the results of sirolimus implementation in the management of three PTLD cases which occurred among 712 kidney graft recipients transplanted at our center between 2000 and 2007. The patients – Caucasian, aged 26 (A), 53 (B), and 34 (C) years, had received cadaveric kidney grafts. For patient B, the transplant was a second one – the first graft had survived 108 months and been treated with cyclosporine/azathioprine/prednisone. The causes of end-stage renal disease were diabetic nephropathy in patient A and glomerulonephritis in patients B and C. Patient A developed PTLD at 6 months post-transplant, patient B at 38 months (from last transplantation), and patient C at 50 months post-transplant. None of the patients had any evidence of malignancy before transplantation. All were HIV negative and cytomegalovirus (CMV) seropositive. Patients B and C were HBV positive. Patient A developed CMV disease 6 weeks post-transplant and was treated with gancyclovir. The PTLD diagnosis was based on biopsies of either enlarged lymph nodes or nodular mass. Biopsies were histologically evaluated in accordance with WHO criteria [3]. Immunohistochemistry was performed to establish lymphoma cell phenotypes. Radiologic evaluation included chest radiographs, ultrasound, contrast enhanced computerized tomography scans of the chest and abdomen. Ultrasound and CT scans were repeated during treatment and remission. A bone marrow trephine biopsy was performed in all patients. The patients showed systemic PTLD symptoms: fever, night sweats, abdominal pain, and weight loss. Additionally, patient A presented throat-ache with tonsillar enlargement as well as lymphadenopathy and splenomegaly. In patient B, CT scans showed a nodular tumor between liver and stomach (Fig. 1) and para-aortic lymph nodes enlargement. In patient C, enlargement of spleen, liver as well as peripheral, mediastinal and abdominal lymph nodes were found. Patient A and C had impaired performance status (3 score). All patients had positive EBV serology, both pretransplant and during PTLD diagnosis. Bone marrow examination proved normal in all patients. Histological examination and immunophenotyping showed large B-cell lymphomas with positive proliferation markers (Ki67) in patients A and B, and T-cell lymphoma in patient C. International Prognostic Index (IPI) was high in patient A and C. Clinical data are presented in Table 1 and Figs 1 and 2. Primary treatment was the immediate discontinuation of calcineurin inhibitors and mycophenolate mofetil or azathioprine and administration of sirolimus at a dose of 2–3 mg/day, to maintain trough blood level between 5–8 ng/ml. Two patients with large B-cell lymphomas completely regressed during sirolimus therapy. Within 2 weeks, clinical PTLD symptoms (i.e., fever, abdominal pain) disappeared. Repeated ultrasound and CT scans showed gradual regression of splenomegaly and resolution of lymphadenopathy in patient A. Graft function since conversion to sirolimus has been stable and GFR is 50.5 ml/min after 44 months of treatment. In patient B, the nodular epigastric tumor diminished by half after 1 month of treatment and disappeared within 5 months (Fig. 1). In this patient proteinuria increased from 0.8– 1.0 g/24 h to 4–6 g/24 h after conversion, GFR however did not change during 14 months of follow-up, and is now at 48 ml/min. In patient C, with a T-cell lymphoma, fever and abdominal pain vanished and the general condition improved. Serum creatinine levels were 2.6–2.8 mg/dl at the onset of sirolimus treatment. He developed endstage renal disease 3 months later. Sirolimus was discontinued at his return to hemodialysis treatment. His PTLD progressed and he began chemotherapy with courses of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), to which he did not respond. The patient died 15 months from PTLD diagnosis.