Evaluation of the Relationship Between Concentrations of Tacrolimus Metabolites, 13-O-Demethyl Tacrolimus and 15-O-Demethyl Tacrolimus, and Clinical and Biochemical Parameters in Kidney Transplant Recipients.

BACKGROUND: Tacrolimus (Tac), an essential component of immunosuppressive therapy after solid-organ transplantation, has a narrow therapeutic index and requires therapeutic drug monitoring. Monitoring of Tac predose blood concentrations seems to be not always sufficient to avoid adverse effects. The aim of the study was to evaluate the levels of main Tac metabolites, 13-O-demethyl tacrolimus (13-DMT), 31-O-demethyl tacrolimus (31-DMT), and 15-O-demethyl tacrolimus (15-DMT), in kidney transplant recipients and to link them to clinical and biochemical parameters. METHODS: In 63 kidney transplant patients, concentrations of 13-DMT, 31-DMT, and 15-DMT were quantified using liquid chromatography combined with tandem mass spectrometry (LC/MS/MS). RESULTS: None of the patients had detectable 31-DMT blood levels. There was a positive correlation between 13-DMT/Tac and alanine aminotransferase (ALAT) (r = 0.29, P = .046) and a negative correlation between 13-DMT/Tac and hemoglobin (r = -0.33, P = .008). Tac level did not correlate with ALAT nor with hemoglobin. There was no relationship between 13-DMT/Tac or 15-DMT/Tac and other biochemical or hematologic parameters or data, such as age, body mass index, arterial pressure, or time posttransplant. We observed significantly higher Tac concentrations in patients with hypercholesterolemia or hypertriglyceridemia compared with those without these comorbidities (6.45 ± 2.32 vs 5.16 ± 2.12 ng/mL, P = .043; 6.60 ± 2.30 vs 5.34 ± 2.20 ng/mL, P = .033, respectively). CONCLUSION: Our data may reflect 13-DMT accumulation in liver dysfunction and higher Tac clearance in anemia. However, these results may suggest that 13-DMT/Tac ratio is a marker of myelotoxicity and hepatotoxicity. Further studies should be carried out to determine whether monitoring of 13-DMT could be beneficial in minimizing the adverse effects.

Cardiovascular Disease in Kidney Transplantation and Its Association With Blood Concentrations of Cyclosporine and Cyclosporine Metabolites.

Cyclosporine A (CsA) is the first calcineurin inhibitor used as immunosuppressive agent. Its administration is associated with multiple adverse effects including cardiovascular diseases (CVDs), but their mechanisms have not been fully elucidated. Cyclosporine metabolites are not well studied in this context. This study was aimed at analysis of the incidence of CVDs and their association with concentrations of cyclosporine and its metabolites. Sixty patients after kidney transplantation (KTX) taking an immunosuppressive regimen including CsA participated in the study. There were 22 women (36.67%) and 38 men (63.33%), mean age 51.73 years, mean 109.38 months after KTX. We observed a correlation between mean diastolic blood pressure and concentrations of metabolite to parent drug ratios of AM1-CsA/CsA (r = 0.35, P = .006), dihydroxy-CsA/CsA (r = 0.42, P = .001), trihydroxy-CsA/CsA (r = 0.42; P = .003) and desmethyl-carboxy-CsA/CsA (r = 0.65, P = .003). There were no significant associations of other CsA metabolites' parameters with CVDs (coronary disease, hypertension, stroke, arrhythmia, diabetes mellitus, obesity). Study results suggest that blood pressure increases associated with CsA therapy could be caused by CsA metabolites that influence mainly diastolic blood pressure levels. A lack of such differences in relation with other CVDs may suggest that more complex mechanisms are involved in the development of cardiovascular injury and disease after kidney transplantation.

Tacrolimus Metabolite M-III May Have Nephrotoxic and Myelotoxic Effects and Increase the Incidence of Infections in Kidney Transplant Recipients.

BACKGROUND: Tacrolimus (Tac) is one of the most commonly used immunosuppressive drugs after solid organ transplantation. Eight Tac metabolites have been described, but their clinical importance remains unclear. The aim of this study was quantification of the 2 major Tac metabolites, 13-O-demethyl (M-I) and 15-O-demethyl (M-III), in kidney transplant recipients and to link them with parameters of kidney and liver function, peripheral blood cell counts, and infection incidence. METHODS: In 81 kidney transplant recipients, concentrations of Tac, M-I, and M-III were measured with the use of liquid chromatography combined with tandem mass spectrometry (LC-MS-MS). RESULTS: There was a negative correlation between M-III levels and estimated glomerular filtration rate (eGFR; r = -0.244; P < .05). Also, a negative correlation between M-III concentrations and red blood cell count (RBC) was found (r = -0.349; P < .05). Neither concentrations of Tac nor of M-I correlated with eGFR or RBC. M-I, M-III, and Tac were not related to alanine aminotransferase activity. Significantly higher Tac and M-III concentrations in the group with all types of infections in comparison with the group without infections were observed (6.95 ± 2.09 ng/mL vs 5.73 ± 2.43 ng/mL [P = .03] and 0.27 ± 0.17 ng/mL vs 0.20 ± 0.11 ng/mL [P = .04], respectively). CONCLUSIONS: The results suggest that higher concentrations of M-III may have a nephrotoxic or myelotoxic effect and result in higher incidence of infections. Further studies are needed to confirm if monitoring of M-III could minimalize adverse effects such as nephrotoxicity or infections.

Hydroxylated, Hydroxymethylated, Dihydroxylated, and Trihydroxylated Cyclosporine Metabolites Can Be Nephrotoxic in Kidney Transplant Recipients.

BACKGROUND: Cyclosporine (CsA) is an immunosuppressive agent whose use is associated with adverse effects, including nephrotoxicity. There are reports indicating that some CsA metabolites may contribute to these effects. This study was aimed at evaluation of CsA metabolites and correlating them with kidney function. METHODS: In 62 kidney transplant recipients (41.9% women; overall mean age, 48.44 ± 11.75 years), concentrations of CsA and 4 groups of metabolites were assessed: hydroxylated (HCsA), hydroxymethylated (HMCsA), dihydroxylated (DHCsA), and trihydroxylated (THCsA). The results were normalized with the use of the metabolite-to-parent drug ratio, and results were linked with estimated glomerular filtration rate (eGFR) at 3 months before (-3M), point zero (0M), and after 3 (+3M) and 12 (+12M) months. RESULTS: Multivariate analysis demonstrated the negative influence of eGFR -3M on HMCsA/CsA (ß = -0.266; P < .05) and the negative influence of HCsA/CsA, HMCsA/CsA, DHCsA/CsA, and THCsA/CsA on eGFR +3M (ß = -0.339, ß = 0.396, ß = -0.314, and ß = -0.321, respectively; P < .005) and eGFR +12M (ß = -0.363, ß = -0.316, ß = -0.267, and ß = -0.312, respectively; P < .05). We did not detect such influence of CsA concentrations on eGFR +3M and +12M. The THCsA/CsA receiver operating characteristic cutoff value for prediction of improvement of kidney function at +12M was 0.143. CONCLUSIONS: Our results suggest that impaired function of the transplanted kidney affects the accumulation of HMCsA. It is possible that the increased metabolite (HCsA, HMCsA, DHCsA, and THCsA) to cyclosporine ratio could influence or could be a marker of cyclosporine nephrotoxicity. In this context, the most promising marker seems to be THCsA/CsA ratio, but its real significance requires further studies to determine.

Determination of Concentrations of Azathioprine Metabolites 6-Thioguanine and 6-Methylmercaptopurine in Whole Blood With the Use of Liquid Chromatography Combined With Mass Spectrometry.

BACKGROUND: 6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are used in many autoimmune diseases and after solid-organ transplantation. Their properties are mediated by active metabolites, 6-thioguanine nucleotides (6-TGN), and 6-methylmercaptopurine (6-MMP). The most common adverse effects are myelo- and hepato-toxicity. The aim of the study was quantification of 6-TG and 6-MMP, with the use of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) in solid-organ transplant recipients. METHODS: In 33 patients, kidney transplant recipient (n = 25) and liver transplant recipient (n = 8) intra-erythrocyte concentrations of 6-TG and 6-MMP were measured with the use of LC/MS/MS. RESULTS: The mean concentration of 6-TG was 205.35 ± 157.62 pmol/8 × 10(8) red blood cells (RBC); median concentration of 6-MMP was 1064.1 (35.78-11,552.9) pmol/8 × 10(8) RBC. There were no correlations between 6-TG levels and peripheral blood parameters (white blood cell count, WBC; hemoglobin, Hb concentration; PLT, blood platelet count) or alanine aminotransferase activity (AlAT) activity. Relationships between 6-MMP concentrations and peripheral blood parameters (WBC, Hb, PLT) or AlAT activity have not been found. Subgroups with leukopenia, anemia, thrombocytopenia, and liver dysfunction did not differ in concentrations of 6-TG or 6-MMP. We have observed a negative correlation between daily azathioprine dose and WBC count (r = -0.37, P = .04). CONCLUSIONS: Relationships between concentrations of azathioprine metabolites and myelotoxicity or hepatotoxicity have not been confirmed. Further studies on larger groups of patients would be helpful in a more accurate understanding of the impact of azathioprine metabolites on parameters of bone marrow and liver function.

Arterial hypertension in liver transplant recipients.

Hypertension is an important cardiovascular risk factor that influences patient survival. This study sought to evaluate hypertension incidence and circadian rhythms of blood pressure (BP) among liver transplant recipients during the first posttransplant month. We also compared hypertension incidence according to clinical and automated blood pressure monitoring methods. BP was determined by clinical blood pressure monitoring (CBPM) methods and by automated blood pressure monitoring (ABPM) using the SpaceLabs device. We also assessed blood biochemistry, particularly kidney function parameters and immunosuppressive drug blood trough levels, among 32 white subjects (10 women and 22 men) of average age 47.58±14.19 years. The leading cause for transplantation was liver insufficiency due to viral hepatitis B and/or C infection (43.75%). The majority (93.75%) of patients was prescribed immunosuppressive treatment with tacrolimus. Although we observed hypertension in 28 patients (87.5%) by ABPM measurements and in 25 (78.12%) using CBPM method, the difference did not reach statistical significance. However, BP control was inadequate in 28 patients (87.5%) by ABPM assessment versus 3 (9.38%) according to CBPM readings (P=.025). The BP circadian rhythm was altered in 30 patients (93.75%) including 15 with higher nighttime BP readings. There was no correlation between tacrolimus blood levels and BP values or with kidney function as assessed by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. We concluded that prevalence of arterial hypertension among liver transplant recipients within 1 month after transplantation is high. The majority of the patients show disturbed circadian rhythms in the early period after liver transplantation with loss or even reversal of the normal nocturnal decrease in BP. Owing to the fact that ABPM enables more adequate daily assessment of BP values, it is an optimal method to adjust antihypertensive therapy to optimal levels.

Increased mRNA expression of transforming growth factor beta in the arterial wall of chronically rejected renal allografts in humans.

INTRODUCTION: Transforming growth factor beta (TGF-beta) has an established role in interstitial damage of renal transplants during chronic rejection (CR). However, its involvement in transplant vasculopathy is not clear. The aim of the study was to assess TGF-beta gene expression in the walls of large-caliber arteries within chronically rejecting renal allografts. We evaluated associations between gene expression of this factor and intimal thickness or clinical data. MATERIAL AND METHODS: Renal artery samples of kidney allografts were obtained from 20 hemodialysis patients with end-stage renal graft disease due to CR, who were undergoing graftectomy. The control group included 32 hemodialysis patients with end-stage renal disease, undergoing nephrectomy due to autosomal dominant polycystic kidney disease (n = 12), chronic pyelonephritis (n = 13), or kidney limited tumor (n = 7). Gene expression of TGF-beta was measured using real-time PCR. RESULTS: TGF-beta mRNA expression was 3.25-fold higher in CR than in control patients (P < .001). Expression of mRNA for this cytokine was not influenced by the following factors: intimal thickness; age; serum cholesterol, triglycerides and glucose; BMI; graft survival; time of dialysis before transplantation; total ischemic time; immunosuppressive regimen; incidence of acute rejection episode; panel reactive antibodies; and period of dialysis before graftectomy. TGF-beta is involved in neointimal formation in CR.

Polyoma BK virus reactivation in kidney and pancreas-kidney recipients.

BK virus infection has become important factor affecting graft function in renal transplant recipients. One of the most important complication of BK infection is nephropathy in patients after renal transplantation. The aim of this study was to evaluate incidence of BK reactivation and nephropathy in our population of renal allograft recipients. One hundred twelve renal or pancreas-kidney allograft recipients were included for the 24 months follow-up. The incidence of BK nephropathy was 7.85% and viremia 27.96%. In the second study group there were 28 patients with graft function deterioration evaluated at the time of biopsy. In this group incidence of BK nephropathy was 7.1% and viral reactivation was diagnosed in 10.7% of patients. In our center, the incidence of BK nephropathy is the same as worldwide. The risk of BK virus replication is highest during first 15 months after the surgical procedure.

Extracellular matrix proteins, proteolytic enzymes, and TGF-beta1 in the renal arterial wall of chronically rejected renal allografts.

Chronic rejection (CR) is the leading cause of long-term failure of transplanted kidneys. The vascular hallmark is intimal hyperplasia, accompanied by macrophage, foam cell, and T-cell infiltration. Intimal thickening results from the migration and proliferation of smooth muscle cells and increased deposits of extracellular matrix (ECM) proteins, due to release of growth factors and cytokines as well as altered ECM protein turnover. We assessed the content of fibronectin (FN) and transforming growth factor-beta1 (TGF-beta1) as well as the activities of collagenase and cathepsin B and L in renal artery walls of chronically rejected human renal allografts. We investigated renal artery samples from 8 patients with CR undergoing graftectomy, 12 patients undergoing nephrectomy, and 7 organ donors. The results were related to the DNA content of homogenates. Cathepsin B and L activities were significantly higher among those with compared with donors (P =.022). There was a trend toward higher collagenase activity in CR compared with donors and the nephrectomy group. TGF-beta1 was significantly enhanced in CR compared with donors (P =.010), and showed a trend toward higher concentrations in CR compared with the nephrectomy group. The trend was toward lower FN concentrations in CR compared with the nephrectomy group and toward higher concentrations compared with donors. Summarizing, renal CR is accompanied by enhanced proteinase activity, alterations of ECM proteins, and increased TGF-beta1 in the renal artery wall. We conclude that ECM turnover and cytokines play an important role in neointimal formation and CR pathogenesis.

Effect of immunosuppressive regimen on acute rejection and liver graft function.

Despite the use of modern immunosuppressive drugs, acute liver rejection (AR) continues to affect up to 70% of transplant recipients. The aim of this retrospective study was to assess the incidence of acute rejection episodes in patients treated with different immunosuppressive protocols. In our series, 37.3% of patients developed a clinical episode of AR. Analysis of immunosuppression has shown that the most effective immunosuppressive protocols, with regard to prevention of AR, include: antibody anti-IL-2R (anti-IL-2R) + tacrolimus (Tac) + mycophenolate mofetil (MMF) + prednisolone (Pred); anti-IL-2R + tacrolimus (Tac) + Pred; or Tac + Pred (25% vs 28.6% vs 30.4%, respectively). The highest rate of AR (66.6%) was observed among patients with anti-IL-2R and Tac but no steroid treatment, mostly (77.7%) in the initial period after liver transplantation. There were no statistical differences in liver function tests between the group treated with a CsA-based versus a Tac-based therapy. Strong immunosuppression contributed to a relatively low incidence of clinical AR in our series. The lowest rate of AR was observed among patients treated with anti-IL-2R antibody. Tac, and Pred. Deprivation of steroids in the early phase after liver transplantation substantially increased the risk of acute rejection episodes despite the use of anti-CD25. There were no statistically significant differences in liver function tests among those treated with Tac versus CsA in the short-term follow-up.

Acute liver transplant rejection: incidence and the role of high-doses steroids.

The aim of this study was to assess the incidence of acute rejection (AR), and the efficacy of high doses of steroids during induction of immunosuppression for AR treatment. Fifty-five patients (33.5%) experienced AR episodes in our series; but, there were no deaths or retransplantations related to AR. The median time from liver transplantation to AR was 18.5 days (range, 2-351 days). In the group with the initial dose of methylprednisolone (MP) 0.05). After 1-year observation, liver function tests were similar in both AR and non-AR groups. The only biochemical parameter that was significantly lower in the non-AR group was the aspartate aminotransferase (AST). Liver function tests determined after 1-year follow-up were not significantly different between the groups with AR treated with doses of MP lower versus higher than 1.25 g. However, liver function tests in the group treated for AR with higher doses of MP were slightly better than in the remaining subjects. Recurrence of AR occurred in 5 cases in the group with lower doses of MP (1.25 g). A relatively low dose of MP was effective to treat AR. The tendency of AR patients treated with higher dose of MP to display better liver function needs further investigation. However, AR does not seem to affect later liver function.