Inosine 5'-Monophosphate Dehydrogenase Activity for the Longitudinal Monitoring of Mycophenolic Acid Treatment in Kidney Allograft Recipients.

BACKGROUND: Mycophenolic acid (MPA) is a standard immunosuppressant in organ transplantation. A simple monitoring biomarker for MPA treatment has not been established so far. Here, we describe inosine 5'-monophosphate dehydrogenase (IMPDH) monitoring in erythrocytes and its application to kidney allograft recipients. METHODS: IMPDH activity measurements were performed using a high-performance liquid chromatography assay. Based on 4203 IMPDH measurements from 1021 patients, we retrospectively explored the dynamics early after treatment start. In addition, we analyzed the influence of clinically relevant variables on IMPDH activity in a multivariate model using data from 711 stable patients. Associations between IMPDH activity and clinical events were evaluated in hospitalized patients. RESULTS: We found that IMPDH activity reflects MPA exposure after 8 weeks of constant dosing. In addition to dosage, body mass index, renal function, and coimmunosuppression affected IMPDH activity. Significantly lower IMPDH activities were found in patients with biopsy-proven acute rejection as compared to patients without rejection (median [interquartile range]: 696 [358-1484] versus 1265 [867-1618] pmol xanthosine-5'-monophosphate/h/mg hemoglobin, P < 0.001). The highest IMPDH activities were observed in hospitalized patients with clinically evident MPA toxicity as compared to patients with hospitalization not related to MPA treatment (1548 [1021-2270] versus 1072 [707-1439] pmol xanthosine-5'-monophosphate/h/mg hemoglobin; P < 0.001). Receiver operating characteristic curve analyses underlined the usefulness of IMPDH to predict rejection episodes (area, 0.662; confidence interval, 0.584-0.740; P < 0.001) and MPA-associated adverse events (area, 0.632; confidence interval, 0.581-0.683; P < 0.001), respectively. CONCLUSIONS: IMPDH measurement in erythrocytes is a novel and useful strategy for the longitudinal monitoring of MPA treatment.

Improved assay for the nonradioactive determination of inosine 5'-monophosphate dehydrogenase activity in peripheral blood mononuclear cells.

Mycophenolic acid (MPA) inhibits the enzyme inosine 5'-monophosphate dehydrogenase (IMPDH). Thus, the measurement of IMPDH activity could serve as a specific pharmacodynamic (PD) tool for monitoring MPA therapy. At present, however, monitoring of pharmacokinetic parameters is preferred over that of PD parameters because, in general, PD assays are labor-intensive and poorly reproducible. Currently, cell count or protein concentration is widely accepted as methods to normalize enzyme activity. In the present study, we have attempted to further improve a method for the determination of IMPDH activity to increase the robustness and reproducibility of the IMPDH activity assay itself, without making the assay more labor-intensive. Therefore, several aspects of the IMPDH method were investigated regarding their influence on the reproducibility and also modified to increase the feasibility and consistency of the assay. The isolation of peripheral blood mononuclear cells (PBMCs) of whole blood samples was found to be the most variable step. Normalization on cell count is labor-intensive and at the same time has a poor reproducibility. Determination of the protein content in cell extracts is impaired by contamination with extracellular proteins and non-PBMCs. Alternatively, the intracellular substance adenosine monophosphate (AMP) was investigated to normalize the newly generated xanthosine monophosphate. Among various subject groups, no significant differences in mean AMP concentration were found. To simplify the procedure, PBMCs were diluted to a fixed volume after isolation from sample of whole blood, and the IMPDH activity was normalized to the AMP concentration quantified in the same high-performance liquid chromatography run as xanthosine monophosphate was quantified. The within-run and total imprecision (coefficient of variation) ranged from 4.2% to 10.6% and from 6.6% to 11.9%, respectively. In conclusion, the modified method described here for the measurement of IMPDH activity can be used reliably in multicenter trials and in longitudinal studies to evaluate the additional value of any PD monitoring among a diversity of patients treated with MPA.

Pre-transplant inosine monophosphate dehydrogenase activity is associated with clinical outcome after renal transplantation.

UNLABELLED: Mycophenolate mofetil (MMF), an inhibitor of inosine monophosphate dehydrogenase (IMPDH) activity, is usually administered as a standard dose of 1 g b.i.d. after renal transplantation. Because MMF dose reductions are associated with inferior outcome, we investigated pre-transplant IMPDH activity, MMF dose reductions and outcome. IMPDH activity was determined in isolated peripheral mononuclear cells immediately prior to renal transplantation. We observed considerable inter-individual variability in pre-transplant IMPDH activity (9.35 +/- 4.22 nmol/mg/h). Thirty of 48 patients (62.5%) with standard MMF dose (1 g b.i.d.) had dose reductions within 3 years post-transplant; these patients also had significantly lower IMPDH activity. The area under the receiver-operating characteristics curve (AUC-ROC) for prediction of dose reduction within 6 months post-transplant was 0.75 (95% CI, 0.61-0.89; p < 0.004). IMPDH activity above the cut-off value, MMF dose reduction and age of recipient were significant contributors for the occurrence of acute rejection in the multivariate logistic regression. Patients with high IMPDH activity and MMF dose reduction had the highest rejection rate (81.8% vs. 36.4%; p < 0.01). CONCLUSION: Patients with low IMPDH activity experienced more complications of MMF therapy. High pre-transplant IMPDH activity and MMF dose reductions were associated with rejection. Determination of IMPDH activity prior to transplantation may help to improve MMF therapy after renal transplantation.

Combined ETA/ETB receptor blockade of human peritoneal mesothelial cells inhibits collagen I RNA synthesis.

BACKGROUND: Peritoneal fibrosis is a serious complication of peritoneal dialysis; however, the mechanisms are poorly understood. We studied osmolarity and physical stress-induced effects on collagen I RNA synthesis in human peritoneal mesothelial cells (HPMCs) and focused on endothelin as a possible mediator. METHODS: HPMCs were grown in a medium containing either d-glucose or glycerol to analyze the impact of osmolarity on mesothelial endothelin-1 (ET-1) release and on collagen I RNA synthesis [reverse transcription-polymerase chain reaction (RT-PCR)]. A cellular model of nonlaminar fluid shear stress and cellular stretch was used to analyze the effects of physical forces. To neutralize the endothelin effects, a combined ETA/ETB receptor antagonist (LU 302 872) was chosen. RESULTS: Glucose, but not glycerol, increased mesothelial ET-1 release in a concentration and time-dependent manner (P < 0.05 vs. controls). Collagen I RNA synthesis was significantly higher in glucose-challenged cell cultures (P < 0.05 vs. controls). The glucose-mediated collagen I RNA synthesis was completely inhibited by adding the combined ETA/ETB receptor antagonist to the medium. Fluid shear stress, but not cellular stretch, led to a significant increase in the mesothelial ET-1 release (P < 0.005 vs. controls) and collagen I RNA synthesis (P < 0.05 vs. controls). LU 302 872 completely inhibited these effects. CONCLUSION: We found that glucose and fluid shear stress are potent stimuli for ET-1 release and collagen I RNA synthesis in a model cellular system. Although our system is highly artificial, our findings raise the hypothesis that similar effects may occur in the peritoneal membranes of peritoneal dialysis patients and suggest that endothelin might be involved.