Human kidney graft survival correlates with structural parameters in baseline biopsies: a quantitative observational cohort study with more than 14 years' follow-up.

This prospective cohort study evaluates associations between structural and ultrastructural parameters in baseline biopsies from human kidney transplants and long-term graft survival after more than 14 years' follow-up. Baseline kidney graft biopsies were obtained prospectively from 54 consecutive patients receiving a kidney transplant at a single institution. Quantitative measurements were performed on the baseline biopsies by computer-assisted light microscopy and electron microscopy. Stereology-based techniques estimated the fraction of interstitial tissue, the volume of glomeruli, mesangial fraction, and basement membrane thickness of glomerular capillaries. The fraction of occluded glomeruli and scores according to the Banff classification were achieved. Kidney graft survival was analyzed by Kaplan-Meier estimates and Cox regression. Association to long-term kidney function was also analyzed. The long-term surviving kidney transplants were characterized at implantation by less arteriolar hyaline thickening (P < 0.001) and less interstitial fibrosis (P = 0.001), as well as a lower fraction of occluded glomeruli (P = 0.004) and lower glomerular volume (P = 0.03). At the latest follow-up, eGFR was decreased by 12 ml/min/1.73 m2 per unit increase in the score for arteriolar hyalinosis at implantation (P = 0.02), and eGFR was decreased by 19 ml/min/1.73 m2 per 106 µm3 increase in glomerular volume at baseline (P = 0.03). The unbiased Cavalieri estimate of glomerular volume and the ultrastructural parameters are the first to be evaluated in a cohort study with prospective follow-up for more than 14 years. The study shows that baseline biopsies from human kidney grafts contain extraordinary long-term prognostic information, and it highlights the importance of these intrinsic graft factors.

n-3 polyunsaturated fatty acids and adiponectin in patients with end-stage renal disease.

BACKGROUND AND AIM: In subjects without kidney disease, adiponectin appears to have anti-inflammatory, anti-diabetic, and anti-atherogenic effects. n-3 polyunsaturated fatty acids (PUFA) from seafood have several beneficial effects in patients with endstage renal disease (ESRD) and the aim of the present study was to assess the effect of n-3 PUFA supplementation on plasma adiponectin levels in ESRD patients. METHODS: In a double blinded intervention trial, 162 ESRD patients (mean age 67 years  ± 13, 56 women and 106 men) undergoing chronic hemodialysis were randomized to 1.7 g n-3 PUFA daily or placebo for 3 months. Adiponectin, plasma lipids and lipoproteins were measured at baseline and after the intervention period. RESULTS: At baseline, adiponectin was positively correlated to HDL-cholesterol (r = 0.55, p < 0.001) and inversely correlated to plasma triglycerides, body mass index (BMI) and high sensitive C-reactive protein (Hs-CRP) (r = -0.32, p < 0.01, r = -0.43, p < 0.01, and r = -0.21, p < 0.01, respectively). Furthermore, adiponectin was inversely correlated to the plasma levels of the two major n-3 PUFA docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (r = -0.19, p < 0.001, and r = -0.30, p < 0.001, respectively). Baseline plasma adiponectin levels were high in both groups but after 3 months of supplementation no significant change was observed in the groups. Thus, n-3 PUFA supplementation did not change adiponectin levels. CONCLUSION: We found an elevated plasma adiponectin level, which was inversely associated with plasma levels of DHA and EPA at baseline. Supplementation with n-3 PUFAs for 3 months did not change adiponectin levels. The negative result in this study may be related to a relatively low dose and future studies with higher dose and longer duration are needed to explore this mechanism.

The Effect of n-3 Fatty Acids on Small Dense Low-Density Lipoproteins in Patients With End-Stage Renal Disease: A Randomized Placebo-Controlled Intervention Study.

OBJECTIVE: Patients with end-stage renal disease (ESRD) have a high risk of cardiovascular disease. Small dense low-density lipoprotein (sdLDL) particles are particularly atherogenic. Marine n-3 polyunsaturated fatty acids (PUFA) may have a beneficial effect on numbers of sdLDL particles, and the aim of this study was to investigate the effect of n-3 PUFA on plasma levels of sdLDL in patients with ESRD. METHODS: ESRD patients with cardiovascular disease (n = 161) on chronic hemodialysis were randomized to treatment with 1.7 g of n-3 PUFA (n = 81) or 2 g of placebo (olive oil; n = 80) for 3 months. The study was double-blinded. Densities of LDL and percentages of sdLDL (sdLDL%) of total LDL were measured before and after intervention. On the basis of sdLDL%, patients were classified as having lipid pattern A, I (intermediate), or B defined by a successive increase in sdLDL concentration and decrease in lipid particle size. RESULTS: n-3 PUFAs significantly reduced triglycerides. However, LDL cholesterol remained unchanged. In the n-3 group, the LDL density did not change significantly during follow-up. Similarly, the LDL density remained unchanged in the placebo group. In the n-3 group, the sdLDL% was 34% at baseline and unchanged at follow-up. At baseline 71% had LDL pattern A, 9% had pattern I, and 20% had pattern B, and none of these patterns were significantly changed by n-3 PUFA supplementation. CONCLUSION: Dietary supplementation with 1.7 g of n-3 PUFA had no effect on LDL density or sdLDL levels in patients with ESRD.

Osteoprotegerin and mortality in hemodialysis patients with cardiovascular disease.

BACKGROUND: Patients treated with hemodialysis (HD) have an increased mortality, mainly caused by cardiovascular disease (CVD). Osteoprotegerin (OPG) is a glycoprotein involved in the regulation of the vascular calcification process. Previous studies have demonstrated that OPG is a prognostic marker of mortality. The aim of this study was to investigate if OPG was a prognostic marker of all-cause mortality in high-risk patients with end-stage renal disease and CVD. METHODS: We prospectively followed 206 HD patients with CVD. OPG was measured at baseline and the patients were followed for 2 years or until reaching the primary endpoint, i.e., all-cause mortality. RESULTS: All-cause mortality during follow-up was 44% (90/206). High OPG was associated with increased mortality, using the first tertile as reference, with an unadjusted HR of 1.70 (CI 1.00 - 2.88) for the second tertile and HR of 1.63 (CI 0.96 - 2.78) for the third tertile. In a multivariate Cox-regression analysis age, CRP and OPG in both the second and third tertile were significantly associated with increased mortality In the unadjusted survival analysis, a test for trend of OPG yielded a p-value of 0.08; in the adjusted analyses, the p-value for trend was 0.03. CONCLUSIONS: In a high-risk population of hemodialysis patients with previously documented cardiovascular disease, a high level of OPG was an independent risk marker of all-cause mortality.

Plasma adiponectin before and after kidney transplantation.

The role of plasma adiponectin (ADPN) in patients with impaired kidney function and following kidney transplantation (Tx) is debated. We aimed to: (i) determine whether pretransplant ADPN level is an independent risk factor for deterioration of glucose tolerance including development of new-onset diabetes mellitus after Tx, (ii) describe which parameters that influence the ADPN concentration before and after Tx. Fifty-seven nondiabetic kidney allograft recipients and 40 nondiabetic uraemic patients were included. The Tx group was examined at baseline and 3 and 12 months after Tx. The uraemic control group was examined twice, separated by 12 months. ADPN levels declined significantly following Tx (P < 0.0001), while estimated glomerular filtration rate (eGFR) increased (P < 0.0005). eGFR, BMI and insulin sensitivity index were independently associated with ADPN in a multivariate regression analysis, whereas an ordinal logistic regression analysis revealed no predictive characteristic of ADPN for aggravation of the glucose tolerance after Tx. In conclusion, kidney transplantation is accompanied by a significant reduction in ADPN concentration. Several factors determine the ADPN concentration before and after Tx including kidney function, insulin resistance, use of immunosuppressive agents and BMI. Pretransplant ADPN level did not predict development of new-onset diabetes mellitus or even deterioration of the glucose tolerance following Tx.

Plasma neutrophil gelatinase associated lipocalin (NGAL) is associated with kidney function in uraemic patients before and after kidney transplantation.

BACKGROUND: Neutrophil gelatinase associated lipocalin (NGAL) is a biomarker of kidney injury. We examined plasma levels of NGAL in a cohort of 57 kidney allograft recipients (Tx group, 39 ± 13 years), a uraemic group of 40 patients remaining on the waiting list (47 ± 11 years) and a control group of 14 healthy subjects matched for age, sex and body mass index (BMI). The kidney graft recipients were studied at baseline before transplantation and 3 and 12 months after transplantation and the uraemic group at baseline and after 12 months. METHODS: NGAL was measured using a validated in-house Time-Resolved Immuno-flourometric assay (TRIFMA). Repeated measurements differed by < 10% and mean values were used for statistical analyses. Spearman rank order correlation analysis and the Kruskal-Wallis non-parametric test were used to evaluate the association of NGAL concentrations with clinical parameters. RESULTS: Plasma NGAL levels before transplantation in the Tx and uraemic groups were significantly higher than in the healthy controls (1,251 µg/L, 1,478 µg/L vs. 163 µg/L, p < 0.0001). In the Tx group NGAL concentrations were associated with serum creatinine (R = 0.51, p < 0.0001), duration of end-stage renal failure (R = 0.41, p = 0.002) and leukocyte count (R = 0.29, p < 0.026). At 3 and 12 months plasma NGAL concentrations declined to 223 µg/L and 243 µg/L, respectively and were associated with homocysteine (R = 0.39, p = 0.0051 and R = 0.47, p = 0.0007). CONCLUSIONS: Plasma NGAL is a novel marker of kidney function, which correlates to duration of end-stage renal failure (ESRD) and serum creatinine in uraemic patients awaiting kidney transplantation. Plasma NGAL is associated with homocysteine in transplanted patients. The prognostic value of these findings requires further studies.

Calcineurin inhibitors acutely improve insulin sensitivity without affecting insulin secretion in healthy human volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: New onset diabetes after transplantation is related to treatment with immunosuppressive medications. Clinical studies have shown that risk of new onset diabetes is greater with tacrolimus compared with ciclosporin. The diabetogenicity of ciclosporin and tacrolimus has been attributed to both beta cell dysfunction and impaired insulin sensitivity. WHAT THIS STUDY ADDS: This is the first trial to investigate beta cell function and insulin sensitivity using gold standard methodology in healthy human volunteers treated with clinically relevant doses of ciclosporin and tacrolimus. We document that both drugs acutely increase insulin sensitivity, while first phase and pulsatile insulin secretion remain unaffected. This study demonstrates that ciclosporin and tacrolimus have similar acute effects on glucose metabolism in healthy humans. AIM The introduction of calcineurin inhibitors (CNIs) ciclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) cause impairment of survival rates. The relative contribution of each CNI to the pathogenesis and development of NODAT remains unclear. We sought to compare the impact of CsA and Tac on glucose metabolism in human subjects. METHODS: Ten healthy men underwent 5 h infusions of CsA, Tac and saline in a randomized, double-blind, crossover study. During infusion glucose metabolism was investigated using following methods: a hyperinsulinaemic-euglycemic clamp, an intravenous glucose tolerance test (i.v.GTT), glucose-stimulated insulin concentration-time series and indirect calorimetry. RESULTS: Clamp derived insulin sensitivity was increased by 25% during CsA (P < 0.0001) and 13% during Tac administration (P = 0.047), whereas first phase and pulsatile insulin secretion were unaffected. Coinciding with the CNI induced improved insulin sensitivity, glucose oxidation rates increased, while insulin clearance rates decreased, only non-significantly. Tac singularly lowered hsCRP concentrations, otherwise no changes were observed in circulating glucagon, FFA or adiponectin concentrations. Mean blood concentrations of CNIs were 486.9 ± 23.5 µg l(-1) for CsA and 12.8 ± 0.5 µg l(-1) for Tac. CONCLUSIONS: Acute effects of i.v. CsA, and to a lesser degree Tac infusions, in healthy volunteers include increased insulin sensitivity, without any effect on first phase or pulsatile insulin secretion.

Marine n-3 fatty acids, atrial fibrillation and QT interval in haemodialysis patients.

Patients treated with haemodialysis are at high risk of sudden cardiac death (SCD) often caused by arrhythmias. Atrial fibrillation (AF) is frequent among haemodialysis patients and is associated with increased mortality. Prolonged QTc is a risk marker of ventricular arrhythmia and is thereby associated with SCD. Studies have suggested that n-3 PUFA may have an antiarrhythmic effect, but the exact mechanism is not clear. The aim of this study was to examine whether AF was associated with n-3 PUFA in plasma phospholipids and whether supplementation with n-3 PUFA would shorten the QTc interval in haemodialysis patients compared to placebo. In a double-blinded randomised, placebo-controlled intervention trial 206 haemodialysis patients with CVD were treated with 1·7 g n-3 PUFA or placebo (olive oil) daily for 3 months. Blood samples and electrocardiogram evaluations were carried out at baseline and after 3 months. The QT interval, PQ interval and heart rate were measured in all patients with sinus rhythm (SR). At baseline 13 % of patients had AF. The content of the n-3 PUFA, DHA, was significantly lower (P < 0·05) in serum of patients with AF compared with patients with SR. Thus, the DHA content was independently negatively associated with AF. Supplementation with n-3 PUFA did not shorten the QT interval significantly compared to the placebo group (P = 0·42), although subgroup analysis within the n-3 PUFA group revealed a shortening effects on QTc (P = 0·01). In conclusion, an inverse association was found between the presence of AF and the plasma DHA in haemodialysis patients. Intervention with n-3 PUFA did not shorten the QTc interval compared to placebo.

Renal transplantation in an HIV-infected patient: pharmacokinetic aspects.

The introduction of highly active antiretroviral therapy in the mid-1990s led to a dramatic reduction in mortality and progression to AIDS, and human immunodeficiency virus (HIV)infection has now turned into a chronic disease with improved survival and prognosis. Hence, patients with well-controlled HIV infection are no longer prevented from receiving transplants, but treatment must be based on knowledge of pharmacokinetics for the drugs involved. The common approach measuring the cyclosporine level after 2 h or trough level is misleading and it was necessary to determine the area under the curve.

The content of docosahexaenoic acid in serum phospholipid is inversely correlated with plasma homocysteine levels in patients with end-stage renal disease.

Patients with end-stage renal disease (ESRD) have a high morbidity and mortality from cardiovascular disease. An elevated homocysteine level is an independent predictor of cardiovascular events in patients with ESRD. Interestingly, some studies have found an inverse relationship between the content of marine n-3 polyunsaturated fatty acids (PUFAs) and homocysteine levels, but data are ambiguous. In patients with ESRD, we hypothesized that serum phospholipid n-3 PUFA content would inversely correlate with homocysteine levels in plasma and that supplementation with n-3 PUFA would reduce plasma homocysteine levels. In a double-blind, randomized, controlled design, 206 patients with documented cardiovascular disease and treated with hemodialysis for a minimum of 6 months were randomized to treatment with daily supplement of 1.7 g n-3 PUFA or placebo (olive oil) for 3 months. The content of n-3 PUFA in serum phospholipids and homocysteine levels in plasma were measured at baseline and after 3 months of intervention. A dietary questionnaire was filled out at baseline, and study participants were divided into groups of low, intermediate, and high fish intake. Docosahexaenoic acid was inversely correlated with homocysteine at baseline (coefficient = -0.161; P = .03). Homocysteine was not related to self-reported fish intake. Supplementation with n-3 PUFA did not reduce homocysteine levels compared with placebo (mean ± SD difference, -0.3 ± 7.8 versus 0.3 ± 7.1; P = .58). The content of docosahexaenoic acid in serum phospholipids is inversely correlated with plasma homocysteine levels, and supplementation with n-3 PUFA does not reduce homocysteine levels in patients with ESRD.

The effect of n-3 fatty acids on levels of methylarginines in patients with end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease (ESRD) have a very high mortality mainly caused by cardiovascular disease (CVD). It has been suggested that plasma concentrations of asymmetric dimethyl arginine (ADMA), an endogenous nitric oxide synthase inhibitor, are markedly elevated in patients with ESRD. Elevation of ADMA is linked to CVD and an adverse prognosis. Supplementation with n-3 fatty acids has previously been shown to prevent CVD, but there is very little data regarding the effect of n-3 fatty acids on levels of ADMA. METHODS: Patients with ESRD and documented CVD were randomized to treatment with 1.7 g of n-3 fatty acids (n=103, 34% women) or olive oil (n=103, 38% women) for three months. ADMA, symmetric dimethyl arginine (SDMA), L-arginine, and the relative content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in serum phospholipids were measured before and after treatment. RESULTS: ADMA was normally distributed with a mean value of 0.56+/-0.13 micromol/L (range 0.21-1.01) and only 14/206 (6.8 %) had elevated levels of ADMA. SDMA was generally elevated with a mean value of 1.88+/-0.64 micromol/L (range 0.67-4.56). Supplementation with n-3 fatty acids for three months did not change plasma levels of ADMA, SDMA or L-arginine. CONCLUSIONS: The present data do not support a beneficial effect of n-3 fatty acids on methylarginines in patients with ESRD.

New-onset diabetes mellitus after kidney transplantation in Denmark.

BACKGROUND AND OBJECTIVES: This study aimed to investigate the development of new-onset diabetes mellitus (NODM) in a prospective study of 97 nondiabetic uremic patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Included were 57 kidney recipients (Tx group, age 39 +/- 13 years) and 40 uremic patients remaining on the waiting list for kidney transplantation (uremic controls, age 47 +/- 11 years). All were examined at baseline before possible transplantation and after 12 months. The prevalence of diabetes, prediabetes, insulin sensitivity index (ISI), and insulin secretion index (Isecr) were estimated using an oral glucose tolerance test with measurements of plasma glucose and plasma insulin. RESULTS: One year after transplantation NODM was present in 14% (8 of 57) compared with 5% (2 of 40) in the uremic control group (P = 0.01). ISI in the Tx group deteriorated from 6.8 +/- 3.9 before transplantation to 4.9 +/- 2.8 at 12 months after transplantation (P = 0.005), and a slight increase in Isecr from 37 +/- 19 to 46 +/- 22 (P = 0.02) was seen. No significant changes occurred in the uremic controls (ISI was 7.9 +/- 5 and 8.5 +/- 5, and Isecr was 31 +/- 17 and 28 +/- 15). Using multivariate ordinal logistic regression, pre-Tx ISI and age predicted NODM (odds ratios: 0.82, P = 0.01 and 1.06, P = 0.02, respectively). CONCLUSIONS: One year after kidney transplantation, NODM was present in 14% of patients. This was mainly caused by an increase in insulin resistance and was observed despite improvement in insulin secretion.

The effect of desmopressin on renal water and solute handling in desmopressin resistant monosymptomatic nocturnal enuresis.

PURPOSE: We sought to evaluate the effect of desmopressin on renal water and solute handling in children with monosymptomatic nocturnal enuresis and desmopressin resistant nocturnal polyuria compared to healthy controls. MATERIALS AND METHODS: A total of 12 patients with enuresis and nocturnal polyuria, normal bladder reservoir function and no response to desmopressin, and 10 age matched controls were enrolled in the study. Children were admitted to the hospital for a 48-hour protocol comprising urine collections and blood sampling. Sodium and water intake was standardized. During the second night children received 40 mug intranasal desmopressin. Parameters characterizing the renal water and solute handling were measured and compared between baseline nights and nights with desmopressin. RESULTS: Desmopressin markedly reduced nocturnal urine output in patients with enuresis, minimizing sodium, urea and overall solute excretion, despite the fact that these children were unresponsive to desmopressin at home. This effect on renal sodium handling was not mediated by atrial natriuretic peptide, angiotensin II, aldosterone or renin. Desmopressin did not influence urinary prostaglandin E(2) excretion. The antinatriuretic effect was seen only in patients with enuresis, and it was directly correlated with the reduction in urine output. CONCLUSIONS: Children with nocturnal enuresis and nocturnal polyuria who do not exhibit adequate response to desmopressin at home seem to respond well to the agent at the clinic. The effect of desmopressin in children with enuresis seems largely dependent on reductions in the amount of sodium excreted. Sodium regulating hormones remained unaffected by desmopressin, indicating a possible direct effect of the agent on renal sodium handling.

The effect of n-3 fatty acids on lipids and lipoproteins in patients treated with chronic haemodialysis: a randomized placebo-controlled intervention study.

BACKGROUND: Patients with end-stage renal disease (ESRD) have an increased mortality, mainly due to cardiovascular disease (CVD). ESRD is accompanied by several lipid abnormalities, which may be responsible for part of the increased risk of CVD in this population. n-3 polyunsaturated fatty acids (PUFA) lower plasma triglycerides in patients with normal renal function. The aim of the present study was to examine the effect of n-3 PUFA on serum lipid and lipoproteins in patients treated with chronic haemodialysis (HD). METHODS: In a double-blind randomized placebo-controlled design, patients with documented CVD, treated with HD for a minimum of 6 months, were randomized to treatment with n-3 PUFA or a control treatment (olive oil). A dietary intake of n-3 PUFA was assessed with a dietary questionnaire. Plasma lipids and lipoproteins and the content of n-3 PUFA in serum phospholipids were measured at baseline and after 3 months. RESULTS: Two hundred and six patients were included. Serum phospholipid levels of n-3 PUFA were significantly higher in patients reporting a high fish intake compared to patients reporting a low fish intake. After 3 months, a significant decrease was seen in serum triglycerides in the n-3 PUFA group compared to the control group (P = 0.01). No significant effect was seen on total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, Lp(a) or apoB. CONCLUSION: In patients treated with HD, consumption of fish increases levels of n-3 PUFA. Additional supplementation with n-3 PUFA for 3 months further increases levels of n-3 PUFA and lowers serum triglycerides, but does not significantly affect other plasma lipids or lipoproteins.

Recurrence of light-chain deposition disease after renal transplantation.

A 51-year-old male with a history of chronic renal disease received a renal allograft, in which disease recurred. Light-chain deposition disease was confirmed through biopsies of the native kidney and graft, and detection of free kappa light chains in serum.

The effect of n-3 fatty acids on heart rate variability in patients treated with chronic hemodialysis.

OBJECTIVE: The aim of the present study was to address the effect of n-3 polyunsaturated fatty acids (PUFAs) on heart rate variability (HRV) in patients treated with chronic hemodialysis. DESIGN: We performed a randomized, placebo-controlled intervention trial. SETTING: The study took place at two hospital-based dialysis centers. PATIENTS: Thirty patients with documented cardiovascular disease who were treated with hemodialysis for at least 6 months were included. INTERVENTION: Treatment consisted of 1.7 g of n-3 PUFA or a control treatment (olive oil). MAIN OUTCOME MEASURE: The outcome measure was 24-hour Holter recordings with time domain HRV measurements at baseline and after 3 months of treatment. Blood samples were obtained to assess the content of n-3 PUFA in serum phospholipids before and after treatment. RESULTS: n-3 PUFA did not significantly affect time domain parameters of HRV, compared with a control group. CONCLUSION: We conclude that treatment with n-3 PUFA does not increase HRV in patients treated with chronic hemodialysis, a result that may have been compromised by a small sample size.

A review of the immunosuppressive activity of cyclosporine metabolites: new insights into an old issue.

Cyclosporine A (CsA) is metabolized into a vast spectrum of metabolites. The potential immunosuppressive action of CsA's metabolites has been studied extensively in the early 1990's, with conflicting and inconclusive results. Since then, the pharmacological and clinical consensus guidelines recommend the use of specific monoclonal assays for measurement of CsA's concentrations thus avoiding metabolite interference. Nevertheless, clinical benefit or superiority of these assays was never convincingly demonstrated. We provide a review of the performed in vivo, in vitro and animal studies and their conclusions. During the last years, many transplantation centres have employed the C(2) monitoring of CsA (2 hours post-dose concentration). The metabolites / parent drug ratio two hours post dose is completely different from trough levels (predose concentration). Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase (CaN). Currently, our laboratory, among others, is working on determination of the enzyme's inhibition and its potential use as a pharmacodynamic biomarker. Utilizing this novel pharmacodynamic approach, we have performed in vitro and in vivo studies investigating the immunosuppressive impact of CsA's metabolites on C2 monitoring and on various monitoring assays (mono-/polyclonal). Interestingly, even though we have estimated in vivo that the potential immunosuppressive action of metabolites is less than 10% of the parent drug, we have found assays that take metabolites into consideration to correlate stronger with calcineurin phosphatase inhibition. We believe that the old controversial issue of metabolite induced immunosuppressive action examined under the light of newer pharmacodynamic approaches is still intriguing. Instead of a priori neglecting CsA's metabolites maybe we should investigate the potential of utilizing them as an additional tool towards better therapeutic drug monitoring of cyclosporine.

The calcineurin activity profiles of cyclosporin and tacrolimus are different in stable renal transplant patients.

Cyclosporin and tacrolimus remain the cornerstone immunosuppressive drugs in organ transplantation. Dosing and monitoring these drugs is based on pharmacokinetic protocols, but measuring a pharmacodynamic parameter, calcineurin phosphatase (CaN) activity, could be a valuable supplement in determining optimal doses. Forty stable renal transplant patients were investigated three times in a 6-month period. Blood samples were drawn at 0, 1, 2, 3 and 4 h after oral intake of tacrolimus (FK) or cyclosporin at days 1 and 180. At day 90, one blood sample at trough level (FK) or C2 level (cyclosporin A, CsA) was drawn. CaN activity was determined in whole blood as the release of 32P from a phosphorylated peptide. Activity of the 32P was quantitated by liquid scintillation and results converted to Units CaN, utilizing a calibration curve with CaN. We demonstrated that calcineurin activity profiles at days 1 and 180 were the same for both drugs. Furthermore, we found that patients treated with tacrolimus or cyclosporin displayed different calcineurin activity profiles. We found that cyclosporin displayed greater calcineurin inhibition than tacrolimus. We have demonstrated that the two drugs exert significantly different effects on calcineurin activity in renal transplant patients with stable, well-functioning grafts and that tacrolimus-treated patients can maintain good, stable graft function with minimal CaN inhibition.

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays.

Cyclosporine exhibits a wide spectrum of metabolites that vary considerably in the extent to which they interfere with the various parent drug monitoring immunoassays. There is no consensus regarding the clinical significance of metabolites. Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites in this perspective? The aim of the present study was to determine the concentration of cyclosporine (by means of three different assay methods) and the four most significant metabolites (AM1, AM4N, AM9, and AM1C) in relation to calcineurin phosphatase inhibition. Twelve randomly selected cyclosporine-treated renal transplant patients were included in the study. Blood samples were drawn before, 1, 2, 3, 4, 6, 8, and 12 hr after oral intake of cyclosporine. Parent drug and metabolites were determined by liquid chromatography/tandem mass spectrometry (LC/MSMS). Additionally, cyclosporine concentration was determined by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition correlates strongly with parent cyclosporine metabolites concentrations determined by all three assay methods. Determination methods that took metabolites into consideration exhibit stronger correlations with calcineurin phosphatase inhibition (sum of cyclosporin plus metabolites r=-0.93, LC/MSMS; pFPIA r=-0.94, P