The impact of calcineurin inhibitors on insulin sensitivity and insulin secretion: a randomized crossover trial in uraemic patients.

AIMS: The calcineurin inhibitors cyclosporine and tacrolimus are implicated in post-transplant complications such as new-onset diabetes after transplantation. The relative contribution of each calcineurin inhibitor to new-onset diabetes after transplantation remains unclear. We sought to compare the impact of cyclosporine and tacrolimus on glucose metabolism in humans. METHODS: Eight haemodialysis patients received 8-10 days of oral treatment followed by 5-h infusions with cyclosporine, tacrolimus and saline in a randomized, investigator-blind, crossover study. Glucose metabolism and ß-cell function was investigated through: a hyperinsulinaemic-euglycaemic clamp, an intravenous glucose tolerance test and insulin concentration time series. RESULTS: Cyclosporine and tacrolimus decreased insulin sensitivity by 22% (P = 0.02) and 13% (P = 0.048), respectively. The acute insulin response and pulsatile insulin secretion were not significantly affected by the drugs. CONCLUSION: In conclusion, 8-10 days of treatment with cyclosporine and tacrolimus impairs insulin sensitivity to a similar degree in haemodialysis patients, while acute insulin responses and pulsatile insulin secretion remain unaffected.

Cyclosporin and tacrolimus impair insulin secretion and transcriptional regulation in INS-1E beta-cells.

BACKGROUND AND PURPOSE: Introducing the calcineurin inhibitors cyclosporin (CsA) and tacrolimus (Tac) has improved the outcome of organ transplants, but complications such as new onset diabetes mellitus after transplantation (NODAT) decrease survival rates. EXPERIMENTAL APPROACH: We sought, in a beta-cell culture model, to elucidate the pathogenic mechanisms behind NODAT and the relative contribution of the calcineurin inhibitors. INS-1E cells were incubated at basal and stimulatory glucose concentrations, while exposed to pharmacologically relevant doses of CsA, Tac and vehicle for 6 or 24 h. RESULTS: Tac inhibited basal (P < 0.05), but not glucose-stimulated insulin secretion (GSIS) after 6 h of exposure. After 24 h, both agents inhibited basal and GSIS (P < 0.05). Calcineurin phosphatase activity was decreased by both drugs during all conditions. Apoptosis was only seen with CsA treatment, which also induced a slight suppression of calcineurin and insulin mRNA, as well as increased levels of the sterol receptor element binding protein (SREBP)-1c, a transcription factor thought to suppress genes essential for beta-cell function and induce insulin resistance. Expression levels of nuclear factor of activated T-cells (NFAT)-c1, -c2, -c3 and -c4 were not decreased notably by either drug. CONCLUSIONS AND IMPLICATIONS: Tac had acute inhibitory effects on basal insulin secretion, but prolonged exposure (24 h) to Tac or CsA revealed similar suppression of insulin secretion. These prolonged effects were mirrored by a total inhibition of calcineurin activity in beta-cells. CsA showed greater inhibition of beta-cell survival and transcriptional markers, essential for beta-cell function.

Abnormal diurnal rhythm of urine output following renal transplantation: the impact of blood pressure and diuretics.

BACKGROUND: Nocturnal polyuria is the excretion at night of an excessive volume of urine. A major problem following renal transplantation is an abnormal diurnal rhythmicity in urine output. The purpose of this study was to elucidate the prevalence of nocturnal polyuria among renal transplant recipients in the early period after transplantation as well as at least 1 year after transplantation. We aimed to explore possible pathophysiological mechanisms behind nocturnal polyuria in this group of patients, focusing on the impact of blood pressure and medication. METHODS: Seventeen recently transplanted patients 17 late transplant recipients, and 17 healthy controls were included in the study. Voiding habits were assessed by completion of a frequency-volume chart recording all fluid intakes and voiding. A concomitant 24-hour blood pressure profile was obtained in all. RESULTS: Renal transplant recipients had a high prevalence of nocturnal polyuria (74%) and a disturbed blood pressure profile with a lack of appropriate nocturnal dipping (P < .0001) compared to controls. We found a positive correlation between increased nocturnal blood pressure and urine output at night (r = .368, P = .008). Patients taking diuretics had a circadian diurnal rhythm of urine output and a blood pressure profile similar to controls. CONCLUSIONS: Nocturnal polyuria was very common among both recent and late transplant recipients. A high fluid intake during daytime and hypervolemia were suggested as causes of a disturbed blood pressure profile, which partly seemed to explain the high urine output at night. Daytime diuretics may be an effective treatment of this inconvenient complication.

Gene expression analysis of calcineurin isoforms in T-lymphocytes--a method applied on kidney-transplant recipients.

BACKGROUND: Tacrolimus exerts its immunosuppressive effect through inhibition of the intracellular enzyme calcineurin phosphatase (CaN). In this study, we set-up a validated real-time PCR method to measure the gene expression of the two major isoforms of the catalytic subunit of CaN in T-lymphocytes. METHODS: 20 stable kidney-transplant recipients, 10 early kidney-transplant recipients and 10 healthy non-medicated subjects had blood drawn and T-lymphocytes were isolated using E-rosette gradient centrifugation method. The cell counts were analyzed by DNA quantification using Hoeschst 33285. Gene expressions were analyzed using real-time PCR for CaN Aalpha, CaN Abeta and the reference genes CD3E and PPIB. RESULTS: The real-time PCR method was found to be with high efficiencies and low intra- and inter-assay variabilities. No statistically significant differences were found in the gene expression levels of the two reference genes among the three groups. The two major isoforms of CaN A were expressed in equal amounts in the T-lymphocytes. CONCLUSION: We found no significant difference in the reference genes between the three groups, but looking at the data there was a trend towards an up-regulation of CD3E. PPIB appears to be the more stable of the two reference genes tested in our study.

A combined experimental and theoretical study on realizing and using laser controlled torsion of molecules.

It is demonstrated that strong laser pulses can introduce torsional motion in the axially chiral molecule 3,5-difluoro-3('),5(')-dibromobiphenyl. A nanosecond laser pulse spatially aligns the stereogenic carbon-carbon (C-C) bond axis allowing a perpendicularly polarized, intense femtosecond pulse to initiate torsional motion accompanied by a rotation about the fixed axis. We monitor the induced motion by femtosecond time-resolved Coulomb explosion imaging. Our theoretical analysis corroborates the experimental findings and on the basis of these results we discuss future applications of laser-induced torsion, viz., time-resolved studies of deracemization and laser controlled molecular junctions based on molecules with torsion.

NT-pro-BNP is an independent predictor of mortality in patients with end-stage renal disease.

AIM: Patients with end-stage renal disease (ESRD) have an increased mortality from cardiovascular disease (CVD). N-terminal pro-brain natriuretic peptide (NT-pro-BNP) is an independent predictor of mortality in patients with ischemic heart disease and congestive heart failure. Previous data have shown markedly elevated levels of NT-pro-BNP in patients with ESRD, while the prognostic value of elevated levels of NT-pro-BNP in patients with ESRD is largely unknown. The aim of the present study was to examine if the level of NT-pro-BNP predicts mortality in patients with ERSD and CVD. METHODS: We prospectively followed 206 patients with ESRD and documented CVD. Levels of NT-pro-BNP were measured at baseline, and patients were followed for 2 years or until they reached the predefined endpoint of all-cause mortality. RESULTS: During follow-up, the total mortality was 44% (90/206). Patients who died were followed for a median of 314 days (interquartile range 179 - 530). Using Cox regression analysis, age, female sex, systolic blood pressure, dialysis efficiency and plasma levels of NT-pro-BNP were independent prognostic risk factors of mortality. In receiver operating characteristic curve analysis a cut off value for NT-pro-BNP was determined. Patients with values of NT-pro-BNP above 12.200 pg/ml had a 3 times higher risk of death than patients below the cut-off value (HR 3.05 95% CI 1.96 - 4.77, p < 0.0001). CONCLUSION: In spite of generally elevated levels of NT-pro-BNP, NT-pro-BNP is still an independent predictor of mortality and might add prognostic information in patients with ESRD and documented CVD.

Manipulating the torsion of molecules by strong laser pulses.

We demonstrate that strong laser pulses can induce torsional motion in a molecule consisting of a pair of phenyl rings. A nanosecond laser pulse spatially aligns the carbon-carbon bond axis, connecting the two phenyl rings, allowing a perpendicularly polarized, intense femtosecond pulse to initiate torsional motion accompanied by an overall rotation about the fixed axis. We monitor the induced motion by femtosecond time-resolved Coulomb explosion imaging. Our theoretical analysis accounts for and generalizes the experimental findings.

Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model.

BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. CONCLUSIONS: CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Calcineurin activity in tacrolimus-treated renal transplant patients early after and 5 years after transplantation.

The pharmacodynamic (PD) action of tacrolimus (FK) within the T-cell is inhibition of calcineurin phosphatase (CaN). Determination of CaN activity provides us with an important PD marker. Eleven renal transplant patients treated with FK were investigated on day 14 following transplantation and 5 years later. Blood samples drawn before as well as 1, 2, 3, and 4 hours after oral intake of FK were analyzed for CaN activity and blood FK concentrations. Twenty healthy subjects had one blood sample drawn for CaN activity, which was measured as the release of (32)P from a phosphorylated peptide. Radioactivity of (32)P was quantitated by liquid scintillation counting with the results converted to units of CaN utilizing a calibration curve. On day 14, we observed significant inhibition of CaN activity at T:1, 2, and 3 compared with the predose level (P = .002; P = .015; P = .015). Furthermore, all measured CaN activities were significantly different from those observed in healthy nonmedicated subjects. In contrast, at 5 years posttransplant only the CaN activity at T:2 was significantly inhibited compared with the predose level (P = .02). Additionally, all CaN activities at this time were not significantly different from CaN activities in the healthy subjects. We were not able to demonstrate individual CaN activity profiles in the patients. The lack of CaN inhibition at 5 years after transplantation despite relevant drug concentrations, probably reflected the lower drug dose used long after transplantation. This result raises the question of whether CaN inhibition is necessary to hold graft function and whether FK possess CaN-independent mechanisms of action.

Nocturnal polyuria in monosymptomatic nocturnal enuresis refractory to desmopressin treatment.

The transition from day to night is associated with a pronounced decline in diuresis with reductions in the amount of excreted water, electrolytes, and other end products of our metabolism. Failure to do so leads to a large urine output at night, a condition known as nocturnal polyuria, encountered in a large proportion of children with nocturnal enuresis. The aim of this study was to clarify the mechanisms responsible for the nocturnal polyuria seen in enuretics with inadequate response to desmopressin (dDAVP). Forty-six enuretics (7-14 yr of age) and fifteen age-matched controls were admitted for a 24-h protocol with standardized fluid and sodium intake, comprising urine collections, blood sampling, and blood pressure monitoring. We included patients with severe enuresis (5 +/- 1 wet nights/wk) showing <50% reduction in wet nights on dDAVP. We characterized the patients on the basis of their nocturnal urine production. The children with nocturnal polyuria excreted larger amounts of sodium and urea at night than nonpolyurics and controls. Solute-free water reabsorption as well as urinary arginine vasopressin and aquaporin-2 excretion were normal in polyurics, and no differences were found in atrial natriuretic peptide, angiotensin II, aldosterone, and renin levels. Urinary prostaglandin E2 (PGE2) excretion was significantly higher in polyurics. The nocturnal polyuria in children with dDAVP-resistant nocturnal enuresis seems to be the result of augmented sodium and urea excretion. The high urinary PGE2 levels found in these children point toward a role for increased prostaglandin synthesis in the pathogenesis of enuresis-related polyuria.

24-h monitoring of calcineurin phosphatase activity in healthy subjects.

The calcineurin inhibitors cyclosporine and tacrolimus are the cornerstone immunosuppressants used in solid organ transplantation. Studies investigating calcineurin (CaN) activity in renal transplanted patients have been published, but basic properties of the enzyme activity in healthy subjects remain to be described. The aim of this study was to investigate whether CaN displays circadian variation or sex difference is present in healthy subjects. Twenty subjects had blood samples drawn every 4 h for a 24-h period. 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. We found no circadian variation in CaN activity and no difference between the two sexes. The clinical importance of these findings is that blood samples for calcineurin activity can be drawn without taking the exact time of day into consideration, but only considering the time of drug intake.

Comparison of the temporal profile of calcineurin inhibition by cyclosporine and tacrolimus in renal transplant patients.

Calcineurin phosphatase (CaN) activity has been the focus of several recent studies on renal transplant patients as the calcineurin inhibitors tacrolimus (FK) and cyclosporine (CsA) are still the cornerstone in the immunosuppressive treatment. The aim of this study was to compare the temporal inhibition profiles of CaN using CsA or FK in two groups of renal transplant patients. Nineteen tacrolimus-treated and 10 cyclosporine-treated renal transplant patients had blood samples drawn before and at 1, 2, 3, 4, and 6 hours after ingestion of drug. CaN activity was measured as the release of 32P from a previously phosphorylated peptide and radioactivity quantitated by liquid scintillation counting. Results were expressed as units CaN. Blood concentrations of tacrolimus were determined with an IMx method and of CsA with an EMIT assay. FK-treated patients showed maximal inhibition of CaN activity at 1 to 3 hours, returning to the predose level 4 hours after drug intake. CsA-treated patients showed a gradual decrease in CaN activity with a nadir after 3 hours, failing to return to predose levels during the observation period. Both groups showed a significant rise in drug blood concentrations. To conclude, we have demonstrated that two widely used immunosuppressants, CsA and FK, show different CaN inhibitory profiles in renal transplant patients.

Comparison of the pharmacokinetics of tacrolimus and cyclosporine at equivalent molecular doses.

Even though calcineurin inhibitors, namely Tacrolimus (FK) and Cyclosporine (CsA) share similar physicochemical properties and a common mechanism of action, their pharmacokinetics (pk) are different and unpredictable. Both drugs are metabolized by cytochrome P450-3A4 isoforms in the liver and in the mucosa of the upper gastrointestinal tract. FK in clinical practice is given in doses up to 50-fold lower than those of CsA due to its greater potency. It is often assumed that the diverse dosing contributes to the observed pharmacokinetic differences between the two drugs. The objective of the present study was to compare single-dose pk profiles of the two drugs, following oral and intravenous administration, on the basis of equivalent molecular dosing, thus ruling out the quantitative factor. Five healthy volunteers and 14 dialysis patients (7 hemodialysis, 7 peritoneal dialysis) were included in the study. Comparing the pharmacokinetic parameters obtained from the drugs, it appeared that cyclosporine has an greater primary volume of distribution and clearance rate compared to tacrolimus. No other statistically significant differences were observed regarding bioavailability, absorption rate, or elimination rate. The only significant correlation between the pk values of the drugs was in primary volume of distribution. We conclude that even at equivalent molecular doses the pk of each drug remains unique and unpredictable. Furthermore our data fail to reveal significant correlations between the bioavailability, clearance, absorption, and elimination rates of the two drugs.

Calcineurin phosphatase activity and immunosuppression. A review on the role of calcineurin phosphatase activity and the immunosuppressive effect of cyclosporin A and tacrolimus.

The mode of immunosuppressive action of tacrolimus (FK506) and cyclosporin A has been elucidated. Both drugs bind to proteins in the cytoplasm to form complexes, which in turn inhibit the phosphatase activity of calcineurin, an important limiting step in the activation of T cells. The association between drug uptake (pharmacokinetics) and enzyme inhibition (pharmacodynamics) is under current investigation. Great variations in the correlation between blood drug levels and enzyme inhibition could indicate that monitoring calcineurin phosphatase activity for treatment might be superior to monitoring blood drug levels.

Catalytic, highly enantioselective Friedel-Crafts reactions of aromatic and heteroaromatic compounds to trifluoropyruvate. A simple approach for the formation of optically active aromatic and heteroaromatic hydroxy trifluoromethyl esters.

A new catalytic enantioselective synthetic method for the formation of optically active aromatic and heteroaromatic hydroxy-trifluoromethyl ethyl esters is presented. This catalytic enantioselective Friedel-Crafts reaction of trifluoromethyl pyruvate with aromatic and heteroaromatic compounds is catalyzed by a chiral bisoxazoline copper(II) complex and proceeds in good yield and with high enantiomeric excess. For a series of substituted indoles, the corresponding 3-substituted hydroxy-trifluoromethyl ethyl esters are formed in up to 93% yield and 94% ee. Pyrrole and 2-substituted pyrroles also react with trifluoromethyl pyruvate in a highly enantioselective aromatic electrophilic reaction and up to 93% ee and good yields are obtained. Furanes and thiophenes give the corresponding 2-hydroxy-trifluoromethyl ethyl esters in high enantiomeric excess; however, the yields of the products are only moderate. Various types of aromatic compounds react in this catalytic reaction with trifluoromethyl pyruvate to give the aromatic electrophilic addition product in good yield. To obtain high enantiomeric excess (> 80% ee) it is necessary that aromatic amines are protected with sterically demanding protecting groups such as benzyl or allyl. This prevents coordination of the amine nitrogen atom to the catalyst, as aromatic amines having a N,N-dimethyl group probably coordinate to the catalyst, leading to a significant reduction of the enantioselective properties of the catalyst. On the basis of the experimental results and the absolute configuration of the formed chiral center, the mechanism for the catalytic enantioselective Friedel-Crafts reaction is discussed.