Cyclosporine A Exposure Research Articles

Renal hemodynamics and pharmacokinetics of bosentan with and without cyclosporine A

Renal hemodynamics and pharmacokinetics of bosentan with and without cyclosporine A. Endothelins may play an important role in cyclosporine A (CsA)-induced renal vasoconstriction. Therefore, the effects of a mixed endothelin A and B receptor antagonist, bosentan (BO), on CsA were studied. BO was given either alone or combined with CsA to healthy subjects in a double-blind, placebo-controlled, cross-over study. Standardized renal hemodynamics took place after a single dose of BO or placebo and after seven days of regular intake of CsA + BO or CsA + placebo. CsA was administered as a dose-adjusted regimen to achieve predetermined target trough levels. A pharmacokinetic study of CsA and BO was performed. A single dose of BO did not affect renal hemodynamics. After seven days of coadministration with CsA, BO significantly attenuated both the overall CsA-induced fall of renal plasma flow (RPF; placebo, 594 +/- 85; CsA + placebo, 490 +/- 93; CsA + BO, 570 +/- 106* mL/min, *P < 0.01) and the maximal RPF fall (P < 0.01) observed five hours after CsA intake. The CsA-induced rise of blood pressure and the decrease of glomerular filtration rate (GFR) were not influenced by comedication with BO. After seven days of CsA + BO, the area under the curve (AUC) of BO was nearly doubled compared with the AUC after a single dose of BO (P < 0.05). To reach the CsA target trough levels after seven days, the average CsA dose was increased by 35% when given with BO, as compared with placebo (P = 0.01). CsA exposure (trough levels, AUC) was not statistically different after CsA + placebo and after CsA + BO. Assuming CsA nephrotoxicity is mainly due to vasoconstriction, BO has the potential to attenuate the CsA renal toxicity by markedly blunting the renal hypoperfusion effect of CsA. A complex drug interaction between BO and CsA was observed.

Calcineurin Enhances Acetylcholinesterase mRNA Stability during C2-C12 Muscle Cell Differentiation

Treatment of C2-C12 mouse myoblasts with the immunosuppressant drug cyclosporin A (CsA) enhances the increase in acetylcholinesterase (AChE) expression observed during skeletal muscle differentiation. The enhanced AChE expression is due primarily to increased mRNA stability because CsA treatment increases the half-life of AChE mRNA, but not the apparent transcriptional rate of the gene. Neither tacrolimus (FK506), an immunosuppressive agent with a distinct structure, nor cyclosporine H, an inactive congener of CsA, alters AChE expression. The enhanced AChE expression is associated with the muscle differentiation process, but cannot be triggered by CsA exposure before differentiation. Myoblasts and myotubes of C2-C12 cells express similar amounts of cyclophilin A and FKBP12, immunophilins known to be intracellular-binding targets for CsA and tacrolimus, respectively. However, cellular levels of calcineurin, a calcium/calmodulin-dependent phosphatase known to be the cellular target of ligand-immunophilin complexes, increase 3-fold during myogenesis. Overexpression of constitutively active calcineurin in differentiating cells reduces AChE mRNA levels and CsA antagonizes such an inhibition. Conversely, overexpression of a dominant negative calcineurin construct increases AChE mRNA levels, which are further enhanced by CsA. Thus, a CsA sensitive, calcineurin mediated pathway appears linked to differentiation-induced stabilization of AChE mRNA during myogenesis.

Cyclosporine A-induced alterations in magnesium homeostasis in the rat

The immunosuppressive drug cyclosporine A (CsA) exhibits significant nephrotoxicity. Disturbance of magnesium (Mg) homeostasis may be an important component of this nephrotoxicity. It has been suggested that transmigration of Mg from plasma to tissues may be an important component of CsA-induced alterations in Mg homeostasis. In this study, CsA nephrotoxicity in male Wistar rats was investigated and alterations in Mg homeostasis along with other indices of toxicity were assessed. Animals were dosed daily for 14 days I.P. With CsA (20 mg kg body weight). Control animals received vehicle alone. CsA toxicity was evidenced by i) lower gain in body weight, ii) reduced thymus body weight ratio, iii) increased blood urea nitrogen and creatinine, iv) a tendency for reduced plasma magnesium and v) increased urinary Mg excretion and greatly increased fractional excretion of Mg. Tissue Mg analysis did not reveal any changes in thymus or skeletal muscle Mg while Mg in kidney tissue tended to be reduced. Electron microscopy revealed some damage in renal tubules of rats treated with cyclosporine including translucent cytoplasm, vacuolization, rounded and swollen mitochondria, damage to brush border and disruption of basal infoldings. These results indicate that direct renal tubular damage may result from CsA exposure. No evidence was found for CsA-induced movement of Mg from plasma to tissues. CsA-induced altered renal handling of Mg and this renal Mg wasting may be an important consequence of the nephrotoxicity.

Effects of cyclosporine A on the regenerating endothelium following direct arterial injury

Cyclosporine A (CyA) is still considered the immunosuppressive drug of choice in human solid organ transplantation, although significant adverse side effects such as systemic hypertension and nephrotoxicity have been reported with its use. Experimentally, CyA has been reported to increase renal release of endothelin and thromboxane and also to enhance sensitivity to circulating catecholamines. Human histologic studies have shown that vessels of transplanted organs could be colonized with recipient endothelial cells suggesting a posttransplant regenerating process. Experimental models have demonstrated that regenerated endothelium does not resume its initial vasoactive characteristics but, rather, displays an increased tendency to cause vasospasm. Chronic use of CyA has also been reported to interfere with endothelial release of nitric oxide (NO). Therefore, the association of endothelial regeneration and CyA exposure as seen after an episode of acute rejection could alter the vascular reactivity of the graft and perhaps contribute to early coronary arteriosclerosis seen among heart transplant recipients. The purpose of this experiment was to study the effects of oral administration of CyA on endothelium-dependent and independent relaxation of regenerated rat thoracic aorta.

Sublethal heat shock and cyclosporine exposure produce tolerance against subsequent cyclosporine toxicity.

Sublethal heat shock has been shown to produce tolerance in cells and tissues subsequently exposed to heat or ischemia/ATP depletion. We tested whether heating LLC-PK1 cells for 2 h at 42 degrees C induced heat shock protein-70 (HSP-70) gene expression and conferred tolerance against subsequent cyclosporine A (CyA) toxicity. HSP-70 mRNA was increased immediately after heat shock, returning to baseline by 4 h. HSP-70 protein increased by 1 h after heat shock and declined thereafter, approaching baseline after 72 h. Cells heat shocked at 4 and 24 h prior to CyA exposure were significantly more viable than controls, at CyA concentrations near the median lethal dose (LD50). Cytoprotection declined with time after heat shock, concurrent with declining HSP-70 protein levels. Sublethal CyA exposure (50 micrograms/ml) for 24 h produced upregulation of HSP-70 mRNA and protein. Pretreatment with 50 micrograms/ml CyA for 24 h followed by exposure to a toxic concentration of CyA (200 micrograms/ml) produced significant cytoprotection compared with untreated controls. In conclusion, HSP-70 protein induction by sublethal heat shock or CyA exposure was associated with tolerance against subsequent lethal CyA exposure.

Immunotoxicological investigation using pharmaceutical drugs. In vitro evaluation of immune effects using rodent or human immune cells

In order to evaluate the relevance of in vitro methods for immunotoxicity assessment, the effects of pharmaceutical drugs on lymphoproliferative and cytotoxic functions of mouse splenocytes and human peripheral blood mononuclear cells (hPBMC) were studied. A comparison of sensitivity of immune cells from different origins to an in vitro exposure to different xenobiotics was performed using non-immunosuppressive (cimetidine and furosemide) and immunosup-pressive (azathioprine (AZA), cyclosporine A (CSA), and dexamethasone (DEX)) drugs. For CSA, sensitivity of both rat and mouse splenocytes following in vitro exposure was compared to the one of hPBMC. Immune function tests included lymphoproliferative response to mitogenic lectins (concanavalin A (Con A) and phytohemagglutinin (PHAP)) or to allogeneic cells (mixed leukocyte response (MLR)) and cytotoxicity assays (cytotoxic-T lymphocyte (CTL) and natural killer (NK) cell-mediated cytolysis). Additionally, to evaluate how well in vitro assays represent the in vivo situation, a comparison of the effect of cyclosporine A on the same immune function tests following in vivo or in vitro exposure was performed. The data obtained show numerous similarities in the effects observed following in vitro exposure of rodent or human cells to the drugs and a very similar sensitivity of rat and mouse cells to CSA in vitro. Discrepancies between human and rodent cells such as lymphoproliferative response to PHA-P following exposure to DEX or sensitivity of CTL-mediated cytolysis to CSA do exist. In vitro assays were very representative of the in vivo situation, both in the rat and in the mouse, following CSA exposure, except for NK cell activity in the rat. These data show the usefulness of in vitro systems for immunotoxicity assessment. They allow direct comparison of rodent and human systems, and could be representative, for drugs altering specifically the immune system like CSA does, of the in vivo situation.

Cyclosporin A induces glomerular endothelial cell injury in vitro

The chronic administration of cyclosporin A (CyA) could be associated with renal arteriolar damage. Endothelial cell injury is considered to be the first event in this pathological change. Bovine glomerular endothelial cells (GEN) were used to investigate whether CyA induces microvascular injury in vitro. The damage induced by CyA was demonstrated by cell detachment from the culture plate and cell lysis as characterized by the increase in lactate dehydrogenase (LDH) at the same time after CyA treatment. One mumol/L of CyA induced detachment of GEN within 3 h and lysis after 6 h. Cyclosporin A-induced GEN lysis appeared in a dose and time-dependent manner. These results indicate that GEN are vulnerable to CyA exposure, suggesting that GEN damage might be related to CyA-mediated glomerular injury.

Effects of endothelin receptor antagonist on cyclosporine-induced vasoconstriction in isolated rat renal arterioles.

Recent evidence suggests that the potent constrictor peptide, endothelin (ET) has a mediating role in cyclosporine A (CsA)-related renal vasoconstriction. However, the nature of the CsA-ET interaction and effect on the renal vasculature is uncertain. The purpose of the present study was twofold: (a) to determine if CsA exposure caused direct local release of ET from the endothelium of the renal microvasculature and (b) to determine if locally generated ET has paracrine effects on the underlying vascular smooth muscle to induce vasoconstriction. Experiments were performed in isolated rat renal arterioles. First it was determined that both afferent arteriole (AA) and efferent arteriole (EA) exhibited concentration-dependent decreases in lumen diameter to increasing molar concentrations of CsA. The AA was more sensitive to the vasoconstrictive effects of CsA than the EA. Next, the blocking effect of a recently synthesized putative ETA receptor antagonist was verified in both the AA and EA, where it was found that the cyclic peptide cyclo D-Asp-L-Pro-D-Val-L-Leu-D-Trp totally inhibited the vasoconstriction observed with ET addition. Finally, the role of locally stimulated ET in CsA-induced vasoconstriction was tested by determining the effect of the ETA receptor antagonist on CsA-induced AA and EA constriction. In the AA the vasoconstrictor effect of 10(-11) M CsA was completely blocked by the ETA receptor antagonist. However, in contrast to AA, 10(-11) M CsA in EA in the presence of the ETA receptor antagonist decreased EA lumen diameter by a mean of 41% from baseline (4.80 +/- 0.75 microns vs 7.80 +/- 0.84 microns, P < 0.05). This change in lumen diameter was similar to that induced by CsA alone. These data suggest that CsA directly constricts renal microvessels. This effect is mediated by ET in the AA but not the EA.

Cyclosporine A Induces an Early and Transient Rigidification of Lymphocyte Membranes

The effect of cyclosporine A (CsA) on peripheral blood lymphocyte (PBL) membranes was studied using fluorescence techniques. Light scattering and intercalation experiments indicate CsA has a critical micelle concentration of 3 x 10(-5) M. Doses above this critical concentration were avoided during these in vitro studies. Steady-state fluorescence polarization of the lipid probe DPH in peripheral blood lymphocyte membranes revealed that 10(-5) M CsA caused a significant increase in the fluorescence polarization 1-5 min after exposure (15-16% above control DPH polarization values). This initial increase in polarization plateaued at 10-11% above control values within roughly 40 min. Fluorescence lifetime measurements of DPH during this response reveal a slight increase in DPH lifetime. The increased DPH lifetimes showed that polarization measurements alone underestimate the rigidity of DPH's microenvironment. Measurements of DPH polarization and lifetime were analyzed using the Perrin equation to calculate the apparent microviscosity. Control PBL membranes at 37 degrees C exhibited a microviscosity (eta) equal to 1.89 poise (P). Five to 10 min after CsA exposure eta = 2.62-2.68 P and plateaus at eta = 2.31-2.51 P 20-120 min after exposure. Dose-response studies show that prolonged (greater than 90 min) exposure to CsA alters lymphocyte membrane fluidity. This early response in lymphocytes to CsA may correspond to other events associated with the initial binding of CsA to lymphocyte membranes such as membrane depolarization and alterations in phospholipid metabolism. Intravenous delivery of CsA in clinical studies has shown that a similar CsA dose of near 10(-5) M is available in the blood during the first hour after injection. The evidence presented here suggests that peripheral blood lymphocyte membranes are rigidified when exposed to CsA doses similar to those found in patient's blood immediately following intravenous administration.

Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages

Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages.