Molecular basis of the counteraction by calcium channel blockers of cyclosporine nephrotoxicity.

Nephrotoxicity is a serious side effect for the immunosuppressant drug cyclosporine A(CSA). In this study, we tested the hypothesis that administration of calcium channel blockers such as verapamil or nifedipine ameliorates renal CSA-induced renal dysfunction. Furthermore, our study investigates the roles of inflammatory, oxidative, and fibrotic pathways in CSA-induced renal dysfunction. Six groups of male rats ( n = 6/group) were used and received one of the following treatments for seven consecutive days: vehicle (Cremophor EL ip), CSA (25 mg·kg-1·day-1 ip), verapamil (2 mg·kg-1·day-1 ip), nifedipine (3 mg·kg-1·day-1 ip), CSA in the presence or absence of either verapamil, or nifedipine. Biochemical and histomorphometric analyses showed that rats treated with CSA exhibited clear signs of nephrotoxicity that included 1) proteinuria and elevations in serum creatinine and blood urea nitrogen, 2) mesangial expansion, 3) increases in glomerular and tubular type IV collagen expression, and 4) increases in the glomerulosclerosis and tubulointerstitial fibrosis indices. Although the single administration of nifedipine or verapamil had no significant effect on renal pathology, or its biochemical and physiological function, the concurrent use of either calcium channel blockers significantly and equipotently ameliorated the biochemical, morphological, and functional derangements caused by CSA. More importantly, we report that the oxidative (reactive oxygen species production, NADPH-oxidase activity, and dual oxidase 1/2 levels), fibrotic (transforming growth factor-ß1 expression), and inflammatory (NF-κB expression) manifestations of renal toxicity induced by CSA were significantly reversed upon administration of nifedipine or verapamil. Together, these results highlight the efficacy of calcium channel-blocking agents in attenuating CSA-induced nephrotoxicity and predisposing biochemical and molecular machineries.

CYP4A/CYP2C modulation of the interaction of calcium channel blockers with cyclosporine on EDHF-mediated renal vasodilations in rats.

The endothelium-derived hyperpolarizing factor (EDHF) serves as a back-up mechanism that compensates for reduced nitric oxide (NO)/prostanoids bioavailability. Here we investigated whether (i) under conditions of vascular endothelium dysfunction, the immunosuppressant drug cyclosporine (CSA) upregulates EDHF-dependent renal vasodilations through altering CYP4A/CYP2C signaling, and (ii) calcium channel blockers modulate the CSA/EDHF/CYP interaction. Rats were treated with CSA, verapamil, nifedipine, or their combinations for 7days. Blood pressure (BP) was measured by tail-cuff plethysmography. Kidneys were then isolated, perfused with physiological solution containing L-NAME (NOS inhibitor) and diclofenac (cyclooxygenase inhibitor, DIC), and preconstricted with phenylephrine. CSA (25mgkg-1day-1 for 7days) increased BP and augmented carbachol renal vasodilations. The co-treatment with verapamil (2mgkg-1day-1) or nifedipine (3mgkg-1day-1) abolished CSA hypertension and conversely affected carbachol vasodilations (increases vs. decreases). Infusion of MSPPOH (epoxyeicosatrienoic acids, EETs, inhibitor) reduced carbachol vasodilations in kidneys of all rat groups, suggesting the importance of EETs in these responses. By contrast, 20-Hydroxyeicosatetraenoic Acid (20-HETE) inhibition by HET0016 increased carbachol vasodilations in control rats, an effect that disappeared by CSA treatment, and reappeared in rats treated with CSA/verapamil or CSA/nifedipine. Renal protein expression of CYP2C and CYP4A as well as their vasoactive products (EETs/20-HETE) were increased in CSA-treated rats. Whereas the CYP2C/EETs effects of CSA were abolished by verapamil and intensified by nifedipine, the CYP4A/20-HETE effects were reduced by either CCB. Overall, nifedipine and verapamil blunts CSA hypertension but variably affected concomitantly enhanced EDHF-dependent renal vasodilations and alterations in CYP2C/CYP4A signaling.

Phytotherapeutic activity of curcumol: Isolation, GC-MS identification, and assessing potentials against acute and subchronic hyperglycemia, tactile allodynia, and hyperalgesia.

CONTEXT: Curcumol has recently attracted special attention due to its potential activities in many chronic disorders. Moreover, the traditional role of turmeric [Curcuma longa L. (Zingiberaceae)] in suppression of hyperglycemia is of great interest. OBJECTIVES: The present work explores the potential acute and subchronic antihyperglycemic, antinociceptive, and in vivo antioxidant effects of curcumol in alloxan-diabetic mice. MATERIALS AND METHODS: Bio-guided fractionation, column-chromatography, and GC-MS were utilized to identify the most active compound of turmeric (curcumol). Turmeric (25, 50, and 100 mg/kg), the curcumol rich fraction (CRF) (7 mg/kg), and curcumol (20, 30, and 40 mg/kg) were assessed for their acute (6 h) and subchronic (8 d) antihyperglycemic potentials and antinociceptive effects (8 weeks) were measured, using hot-plate and tail-flick latencies and von-Frey filaments method and in vivo antioxidant effects in alloxan-diabetic mice. RESULTS: The most-active turmeric fraction was found to be rich in curcumol (45.5%) using GC-MS analysis method. The results proved that the highest dose levels of turmeric extract and curcumol exerted remarkable hypoglycemic activity with 41.4 and 39.3% drop in the mice glucose levels after 6 h, respectively. Curcumol (40 mg/kg) was found to be 9.4% more potent than turmeric extract (100 mg/kg) in subchronic management of diabetes. Curcumol also showed a significant improvement of peripheral nerve function as observed from the latency and tactile tests. DISCUSSION: The antioxidant potential of curcumol may cause its ability to ameliorate diabetes and diabetes-related complications. CONCLUSIONS: Curcumol, a natural metabolite with a good safety-profile, showed results comparable with tramadol in reversing diabetes-induced tactile allodynia and hyperalgesia.

Evaluation of l-arginine on kidney function and vascular reactivity following ischemic injury in rats: protective effects and potential interactions.

BACKGROUND: There is an interaction between many cell types involved in the pathophysiology of ischemic acute renal failure. Nitric oxide (NO) precursors, especially l-arginine, may have protective effects on tissue ischemia/reperfusion injury (IRI); however, their molecular mechanisms are unclear. In the present study, the interaction between l-arginine, cyclo-oxygenase (COX)-2 and reactive oxygen species (ROS) in the pathogenesis of ischemic acute renal failure was investigated. METHODS: Ischemia/reperfusion injury model in rats was used and various biochemical parameters examined. The rat isolated aortic rings served as model for hypoxia/reoxygenation where endothelium dependent and independent relaxations were exerted. RESULTS: Pre-treatment of rats subjected to IRI with l-arginine (125mg/kg) significantly reduced kidney MDA levels, elevated kidney SOD activity, GSH level and total NO levels at 24 and 48h after reperfusion. Kidney COX-2 level was only different in the l-arginine-treated group 48h after reperfusion compared to the IRI group. Pre-treatment with l-arginine (10(-2)M) alone or in combination with celecoxib significantly potentiated the acetylcholine (Ach)-induced relaxations in control and hypoxic rings. The effect of the combination was synergistic only in hypoxic rings. Addition of ascorbic acid to the celecoxib-arginine combination did not produce further potentiation. Sodium nitroprusside-induced relaxations in control and hypoxic rings were potentiated by l-arginine or celecoxib-arginine combination but not by ascorbic acid. CONCLUSIONS: The protective effect of l-arginine may result from the interaction between NO and ROS and increased NO bioavailability. The protective effects of combined celecoxib and l-arginine against IRI could be attributed to their antioxidant activity which exceeded that of ascorbic acid.

The involvement of K(ATP) channels in morphine-induced antinociception and hepatic oxidative stress in acute and inflammatory pain in rats.

This study investigated the role of K(ATP) channels in morphine-induced antinociception and hepatic oxidative stress in acute and inflammatory pain. The K(ATP) channel modulators (K(ATP) channel opener, diazoxide 100 mg/kg, p.o, and K(ATP) channel blocker, glibenclamide, 3 mg/kg i.p.) were administered with morphine (80 mg/kg, i.p.). Antinociception was assessed by the tail-flick and formalin tests in rats and measured by the area under the curve values and the maximum percent effect for 3 h. The indices of hepatic oxidative stress: glutathione, glutathione peroxidase, and malondialdehyde were then determined in the liver homogenates obtained from the treated animals. In both tests, glibenclamide antagonized morphine-induced antinociception, whereas diazoxide augmented it in the tail-flick test only. In the formalin test, glibenclamide alone has a significant hyperalgesic effect, whereas diazoxide decreased the number of flinches. Coadministration of glibenclamide with morphine antagonized the hepatotoxic effect of morphine in both animal models. In the tail-flick test, glibenclamide administered alone significantly increased malondialdehyde's level. Coadministration of diazoxide with morphine increased glutathione level in the formalin test. Diazoxide administered alone exacerbated the hepatic oxidative stress in both animal models. These findings suggest a role of K(ATP) channel modulators on morphine-induced antinociception and hepatic oxidative stress. The administration of glibenclamide may prevent morphine-induced hepatotoxicity. The effectiveness of diazoxide in the management of pain is limited due to its deleterious effect on the liver. However, the interaction of the K(ATP) channel modulators with morphine depends on the differential sensitivity to the pain stimulus.

Redox imbalances incite the hypertensive, baroreflex, and autonomic effects of cyclosporine in rats.

Previous studies including ours showed that cyclosporine (CSA) causes baroreflex dysfunction and hypertension. Here we tested the hypothesis that oxidative damage in central and peripheral tissues underlies the hypertensive, baroreflex and autonomic actions elicited by CSA in rats. We investigated the effects of individual and combined 7-day treatments with CSA (25 mg/kg/day, n=7) and 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol, superoxide dismutase mimetic, 100 mg/kg/day, n=7) on blood pressure, reflex heart rate responses to peripherally mediated pressor and depressor responses, and biomarkers of oxidative stress. CSA elevated blood pressure and reduced reflex bradycardic (phenylephrine) and tachycardic (sodium nitroptrusside) responses. The ability of muscarinic (atropine, 1 mg/kg i.v.) or ß-adrenoceptor blockade (propranolol, 1 mg/kg i.v.) to reduce reflex heart rate responses was reduced in CSA-treated rats, suggesting the impairment by CSA of reflex cardiac autonomic control. Concurrent administration of tempol abolished CSA-induced hypertension and normalized the associated impairment in baroreflex gain and cardiac autonomic control. Tempol also reversed the CSA-induced increases in aortic and brainstem nitrite/nitrate and malondialdehyde (MDA) and decreases in aortic superoxide dismutase (SOD). These findings implicate oxidative stress in peripheral and central cardiovascular sites in the deleterious actions of CSA on blood pressure and baroreceptor control of heart rate.

Crosstalk between central pathways of nitric oxide and carbon monoxide in the hypertensive action of cyclosporine.

Although the intermediary role of central neurons in the hypertensive and sympathoexcitatory actions of cyclosporine (CSA) has been recognized in previous studies including our own, the underlying mechanism remains obscure. In this study, we tested the hypothesis that central pathways of nitric oxide (NO) and carbon monoxide (CO) modulate the blood pressure (BP) response elicited by CSA in conscious rats. Hemodynamic effects of CSA were evaluated in absence and presence of maneuvers that inhibit or facilitate biosynthesizing enzymes of NO (NOS) or CO (heme oxygenase, HO). CSA (20mg/kg i.v.) produced abrupt increases in BP that peaked in 5min and maintained for at least 45min. The hypertensive effect of CSA disappeared in rats pretreated intracisternally (i.c.) with N(ω)-nitro-l-arginine methyl ester (L-NAME, nonselective NOS inhibitor), N(5)-(1-iminoethyl)-l-ornithine (L-NIO, selective eNOS inhibitor), N(ω)-propyl-l-arginine (NPLA, selective nNOS inhibitor), or 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, guanylate cyclase inhibitor), suggesting the importance of central eNOS/nNOS/GC cascade in CSA-induced hypertension. L-NAME also abolished the hypotension caused by the sympatholytic drug moxonidine, indicating a tonic sympathoinhibitory action for NO. The inhibition of HO activity by zinc protoporphyrin IX (ZnPP) abrogated the hypertensive action of CSA. The abolition by L-NAME or ZnPP of CSA hypertension was compromised upon simultaneous i.c. exposure to hemin (HO substrate) and l-arginine (NOS substrate), respectively. Together, the interruption of the mutually facilitated NOS/NO and HO/CO pathways and coupled GC/cGMP in central neuronal pools accounts, at least partly, for the hypertensive and perhaps sympathoexcitatory actions of CSA.

Interruption of central neuronal pathway of imidazoline I1 receptor mediates the hypertensive effect of cyclosporine in rats.

Increased central sympathetic outflow secondary to afferent sympathetic excitation has been implicated in the hypertensive effect of the immunosuppressant drug cyclosporine (CSA). The present study investigated the roles of central alpha(2)-adrenoceptors and I(1)-imidazoline receptors in modulating the hypertensive action of CSA. The blood pressure (BP) response to CSA in conscious rats was assessed in the absence and presence of peripherally or centrally acting sympatholytic drugs. Also, the effect of selective pharmacologic blockade of alpha(2) or I(1) receptors by yohimbine and efaroxan, respectively, on the pressor response to CSA was evaluated. CSA (20 mg/kg i.v.) produced a rapid increase in BP that peaked (25+/-4 mm Hg) after approximately 4 min and continued for the 45 min study duration. Ganglionic (hexamethonium 20 mg/kg) or alpha(1)-adrenoceptor (prazosin 1 mg/kg) blockade reduced the pressor effect of CSA. Pressor responses to phenylephrine (alpha(1)-adrenoceptor agonist) were not affected by CSA, thereby eliminating a possible role for alterations of vascular alpha(1)-adrenoceptor responsiveness in CSA hypertension. CSA hypertension was attenuated in rats pretreated intravenously with drugs that reduce central sympathetic tone including clonidine (mixed alpha(2)/I(1)-receptor agonist, 30 microg/kg) or moxonidine (selective I(1)-receptor agonist, 100 microg/kg) in contrast to no effect for guanabenz (selective alpha(2)-receptor agonist, 30 microg/kg). Intracisternal (i.c.) administration of moxonidine also reduced CSA hypertension. Selective blockade of central I(1) (efaroxan, 0.15 microg/rat, i.c.) but not alpha(2) (yohimbine, 25 microg/5 microl/rat, i.c.) receptors abolished the hypertensive response to CSA. Together, these findings highlight that CSA elicits its hypertensive effect via disruption of central sympathoinhibitory pathways which include I(1)-imidazoline receptors.

Time-domain evaluation of cyclosporine interaction with hemodynamic variability in rats.

This study investigated the effects of chronic exposure of Wistar rats to the immunosuppressant drug cyclosporine on blood pressure, heart rate, and their variability and the role of sympathovagal balance in this interaction. The blood pressure variability was determined as the standard deviation of the mean arterial pressure (SDMAP). Two time-domain heart rate variability indices were employed, the standard deviation of beat-to-beat intervals (SDRR) and the root mean square of successive beat-to-beat differences in R-R interval durations (rMSSD). Subcutaneous cyclosporine administration (20 mg/kg/day) for 12 days had no effect on blood pressure or its variability index (SDMAP). In contrast, the average level of heart rate and its variability indices (SDRR and rMSSD) showed significant increases and decreases, respectively, in cyclosporine- compared with vehicle-treated rats. Vagal (atropine) or beta -adrenergic (propranolol) blockade had no effect on blood pressure but elicited increases and decreases, respectively, in heart rate. Compared with control rats, cyclosporine-treated rats exhibited lesser tachycardic responses to atropine and greater bradycardic responses to propranolol, suggesting alterations of cardiac vagal (attenuation) and sympathetic (enhancement) activity by cyclosporine. Further, atropine reduced indices of heart rate variability (rMSSD and SDRR) in control rats, effects that were blunted by cyclosporine treatment. On the other hand, propranolol had no effect on heart rate variability in either cyclosporine-treated or control rats. These findings implicate vagally-mediated alterations in the cardiac sympathovagal balance in the cyclosporine-induced impairment of heart rate oscillations.

Testosterone depletion contributes to cyclosporine-induced chronic impairment of acetylcholine renovascular relaxations.

The immunosuppressant drug cyclosporine causes nephrotoxicity mainly via alterations of renovascular reactivity. This study investigated whether this effect of cyclosporine is modulated by the male gonadal hormone testosterone. The endothelium-dependent and -independent relaxations evoked by acetylcholine and sodium nitroprusside, respectively, were evaluated in phenylephrine-preconstricted isolated perfused kidneys obtained from sham-operated, castrated, and testosterone-replaced castrated (CAS+T) male rats in the absence and presence of cyclosporine. Compared with sham-operated values, short-term (10 days) castration or cyclosporine treatment caused significant and equivalent reductions in plasma testosterone levels and vasorelaxant responses to acetylcholine. Treatment of castrated rats with cyclosporine caused no further attenuation of acetylcholine relaxations. Testosterone replacement of castrated (CAS+T) or cyclosporine-treated castrated (CAS+CyA+T) rats restored plasma testosterone and acetylcholine relaxations to near-sham-operated levels. On the other hand, castration caused significant increases in nitroprusside relaxations versus no effect for cyclosporine. The relaxant responses to nitroprusside in castrated rats were restored to sham-operated levels after testosterone replacement. Plasma urea and creatinine were not affected by castration but were significantly increased by cyclosporine. These findings suggest that testosterone exerts directionally opposite modulatory effects on endothelium-dependent and -independent renal relaxations. Further, the results demonstrate that testosterone depletion may contribute, at least partly, to the inhibitory effect of cyclosporine on renovascular endothelial function. These data are clinically important because endothelial dysfunction contributes to vascular abnormalities associating cyclosporine therapy.

Cyclosporine attenuates the autonomic modulation of reflex chronotropic responses in conscious rats.

Cyclosporine A (CyA), an immunosuppressant drug, has been shown to attenuate the baroreflex control of heart rate (HR). This study investigated whether or not the CyA-induced baroreflex dysfunction is due to alterations in the autonomic (sympathetic and parasympathetic) control of the heart. We evaluated the effect of muscarinic or beta-adrenergic blockade by atropine and propranolol, respectively, on reflex HR responses in conscious rats treated with CyA (20 mg x kg(-1) x day(-1) dissolved in sesame oil) for 11-13 days or the vehicle. Baroreflex curves relating changes in HR to increases or decreases in blood pressure (BP) evoked by phenylephrine (PE) and sodium nitroprusside (NP), respectively, were constructed and the slopes of the curves were taken as a measure of baroreflex sensitivity (BRS(PE) and BRS(NP)). Intravenous administration of PE and NP produced dose-related increases and decreases in BP, respectively, that were associated with reciprocal changes in HR. CyA caused significant (P < 0.05) reductions in reflex HR responses as indicated by the smaller BRS(PE) (-0.97 +/- 0.07 versus -1.47 +/- 0.10 beats x min(-1) x mmHg(-1) (1 mmHg = 133.322 Pa)) and BRS(NP) (-2.49 +/- 0.29 versus -5.23 +/- 0.42 beats x min(-1) x mmHg(-1)) in CyA-treated versus control rats. Vagal withdrawal evoked by muscarinic blockade elicited significantly lesser attenuation of BRS(PE) in CyA compared with control rats (40.2 +/- 8.0 versus 57.7 +/- 4.4%) and abolished the BRS(PE) difference between the two groups, suggesting that CyA reduces vagal activity. CyA also appears to impair cardiac sympathetic control because blockade of beta-adrenergic receptors by propranolol was less effective in reducing reflex tachycardic responses in CyA compared with control rats (41.6 +/- 4.2 versus 59.5 +/- 4.5%). These findings confirm earlier reports that CyA attenuates the baroreceptor control of HR. More importantly, the study provides the first pharmacological evidence that CyA attenuates reflex chronotropic responses via impairment of the autonomic modulation of the baroreceptor neural pathways.

Cyclosporine adversely affects baroreflexes via inhibition of testosterone modulation of cardiac vagal control.

Previous studies have shown that the immunosuppressant drug cyclosporine A attenuates arterial baroreceptor function. This study investigated whether the modulatory effect of cyclosporine on baroreceptor function involves inhibition of the baroreflex-facilitatory effect of testosterone. The role of cardiac autonomic control in cyclosporine-testosterone baroreflex interaction was also investigated. Baroreflex curves relating bradycardic responses to increments in blood pressure evoked by phenylephrine were constructed in conscious, sham-operated, castrated rats and in testosterone-replaced castrated (CAS + T) rats in the absence and presence of cyclosporine. The slopes of the curves were taken as an index of the baroreflex sensitivity (BRS). Short-term (11-13 days) cyclosporine treatment or castration reduced plasma testosterone levels and caused similar attenuation of the reflex bradycardia, as indicated by the significantly smaller BRS compared with sham-operated values (-0.97 +/- 0.07, -0.86 +/- 0.06, and -1.47 +/- 0.10 beats/min/mm Hg, respectively). The notion that androgens facilitate baroreflexes is further confirmed by the observation that testosterone replacement of castrated rats restored plasma testosterone and BRS to sham-operated levels. Cyclosporine had no effect on BRS in castrated rats but caused a significant reduction in CAS + T rats. Muscarinic blockade by atropine caused approximately 60% reduction in the BRS in sham-operated rats, an effect that was significantly and similarly diminished by castration, cyclosporine, or their combination. beta-Adrenergic blockade by propranolol caused no significant changes in BRS. These findings suggest that cyclosporine attenuates baroreflex responsiveness via, at least partly, inhibition of the testosterone-induced facilitation of cardiomotor vagal control.