Legal Performance-enhancing Drugs Alter Course and Treatment of Rhabdomyolysis-induced Acute Kidney Injury.

INTRODUCTION: Rhabdomyolysis-induced acute kidney injury (RIAKI) can interrupt physical training and increase mortality in injured warfighters. The legal performance-enhancing drugs caffeine and ibuprofen, which can cause renal injury, are widely used by service members. Whether caffeine or ibuprofen affects RIAKI is unknown. Cilastatin treatment was recently identified as an experimental treatment to prevent RIAKI at injury. To determine potential interacting factors in RIAKI treatment, we test the hypothesis that caffeine and ibuprofen worsen RIAKI and interfere with treatment. MATERIALS AND METHODS: In mice, RIAKI was induced by glycerol intramuscular injection. Simultaneously, mice received caffeine (3 mg/kg), ibuprofen (10 mg/kg), or vehicle. A second cohort received volume resuscitation (PlasmaLyte, 20 mL/kg) in addition to caffeine or ibuprofen. In a third cohort, cilastatin (200 mg/kg) was administered concurrently with drug and glycerol administration. Glomerular filtration rate (GFR), blood urea nitrogen (BUN), urine output (UOP), renal pathology, and renal immunofluorescence for kidney injury molecule 1 were quantified after 24 hours. RESULTS: Caffeine did not worsen RIAKI; although BUN was modestly increased by caffeine administration, 24-hour GFR, UOP, and renal histopathology were similar between vehicle-treated, caffeine-treated, and caffeine + PlasmaLyte-treated mice. Ibuprofen administration greatly worsened RIAKI (GFR 14.3 ± 19.5 vs. 577.4 ± 454.6 µL/min/100 g in control, UOP 0.5 ± 0.4 in ibuprofen-treated mice vs. 2.7 ± 1.7 mL/24 h in control, and BUN 264 ± 201 in ibuprofen-treated mice vs. 66 ± 21 mg/dL in control, P < .05 for all); PlasmaLyte treatment did not reverse this effect. Cilastatin with or without PlasmaLyte did not reverse the deleterious effect of ibuprofen in RIAKI. CONCLUSIONS: Caffeine does not worsen RIAKI. The widely used performance-enhancing drug ibuprofen greatly worsens RIAKI in mice. Standard or experimental treatment of RIAKI including the addition of cilastatin to standard resuscitation is ineffective in mice with RIAKI exacerbated by ibuprofen. These findings may have clinical implications for the current therapy of RIAKI and for translational studies of novel treatment.

Cilastatin Ameliorates Rhabdomyolysis-induced AKI in Mice.

BACKGROUND: Rhabdomyolysis, the destruction of skeletal muscle, is a significant cause of AKI and death in the context of natural disaster and armed conflict. Rhabdomyolysis may also initiate CKD. Development of specific pharmacologic therapy is desirable because supportive care is nearly impossible in austere environments. Myoglobin, the principal cause of rhabdomyolysis-related AKI, undergoes megalin-mediated endocytosis in proximal tubule cells, a process that specifically injures these cells. METHODS: To investigate whether megalin is protective in a mouse model of rhabdomyolysis-induced AKI, we used male C57BL/6 mice and mice (14-32 weeks old) with proximal tubule-specific deletion of megalin. We used a well-characterized rhabdomyolysis model, injection of 50% glycerol in normal saline preceded by water deprivation. RESULTS: Inducible proximal tubule-specific deletion of megalin was highly protective in this mouse model of rhabdomyolysis-induced AKI. The megalin knockout mice demonstrated preserved GFR, reduced proximal tubule injury (as indicated by kidney injury molecule-1), and reduced renal apoptosis 24 hours after injury. These effects were accompanied by increased urinary myoglobin clearance. Unlike littermate controls, the megalin-deficient mice also did not develop progressive GFR decline and persistent new proteinuria. Administration of the pharmacologic megalin inhibitor cilastatin to wild-type mice recapitulated the renoprotective effects of megalin deletion. This cilastatin-mediated renoprotective effect was dependent on megalin. Cilastatin administration caused selective proteinuria and inhibition of tubular myoglobin uptake similar to that caused by megalin deletion. CONCLUSIONS: We conclude that megalin plays a critical role in rhabdomyolysis-induced AKI, and megalin interference and inhibition ameliorate rhabdomyolysis-induced AKI. Further investigation of megalin inhibition may inform translational investigation of a novel potential therapy.

Dopamine Evokes a Trace Amine Receptor-dependent Inward Current that is Regulated by AMP Kinase in Substantia Nigra Dopamine Neurons.

We reported recently that activators of AMP-activated protein kinase (AMPK) slow the rundown of current evoked by the D2 autoreceptor agonist quinpirole in rat substantia nigra compacta (SNC) dopamine neurons. The present study examined the effect of AMPK on current generated by dopamine, which unlike quinpirole, is a substrate for the dopamine transporter (DAT). Using whole-cell patch-clamp, we constructed current-voltage (I-V) plots while superfusing brain slices with dopamine (100 µM) for 25 min. Two minutes after starting superfusion, dopamine evoked a peak current with an average slope conductance of 0.97 nS and an estimated reversal potential (Erev) of -113 mV, which is near that expected for K+. But after 10 min of superfusion, dopamine-evoked currents had shifted to more depolarized values with a slope conductance of 0.64 nS and an Erev of -83 mV. This inward shift in current was completely blocked by the DAT inhibitor GBR12935. However, an AMPK blocking agent (dorsomorphin) permitted the emergence of inward current despite the continued presence of the DAT inhibitor. When D2 autoreceptors were blocked by sulpiride, I-V plots showed that dopamine evoked an inward current with an estimated slope conductance of 0.45 nS with an Erev of -57 mV. Moreover, this inward current was completely blocked by the trace amine-associated receptor 1 (TAAR1) antagonist EPPTB. These results suggest that dopamine activates a TAAR1-dependent non-selective cation current that is regulated by AMPK.

AMP-activated protein kinase slows D2 dopamine autoreceptor desensitization in substantia nigra neurons.

Dopamine neurons in the substantia nigra zona compacta (SNC) are well known to express D2 receptors. When dopamine is released from somatodendritic sites, activation of D2 autoreceptors suppresses dopamine neuronal activity through activation of G protein-coupled K+ channels. AMP-activated protein kinase (AMPK) is a master enzyme that acts in somatic tissues to suppress energy expenditure and encourage energy production. We hypothesize that AMPK may also conserve energy in central neurons by reducing desensitization of D2 autoreceptors. We used whole-cell patch-clamp recordings to study the effects of AMPK activators and inhibitors on D2 autoreceptor-mediated current in SNC neurons in midbrain slices from rat pups (11-23 days post-natal). Slices were superfused with 100 µM dopamine or 30 µM quinpirole for 25 min, which evoked outward currents that decayed slowly over time. Although the AMPK activators A769662 and ZLN024 significantly slowed rundown of dopamine-evoked current, slowing of quinpirole-evoked current required the presence of a D1-like agonist (SKF38393). Moreover, the D1-like agonist also slowed the rundown of quinpirole-induced current even in the absence of an AMPK activator. Pharmacological antagonist experiments showed that the D1-like agonist effect required activation of either protein kinase A (PKA) or exchange protein directly activated by cAMP 2 (Epac2) pathways. In contrast, the effect of AMPK on rundown of current evoked by quinpirole plus SKF38393 required PKA but not Epac2. We conclude that AMPK slows D2 autoreceptor desensitization by augmenting the effect of D1-like receptors.

Phosphoinositol metabolism affects AMP kinase-dependent K-ATP currents in rat substantia nigra dopamine neurons.

We reported recently that ligand-gated ATP-sensitive K+ (K-ATP) current is potentiated by AMP-activated protein kinase (AMPK) in rat substantia nigra compacta (SNC) dopamine neurons. Because phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) regulates K-ATP current, we explored the hypothesis that changes in PI(4,5)P2 modify the ability of AMPK to augment K-ATP current. To influence PI(4,5)P2 levels, we superfused brain slices with phospholipase C (PLC) activators and inhibitors while recording whole-cell currents in SNC dopamine neurons. Diazoxide, superfused for 5 min every 20 min, evoked K-ATP currents that, on average, increased from 38 pA at first application to 122 pA at the fourth application, a 220% increase. This enhancement of diazoxide-induced current was AMPK dependent because K-ATP current remained at baseline when slices were superfused with either the AMPK inhibitor dorsomorphin or the upstream kinase inhibitor STO-609. The PLC inhibitor U73122 significantly increased diazoxide current over control values, and this increase was blocked by dorsomorphin. Enhancement of diazoxide-induced current was also completely prevented by the PLC activator m-3M3FBS. Agonists at 5-HT2C and group I metabotropic glutamate receptors, both of which activate PLC, also prevented augmentation of diazoxide-induced current. Finally, inhibition of spike discharges by diazoxide was significantly antagonized by m-3M3FBS. These results suggest that PLC activity significantly influences the inhibitory effect of K-ATP channels by altering PI(4,5)P2 content. Results also suggest that modification of K-ATP current by PLC requires AMPK activity.

AMP kinase regulates ligand-gated K-ATP channels in substantia nigra dopamine neurons.

AMP-activated protein kinase (AMPK) is a master enzyme that regulates ATP-sensitive K(+) (K-ATP) channels in pancreatic beta-cells and cardiac myocytes. We used patch pipettes to record currents and potentials to investigate effects of AMPK on K-ATP currents in substantia nigra compacta (SNC) dopamine neurons in slices of rat midbrain. When slices were superfused repeatedly with the K-ATP channel opener diazoxide, we were surprised to find that diazoxide currents gradually increased in magnitude, reaching 300% of the control value 60min after starting whole-cell recording. However, diazoxide current increased significantly more, to 472% of control, when recorded in the presence of the AMPK activator A769662. Moreover, superfusing the slice with the AMPK blocking agent dorsomorphin significantly reduced diazoxide current to 38% of control. Control experiments showed that outward currents evoked by the K-ATP channel opener NN-414 also increased over time, but not currents evoked by the GABAB agonist baclofen. Delaying the application of diazoxide after starting whole-cell recording correlated with augmentation of current. Loose-patch recording showed that diazoxide produced a 34% slowing of spontaneous firing rate that did not intensify with repeated applications of diazoxide. However, superfusion with A769662 significantly augmented the inhibitory effect of diazoxide on firing rate. We conclude that K-ATP channel function is augmented by AMPK, which is activated during the process of making whole-cell recordings. Our results suggest that AMPK and K-ATP interactions may play an important role in regulating dopamine neuronal excitability.

NMDA alters rotenone toxicity in rat substantia nigra zona compacta and ventral tegmental area dopamine neurons.

Previous patch-clamp studies by our laboratory showed that acute exposure to the pesticide rotenone augments inward currents evoked by N-methyl-d-aspartate (NMDA) in substantia nigra zona compacta (SNC) dopamine neurons in slices of rat brain. The present experiments were done to search for histological evidence of increased neurotoxicity produced by combined rotenone and NMDA treatments. In horizontal slices of rat midbrain, we found that a 30 min superfusion with 100 nM rotenone caused significant injury to tyrosine hydroxylase (TH)-positive proximal dendrites in dorsal and ventral regions of the SNC and ventral tegmental area (VTA). Moreover, treatment with 100 µM NMDA potentiated rotenone toxicity. In contrast, treatment with 30 µM NMDA protected against rotenone-induced injury to dendrites in the ventral SNC and ventral VTA. Interestingly, treatment with 30 µM NMDA-alone produced an apparent increase in proximal dendrite scores in ventral SNC and dorsal VTA. We conclude that NMDA has concentration-dependent actions on rotenone toxicity that differ according to regional subtype of dopamine neuron.

Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons.

Mild uncoupling of oxidative phosphorylation has been reported to reduce generation of reactive oxygen species (ROS) and therefore may be neuroprotective. We reported previously that the mitochondrial poison rotenone enhanced currents evoked by N-methyl-D-aspartate (NMDA) by a ROS-dependent mechanism in rat midbrain dopamine neurons. Thus, rotenone, which produces a model of Parkinson's disease in rodents, may increase the risk of dopamine neuron excitotoxicity. The purpose of this study was to test the hypothesis that oxidative phosphorylation uncoupling agents would antagonize the effect of rotenone on NMDA current. We used patch pipettes to record whole-cell currents under voltage-clamp (-60 mV) in substantia nigra dopamine neurons in slices of rat brain. Rotenone, NMDA and uncoupling agents were added to the brain slice superfusate. Inward currents evoked by NMDA (30 µM) more than doubled in amplitude after slices were superfused for 30 min with 100 nM rotenone. Continuous superfusion with the uncoupling agent carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (1-3 nM) or 2,4-dinitrophenol (100 nM) significantly antagonized and delayed the ability of rotenone to potentiate NMDA currents. Coenzyme Q10 (1-10 nM), which has been reported to facilitate uncoupling protein activity, also antagonized this action of rotenone. These results suggest that mild uncoupling of oxidative phosphorylation may protect dopamine neurons against injury from mitochondrial poisons such as rotenone.

Evaluation of levodopa dose and magnitude of dopamine depletion as risk factors for levodopa-induced dyskinesia in a rat model of Parkinson's disease.

Levodopa dose and severity of Parkinson's disease (PD) are recognized risk factors for levodopa-induced dyskinesia (LID) in humans. The purpose of the present study was to evaluate the ability of these variables to predict severity of LID in a rat model of PD. Varied concentrations of 6-hydroxy-dopamine were injected into the midbrain to produce wide ranges of dopamine depletion in striatum. Three weeks later, rats were given daily injections of levodopa (2-10 mg/kg i.p.) plus benserazide (12.5 mg/kg i.p.) for 15 days. Abnormal involuntary movements (AIMs) were measured for limb, axial, orolingual, and rotatory movements. Dose-response analysis for total AIM scores yielded a levodopa ED50 value of 3.2 mg/kg on treatment day 15. There were strong interrelated correlations between individual AIM categories (rho > 0.7) and for each AIM category in regard to total AIM score (rho > 0.7). In rats that received levodopa doses that were greater than the ED50, rates of amphetamine-induced rotation were significantly correlated with total AIM scores (rho = 0.413). However, of those rotating >5 times/min, 34% had relatively low AIM scores (<8). Likewise, there was a significant correlation between percentages of tyrosine hydroxylase (TH) loss and total AIM scores (rho = 0.388). However, in those rats that had >85% TH loss, 30% had AIM scores <8. Our results show that given an adequate dose and magnitude of striatal dopamine depletion, levodopa produces dyskinesia with a continuous spectrum of severity. Although levodopa dose and level of dopamine depletion are significant risk factors for LID, we conclude that other factors must contribute to LID susceptibility.

Tyramine excites rat subthalamic neurons in vitro by a dopamine-dependent mechanism.

Tyramine, an endogenous ligand for mammalian trace amine-associated receptors, may act as a neuromodulator that regulates neuronal activity in basal ganglia. Using whole-cell patch recordings of subthalamic nucleus (STN) neurons in rat brain slices, we found that bath application of tyramine evoked an inward current in voltage-clamp in over 60% of all STN neurons. The inward current induced by tyramine was mimicked by the D(2)-like dopamine receptor agonist quinpirole, but was only partially blocked by the D(2)-like receptor antagonist sulpiride. In contrast, the D(1)-like receptor agonist SKF38393 evoked no current in STN neurons. Inward current evoked by tyramine was significantly reduced by the catecholamine uptake inhibitor nomifensine, and by exhausting catecholamines in the brain via pretreatment with reserpine. Tyramine also reduced the amplitude of GABA(A) receptor-mediated IPSCs that were evoked by focal electrical stimulation of the slice. Inhibition of IPSCs by tyramine was mimicked by quinpirole and was blocked by sulpiride but not by SCH23390, a D(1) receptor antagonist. Moreover, tyramine-induced inhibition of IPSCs was reduced in slices pretreated with reserpine, and this inhibition could be restored by briefly superfusing the slice with dopamine. These results suggest that tyramine acts as an indirect dopamine agonist in the STN. Although inhibition of IPSCs is mediated by D(2)-like receptors, the dopamine-dependent inward currents evoked by tyramine do not fit a typical dopamine receptor pharmacological profile.

Dopamine-mediated actions of ephedrine in the rat substantia nigra.

Although ephedrine is a centrally active stimulant, its effect on midbrain dopamine neurons is not known. To study the effect of ephedrine on dopamine-containing cells, current-clamp microelectrode recordings were made from substantia nigra pars compacta (SNC) neurons in horizontal brain slice preparations. Ephedrine (100-1000 microM) slowed spontaneous firing and produced a modest concentration-dependent hyperpolarization of membrane potential (EC50 279 microM), with a concomitant net decrease in membrane resistance. These effects were blocked by the D2-like dopamine antagonist sulpiride (1 microM). Electrically evoked inhibitory synaptic potentials mediated by GABAB receptors were reduced 28% by ephedrine. However, ephedrine did not reduce fast synaptic potentials mediated by GABAA or ionotropic glutamate receptors. Inhibition of the GABAB response appeared to be mediated by a postsynaptic mechanism because ephedrine also reduced baclofen-induced hyperpolarization by 28%. Both ephedrine-induced hyperpolarization and inhibition of baclofen-induced hyperpolarization were abolished when slices were superfused with the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT). Despite perfusion with AMPT, the ability of ephedrine to cause hyperpolarization was restored after perfusing the slice with dopamine (30 microM). Taken together, these results suggest that ephedrine causes hyperpolarization and suppresses GABAB receptor-mediated effects by releasing endogenous dopamine. However, the high concentrations required to observe these effects in vitro suggest that biologically relevant central effects of ephedrine are more likely to be mediated either by non-dopamine systems, such as those involving noradrenaline, or by dopamine systems outside the SNC.