Measurement of intracellular generation of hydrogen peroxide by rat glomeruli in vitro

Sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water [1, 2]. These partially reduced oxygen intermediates have been implicated as important mediators in various models of ischemic, toxic and immune-mediated tissue injury including glomerular injury [3]. Reactive oxygen metabolites have been shown to affect several biological processes potentially important in glomerular diseases, and their role in both inflammatory as well as non-inflammatory glomerular diseases has recently been demonstrated [3]. Evidence for the importance of reactive oxygen metabolites in experimental models of glomerular disease is based largely on the protective effects of scavengers of reactive oxygen metabolites

Optimizing epilepsy treatment in children, adults and elderly: clinical perspectives

Epilepsy is a common neurological condition among all age groups. Its diagnosis depends on clinical history and is supported by investigations like electroencephalograph and magnetic resonance imaging. In children, febrile seizures and juvenile myoclonic seizures are common. In adults and elderly, epilepsy could be new onset or present since childhood. Several antiseizure medications (ASMs) are available for treatment of epilepsy. Each of these drugs has different advantages as well as limitations. Several factors including patient related factors (e. g. age, comorbidities, and gender), seizure characteristics and epilepsy type, and drug related factors (namely, pharmacokinetics, pharmacodynamics, efficacy, side effects, and risk of drug-drug interactions) are considered while selecting drug therapy. There are concerns related to teratogenicity due to pharmacotherapy in females of child-bearing potential. Elderly patients also need special considerations while selecting an appropriate treatment option for epilepsy. Advisory board meetings with a group of expert neurologists were conducted at eight cities across India to gain clinical insights on management of epilepsy in different age groups. Consensus was supported by relevant literature obtained from PubMed and Google scholar using the keywords ‘epilepsy’, ‘diagnosis’, ‘children’, ‘adults’, ‘elderly’, and ‘management’. According to the experts, choice of appropriate ASM is driven by patient- and drug-related factors and seizure characteristics. In India, valproate remains a commonly prescribed agent except in females of childbearing age. Levetiracetam is preferred because of its safety profile whereas clobazam is an effective initial add-on therapy. Newer drugs are largely useful as adjuvants

Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats1

Meprin, a brush-border enzyme, plays an important role in hypoxic/ischemic acute renal tubular injury in rats.BackgroundIt has been shown that non-congenic mice strains with lower levels of renal meprin develop less renal injury following renal ischemia and reperfusion. We have demonstrated that following ischemia-reperfusion renal injury, there is a rapid shift of meprin localization and intensity from the brush border to the cytoplasmic compartment, tubular lumens and the tubular basement membranes. Radical shifts in the localization of an activated enzyme to potentially sensitive areas of the tubule suggest a toxic role for meprin in ischemia-reperfusion injury. Though meprin degrades extracellular matrix components and other substrates, to our knowledge meprin cytotoxicity has never been examined. Therefore, the first objective of this study was to determine if meprin is directly cytotoxic to renal cells in vitro. The second objective was to determine if inhibition of meprin is protective against hypoxia-reoxygenation injury in vitro and ischemia-reperfusion injury in vivo.MethodsThe immortalized porcine epithelial cell line (LLC-PK1) and Madin-Darby canine kidney (MDCK) cells in culture were exposed to meprin in various concentrations and for various times. Cell death was determined by Trypan Blue exclusion, lactate dehydrogenase (LDH) release and the 3-4, 5 dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay. Renal slices were used to examine the effect of the meprin inhibitor, actinonin, on hypoxic injury in vitro. Male Sprague-Dawley rats were used in ischemia-reperfusion injury studies to determine the effect of actinonin on renal function as measured by plasma urea nitrogen, creatinine and renal histology.ResultsMeprin is cytotoxic to LLC-PK1 and MDCK cells in a concentration and time dependent manner. The meprin inhibitor 1,10-phenanthroline completely abolished the cytotoxic effect. Renal slices exposed to hypoxia and hypoxia followed by reoxygenation showed marked cell death. Pre-treatment with the actinonin was markedly protective while not interfering with the hypoxia-induced fall in adenosine 5′-triphosphate (ATP) levels. In in vivo studies, rats exposed to ischemia/reperfusion injury were markedly protected against acute renal failure by IP treatment with actinonin.ConclusionsMeprin is cytotoxic to cultured renal tubular epithelial cells in vitro. Renal slices are protected from hypoxia-reoxygenation injury in vitro by the meprin inhibitor actinonin. Meprin inhibition is protective against rat renal hypoxia-reoxygenation injury. These data strongly support the concept that meprin is cytotoxic and may play a key role in renal ischemia-reperfusion induced renal injury

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity.BackgroundIron plays a role in free radical-mediated tissue injury, including cisplatin-induced nephrotoxicity. However, the source of iron (catalyzing free radical reactions) is not known. We examined the role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity both in vivo and in vitro.MethodsCisplatin-induced acute renal failure was produced in rats by intraperitoneal injection of cisplatin (10mg/kg body wt). Piperonyl butoxide, a cytochrome P-450 inhibitor, was administered intraperitoneally (400mg/kg body wt twice at 48-hr intervals) prior to cisplatin injection. The effects of cisplatin in the absence or presence of piperonyl butoxide on the belomycin-detectable iron, cytochrome P-450 content in the kidney, and renal functional and histological changes were evaluated. In an in vitro study, the effect of cytochrome P-450 inhibitors, cimetidine or piperonyl butoxide, on cisplatin-induced cytotoxicity and catalytic iron release from LLC-PK1 cells was examined.ResultsIn cisplatin-treated rats, there was a marked decrease in the cytochrome P-450 content specifically in the kidney, accompanied by increased bleomycin-detectable iron content in the kidney. Piperonyl butoxide prevented cisplatin-induced loss of cytochrome P-450 as well as the increase of bleomycin-detectable iron in the kidney, along with both functional and histological protection. Both cimetidine and piperonyl butoxide prevented cisplatin-induced increase in bleomycin-detectable iron and cytotoxicity in LLC-PK1 cells. Treatment of cimetidine did not affect cellular uptake of cisplatin.ConclusionCytochrome P-450, a group of heme proteins, may serve as a significant source of catalytic iron in cisplatin-induced nephrotoxicity

In vitro and in vivo evidence suggesting a role for iron in cisplatin-induced nephrotoxicity

In vitro and in vivo evidence suggesting a role for iron in cisplatin-induced nephrotoxicity. Cisplatin is a widely used antineoplastic agent that has nephrotoxicity as a major side effect. The underlying mechanism of this nephrotoxicity is still not well known. Iron has been implicated to play an important role in several models of tissue injury, presumably through the generation of hydroxyl radicals via the Haber-Weiss reaction or other highly toxic free radicals. In the present study we examined the catalytic iron content and the effect of iron chelators in an in vitro model of cisplatin-induced cytotoxicity in LLC-PK1 cells (renal tubular epithelial cells) and in an in vivo model of cisplatin-induced acute renal failure in rats. Exposure of LLC-PK1 cells to cisplatin resulted in a significant increase in bleomycin-detectable iron (iron capable of catalyzing free radical reactions) released into the medium. Concurrent incubation of LLC-PK1 cells with iron chelators including deferoxamine and 1,10-phenanthroline significantly attenuated cisplatin-induced cytotoxicity as measured by lactate dehydrogenase (LDH) release. Bleomycin-detectable iron content was also markedly increased in the kidney of rats treated with cisplatin. Similarly, administration of deferoxamine in rats provided marked functional (as measured by blood urea nitrogen and creatinine) and histological protection against cisplatin-induced acute renal failure. In a separate study, we examined the role of hydroxyl radical in cisplatin-induced nephrotoxicity. Incubation of LLC-PK1 cells with cisplatin caused an increase in hydroxyl radical formation. Hydroxyl radical scavengers, dimethyl sulfoxide, mannitol and benzoic acid, significantly reduced cisplatin-induced cytotoxicity and, treatment with dimethyl sulfoxide or dimethylthiourea provided significant protection against cisplatin-induced acute renal failure. Taken together, our data strongly support a critical role for iron in mediating tissue injury via hydroxyl radical (or a similar oxidant) in this model of nephrotoxicity

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2. Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration ([Ca2+]i) in cultured LLC-PK1, cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10-7 M) increased [Ca2+]i from 148 ± 16 to 288 ± 39 nM and from 130 ± 8 to 240 ± 18 nM, with the values of EC50 for PAF and ONO11113 being 17 ± 4 and 17 ± 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO1113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(β-aminomethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). In the absence of extracellular Ca2+ 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (which has extensively been used to study the contribution of Ca2+ released from intracellular storage sites in the increase in [Ca2+]i), blocked the increment in [Ca2+]i induced by PAF or ONO1113. These results indicate that LLC-PKi cells express discrete receptors for PAF and TXA2 that are coupled to an increase in [Ca2+]i through mobilization of calcium from both intracellular storage sites and extracellular milieu, and suggest the possible importance of PAF and TXA2 in tubular pathophysiology

Action of serotonin (5-hydroxytryptamine) on cyclic nucleotides in glomeruli of rat renal cortex

Action of serotonin (5-hydroxytryptamine) on cyclic nucleotides in glomeruli of rat renal cortex. Serotonin (5-hydroxytryptamine) is known to influence glomerular function and may have an important role in the pathogenesis of glomerulopathies. Because serotonin acts in nonrenal tissues through mediation of cyclic nucleotides, we investigated in vitro its effect on cAMP and cyclic guanosine monophosphate (cGMP) in tissue slices and isolated glomeruli from rat kidney. Serotonin increased cAMP 161 ± 35% but not cGMP in renal cortex; it had no effect on cyclic nucleotides in medulla and papilla. In isolated glomeruli, serotonin elicited a dose-dependent (in the range of 10−7 to 10−4M) increase in cAMP; the maximum increase over basal values was 376 ± 45%. Serotonin increased cAMP either in the presence or in the absence of a cAMP phosphodiesterase inhibitor. In tubular fraction, serotonin elevated cAMP to a much lesser degree (82 ± 15%). Neither in glomeruli nor in tubules did cGMP concentrations change in response to serotonin, but carbamylcholine, a known cGMP agonist, significantly increased cGMP concentrations. The increase in cAMP in response to serotonin was blocked (>85% inhibition) by equimolar concentrations of serotonin antagonists methysergide and cinanserine. Results of this study demonstrate that interaction of serotonin with receptors in the kidney, particularly in the glomeruli, cause a striking increase in cAMP concentrations without detectable changes in cGMP concentrations. These findings suggest that serotonin, either synthesized in the kidney or released locally from platelets aggregated in glomeruli (for example, in association with immu-nopathologic injury) may exert or modulate its physiologic or pathologic effects via mediation of cAMP.Action de la sérotonine (5-hydroxytryptamine) sur les nucléotides cycliques des glomérules du cortex rénal du rat. Il est connu que la sérotonine (5-hydroxytryptamine) influence la fonction glomérulaire et peut avoir un rôle important dans la pathogénie des glomérulopathies. Puisque la sérotonine agit sur d'autres tissus que le rein par l'intermédiaire des nucléotides cycliques, nous avons étudié son effet in vitro sur le contenu en cAMP et cGMP des tranches de rein et des glomérules isolés du rein de rat. La sérotonine augmente cAMP 161 ± 35%, mais pas cGMP du cortex rénal. Elle n'a pas d'effet sur les nucléotides cycliques de la médullaire et de la papille. Dans les glomérules isolés la sérontonine détermine une augmentation de cAMP dose dépendante (de 10−7 à 10−4M), l'augmentation maximale est de 376 ± 45% par rapport à la valeur basale. La sérotonine augmente cAMP aussi bien en présence qu'en l'absence d'inhibiteur de la cAMP phosphodiestérase. Dans les fractions tabulaires la sérotonine n'augmente cAMP qu'à un moindre degré (82 ± 15%). La sérotonine n'augmente cGMP ni dans les glomérules ni dans les tubules mais la carbamylcholine, un agoniste connu de cGMP augmente celui-ci significativement. L'augmentation de cAMP en réponse à la sérotonine est bloquée (inhibition supérieure à 85%) par des concentrations équimolaires des antagonistes méthysergide et cinansérine. Les résultats de ce travail démontrent que l'interaction de la sérotonine avec les récepteurs rénaux, particulièrement glomérulaires, détermine une augmentation importante de cAMP sans modification détéctable de cGMP. Ces constatations suggèrent que la sérotonine, qu'elle soit synthétisée dans le rein ou libérée localement par des aggrégats plaquettaires formés dans les glomérules (c'est à dire en association avec une lésion immunopathologique), peut exercer ou moduler ses effets physiologiques ou pathologiques par l'intermédiaire de cAMP

NADPH-induced chemiluminescence and lipid peroxidation in kidney microsomes

NADPH-induced chemiluminescence and lipid peroxidation in kidney microsomes. Lipid peroxidation and reactive oxygen species have been shown to affect diverse biological processes potentially important in renal disease. We therefore examined NADPH-induced chemiluminescence (CL) and lipid peroxidation (LP) by renal cortical and, in some experiments, medullary microsomes. We further examined the role of reactive oxygen species in these processes by the use of enzymatic and chemical scavengers. Cortical microsomes gave a marked NADPH-induced CL accompanied by LP. The time course of LP closely paralleled the CL response. Cortical microsomal CL and LP increased with increasing concentrations of protein (0.3 to 1.8 mg) and NADPH (0.1 to 3.0mM); NADH could not substitute for NADPH. Using similar amounts of protein and NADPH concentrations, cortical CL was significantly higher than medullary CL at all time points examined (peak cortical CL: 490 ± 25 × 103 cpm/mg protein, N = 4; peak medullary CL: 226 ± 61 × 103 cpm/mg protein, N = 4). Cortical LP was similarly higher at all time points, values corresponding to peak CL being 44.7 ± 3 nmoles/mg protein for cortex and 29.9 ± 0.8 nmoles/mg protein for medulla. Para-chloromercuribenzoate (PCMB), an inhibitor of NADPH-cytochrome P450 reductase, caused a marked inhibition of the microsomal CL and LP whereas SKF 525A, an inhibitor of cytochrome P450, had a relatively minor effect. Marked inhibition of NADPH induced CL and LP was observed with chelators EDTA and 1,10-phenanthroline. The addition of NADPH to microsomes prepared with sucrose-EDTA and washed in a Chelex-treated buffer gave a negligible CL and LP response; the responses were restored by the addition of iron. Scavengers of superoxide anion (superoxide dismutase), hydrogen peroxide (catalase), and the hydroxyl radical (sodium benzoate, tryptophan) had a relatively minor effect on CL and LP. Singlet oxygen quenchers sodium azide and 1,4-diazabicyclo (2.2.2) octane (DABCO) caused an incomplete inhibition of CL and LP responses and then only after the first 30min. Taken together, these data suggest that other free radical mechanisms are likely to be important in the CL and LP responses. These results which demonstrate the production of excited states and LP by renal microsomes suggest that their role in renal disease deserves further study.Chemiluminescence induite par le NADPH et peroxydation des lipides dans les microsomes de rein. Il a été montré que la peroxydation des lipides et le type d'oxygène réactif modifient diversement les processus biologiques potentiellement importants dans les maladies rénales. C'est pourquoi nous avons examiné la chemiluminescence (CL) induite par le NADPH, et la peroxydation (LP) des lipides par des microsomes corticaux et dans quelques expériences, médullaires rénaux. Nous avons ensuite examiné le rôle du type d'oxygène réactif dans ces processus en utilisant des agents métabolisants enzymatiques et chimiques. Les microsomes corticaux donnaient une CL marquée induite par le NADPH accompagnée par une LP. Dans le temps, la LP était étroitement parallèle à la réponse CL. La CL et la LP microsomiales corticales s'élevaient avec l'accroissement des concentrations de protéines (0, 1 à 1,8 mg) et de NADPH (0,1 à 3,0mM); le NADH ne pouvait so substituer au NADPH. En utilisant des quantités identiques de protéines et de NADPH, la CL corticale était significativement plus élevée que la CL médullaire à tous les temps examinés (pic de CL corticale: 490 ± 25 × 103 cpm/mg protéine, N = 4, pic de CL médullaire: 226 ± 61 × 103 cpm/mg protéine, N = 4). De même la LP corticale était plus élevée à tous les temps, les valeurs correspondant au pic de CL étant de 44,7 ± 3 nmoles/mg protéine pour le cortex et de 29,9 ± 0,8 nmoles/mg protéine pour la médullaire. Le parachloromercuribenzoate (PCMB), un inhibiteur de la réductase NADPH-cytochrome P450a entraîné une inhibition marquée de la CL et de la LP microsomiales tandis que le SKF 525 A, un inhibiteur du cytochrome P450, avait un effet relativement minime. Une inhibition marquée de la CL induite par le NADPH et de la LP a été observée avec des chélateurs tels l'EDTA et le 1,10-phénanthroline. L'addition de NADPH à des microsomes préparés dans du sucrose-EDTA et lavés dans un tampon traité au Chelex entraînait une réponse CL et LP négligeable; les réponses étaient restaurées par l'addition de fer. Les agents métabolisants l'anion superoxide (dismutase superoxide), le peroxide d'hydrogène (catalase), et le radical hydroxylé (benzoate de sodium, tryptophane) avaient un effet relativement minime sur CL et LP. Les capteurs d'oxygène singulet comme l'azide de sodium et le 1,4-diazabicyclo (2.2.2) octane (DABCO) entraînaient une inhibition incomplète des réponses CL et LP et cela seulement après les 30 premières min. Prises dans leur ensemble, ces données suggèrent que d'autres mécanismes par radicaux libres sont probablement importants dans les réponses CL et LP. Ces résultats, qui démontrent la production d'états excités et de LP par des microsomes rénaux, suggèrent que leur rôle dans les maladies rénales nécessite d'autres études