Modulation by cyclic AMP and phorbol myristate acetate of cephaloridine-induced injury in rat renal cortical slices.

Intracellular signaling pathways of cAMP and protein kinase C (PKC) have been suggested to modulate the generation of free radicals. We investigated the effects of cAMP and phorbol myristate acetate (PMA), a PKC activator, on cephaloridine (CER)-induced renal cell injury, which has been reported to be due to the generation of free radicals. Incubation of rat renal cortical slices with CER resulted in increases in lipid peroxidation and lactate dehydrogenase (LDH) release and in decreases in gluconeogenesis and p-aminohippurate (PAH) accumulation in rat renal cortical slices, suggesting free radical-induced injury in slices exposed to CER. A derivative of cAMP ameliorated not only the increase in lipid peroxidation but also the renal cell damage induced by CER. This amelioration by a cAMP derivative of lipid peroxidation and renal cell damage caused by CER was blocked by KT 5720, a protein kinase A (PKA) inhibitor. Lipid peroxidation and the indices of cell injury were increased by PMA. PMA also enhanced CER-induced lipid peroxidation and cell damage in the slices. This enhancement by PMA of CER-induced injury was blocked by H-7, a PKC inhibitor. These results indicated that intracellular signaling pathways of cAMP and PKC modulate free radical-mediated nephrotoxicity induced by CER.

Role of prostaglandin E2 and leukotriene B4 in skin reaction induced by transdermal application of propranolol.

Dermal application of propranolol (PRL) induced formation of erythema and edema, and pseudoeosinophil infiltration in epidermis and dermis at the application site in guinea pigs. We investigated the production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) at the application site of PRL and the role of these inflammatory chemical mediators in the occurrence of the skin reactions. PGE2 was found to be produced at the application site slightly after the accumulation of PRL released from the adhesive bandage in the patch test, and the amount of PGE2 increased continuously, with a peak value obtained at 24 h after application. The time-course changes resembled those of delta a* value, the index of erythema formation determined by colorimetric measurement, and edema formation. The production of PGE2 by dermal application of PRL was suppressed by local pretreatment with dexamethasone or indomethacin. However, no notable production of LTB4 was observed at the application site of PRL.

Ameliorative effect of adenosine on hypoxia-reoxygenation injury in LLC-PK1, a porcine kidney cell line.

We studied the effects of adenosine on injury caused by hypoxia and reoxygenation in LLC-PK1 cells. Lactate dehydrogenase and gamma-glutamyltranspeptidase were released from cells exposed to hypoxia for 6 hr and then reoxygenation for 1 hr. The addition of adenosine at 100 microM to the medium before hypoxia began significantly decreased enzyme leakage into medium during both hypoxia and reoxygenation. The adenosine A1-receptor agonist, R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), at the concentration of 100 microM, did not affect enzyme release, but the adenosine A2-receptor agonist 2-p-[2-car-boxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosi ne hydrochloride (CGS 21680) at the concentration of 100 nM, suppressed the injury caused by hypoxia and reoxygenation. There were decreases in cAMP contents and ATP levels in LLC-PK1 cells injured by hypoxia and reoxygenation. Adenosine (100 microM) restored ATP levels in the cells during reoxygenation. With adenosine, the intracellular cAMP level was increased prominently during reoxygenation. These results suggest that adenosine protects LLC-PK1 cells from injury caused by hypoxia and reoxygenation by increasing the intracellular cAMP level via adenosine A2 receptor.

Skin toxicity of propranolol in guinea pigs.

The skin toxicities of propranolol were studied in guinea pigs. In the primary and cumulative skin irritation studies, the skin reactions and the histopathological changes were observed in all animals treated with propranolol, and those tended to increase with the increase of propranolol dosage. The skin reactions increased with the application times of propranolol up to 7 days in the cumulative skin irritation study. In the skin sensitization, the phototoxicity and the skin photosensitization studies, no skin reactions were observed in any animals used in the studies. These results indicate that propranolol caused skin irritation, but was negative for skin sensitization, phototoxicity and skin photosensitization in guinea pigs.

Protective effects of some flavonoids on the renal cellular membrane.

By assaying lactate dehydrogenase and malondialdehyde leakage from LLC-PK1 cells in culture, a study was conducted to clarify whether flavonoid compounds ameliorate renal cellular injury. The cells were cultured with various concentrations of samples under routine conditions. The results demonstrated that baicalin, cirsimaritin, 6-hydroxyluteolin, luteolin, plantaginin, rhoifolin, sorbarin, afzelin, hyperin, isoquercitrin, isorhamnetin, kaempferitrin, kaempferol-7-glucoside, oxyayanin A, quercetin, quercitrin, rhamnetin and rutin exerted marked protective effects on the cells, whereas acacetin, apigenin, apiin, cirsilineol, genkwanin, pectolinarin and tetramethylquercitin had virtually no effect. In the light of these findings, we propose that the general capability of these compounds is largely decided by the number and position of phenolic hydroxyl groups linked to the structural backbone.

Relationship between the skin permeation movement of propranolol and skin inflammatory reactions.

We studied inflammatory reactions induced by dermal application of the beta-blocker propranolol (PRL) in ethanol to guinea pigs in order to elucidate the relation of the reactions with the cumulative PRL permeating amount through the stratum corneum or the PRL content in the stripped skin, and to investigate the chemical mediators responsible for the reactions. The cumulative PRL permeating amount through the stratum corneum increased rapidly up to 2 h after dermal application, then increased linearly with time up to 24 h after application. Visual observation revealed formation of erythema and edema at the applied site of PRL, and histopathological examination revealed infiltration of pseudoeosinophiles of dermis and epidermis and degeneration/necrosis of epidermis. In general, it was considered that the duration and the extent of these reactions were dependent on the PRL dosage and application time. It was expected that the cumulative PRL permeating amount through the stratum corneum could be used to predict possible inflammatory reactions during development of transdermal drug delivery systems. On the other hand, contact of PRL with guinea pig skin tissues released histamine, and intradermal injection of PRL caused an increase of capillary permeability at the site of application. Also, the inhibitory effects of anti-inflammatory agents (diphenhydramine, dexamethasone, indomethacin, cyproheptadine hydrochloride, CV3988 and AA-861) to PRL-induced erythema formation demonstrated that histamine and prostaglandins were responsible for the inflammatory reactions induced by PRL.

Comparative toxic effects of iobitridol and iohexol on the kidney.

RATIONALE AND OBJECTIVES: The authors compare the toxic effects of iobitridol and iohexol, which are nonionic contrast media with equivalent osmolalities and viscosities on the kidney. METHODS: In a rat acute renal failure (ARF) model, iobitridol or iohexol (both at the dose of 2.87 g I/kg) were injected to rats after pretreatment with indomethacin and N omega-nitro-L-arginin methyl ester. The effects on histopathology, creatinine clearance, and urinary N-acethyl-beta-D-glucosaminidase (NAG) activity were assessed. In a rat renal slice system, the slices were exposed to iobitridol or iohexol (both at the concentration range of 17.5-70 mg iodine/mL) for 60 min. The accumulation of para-aminohippuric acid (PAH), an organic anion, and the intracellular potassium content as the indicators of renal tubular injury were measured to assess the direct effects of iobitridol and iohexol on renal tubules. RESULTS: In the ARF model, no significant difference was detected between the effects of iobitridol and those of iohexol on the creatinine clearance and urinary NAG activity 24 hours after the injection. However, iobitridol produced a lower degree and incidence of renal tubular injury of renal proximal tubules (P < 0.001) and distal tubules (P < 0.05) compared with iohexol. In the rat renal slice system, the iobitridol treatment had significantly less effect on the PAH accumulation compared with iohexol (P < 0.001). There were no changes in the intracellular potassium content. CONCLUSIONS: These findings suggest that iobitridol has significantly less toxic effects on the kidney compared with iohexol under the condition of our experiment.

Relationship between amount of beta-blockers permeating through the stratum corneum and skin irritation after application of beta-blocker adhesive patches to guinea pig skin.

We evaluated the relationship between the cumulative amounts of 5 kinds of beta-blockers (alprenolol, oxprenolol, timolol, acebutolol and atenolol) permeating through the stratum corneum and a* values obtained by measuring the formation of erythema, a skin irritation reaction, with a chromameter after transdermal application of adhesive patches containing 2 beta-blocker to the skin of guinea pigs. The cumulative amount of beta-blocker released from each adhesive patch to the skin increased with the increase in application time. The contents of alprenolol, oxprenolol and timolol in the stratum corneum and in the stripped skin increased markedly up to 4 h after application and thereafter were maintained at high levels up to 24 h. The contents of acebutolol and atenolol, on the other hand, increased up to 24 h, but these values were low. a* values of all adhesive patches 24 h after application were higher than those before application. The correlation coefficients between the cumulative amounts of alprenolol, oxprenolol, timolol, acebutolol or atenolol permeating through the stratum corneum and (delta a* -delta a*Placebo) values were 0.739, 0.717, 0.722, 0.551 and 0.633, respectively. The correlation coefficient calculated by averaging the cumulative amounts of 6 kinds of beta-blockers permeating through the stratum corneum [including propranolol which was reported previously (Kobayashi I., et al., Biol. Pharm. Bull., 19, 839-844 (1996))] was 0.731, higher than the correlation coefficient between contents of these beta-blockers in the stripped skin and (delta a* -delta a*Placebo) values (r = 0.552). This suggests that there was a high correlation between the cumulative amounts of beta-blockers permeating through the stratum corneum and (delta a* -delta a*Placebo) values.

Effects of ischemia-reperfusion on individual cytochrome P450 isoforms in the rat kidney.

Ischemia-reperfusion of organs such as the kidney produces reactive oxygen and free radical species in tissues and leads to injury of intracellular molecules critical to cell homeostasis. Ischemia-reperfusion affects the NADPH-dependent monooxygenase system including P450 system, which is also a source of reactive oxygen species. In this study, the effects of ischemia-reperfusion on monooxygenase activity and levels of individual P450 isoforms including CYP2C23, 4A2, and 4A8 in the rat kidney were investigated. Ischemia of the rat kidney for 30 min had little effect on lauric acid hydroxylation activity and levels of P450 isoforms but ischemia for 60 min significantly decreased lauric acid omega- and (omega-1)-hydroxylation activities and also decreased the levels of CYP2C23, 4A2, and 4A8. Reperfusion for 60 min after 30-min ischemia decreased the levels of CYP2C23 and 4A2 in the rat kidney although 30-min ischemia did not. Reperfusion for 240 min after 30-min or 60-min ischemia recovered the decreased levels of lauric acid hydroxylation activity and the levels of CYP2C23 and 4A2. Changes in the levels of monooxygenase activity and the levels of P450 isoforms in kidneys by ischemia-reperfusion are faster than those in the liver; it takes several hours for ischemia-reperfusion to affect the levels of monooxygenase activity and the levels of P450 in the rat liver. Our findings suggest that damage of P450 isoforms in the kidney by ischemia-reperfusion occurs by a mechanism different from that in the liver and that active oxygen or free radical species directly attack proteins.

Protective effects of Wen-Pi-Tang against cultured renal epithelial cellular injury.

A study was conducted to clarify whether extracts of Wen-Pi-Tang and its component crude drugs ameliorate renal cellular injury by assaying lactate dehydrogenase and malondialdehyde leakage from LLC-PK(1) cells in culture. The cells were cultured with various concentrations of the samples under two sets of conditions: routine and hypoxia-reoxygenation. The results demonstrated that Wen-Pi-Tang, Rhei rhizoma, Glycyrrhizae radix exerted marked protective effects on the cells; Ginseng radix showed moderate activity, whereas Zingiberis rhizoma and Aconiti tuber had virtually no such effect. Two pure compounds, epicatechin 3-O-gallate and licochalcone A isolated from Rhei rhizoma and Glycyrrhizae radix, respectively, exerted the same marked effects as the parent crude drugs. In the light of these findings, we concluded that Wen-Pi-Tang and its major components protect renal epithelial cells against injury mediated by hypoxia-reoxygenation and/or prevent such injury. The primary mechanism of these effects appeared to be antilipid peroxidant activity present in the preparation.

Synthesis and opiate activity of pseudo-tetrapeptides containing chiral piperazin-2-one and piperazine derivatives.

Enantiomeric piperazin-2-one derivatives, N,N'-ethylene-bridged alanylphenylalanines (1a or 1b), were synthesized using (S)- or (R)-alanine and phenylalanine as starting materials, and were inserted into the second and third positions of enantiomeric pseudo-tetrapeptides (P1a- or P1b-OEt). The corresponding piperazine derivatives (1a- or 1b-sRed) obtained by selective BH3 reduction of the amide carbonyl groups of 1a or 1b were similarly inserted into the same positions of tetrapeptides (P1a- and P1b-sRed). Enantiomeric N,N'-ethylene-bridged tyrosyltyrosine derivatives (2a or 2b) obtained from (S)- or (R)-tyrosine were also inserted into the first and second positions of two pairs of enantiomeric tetrapeptides (P2a- and P2b-OEt or P'2a- and P'2b-OEt). The opiate activities of the eight peptides thus obtained were studied by use of the mouse vas deferens and the guinea pig ilcum assays in order to elucidate the structure-activity relationships of these peptides, especially with respect to stereochemistry.

Amelioration by cAMP of cephaloridine-induced injury in the porcine kidney cell line LLC-PK1.

We investigated the effects of a phosphodiesterase inhibitor and dibutyryl cAMP (dBcAMP) on the cell injury induced by cephaloridine (CER) in an established renal epithelial cell LLC-PK1. CER increased the leakage of lactate dehydrogenase (LDH) from LLC-PK1 cells to the medium and the level of lipid peroxidation in the cells. 3-Isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, increased cAMP content in LLC-PK1 cells and ameliorated the increase in LDH leakage induced by CER, dBcAMP reduced the cell injury induced by CER. Our results suggest that a signalling pathway of cAMP protects against CER-induced renal cell injury, which is probably due to generation of oxygen radicals.

Relationship between the amount of propranolol permeating through the stratum corneum of guinea pig skin after application of propranolol adhesive patches and skin irritation.

In the present study we evaluated the relationship between the cumulative amount of propranolol permeating through the stratum corneum and the formation of erythema, a skin irritation reaction, after transdermal application of adhesive patches containing propranolol to the skin of guinea pigs. The intensity of erythema was expressed in terms of a* values measured with a chromameter. The a* values increased in guinea pigs after application of the adhesive patches containing 0.4 mg/cm2 of propranolol to the skin. Since the adhesive patches showed good adhesion to the skin (propranolol content is less than the saturated concentration in the adhesive base) and the cumulative amount of propranolol permeating through the stratum corneum is small, the development of erythema was considered to be mainly due to physical factors such as peeling. Even in adhesive patches containing 0.8 mg/cm2 or 1.2 mg/cm2 of propranolol, a* values increased, although adhesion to the skin is low because of crystallization of propranolol in the adhesive base. On the other hand, in these two adhesive patches, the cumulative amount of propranolol permeating through the stratum corneum increased up to 24 h after application. These findings suggest that the skin irritation reaction is due to propranolol mainly absorbed transdermally, because there is a high correlation between the cumulative amount of propranolol permeating through the stratum corneum and the a* values (r = 0.928).

Structure-activity relationships of dermorphin analogues containing chiral piperazin-2-one and piperazine derivatives.

The amide and ester carbonyl groups of four piperazin-2-one derivatives (N,N'-ethylene-bridged dipeptide ethyl esters) constructed from (R)- or (S)-phenylalanine and glycine were reduced with borane-tetrahydrofuran complex to produce the corresponding piperazine derivatives in 70-80% yields. These piperazin-2-one or piperazine derivatives were used as the carboxyl-terminal residues of eight dermorphin analogues (H-tyrosyl-D-alanyl-piperazine derivatives were used as the carboxyl-terminal residues of eight dermorphin analogues (H-tyrosyl-D-alanyl-piperazin-2-one or piperazine derivatives) whose opiate activities were examined in vitro by use of the guinea pig ileum and the mouse vas deferens assays. It was found in the guinea pig ileum assay that the configuration of pheylalanine and the replacement of the piperazin-2-one ring with a piperazine ring are important for enhancing or reducing the opiate activities of these analogues.

Hypoxia and reoxygenation-induced injury of renal epithelial cells: effect of free radical scavengers.

The aim of this study was to characterize injuries of LLC-PK1 and MDCK cells exposed to hypoxia and reoxygenation. Exposure of LLC-PK1 cells to hypoxia reduced the ATP contents and increased the leakage of lactate dehydrogenase (LDH), but MDCK cells had no such injuries. Hypoxia-reoxygenation of LLC-PK1 cells dramatically increased LDH leakage, which was suppressed by free radical scavengers, N,N'-diphenyl-p-phenylenediamine, superoxide dismutase and N,N'-dimethylthiourea. These results suggest that use of LLC-PK1 cells has advantages for the investigation of ischemia-reperfusion injury of the kidney as an in vitro model and that generation of oxygen radicals is involved in the cellular injury induced by hypoxia-reoxygenation.

In vitro oxygenation injury to slices prepared from ischemic kidney in rats.

When cortical slices prepared from rat kidneys made ischemic were incubated under a 100% oxygen atmosphere, lipid peroxidation increased and the ATP level decreased. Such oxygenation of the slices was accompanied by decreases in gluconeogenesis and the glutathione level, but an antioxidant, N,N'-diphenyl-p-phenylenediamine, prevented the increase in lipid peroxidation without affecting decreases in ATP and glutathione levels, and gluconeogenesis. The results suggest that postischemic oxygenation of slices generates free radicals that cause the production of lipid peroxidation not associated with tissue injury.

Increases in urinary enzyme excretion in rats depleted of glutathione inhibited by scavenger of oxygen free radicals.

Urinary excretion of enzymes by rats was assessed after glutathione (GSH) was depleted by treatment with a mixture of the GSH depletors D,L-buthionine-S,R-sulfoximine (BSO) and diethylmaleate (DEM). Renal GSH was low 2 h after treatment and later returned to the control level. The urinary excretion of gamma-glutamyltranspeptidase (gamma-GTP) and N-acetyl-beta-D-glucosaminidase (NAG) remained high for at least 3 d after the injection of BSO (100 mg/kg) and DEM (0.5 ml/kg), with no effect on the blood urea nitrogen level. N,N'-Dimethylthiourea (DMTU), a scavenger of oxygen free radicals, inhibited this increase in the urinary excretion of gamma-GTP. DMTU also inhibited the increase in cisplatin-induced NAG excretion caused by the GSH depletors. These results suggested that the urinary excretion of these enzymes is an index of renal tubular injury caused by short-term depletion of renal GSH, and that the generation of free radicals may be involved in renal tubular injury during GSH depletion or caused by cisplatin together with GSH depletors.

The iron chelator deferoxamine prevents cisplatin-induced lipid peroxidation in rat kidney cortical slices.

We evaluated the effect of the iron chelator deferoxamine on lipid peroxidation produced by the nephrotoxic antineoplastic drug cisplatin in rat kidney cortical slices. The addition of deferoxamine to the incubation medium prevented such lipid peroxidation in the incubated slices. Treatment of rats with deferoxamine inhibited the increase in lipid peroxidation caused by cisplatin in the medium. These results suggest that iron may be a causal agent of cisplatin-induced lipid peroxidation.

Cisplatin-induced injury to calcium uptake by mitochondria in glutathione-depleted slices of rat kidney cortex.

Changes in functional activities of mitochondria from kidney cortical slices incubated with cisplatin and the glutathione depletor diethylmaleate were examined. Diethylmaleate, which decreased the glutathione level in the slices, enhanced the cisplatin-induced decreases in glutathione level and calcium uptake in the mitochondria. The movement of cisplatin into mitochondria in the slices was not affected by diethylmaleate. These results suggest that the depressions in glutathione level and calcium uptake by cisplatin in mitochondria are enhanced by a decrease in cytoplasmic glutathione.