Munekazu Gemba

Nephrotoxicity of a new platinum compound, 254-S, evaluated with rat kidney cortical slices

The addition of a new compound containing platinum, 254-S, an antineoplastic agent, to medium had no effect on p-aminohippurate (PAH) accumulation, gluconeogenesis, or potassium and ATP concentrations in rat kidney cortical slices at the concentrations tested, up to 10 mM. At 1 mM, cisplatin, used for comparison, significantly decreased all of these biochemical indices in the slices. Administration of 254-S at a low dose (10 mg/kg i.v.) to rats decreased the ability of the slices to accumulate PAH and to maintain the potassium concentration, without affecting levels of urea or creatinine in blood plasma. 254-S at a high dose (20 mg/kg i.v.) or cisplatin at 5 mg/kg (i.v.) also decreased these indices in the slices, and affected urea and creatinine in blood plasma. These results suggested that use of the renal slice technique gives data useful for the evaluation of the nephrotoxicity of 254-S, and that PAH accumulation and the potassium concentration in slices from rats treated with 254-S are indicators of nephrotoxic damage.

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.

Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies.

Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin‐biotin sandwich ELISA (ABS‐ELISA) system for SEA, SEB, and SEC1 using newly‐developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78–25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantification and simplicity in detecting SEs.

Effect of cisplatin on calcium uptake by rat kidney cortical mitochondria

The effect of the nephrotoxic antineoplastic drug, cisplatin, on mitochondrial calcium uptake was examined in rat kidney cortical mitochondria. We treated rats with cisplatin (5 mg/kg, i.p.), and prepared and incubated the mitochondria. Uptake of calcium decreased after 24 h. The mitochondria contained platinum even 3 days after injection. Cisplatin (0.5 mM) added to incubation medium inhibited calcium uptake. Platinum accumulated in the mitochondria during incubation. Mitochondria accumulated less of another divalent cation, magnesium, in rats given cisplatin and in incubation medium with cisplatin added. The results suggest that cisplatin taken up into kidney cortical mitochondria inhibited divalent cation uptake there, which may contribute to cisplatin nephrotoxicity.

Involvement of MEK/ERK pathway in cephaloridine-induced injury in rat renal cortical slices.

We have previously reported that free radical-mediated injury induced by cephaloridine (CER) is enhanced by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in rat renal cortical slices. We have also shown that PKC activation in mitochondria is involved in CER-induced nephrotoxicity in rats. We investigated the role of a downstream PKC pathway, a MEK/ERK pathway, in free radical-induced injury in rat renal cortical slices exposed to CER. Immediately after preparing slices from rat renal cortex, the slices were incubated in the medium containing MEK inhibitors. ERK1/2 activation was determined by Western blot analysis for phosphorylated ERK (pERK) 1/2 protein in nucleus fraction prepared from the slices exposed to CER. Prominently, CER caused not only increases in lipid peroxidation as an index of free radical generation and in LDH leakage as that of cell injury in the slices, but also marked activation of ERK1/2 in nucleus fraction. PD98059 and U0126, MEK1/2 inhibitors, significantly attenuated CER-induced increases in lipid peroxidation and LDH leakage in the slices. PD98059 also suppressed ERK1/2 activation in nucleus fraction prepared from the slices treated with CER. Inhibition of other MAP kinase pathways, p38 MAP kinase and c-Jun N-terminal kinase (JNK) had no effect on CER-induced increases in lipid peroxidation level and LDH leakage in the slices. The present results suggest that a MEK/ERK pathway down stream of a PKC pathway is probably involved in free radical-induced injury in rat renal cortical slices exposed to CER.

Inhibitory Effect of Lithium on p-Aminohippurate Transport in Rat Kidney Cortex in vitro

The effect of lithium on p-aminohippurate (PAH) transport was studied using slices and basolateral membrane vesicles prepared from rat kidney cortex. The addition of lithium in concentrations ranging from 0.5 to 5 mM caused a concentration-dependent inhibition of PAH accumulation in the slices. Lithium inhibited PAH accumulation in the slices, not only during the rapid uptake period (after 10 min) but also during the approach to equilibrium (after 30 min). The effect of lithium (2 mM) in the slices was irreversible. The inhibitory effect of lithium was not the result of changes in the water distribution and the concentrations of ATP, sodium and potassium in the slices during incubation. The effect of lithium on the kinetic parameters for PAH accumulation was to decrease Vmax, while apparent Km remained constant. There was no lithium effect on the efflux of PAH from the slices back into the incubation medium, indicating that lithium inhibited PAH influx to the kidney cell. No evidence was obtained to indicate that lithium (1 mM) directly affected PAH uptake by isolated basolateral membrane vesicles. These results suggest that lithium seems to affect metabolism linked to the carriers for PAH transport other than ATP production and sodium gradient and then seems to decrease the mobility of the carriers in the membranes.

Chronotoxicity of nedaplatin in rats

Chronotoxicologic profiles of nedaplatin, a platinum compound, were evaluated in rats maintained under a 12 light/12 dark cycle with light from 07:00 h to 19:00 h. Nedaplatin (5 mg/kg) was injected intravenously, once a week for 5 weeks at 08:00 h or 20:00 h. The suppression of body weight gain and reduction of creatinine clearance were significantly greater with the 20:00 h than 08:00 h treatment. Accumulation of nedaplatin in the renal cortex and bone marrow were also greater with 20:00 h treatment. There were significant relationships between the nedaplatin content in the kidney and bone marrow and degree of injury to each. These results suggest that the nedaplatin-induced toxicity depends on its dosing-time, and it is greater with treatment at 20:00 h, during the active phase. The dosing-time dependency in the accumulation of nedaplatin in the tissue of the organs might be involved in this chronotoxicologic phenomenon.

Enhancement of protein kinase C activity and chemiluminescence intensity in mitochondria isolated from the kidney cortex of rats treated with cephaloridine.

The development of nephrotoxicity induced by cephaloridine (CER) has been reported to be due to reactive oxygen species (ROS). Protein kinase C (PKC) has been suggested to modulate the generation of ROS. We investigated the possible participation of ROS generation assessed by chemiluminescence (CL) and PKC activity in rat kidney cortical mitochondria in the development of CER-induced nephrotoxicity. We first evaluated the magnitude of the nephrotoxic damage caused by CER in rats. The plasma parameters and ultrastructural morphology changes were increased markedly 24hr after the treatment of rats with CER. We demonstrated that the treatment of rats with CER clearly evoked not only enhancement of Cypridina luciferin analog (CLA)-dependent CL intensity, but also the activation of PKC in mitochondria isolated from the kidney cortex of rats 1.5 and 3.5 hr after injection of the drug. These changes were detected in advance of those observed in plasma and by electron microscopy. The increase in CLA-dependent CL intensity detected in the kidney cortical mitochondria 1.5 and 3.5 hr after injection of CER was inhibited completely by the addition of superoxide dismutase, suggesting the generation of superoxide anion in these mitochondria during the early stages of CER-induced nephrotoxicity. These results suggest that the activation of PKC and the enhancement of superoxide anion generation in kidney cortical mitochondria precede the increases in plasma parameters and the electron micrographic changes indicative of renal dysfunction in rats treated with CER. Additionally, they suggest a possible relationship between PKC activation in mitochondria and free radical-induced CER nephrotoxicity in rats.

Stimulatory Effect of Calcium Ions on p-Aminohippurate Accumulation by Rat Kidney Cortical Slices

The effect of external Ca2+ on p-aminohippurate (PAH) transport was examined using rat kidney cortical slices. External Ca2+ increased the initial rate of PAH uptake, markedly enhanced its maximal accumulation at steady-state conditions and depressed the efflux. Kinetic studies revealed that increasing external Ca2+ from 0 to 2.5 mM resulted in an increase in the Vmax of transport, while the apparent Km values remained constant. When the intracellular Na+ levels of the slices were equal, PAH accumulation was increased in the presence of Ca2+. A divalent cation such as Ba2+, Mn2+ or Sr2+ enhanced PAH accumulation to at least the same degree as Ca2+. While Mg2+ had no effect, La3+ exhibited a little stimulatory effect on the PAH accumulation. We suggest that the stimulatory action of Ca2+ on PAH accumulation is in part dissociated from that of Na+ gradient and that Ca2+ can be substituted by Ba2+, Mn2+ and Sr2+ with regard to the stimulation of PAH accumulation.

Dissipation by tetraethylammonium of verapamil-stimulated p-aminohippurate accumulation in rat kidney cortical slices

The stimulatory effect of verapamil on p-aminohippurate (PAH) accumulation was studied in rat kidney cortical slices. PAH accumulation was stimulated by the presence of 0.3 mM verapamil in the incubation medium, but this stimulatory action of verapamil was dissipated by adding tetraethylammonium (TEA) to the medium. TEA accumulation by the kidney cortical slices was inhibited by verapamil in a concentration-dependent fashion. In addition, kinetic studies revealed that verapamil resulted in an increase in the Michaelis constant (Km) of TEA transport, with the Vmax remaining constant, suggesting that verapamil competitively inhibited TEA transport. These results suggest that the transport system for verapamil is the same as that for TEA and that verapamil taken up within the cells may stimulate PAH transport.