Junichi Sudo

Mechanism of nephrotoxicity induced by repeated administration of cadmium chloride in rats

To explore the mechanism of Cd nephrotoxicity, CdCl2 was subcutaneously injected to rats, at 3 mg Cd/kg body weight once a day, for 8 d. In the liver, Cd bound to metallothioneins (MTs-Cd) rose from d 1 after the initiation of CdCl2 administration, and reached a plateau after the administration ceased. In the plasma, MTs-Cd rose from d 4, peaked on d 8, and gradually fell thereafter. In the kidneys, leucine aminopeptidase (LAP) and N-acetyl beta-D-glucosaminidase (NAG) fell during d 6-20, and Cd bound to cellular membranes (Mem-Cd) rose from d 1 and reached a plateau during d 6-20. The Mem-Cd levels were significantly correlated with the reduction in the LAP and NAG activity; the values of MTs-Cd plus Mem-Cd were almost equivalent to those of total Cd. These findings showed that the hepatic synthesis of MTs-Cd occurred followed by its release into plasma; the extent of renal injury was aggravated as the plasma level of MTs-Cd rose; and a greater part of the renal Cd distributed intracellularly as the MTs-binding form, while the residual Cd distributed as the cellular membrane-binding form. Also, it was suggested that Cd that occurred as the cellular membrane- binding form in the kidneys was involved in manifestation of renal injury.

Assay of ATPase and Na, K-ATPase activity using high-performance liquid chromatographic determination of ADP derived from ATP

An HPLC assay for determination of ATPase activity was developed and validated. After stopping the enzyme reaction of the enzyme source (rat renal cortical basolateral membranes) with ATP, products derived from ATP were analyzed by two methods; HPLC determination of ADP derived from ATP, and colorimetry of inorganic phosphorus (Pi) released from ATP. This HPLC procedure was precise and linear over the range of protein amount of the enzyme source studied, and the intra-and inter-assay variations were lower than 10%. The values that were obtained by the two methods revealed a significant correlation. Also, even when the samples contained Pi or were contaminated with Pi, this HPLC method allowed determination of ATPase activity. In addition, when ouabain was used as an inhibitor, the HPLC method was found to be applicable for Na,K-ATPase determination. This indicated that this HPLC assay would enable determination of ATPases other than Na,K-ATPase, when other inhibitors are employed instead of ouabain.

Transdermal absorption of L-dopa from hydrogel in rats.

To improve compliance in administration of l-dopa, transdermal absorption of the agent was investigated in rats in vitro employing two-chamber diffusion cells in which the excised rat abdominal skin was mounted, and in vivo using an alcoholic hydrogel containing l-menthol. The in vitro study revealed that in presence of l-menthol (2%, W/W), ethanol (20 and 40%, V/V) accelerated transdermal penetration of l-dopa with an increase of its percentages. The in vivo study showed that when the l-dopa-hydrogel containing 2% l-menthol and 40% ethanol was attached on the skin, plasma levels of l-dopa and norepinephrine increased with the time elapsed; the level of dopamine increased and reached a plateau thereafter; and the level of epinephrine was unchanged. These in vitro and in vivo findings indicated that the hydrogel formulation of l-dopa provides new direction in treating Parkinsons disease.

Transdermal Absorption of L-Dopa from a New System Composed of two Separate Layers of L-Dopa and Hydrogel in Rats

To maintain the stability of L-dopa in hydrogel, a new system composed of two separate layers of L-dopa and hydrogel was developed. L-Dopa sheets were made by immersing L-dopa solution into wiper sheets and by lyophilizing them. Examination for stability of L-dopa in the L-dopa sheet revealed that its stability was maintained for at least 12 weeks, providing the sheet was kept at room temperature in a dark box. In a cutaneous absorption study of L-dopa in rats, an L-dopa sheet was attached to the shaved abdominal skin. A hydrogel composed of cutaneous absorption enhancers, water and ethanol, was spread on vinyl tape (hydrogel sheet), and this sheet was placed over the L-dopa sheet. L-Dopa that was administered transdermally effectively penetrated through the skin: The plasma level of L-dopa peaked at 30 min and remained high between 60 and 180 min after the cutaneous application. Our system, composed of two separated layers of L-dopa and hydrogel, enabled the stability of L-dopa to be maintained without losing transdermal absorption of L-dopa.

Elevation of plasma levels of L-dopa in transdermal administration of L-dopa-butylester in rats.

ABSTRACT To increase delivery of L-dopa in its transdermal absorption, a new lipophilic derivative of L-dopa, L-dopa-butylester, was synthesized. An in-vitro study employing two-chamber diffusion cells, in which the excised rat abdominal skin was mounted, revealed that, in the presence of L-menthol and ethanol, L-dopa-butylester penetrated in its original form more effectively than L-dopa. L-Dopa-butylester sheets were made by immersing wiper sheets in methanol containing the compound, and then evaporating the methanol. An extraction study of the compound from the sheets revealed that its stability was maintained for at least 12 weeks. In an in-vivo cutaneous absorption study, an L-dopa-butylester sheet was attached to the shaved rat abdominal skin. A hydrogel containing L-menthol and ethanol was spread on vinyl tape, and this sheet was placed over it. In plasma, the L-dopa level rose linearly between 30 and 180 min after the cutaneous application; L-dopa-butylester was not detected. The L-dopa level was higher than that in which L-dopa was applied. These findings indicated that the lipophilic nature of L-dopa-butylester further increased its penetration through the skin, and that L-dopa-butylester that was taken up into the general circulation system was rapidly converted to L-dopa by hydrolysis in the body.

Further accumulation of Cd in renal cellular membranes caused by administration of desferrioxamine to Cd-burdened rats

AbstractBackground. Desferrioxamine (DFO) a chelating agent, is used to treat metal toxicity caused by iron and aluminum in patients on hemodialysis. We hypothesized that DFO could also be used to treat cadrium-induced nephropathy. Animal experiments were therefore performed to explore whether DFO removed cadmium (Cd) from the kidneys of rats with a Cd burden. Methods. Rats received subcutaneous injections of Cd chloride (3 mg Cd/kg per day, days 0–7) followed by DFO (50 mg/kg per day, days 8–14). Levels of Cd were determined in liver, kidneys, and plasma. Enzymes assays and histopathological examination were performed in kidneys. Results. In liver, Cd injections elevated Cd levels; subsequent injections of DFO lowered the Cd levels compared with levels after injections of Cd alone. In kidneys, Cd injections increased levels of total Cd and Cd bound to cellular membranes (Mem-Cd), and decreased leucine aminopeptidase (LAP) activity (a marker of renal injury); subsequent injections of DFO elevated levels of total Cd and Mem-Cd, and lowered LAP activity compared with fundings after the injection of Cd alone. After the injections of Cd alone and DFO following Cd the renal levels of Cd were below the critical concentration required to cause renal injury, since no histopathological changes were observed in the kidney. Conclusion. DFO administration to Cd-burdened rats removed Cd from the liver, but led to accumulation of Cd in the kidneys, particularly in the cellular membranes. These results suggest that if DFO is given long-term to Cd-burdened patients, the Cd level in kidneys, particularly in renal cellular membranes, could reach concentrations that could cause manifest renal injury.