Fumiaki Yamasaki

Protective action of ulinastatin against cisplatin nephrotoxicity in mice and its effect on the lysosomal fragility.

The development of azotemia after cisplatin injection in mice was inhibited by ulinastatin treatment in a dose-dependent manner. Reduction in creatinine clearance and elevation in fractional excretion of sodium in mice receiving cisplatin was ameliorated by ulinastatin administration. Epithelial necrosis and hyaline cast formation in the proximal tubule were also suppressed. Ulinastatin showed no influence on the kidney platinum level after cisplatin injection. In LLC-PK1 cells, addition of ulinastatin to the incubation medium markedly reduced the release of N-acetyl-beta-D-glucosaminidase, on of the lysosomal enzymes, during hypotonic treatment only when cells were damaged with cisplatin. On the other hand, ulinastatin showed no effect on the elevation of malondialdehyde concentration in the murine kidney cortical slices after the treatment with cisplatin. These results indicate that ulinastatin has a protective effect against cisplatin nephrotoxicity, and its prevention of the increase in lysosomal fragility is a probable mechanism involved in the renal protection.

A novel quinolinone diuretic, M12285, and its activation mechanism through sulfate conjugation.

The diuretic activity of a quinolinone oxime diuretic, M12285, was examined after renal arterial, i.v. and portal injection in rats. M12285 injected into the renal artery at a dose of 1 mg/kg caused no diuretic effect, whereas i.v. and portal injections induced marked diuresis dose dependently. The minimum effective dose with portal injection was lower (1 mg/kg) than that with i.v. injection (3 mg/kg) and the start of the effect was faster with portal injection. These results indicated that some metabolic modification in the liver is necessary for the diuretic activity to appear. Accordingly, we performed in situ rat liver perfusion with M12285 and obtained several metabolites. Renal arterial injection of each fractionated metabolite of M12285 revealed that all the diuretic activity derived from one of these metabolites. From IR and 1H-nuclear magnetic resonance (1HNMR) measurements, the chemical structure of this active metabolite was assumed to be a sulfate-conjugated form of M12285 at the oxime moiety. Based on this tentative chemical structure, we synthesized the oxime sulfate of M12285 (potassium salt, M17000) and confirmed the identity of IR and 1HNMR spectra. Administration of M17000 into the renal artery induced apparent diuresis in a dose-dependent manner in both rats and dogs. These results indicate that the oxime sulfate of M12285 is responsible for the diuretic activity of M12285. Therefore, we synthesized several derivatives of M17000 and confirmed their possible therapeutic value as a novel family of diuretics, namely quinolinone oxime sulfonic acids.

Uptake of human urinary trypsin inhibitor by the kidney epithelial cell line, LLC-PK1

Abstract We investigated the uptake of human urinary trypsin inhibitor (UTI) by the kidney epithelial cells, LLC-PK1. Indirect immunogold techniques with an electron microscope demonstrated the localization of UTI within the cells after an incubation during which UTI was added to the apical side. Immunoreactivities were found in endocytic vesicles, vacuoles and lysosomes. Subsequently, we tried to characterize the property of the uptake of UTI using the fluorescein isothiocyanate-labelled UTI (FITC-UTI). FITC-UTI uptake was decreased by an incubation with an excess of unlabelled UTI and showed concentration-dependent saturation. This process was markedly suppressed during the incubation at 4°C. The uptake was significantly lessened with 2,4-dinitrophenol and antimycin A, inhibitors of oxidative phosphorylation, and colchicine, a microtubule-depolymerizing agent. These results indicate that exogenous UTI is internalized by LLC-PK1 cells through an endocytic pathway. From uptake studies, it is suggested that an adsorptive process is partially involved in the mechanisms of endocytosis.

Cytoprotective Effect of Ulinastatin on LLC-PK1 Cells Treated with Antimycin A, Gentamicin, and Cisplatin

The cytoprotective effect of ulinastatin was studied in LLC-PK1 cells treated with antimycin A, gentamicin, or cisplatin. All of the three agents induced a concentration-dependent increase in the release of lactate dehydrogenase and a decrease in the amount of remaining protein. In the cell injury models treated with 1.5 microM antimycin A, 10 mM gentamicin, and 0.3 mM cisplatin, ulinastatin tended to show a cytoprotective effect at a concentration of 3,000 U/ml and provided a significant protective effect at 10,000 U/ml. LLC-PK1 cells treated with 0.3 mM cisplatin, bovine serum albumin, and alpha1-acid glycoprotein at a concentration of 3.54 mg/ml, which is a comparable protein concentration to that of 10,000 U/ml ulinastatin, showed no protective effect but rather enhanced cell injury. These results suggest that ulinastatin exerts a direct protective effect on LLC-PK1 cells against various renal toxicities.

Electrophysiological Localization of Transporters in the Distal Nephron Segments

The distal convoluted tubule (DCT) and the connecting tubule (CNT) from the rabbit kidney were investigated by electrophysiological techniques, isotopic flux measurement, and microfluorometry, in order to characterize the ion transport properties of cell membranes. When the DCT and the CNT were per-fused in vitro, the transepithelial voltage (VT) displayed lumen negative, and the transepithelial resistances (RT) were relatively low, as in the category of leaky epithelia. Random cellular impalement revealed that the basolateral membrane voltage (VB) of the DCT showed Gaussian distribution, whereas the CNT consisted of two cell populations, having different VB and different fractional resistance of the apical membrane (fRA). The CNT cell had a high VB. and lower fRA and the intercalated (IC) cell in the CNT had a low VB and higher fRA Using ion substitution and channel inhibitors, the conductive properties of DCT cells and CNT cells revealed that the luminal membrane had both Na+ and K+ conductances and that the basolateral membrane had both K+ and Cl-conductances. Intercalated cells of CNT had only a Cl-conductance in the basolateral membrane. Besides Na+ conductance in the luminal membrane of CNT cells, two other modes of Na+ entry process were observed. One pathway was the Na/Cl cotransporter, revealed by isotopic ion flux studies, which was inhibitable with thiazide diuretics, and the other was a nonselective cation conductance, which was not sensitive to amiloride and was opened by parathyroid hormone (PTH). From intracellular calcium measurement and calcium flux studies, it was found that the latter mode serves as a route for calcium entry pathway in CNT cells.