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The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.

Aminoglycosides such as gentamicin and amikacin are the most commonly used antibiotics worldwide in the treatment of Gram-negative bacterial infections. However, serious complications like nephrotoxicity and ototoxicity are dose-limiting factors in the use of aminoglycosides. A relatively large amount of the intravenously administered dose is accumulated in the kidney (about 10% of dose), whereas little distribution of aminoglycosides to other tissues is observed. Aminoglycosides are taken up in the epithelial cells of the renal proximal tubules and stay there for a long time, resulting in nephrotoxicity. Acidic phospholipids are considered as a binding site for aminoglycosides in the brush-border membrane of the proximal tubular cells. More recently, it has been reported that megalin, a giant endocytic receptor abundantly expressed at the apical membrane of renal proximal tubules, plays an important role in binding and endocytosis of aminoglycosides in the proximal tubular cells. The elucidation of the aminoglycoside-binding receptor would help design a strategy to prevent against aminoglycoside-induced nephrotoxicity. In this review, we summarize recent advances in the understandings of the molecular mechanisms responsible for renal accumulation of aminoglycosides, especially megalin-mediated endocytosis. In addition, approaches toward prevention of aminoglycoside-induced nephrotoxicity are discussed, based on the molecular mechanisms of the renal accumulation of aminoglycosides.

The role of megalin in tissue distribution of aminoglycosides was examined in normal rats and maleate-treated rats that shed megalin from the renal brush-border membrane. In normal rats, amikacin administered intravenously accumulated most abundantly in the renal cortex, followed by the renal medulla. No amikacin was detected in other tissues. Tissue distributions of amikacin were well correlated with megalin levels in each tissue. Bolus administration of gentamicin increased urinary excretion of megalin ligands (vitamin D binding protein and calcium), suggesting the competition between gentamicin and these megalin ligands in renal tubules. Ligand blotting showed that binding of (45)Ca(2+) to megalin was inhibited by aminoglycosides. Both megalin levels and amikacin accumulation in renal cortex were decreased by maleate injection. Then, amikacin accumulation recovered proportionate to megalin levels. These findings suggest that megalin is involved in the renal cortical accumulation of aminoglycosides in vivo. In addition, the interaction between aminoglycosides and calcium in the kidney may be due to the competition among these compounds to bind to megalin.

-We examined mechanisms of FITC-albumin uptake by alveolar type II epithelial cells using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA, which are characteristic features of alveolar type II epithelial cells, were detected in RLE-6TN cells. The uptake of FITCalbumin by the cells was time and temperature dependent and showed the saturation kinetics of high-and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin, by chemically modified albumin, and by metabolic inhibitors and bafilomycin A 1, an inhibitor of vacuolar H ϩ -ATPase. Confocal laser scanning microscopic analysis after FITCalbumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl-␤-cyclodextrin (inhibitors of caveolae-mediated endocytosis) but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis) in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed. albumin clearance; caveolae-mediated endocytosis; endocytic receptor; modified albumin; protein transport THE DISTAL AIR SPACES OF THE lung (alveolar region) are lined with a continuous epithelium comprising two major types of epithelial cells, type I and type II. Type I epithelial cells have a squamous morphology and cover ϳ90 -95% of the alveolar surface area. Type II cells are cuboidal epithelial cells and cover ϳ5-10% of the surface area, although the number of type II cells in alveolar epithelia is similar to or more than that of type I cells. Type II cells also serve as progenitors of type I cells (13,40). Alveolar lining fluid contains various physiologically important proteins such as albumin, immunoglobulin G, and transferrin. The concentration of albumin in alveolar fluid is usually much lower than that in the blood, whereas the concentration would increase to 40 -65% of plasma level in hydrostatic pulmonary edema and to 75-95% in lung injury pulmonary edema. The clearance of serum proteins from the alveolar space is a critical process in recovery from pulmonary edema and in maintaining the normal alveolar milieu (17,22). Therefore, understanding the mechanisms of protein transport in alveolar epithelial cells is important for the development of better therapeutic approaches. In addition, such information could provide new insights into improved strategies for pulmonary delivery of ex...

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