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Antimicrobial Agents and Chemotherapy, August 2009, p. 3302-3307, Vol. 53, No. 8
0066-4804/09/$08.00+0     doi:10.1128/AAC.01627-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney{triangledown}

Kunio Fujiwara,1* Masashi Shin,1 Hayato Matsunaga,1 Tetsuya Saita,2 and Lars-Inge Larsson3

Department of Applied Life Science, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082,1 Daiichi College of Pharmaceutical Sciences, 22-1, Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan,2 Department of Cell Biology IBHV, Faculty of Life Sciences, University of Copenhagen, Gronnegaardsvej 7, 1870 Frederiksberg C, Denmark3

Received 11 December 2008/ Returned for modification 19 April 2009/ Accepted 8 May 2009

Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin, kanamycin, or amikacin. The antiserum also detected glutaraldehyde-fixed GM, and this enabled us to develop an immunocytochemical method for detecting the uptake of GM in rat kidney. Twelve hours after a single intravenous administration of GM, immunocytochemistry revealed that GM accumulated in the S1, S2, and S3 segments of the proximal tubules, as well as in the distal tubules and collecting ducts. By 12 h after injection, the drug was detected in cytoplasmic granules of the proximal tubule cells. However, early (1 h) after injection, drug accumulation was detected in the microvilli of these cells. The distal tubules and collecting ducts contained scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and the cytoplasm reacted strongly with GM. No staining occurred in the kidneys of saline-injected control rats. These results agree with previous studies showing that GM is endocytosed in the proximal tubules and accumulates in lysosomes. Additionally, our results show that GM also accumulates in the distal tubules and collecting ducts. This was achieved by systematically varying the pretreatment conditions—an approach necessary for detecting GM in different subcellular compartments. This approach should be useful for accurately detecting the uptake and toxicity of the antibiotic in different tissues.

* Corresponding author. Mailing address: Department of Applied Life Science, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan. Phone: 81 963 26 3111, ext. 5251. Fax: 81 963 23 1331. E-mail: fujiwara{at}life.sojo-u.ac.jp

{triangledown} Published ahead of print on 18 May 2009.

Antimicrobial Agents and Chemotherapy, August 2009, p. 3302-3307, Vol. 53, No. 8
0066-4804/09/$08.00+0     doi:10.1128/AAC.01627-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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