Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney
Light-Microscopic Immunocytochemistry for Gentamicin and Its Use for Studying Uptake of the Drug in Kidney
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DOI：
10.1128/aac.01627-08
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发表时间：
2009-08-01
影响因子：
4.9
通讯作者：
Larsson, Lars-Inge
中科院分区：
文献类型：
--
作者：
Fujiwara, Kunio;Shin, Masashi;Larsson, Lars-Inge
庆大霉素（GM）是一种广泛使用的抗生素，但具有肾毒性。我们用N-（γ -马来酰亚胺丁基氧基）琥珀酰亚胺偶联牛血清白蛋白制备了抗GM（抗GM）血清。抗血清对GM是单特异性的，不与类似物链霉素、妥布霉素、卡那霉素或阿米卡星发生交叉反应。抗血清还检测戊二醛固定的GM，这使我们能够开发一种免疫细胞化学方法来检测大鼠肾脏中GM的摄取。单次静脉注射GM 12小时后，免疫细胞化学显示GM在近端小管的S1、S2和S3段以及远端小管和集管中积累。注射后12 h，近端小管细胞细胞质颗粒中检测到药物。然而，注射后早期（1 h），在这些细胞的微绒毛中检测到药物积累。远端小管和集合管中有分散的肿胀细胞，使人联想到坏死细胞，其中细胞核和细胞质与GM强烈反应。注射盐水的对照大鼠肾脏未见染色。这些结果与先前的研究一致，表明GM在近端小管中被内吞并在溶酶体中积累。此外，我们的结果表明GM也在远端小管和集管中积累。这是通过系统地改变预处理条件来实现的，这是在不同亚细胞区室中检测转基因所必需的方法。这种方法应该有助于准确检测抗生素在不同组织中的摄取和毒性。
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.