Giurgea-Marion L, Toubeau G, Laurent G, Heuson-Stiennon J A, Tulkens P M
Toxicol Appl Pharmacol. 1986 Nov;86(2):271-85. doi: 10.1016/0041-008x(86)90058-x.
Gentamicin, an aminoglycoside antibiotic, is known to accumulate within the kidney cortex and to elicit nephrotoxic reactions due to the necrosis of proximal tubules. Female Sprague-Dawley rats, treated for 9 days with gentamicin at a low dose (10 mg/kg ip, once a day), were used to determine the fate of the antibiotic within the proximal tubular cells and its effects on the functional properties of the lysosomes. The analysis of the lysosomes by isopyknic equilibration in sucrose gradient (density 1.10-1.24 g/ml) revealed that gentamicin remains associated with these organelles (marker enzymes sulfatase B and cathepsin B) throughout the treatment duration. Gentamicin treatment markedly decreased the buoyant density of the lysosomes. As was shown by electron microscopic examination of the subcellular fractions collected from the sucrose gradient, the shift of the lysosomes toward lower densities was a result of overloading with undegraded phospholipids (myelin-like figures). The effect of gentamicin on the functional properties of the lysosomes was examined by using horseradish peroxidase (HRP) as a marker of endocytic activity and of the processing by tubular cells of exogenous proteins. Treatment with gentamicin did not significantly modify the intracortical accumulation of HRP, which was estimated to be 2.2% of the amount injected. HRP was shown by isopyknic equilibration to be mostly associated with the lysosomes. This was confirmed by electron microscopic examination of proximal tubular cells after cytochemical demonstration of HRP with diaminobenzidine and H2O2. In rats not exposed to gentamicin, more than half of the lysosomes contained HRP activity. In animals treated with gentamicin, one-third of the lysosomes that retained a normal appearance exhibited HRP activity. In contrast, lysosomes overloaded with phospholipids (identified by the presence of myelin-like figures) were very seldom labeled with HRP activity. Taken altogether, the present observations suggest that the alterations induced by gentamicin treatment impair their ability to fuse with incoming endocytic vesicles.
庆大霉素是一种氨基糖苷类抗生素，已知其会在肾皮质内蓄积，并因近端肾小管坏死引发肾毒性反应。将雌性Sprague-Dawley大鼠以低剂量（10毫克/千克腹腔注射，每日一次）用庆大霉素治疗9天，用于确定该抗生素在近端肾小管细胞内的去向及其对溶酶体功能特性的影响。通过在蔗糖梯度（密度1.10 - 1.24克/毫升）中进行等密度平衡分析溶酶体，结果显示在整个治疗期间庆大霉素都与这些细胞器（标记酶硫酸酯酶B和组织蛋白酶B）相关联。庆大霉素治疗显著降低了溶酶体的漂浮密度。从蔗糖梯度收集的亚细胞组分的电子显微镜检查表明，溶酶体向较低密度的移动是由于未降解的磷脂过载（髓鞘样结构）所致。通过使用辣根过氧化物酶（HRP）作为内吞活性和肾小管细胞对外源蛋白质加工的标志物，研究了庆大霉素对溶酶体功能特性的影响。用庆大霉素治疗并未显著改变HRP在肾皮质内的蓄积，估计其蓄积量为注射量的2.2%。通过等密度平衡显示HRP主要与溶酶体相关联。在用二氨基联苯胺和H2O2对HRP进行细胞化学显示后，对近端肾小管细胞进行电子显微镜检查证实了这一点。在未接触庆大霉素的大鼠中，超过一半的溶酶体具有HRP活性。在用庆大霉素治疗的动物中，外观正常的溶酶体中有三分之一具有HRP活性。相比之下，因存在髓鞘样结构而被磷脂过载的溶酶体很少被HRP活性标记。综上所述，目前的观察结果表明，庆大霉素治疗引起的改变损害了它们与进入的内吞小泡融合的能力。
