Tubular Injury and Regeneration in the Rat Kidney Following Acute Exposure to Gentamicin: A Time-Course Study

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Abstract
Aminoglycoside antibiotics act as nephrotoxic drugs, inducing a lysosomal phospholipidosis and necrotic lesions essentially in convoluted proximal tubules. Previous studies have demonstrated that tubular injury caused by these compounds elicits a process of renal tissue repair (tubular regeneration) involving an increase of cell turnover in tubular epithelium. The present study was performed in order to: (i) achieve further insight into the temporal relationship between aminoglycoside-induced phospholipidosis, tubular necrosis, and tubular regeneration; and (ii) approach the control of tubular regeneration after nephrotoxin-induced insult. To investigate the latter point, we examined by immunocytochemistry the intrarenal distribution of epidermal growth factor (EGF) during tubular regeneration. Five groups of female Sprague-Dawley rats (n = 5) were treated for 4 days with gentamicin i.p. at a daily dose of 50 mg/kg delivered in 2 injections per day. Sham-treated animals (n = 5) received an equivalent amount of vehicle (0.9% NaCl) according to the same protocol. Groups of treated rats, and controls, were terminated 16 h (day 1),4 days, 7 days, 14 days, and 21 days after the end of gentamicin administration. One hour prior to necropsy, each animal was given an i.p. injection of 40 mg 5-bromo-2′-deoxyuridine (BrdU)for the immunocytochemical demonstration ofS-phase cells, using an anti-BrdU monoclonal antibody. Renal tissue was processed for light microscopy analysis, namely: a computer-aided morphometry of lysosomes in proximal tubular cells, a single-blind evaluation of gentamicin-induced tubular injury, the measurement of cell proliferation by immunocyto-chemical detection of BrdU-labeled nuclei, the demonstration of EGF-like immunoreactive material in renal tissue by using anti-rat EGF antiserum and immunogold-silver staining. As revealed by the morphometry of lysosomes in proximal tubular epithelium, the degree of gentamicin-induced phospholipidosis was maximum at day 1 (relative area occupied by lysosomes was increased 25-fold over mean control value) and declined thereafter. In contrast, tubular necrosis reached a peak 4 days after the end of drug administration. In proximal tubular epithelium, the stimulation of cell turnover associated with tubular regeneration showed a peak at day 7 (15-fold the mean control value). Tubular regeneration was also accompanied by mild interstitial hyperplasia. Three weeks after treatment with gentamicin, morphological evidence of drug-induced injury had disappeared due to the tissue repair process, except for the occasional presence of small hyperplastic foci in renal cortex interstitium. In both treated animals and controls, EGF immuno-reactivity as revealed by immunocytochemical staining was associated with distal tubules (renal cortex and outer medulla). However, during tubular necrosis and regeneration there was a transitory reduction of EGF immunolabeling in distal tubules. Thus, the incidence of EGF-positive tubules, as determined by morphometry, decreased markedly to reach a trough by day 4. Afterwards, the distribution of EGF immunoreactivity resumed a normal appearance in parallel with the repair of tubular lesions. Altogether, these data show that aminoglycoside-induced phospholipidosis, tubular necrosis, and regeneration actually occur in succession, and suggest a possible involvement of EGF as an endogenous mediator during renal tubular regeneration.