Acute tubular necrosis induced by aminoglycoside antibiotics and various other nephrotoxins is followed by a regenerative process which leads to the restoration of damaged tubules. Several lines of evidence indicate that tubular regeneration is mediated by polypeptide growth factors such as epidermal growth factor (EGF). Previous studies devoted to cisplatin nephrotoxicity have shown that this agent causes tubular cystic degeneration possibly related to an impairment of renal tissue repair. Thus, we examined on a comparative basis the time course of the regenerative response subsequent to tubular damage induced by tobramycin or cisplatin, particular attention being paid to renal EGF and its receptor. Female Sprague-Dawley rats (160-180 g body weight) were treated during 4 consecutive days with daily doses of 200 mg/kg tobramycin i.p. (BID) or 2 mg/kg cisplatin (once a day). Sham-treated rats were given 0.9% NaCl i.p. following the same protocol. Groups of experimental animals (n = 5-10) were terminated at increasing time intervals (1, 4, 7, 14, 21, 60 days) after cessation of treatment. One hour prior to sacrifice, each individual received i.p. 200 mg/kg 5-bromo-2'-deoxyuridine (BrdU) for the immunohistochemical demonstration of cell proliferation. Blood was collected at the time of sacrifice in order to assess glomerular filtration rate by measuring serum creatinine and BUN levels. Kidneys were analyzed with respect to total EGF determined by RIA in renal tissue homogenates, and soluble EGF was assayed in extracts prepared by centrifugation. Renal tissue was processed for the immunohistochemical detection of S-phase cells, of EGF, of EGF receptors, and of the intermediate filament vimentin, the latter being used as a marker of epithelium dedifferentiation. In absence of nephrotoxic alterations, EGF was immunolocalized in distal tubules, whereas EGF receptor immunostaining was seen in proximal tubules cells. Vimentin immunostaining was confined to glomeruli and blood vessels. Tobramycin and cisplatin caused acute tubular necrosis in proximal convoluted tubules and proximal straight tubules, respectively. Tissue damage was accompanied by renal dysfunction reflected by an elevation of serum creatinine and BUN levels. Tubular necrosis was followed by a proliferative response indicative of tubular regeneration. Regenerative hyperplasia was associated with a reduction of total immunoreactive EGF due to a decrease of tissue-bound proEGF. Tubules undergoing regenerative repair were characterized by a disappearance of EGF receptors and the presence of immunoreactive vimentin. In tobramycin-treated rats, renal dysfunction lasted for 4-7 days and was fully reversible, as indicated by the return of serum markers to normal values.
Normal rat kidney (NRK-52E) cells, an established cell line of renal origin, were used as a bioassay system to reveal a possible mitogenic activity in tissue extracts prepared from kidneys undergoing tubular regeneration. Acute tubular injury was induced in female Wistar rats by a 4-day treatment with gentamicin at daily doses of 50 or 100 mg/kg twice daily. Animals were killed either 1 or 4 days after cessation of gentamicin administration. Proximal tubule regeneration in treated animals was confirmed by morphological examination after proliferating cell nuclear antigen staining. Tissue extracts from regenerating kidneys stimulated DNA synthesis in growth-arrested cells to a higher extent than extracts from intact kidneys. Sera from treated and control animals showed no difference with respect to mitogenic activity. The mitogenic effect of tissue extracts was sensitive to the tyrosine kinase inhibitor tyrphostin A46. The cell proliferative response to regenerating kidney extracts, but not that to intact kidney extracts, was partly suppressed by the addition of anti-insulin-like growth factor I (anti-IGF-I) antiserum. These data indicate that nephrogenic repair entails an elevation of biologically active IGF-I in kidney tissue.
Abstract— Survival curves were obtained for DNA repair‐deficient strains of Escherichia coli K‐12 (polA1, uvrB5, and recA56) exposed to near‐ultraviolet radiation [black light (BL)] in the presence of the DNA cross‐linking agent 8‐methoxypsoralen (8‐MOP) or in the presence of photosensitizers forming primarily monoadducts with DNA [angelicin; 3‐carbethoxypsoralen (3‐CPs); 5,7‐dimethoxycoumarin (DMC)], and after exposure to blue light (BluL) in the presence of 8‐MOP or 3‐CPs. An interpretation of these data suggests that DNA polymerase I is required for the major pathway of monoadduct repair, but appears to play little or no role in the repair of 8‐MOP cross‐links. The uvrB and recA strains were very sensitive, both to the cross‐linking agent and to the monoadduct formers. The markedly different results for BL plus DMC or 3‐CPs compared to angelicin suggests that the DMC and 3‐CPs monoadducts are repaired by a different mechanism than are the angelicin monoadducts, or else DMC and 3‐CPs undergo photochemical side reactions that produce DNA lesions other than the expected monoadducts. From photochemical evidence, we predicted that fewer 8‐MOP monoadducts should be converted to cross‐links by BluL vs BL; this appears to be the case. 3‐CPs showed dramatically different biological results when irradiated with BL vs BluL, suggesting that 3‐CPs may form more types of photoproducts than the expected monoadducts; BluL, however, appears to favor monoadduct formation.
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