Pharmacological investigation of nephrotoxicity of colistin: the potential for nephro-protection afforded by co-administration of antioxidants
thesisposted on 06.02.2017, 05:31 by Yousef, Jumana
Colistin, a cationic polypeptide antibiotic that belongs to the polymyxin family, has been revived as a last-line therapy for life-threatening infections over the last decade due to resistance to all current antibiotics. To minimise resistance to colistin, there is an urgent need to optimise its dosage regimens. However, colistin can cause nephrotoxicity which is a major dose-limiting factor. The nephrotoxicity rate in patients receiving currently recommended dosage regimens is high (up to 50%). Therefore, understanding the mechanism of colistin-induced nephrotoxicity becomes crucial to optimise for clinical use. In this thesis, a rat model was developed to investigate colistin-induced nephrotoxicity. The nephrotoxicity was assessed by measuring the plasma creatinine concentration and urinary N-acetyl-β-D-glucosaminidase (NAG) excretion and histological examination. A range of intravenous dosage regimens were examined. It was revealed that colistin-induced nephrotoxicity was dose- and time-dependent. The dosage regimen used to induce reproducible nephrotoxicity was identified (a cumulative dose of 36.5 mg/kg administered over 7 days). Rats treated with this dosage regimen showed a significant increase (p < 0.05) in the plasma creatinine concentration and urinary NAG excretion compared to the baseline and the control group. Histological examination results showed renal tubules injuries including tubular dilatation, casts and necrosis. This rat model was employed to examine the potential protective effect of co-administration of antioxidants, melatonin and ascorbic acid, against colistin-induced nephrotoxicity. Rats treated with both colistin and melatonin (10 mg/kg/day) or ascorbic acid (400 mg/kg/day) showed significant decrease in urinary NAG excretion (p < 0.05) compared to those treated with colistin alone. Additionally, kidney tissues of rats treated with both colistin and melatonin or ascorbic acid showed normal morphology under histological examination. Colistin-induced apoptosis was examined in rats after 5 days of treatment and in vitro using rat renal proximal tubular cells (NRK-52E) over 24 h with and without melatonin or ascorbic acid. With immunohistochemical examination, it was demonstrated that colistin-induced apoptosis in vivo and in v itro with apoptotic cell percentages of 30.6 ± 7.8% and 51.8 ± 2.0%, respectively. Melatonin and ascorbic acid protected against colistin-induced apoptosis. In addition, the effect of co-administration of antioxidants on colistin pharmacokinetics was also investigated. Melatonin and ascorbic acid altered colistin pharmacokinetics; the total body clearances in colistin/antioxidant-treated groups were lower than in the colistin group. Microarrays were employed to examine the regulation of gene expressions associated with colistin-induced nephrotoxicity. Administration of colistin resulted in up- and down-regulation of gene expressions which play important roles in cell growth/death, cellular functions, inflammation, immune response, angiogenesis and apoptosis. Co-administration of antioxidants, melatonin and ascorbic acid, prevented the alteration of the gene expressions associated with the nephrotoxicity of colistin. Additionally, these antioxidants also altered gene expressions that play important roles in cellular defence system against oxidative stress. Two metabolic pathways were identified in both in vivo and in vitro colistin groups: complement and coagulation cascades and the mitogen activated protein kinase (MAPK) signaling pathways. Both pathways play key roles in regulation of various cellular actions, such as proliferation, differentiation, inflammation and apoptosis. This thesis is the first study to investigate the nephrotoxicity associated with colistin treatment in a validated rat model, examine the potential nephroprotective effect of co-administration of antioxidants, and explore the mechanisms of colistin-induced nephrotoxicity. Importantly, this is the first study that highlights the potential of co-administering an antioxidant to widen the therapeutic window of this very important last-line antibiotic.