Ascorbic acid protects against the nephrotoxicity and apoptosis caused by colistin and affects its pharmacokinetics

Abstract

Objectives: The use of colistin in the treatment of life-threatening Gram-negative infections is associated with a high rate of nephrotoxicity that is dose limiting. This study aimed to examine the nephroprotective effect of ascorbic acid against colistin-induced nephrotoxicity.

Methods: Rats were treated intravenously twice daily with saline, colistin (cumulative dose of 36.5 mg/kg), a combination of ascorbic acid (50 or 200 mg/kg) and colistin, or ascorbic acid (200 mg/kg) over 7 days. Colistin-induced apoptosis was examined in rats over 5 days and in vitro using rat renal proximal tubular cells NRK-52E over 24 h with and without ascorbic acid. The effect of co-administered ascorbic acid on colistin pharmacokinetics was investigated.

Results: The 24 h urinary excretion of N-acetyl-β-D-glucosaminidase, a sensitive marker for tubular damage, was significantly lower (P < 0.0001) in the colistin/ascorbic acid 200 mg/kg group. Significant histological abnormalities (P < 0.01) were detected only in the kidneys of the colistin group, which also had the highest percentage (30.6 ± 7.8%) of apoptotic cells (P < 0.005). In the cell culture studies, the percentage of apoptotic cells was significantly higher in the presence of 0.1 mM colistin alone (51.8 ± 2.0%; P < 0.0001) than in the presence of ascorbic acid, which decreased the apoptotic effect in a concentration-dependent manner. Ascorbic acid (200 mg/kg) altered colistin pharmacokinetics, as the total body clearance decreased from 3.78 ± 0.36 mL/min/kg (colistin group) to 2.46 ± 0.57 mL/min/kg (P = 0.0024).

Conclusions: This is the first study demonstrating the protective effect of ascorbic acid against colistin-induced nephrotoxicity and tubular apoptosis. Co-administration of ascorbic acid has the potential to increase the therapeutic index of colistin.

Figure 1.

Mean (±SD) urinary excretion of NAG in the control, colistin, ascorbic acid 200 mg/kg, colistin/ascorbic acid 50 mg/kg and colistin/ascorbic acid 200 mg/kg groups. *Significantly different from the baseline for the same group and control group on the same day (P < 0.0001).

Figure 2.

Representative histological images of: (a) kidney of a control rat treated with saline for 7 days showing normal renal cortex and glomeruli (G); (b–d) range of changes in kidneys of rats administered colistin alone [(b) mild tubular damage with tubular casts (★), (c) acute tubular necrosis with preservation of glomeruli (G) and (d) acute cortical necrosis with infarction of tubules and glomeruli (★)]; (e) kidney of a rat treated with ascorbic acid 200 mg/kg alone showing mild focal tubular damage (★); and (f) kidney of a rat in the colistin/ascorbic acid 200 mg/kg group showing normal renal cortex (C) and medulla (M). No significant kidney damage seen in (a), (e) and (f). Original magnification was ×100 for (a–e) and ×20 for (f). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

Immunohistochemical images of apoptotic nuclei in sections of kidneys of the rats treated for 5 days with (a) saline, (b) colistin (cumulative dose of 20.5 mg/kg), (c) colistin and ascorbic acid 200 mg/kg and (d) ascorbic acid 200 mg/kg. Original magnification ×100.

Figure 4.

Percentage of TUNEL-positive apoptotic cells in rat proximal tubular cell line treated with 0.1 mM colistin alone or in the presence of various concentrations of ascorbic acid. *P < 0.005 compared with corresponding control. #P < 0.0001 compared with colistin alone.

Figure 5.

Plasma concentration (mean ± SD) of colistin versus time profiles after the first intravenous dose of colistin (0.5 mg/kg) either alone (open triangles) or preceded by 200 mg/kg ascorbic acid (filled squares) (n = 5 per group). In all rats of the colistin group the plasma colistin concentrations at 360 min were below the limit of quantification of the assay (0.125 μg/mL).

Figure 6.

Amount of unchanged colistin excreted in urine on days 1, 3, 5 and 6 following multiple intravenous doses of colistin for 7 days or colistin preceded by ascorbic acid 50 mg/kg or 200 mg/kg. *P < 0.05 as compared with the colistin group.