Colistin has been out of trade since 1970, however, it is possible to see more of this drug due to the increasing gram-negative bacterial infections to multiple drug resistance.1–3 It is used in the treatment of infections caused by carbapenem-resistant Enterobacteria, Acinetobacter baumannii, Pseudomonas aeruginosa.4 Renal failure occurs in 20–60% of colistin users.5 The studies on antioxidant molecules such as hesperidin,6 black garlic extract,7 curcumin,8 N-acetylcysteine,9 ascorbic acid,10 melatonin,11 lycopene,12 luteolin,13 grape seed extract14 found them to be effective in the prevention of colistin-induced nephropathy.

Magnesium, an antioxidant macromineral, also has the advantages of being low in cost and easy to obtain. Various agents such as contrast agents and chemotrophic agents reduce nephrotoxicity with these antioxidant effects.15–18

GSH levels, an endogenous antioxidant molecule, are decreased with colistin treatment8,12 and increased levels of MDA; which are indicative of lipid peroxidation and increased in the case of oxidative damage.19 Therefore, we examined whether magnesium had an antioxidant effect by analyzing different analytes such as GSH and MDA. The effects of colistin on the kidney have been evaluated histopathologically in some studies. The SQS system, which is the sum of tubular damage in renal tissues and the percentage of tissue damage, was used in these studies.9,13 We also planned to evaluate the colistin-induced renal tissue damage rate using this scoring system.

This study aimed to determine whether magnesium decreases nephrotoxicity caused by colistin, which found to have an antioxidant effect in various drug studies of contrast and chemotherapeutic agents.

In the present study, the sample size was calculated based on the “resource equation” proposed by Arifin and Zahiruddin. The sample size in each group should be a minimum of 10/k+1 and a maximum of 20/k+1 (k the number of groups) in this equality. Thereby, we calculated the sample size of each group to be a minimum of four, and a maximum of six.23

Data were analyzed using R Version 3.6.0 (www.r-project.org). Variables were described as mean±standard deviation or median (interquartile range). The two-way repeated-measures ANOVA was performed to assess the groups’ effects and time on urea and creatinine values in rats. The box-plot method showed that there were no extreme outliers. Shapiro–Wilk's test of normality was used to analyze the distribution of data (p>0.05). Levene's test of homogeneity of variances found variances to be homogeneous (p>0.05). A Bonferroni adjustment was used for the interaction and main effects. Because the plasma MDA was not normally distributed, Kruskal–Wallis and Wilcoxon tests were used to compare groups and time effects, respectively. After the Kruskal–Wallis test results were significant, the Conover-Iman posthoc test with Bonferroni adjustment was used for multiple comparisons. One way ANOVA and Welch tests were used to compare groups in terms of GSH and MDA levels. Tukey HSD and Games-Howell posthoc tests were used for multiple comparisons according to these parameters at T1 time. Mann–Whitney-U test was used to compare groups according to the grade of renal histology, renal affected area score, and SQS. A p-value of less than 0.05 was considered as significant.

Colistin has been increasingly used in recent times and is an important nephrotoxic. The present study investigated if the toxic effects of colistin on the kidney decreased when it is combined with the antioxidant mineral Mg. We found that the colistin+Mg group had lower urea and creatinine values and histopathological damage than the Mg group. Additionally, the colistin group had higher plasma MDA and GSH erythrocyte values than the other groups.

Colistin is filtered from glomeruli and secreted from tubules in the kidney.4 Its nephrotoxic effects are observed in the clinic as oliguria, hematuria, acute tubular necrosis.24,25 There are studies showing that oxidative stress plays a role in the formation of nephrotoxicity.9,12,14,19 It causes tubular lysis by causing the entrance of anion, cation, and water through tubular epithelial cell damage and affects in a similar way to bacteriostatic effect. Colistin acts by displacing divalent cations Mg2+ and Ca2+ to disrupt the stability of the bacterial outer membrane. Likewise, it increases the permeability in tubules.26 Many reactive oxygen species that are formed in mitochondria result apoptosis and renal dysfunction. Ozkan et al. determined colistin-related in apoptosis to occur with the activation of caspase I and III.27,28 Many antioxidants have been studied to reduce the nephrotoxicity of colistin.6,10,11,13

Magnesium is a mineral with vital functions such as transport function, enzyme and signaling pathways, energy metabolism, and nucleic acid synthesis.29 It is an antioxidant and anti-inflammatory molecule. It facilitates the intracellular calcium accumulation, which we mentioned in the colistin nephrotoxicity, by inhibiting the l-type calcium channels.30 Magnesium was also found to decrease drug-related cardiotoxicity, apoptosis, and necrosis.31 Moreover, various studies found this molecule to have antioxidant effects. Administration of magnesium sulfate which has an antioxidant effect and dehydration in contrast-associated nephropathy reduced the risk of nephropathy.15 Another study indicated that contrast nephropathy was more common in patients with low magnesium levels.16 There are studies that show the effect of magnesium on nephropathy caused by some chemotherapeutic agents apart from contrast nephropathy. In their experimental study, Saito et al. designed cisplatin nephrotoxicity and showed that giving magnesium to the rats decreased nephrotoxicity by reducing the cisplatin accumulation in the kidney.17 In a study conducted with people who received cisplatin chemotherapy, the supplementation of magnesium to hydration reduced cisplatin nephrotoxicity.18 In another study performed on cyclosporine-treated mice, magnesium supplementation had a renal protective effect by increasing nitric oxide synthase activity in the renal tissue.32

Because there is no study examining the supplementation of Mg with colistin, we referenced other studies for the Mg sulfate doses. In their experiment to reduce the cardiotoxic effects of doxorubicin, Khalilzadeh and colleagues gave rats 120mg/kg for two weeks and magnesium sulfate for three days a week as an IP and found that cardiotoxicity decreased.33 In another study, nephrotoxicity was reduced by administering 40mg/kg Mg sulfate before cisplatin once a week for three weeks in rats given cisplatin.17 However, higher magnesium doses were used in studies that assessed the effects of magnesium in rats with ischemic stroke and in Alzheimer's rat model.34,35

In a review by Heybeli et al. assessing the studies that involved colistin nephrotoxicity in rats, it was observed that colistin was given at 300,000–480,000IU/kg/day or 15–18mg/kg/day or 36–84mg/kg cumulative doses because five of the eleven studies gave 300,000IU/kg/day. In their article which was conducted in 2019 and included views of many institutions on the use of polymyxin, Tsuji et al. suggested 9,000,000IU CMS maintenance dose to be systemically administered every 12–24h after the 9,000,000IU CMS loading dose and this was significantly lower than the dose (300,000IU/kg/day) we administered in the present study.36 The drug was provided for seven days in the majority of the studies.37 In their study in which they administered 300,000IU/day CMS for six days, Ozkan and Ozyılmaz determined that colistin caused a significant level of toxicity in the tissue and biochemical levels of the rats.28,38 Based on this study, we administered colistin at a dose of 300,000IU/kg/day, and for seven days.

We found that urea value decreased significantly in the Mg and Mg+colistin group, creatinine value increased in the colistin and Mg groups and there was no significant increase in the control and Mg+colistin groups. Although the decrease in the urea value of the Mg and Mg+colistin group supported the renal protective effect of magnesium, it was also interesting to find that creatinine levels increased in the Mg group. Cheungpasitporn et al. showed that both hypomagnesemia and hypermagnesemia were associated with an increased risk of acute kidney disease. They considered that overstimulated vasodilatation and vasoconstriction may be effective.39 Hypermagnesemia may occur in the group that was administered only magnesium and may cause creatinine value to be higher than the baseline. The absence of histopathological changes in this group receiving magnesium alone may support the idea that a prerenal event may trigger an increase in creatinine. Severe hypomagnesemia was detected in the colistin-treated groups in two studies in patients who were given colistin in the neonatal intensive care unit.40,41 Hypomagnesemia in the colistin group may lead to increased oxidative stress leading to renal toxicity. Reduction of renal toxicity by adding magnesium to colistin may be a result of regulating hypomagnesemia with colistin.

MDA causes serious damage to the cell membrane structure.42 GSH is a tripeptide that reacts with hydrogen peroxide and organic acids and shows an antioxidant effec.43 In the study by Edrees et al., concurrent administration of curcumin with colistin led to a decrease in the MDA level and an increase in GSH level in renal tissue. Lee, Özkan, and Ghlissi found that some antioxidant molecules used in their studies decreased the MDA levels compared to the colistin group.7,14,44 Ozyilmaz et al. found no change in the MDA level in plasma in the group given N-acetylcysteine together with colistin.38 We found that plasma MDA levels were significantly increased in the colistin group compared to the beginning of the experiment and compared to the other groups and decreased in the Mg and Mg+colistin groups. While the colistin group had significantly lower erythrocyte GSH levels than the other groups, Mg+colistin group was higher than the colistin group, however; it was not statistically significant. Although renal GSH levels were the lowest in the colistin group renal MDA levels were the highest in the colistin group, but it was not statistically significant.

We found tubular injury, renal involvement area, and SQS were found to be high in the colistin-treated group, while the histopathological changes were significantly decreased in the magnesium-treated and colistin-treated rats. This was in line with previous studies.9,10,13

There were some limitations to our study. We did not study the effects of different doses of magnesium and colistin. The fact that we did not study serum magnesium levels at the beginning and end of the study was another limitation of this study. However, this study was the first to examine colistin nephrotoxicity with magnesium which is an easily accessed, inexpensive molecule with many functions in the body. We believe that comprehensive and more detailed studies should be conducted to investigate the relationship between magnesium and colistin nephrotoxicity.

The study was approved by the Ethics Advisory Committee Selçuk University Experimental Medicine Application and Research Center (IRB approval number: 2019-46). All procedures performed in animal studies were in accordance with the ethical standards of the institution or practice in which the studies were conducted.

The authors have declared that no conflict of interest exists.