Colistin-induced renal injury: Amelioration by thymoquinone in rats

INTRODUCTION

Colistin (polymyxin E) is an antibiotic useful in hospitalised patients in the treatment of multidrug-resistant (MDR) Gram-negative bacteria.^[1] It has re-emerged as a last-line option for life-threatening MDR infections in critically ill patients, despite its higher toxicity profile due to the lack of effective alternative choices. The most important and dose-limiting toxicity that occurs with the administration of colistin is renal damage.^[2]

According to Kidney Disease Improving Global Outcomes Guidelines (2012), acute kidney injury is defined as ‘an increase in serum creatinine >0.3 mg/dL over 48 h or 1.5 times the increase in creatinine over 7 days of colistin administration’. The histopathologic findings include tubular dilation, tubular epithelial cell vesiculation and necrosis, tubular casts, and inflammatory cell infiltration.^[1,3] The incidence of nephrotoxicity with colistin is reported to be about 27% in a recent systematic review and meta-analysis.^[4] Another meta-analysis of five randomised clinical trials defined the incidence of colistin-induced renal injury as 36.2%, and some other studies have reported up to 53.5%.^[5,6]

The exact mechanism of colistin-induced renal injury is unknown. An in vitro study electrophysiologically demonstrated that colistin increases transepithelial conduction and is directly toxic to the urothelium.^[7] Recent studies have reported that renal injury due to colistin could be due to oxidative stress, which causes cell death. Oxidative stress resulting in increased reactive oxygen species (ROS) and reduction of superoxide dismutase (SOD), endothelial nitric oxide synthase, and glutathione (GSH) levels may be involved in the pathogenesis of colistin-induced nephrotoxicity.^[8]

Thymoquinone (TQ) is an important bioactive ingredient in Nigella sativa (Kalonji) seeds. It is an oxidised form of quinone with the capability to quench other cellular toxins.^[9,10] TQ has anti-inflammatory, antioxidant, immunomodulatory, anti-tumour, neuroprotective, and nephroprotective effects.^[9-14] Due to its potential nephroprotective activity, TQ or N. sativa extract has been studied in drug-induced (cisplatin, doxorubicin, ifosfamide, and methotrexate) nephrotoxicity models.^[15] Rats and mice are frequently employed as models for studying drug-induced nephrotoxicity, including with colistin, with Sprague-Dawley rats being recognised for their sensitivity to ischemia and chemical-induced kidney injury.^[16,17]

Since nephrotoxicity is a major problem with colistin use, drugs to prevent or ameliorate the same are needed. The prevention of colistin renal injury would enable clinicians to complete the course of therapy, which is usually the last resort in critically ill patients. Hence, this study was planned to evaluate the nephroprotective potential of TQ against colistin-induced nephrotoxicity in Sprague-Dawley rats.

MATERIALS AND METHODS

This study was conducted with the approval of the Institutional Animal Ethics Committee. Approval number MAMC/IAEC/04/2017. Dated 04/04/2017. The study was done in adult albino rats (Sprague-Dawley) weighing 150– 250 g of either sex. Rats were randomly divided into five groups, each containing six animals [Table 1]. They were housed under standard conditions (temperature 27 ± 2°C, humidity 30–70%, and 12-h light/dark cycles) and fed with a standard pellet diet and water ad libitum. All the animals were acclimatised to laboratory conditions for 1 week before the experimental procedure.

The rats were administered injections of colistin 450,000 IU/kg/day (colistimethate sodium dissolved in dimethyl sulfoxide, equivalent to 36 mg/kg/day colistin) intramuscularly on the anterior aspect of the thigh for 7 days to induce nephrotoxicity.^[18,19] TQ was administered in test groups orally using a gavage needle, and the doses were chosen on the basis of previous studies.^[14,20]

Blood was collected on days 0 and 8. On day 0, it was collected from the tail vein with the help of a 23G needle, and on day 8, the rats were euthanized, and blood was collected by cardiac puncture. On each occasion, 2 mL of blood was collected, kept in a vacutainer, and processed for serum separation. The serum was stored at −20°C until further analysis. Twenty-four-hour urine of each rat was collected on days 0, 2, 4, and 6 for the analysis. Both kidneys were isolated. A part of one kidney was weighed and homogenised in phosphate buffer saline for oxidative stress and antioxidant assays. The other kidney was preserved in 10% formalin for histological analysis.

Blood urea was estimated by the enzymatic method. Serum and urine creatinine were estimated by the kinetic method. Oxidative stress was determined by estimating the malondialdehyde (MDA) level in renal tissue using the thiobarbituric acid reactive substances method. Antioxidant activity was determined by estimating reduced GSH level, catalase (CAT) level, and SOD level in renal tissue using the Randox kit. Total protein in renal tissue was estimated by the Pyrogallol red method.

Kidney samples were fixed in 10% formalin for 24 h. After cleaning the kidney tissues, they were fixed in paraffin and sectioned. The paraffin-sectioned tissue was stained with hematoxylin and eosin and was analysed under a microscope for tubular changes, cast, dilatation, necrosis, glomerular cast, necrosis, and haemorrhage.

RESULTS

Histopathology findings

Microscopically, there were no tubular or glomerular changes in the renal tissue of rats in the control groups (Groups I and III) [Figure 1a]. Renal tissues of the colistin-administered group (Group II) showed significant degenerative glomeruli, degenerative changes in tubules with tubular dilation, loss of tubular epithelium or necrosed epithelium and the presence of proteinaceous cast in the lumen in the colistin-treated group [Figure 1b]. These colistin-induced changes were minimal in the renal tissues of rats treated with thymoquinone at 10 mg/kg and 20 mg/kg (Groups IV and V) [Figure 1c].

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Figure 1:

(a) Microphotograph of hematoxylin and eosin (H&E)-stained slides (×20) reveals normal histomorphology of renal parenchyma without any degenerative changes in glomeruli and tubules and cleared urinary space in control groups. (b) Microphotograph of H&E-stained slides (×20) reveals degenerative glomerulus, degenerative changes in tubules with tubular dilation, loss of tubular epithelium or necrosed epithelium, presence of proteinaceous cast in lumen in colistin treated group. (c) Microphotograph of H&E-stained slides (×20) reveals normal histomorphology of renal parenchyma without any degenerative changes in glomeruli and tubules and normal urinary space and with few focal haemorrhages in thymoquinone treated groups. PCT: Proximal convoluted tubule, DCT: Distal convoluted tubule. Black arrows significance are mentioned in the image itself.

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DISCUSSION

Colistin is one of the last-line therapies for MDR Gram-negative organisms, while its dose limiting nephrotoxicity is a major concern.^[21] In this study, we evaluated the protective effects of thymoquinone against colistin-induced nephrotoxicity in rats.

We observed a higher level of MDA in the renal tissues of colistin-administered rats. This may be due to colistin-induced oxidative stress and ROSs-induced lipid peroxidation in renal tissue. In this study, we found that thymoquinone exhibited a protective effect against colistin-mediated oxidative stress in the renal tissues of rats. Thymoquinone at doses of 10 mg/kg/day and 20 mg/kg/day administered along with colistin has significantly attenuated MDA levels in renal tissues compared to the MDA levels in colistin-only-treated rats. Similarly, in a previous study in adult male Wistar rats, coadministration of thymoquinone (10 mg/kg/day) significantly reduced cisplatin-induced increases in MDA levels in renal tissue.^[22] In another study with a higher dose of thymoquinone (50 mg/kg), significant suppression of cadmium-induced elevation of MDA levels in renal tissue was observed.^[23] Similarly, in a study of acetaminophen-induced nephrotoxicity in rats, it was observed that in contrast to the acetaminophen-treated group, tissue MDA levels were found to be significantly lower in the thymoquinone plus acetaminophen-treated group.^[24]

Antioxidants, SOD, and CAT were found to be lower in the renal tissues of colistin-administered rats. This again indicates the oxidative stress induced by colistin on renal tissues. We observed in this study that SOD and CAT levels were normalised in rats treated with thymoquinone at 10 mg/kg and 20 mg/kg, indicating restoration of antioxidant activity in colistin injury. In a previous study, SOD and CAT levels were significantly elevated when thymoquinone was co-administered with cadmium, indicating the nephroprotective activity of thymoquinone through antioxidant activity.^[23] In another recent investigation, thymoquinone at a dose of 20 mg/kg successfully prevented the profound decrease in gentamicin-induced SOD activity in the renal tissue of albino rats.^[25] In a study of dioxin-induced nephrotoxicity in rats, similar elevations of levels of both SOD and CAT were observed in the thymoquinone-treated group.^[26] TQ administration before and during cisplatin therapy significantly ameliorated cisplatin-induced deterioration of SOD and CAT activities in another animal study.^[27] However, the restoration of GSH levels by thymoquinone treatment in colistin-administered rats was non-significant in this study.

In this study, colistin administration resulted in a significant increase in blood urea, serum creatinine, and urine creatinine. Thymoquinone administration attenuated colistin-induced elevations of blood urea levels at both doses and minimised serum creatinine elevation at 20 mg/kg dose. We observed inconsistent changes in serum creatinine in thymoquinone-treated rats at 10 mg/kg. In previous studies involving the investigation of ameliorative effects of thymoquinone in drugs or chemical-induced nephrotoxicity, all the above parameters were attenuated.^[22-28] We assume that rats in Group IV (thymoquinone 10 mg/kg) might have been more sensitive to colistin-induced renal injury, leading to variations in serum creatinine levels despite thymoquinone treatment. We observed attenuation of colistin-induced histopathological changes in the renal tissues of rats treated with thymoquinone at both doses.

From the available literature, it is evident that colistin exposure induces oxidative stress in renal tubular cells. Administration of potential antioxidants could protect the renal cells during colistin treatment. Several such compounds (ascorbate, tocopherol, curcumin, melatonin, cilastatin, etc.) have been studied preclinically for their potential protective activity against colistin-induced nephrotoxicity.^[29] In this study, we investigated whether thymoquinone possesses such protective activity and found that it shows some potential.

Limitations of the study: The study tested only two doses of thymoquinone (10 mg/kg and 20 mg/kg). Exploring a broader range of doses could help in identifying the optimal dose that provides maximum nephroprotection with minimal side effects. We could not find the cause for the significant increase in the blood urea level of normal control rats from the baseline.

CONCLUSION

Colistin is frequently associated with renal toxicity. The use of a nephroprotective agent may help mitigate this adverse effect. In this study, we observed that thymoquinone at both 10 mg/kg/day and 20 mg/kg/day doses significantly reduced oxidative stress and increased antioxidant activity in colistin-administered rats. We conclude that thymoquinone has the potential to attenuate colistin-induced nephrotoxicity in humans. More studies at different doses and durations are needed to corroborate the above finding.