The study was conducted in December 2016 at the Department of Research and Development, Saveetha University, Chennai, India, and ethical clearance was obtained from Department of Research and Development, Saveetha Medical College, Saveetha University (SU/BRULAC/RD/ 001/2014). Thirty male Wistar rats weighing about 160 g were selected randomly, obtained from Biomedical Research Unit and Laboratory Animal Centre (BRULAC), Saveetha University, Chennai and divided into five different groups (n=6) sample size was calculated by resource equation (E) where confidence interval (CI) was 95% with 80% power of the study. There was no attrition or death of animal in this study, therefore the attrition sample size calculation was neglected.

Animals were maintained at standard laboratory conditions in wood shavings lined cages with food and water ad libitum throughout the experimental protocol. Blood samples were collected by bleeding retro-orbital plexuses under isoflurane (USP) inhalation anaesthesia before and after the induction of contrast. Animals were allowed to stabilise for 24 hours before the administration of contrast. About 0.6 mL of contrast (Iohexol) was injected intraperitoneally for each animal dose was calculated on the weight of the animal in accordance with contrast dose given to humans during diagnostic and therauptic procedures per kg body weight.

Pre- and post-contrast blood samples were collected at 3 hours, 6 hours, 12 hours, 24 hours and 48 hours post contrast, centrifuged at 3000 rpm (37°C) for 15 minutes (Rotofix 32A, Hettich) to obtain clear supernatant serum and was stored at -20°C until further analysis. IL-18 before and after contrast was assessed by using sandwich Enzyme-Linked Immunosorbent Assay (ELISA) (RayBio, Rat IL-18 ELISA, Catalog #: ELR-IL18). The optical density (OD) was measured spectrophotometrically at a wavelength of 450±2 nm. The concentration of IL-18 in the sample was proportional to the OD values and was calculated by comparing the OD of the samples to the standard curve.

GraphPad Prism version 7.0 was used to calculate p-value and p<0.05 was considered to be statistically significant. Paired t-test was used to compare the data, before and after administration of contrast in rats. Data were presented as mean±SE.

In the current study, the serum IL-18 levels increased by 48% at 3 hours, 100% at 6 hours, 37% at 12 hours, and 18% at 24 hours. The percent increase in the IL-18 levels suggests an early ischaemia of the renal tubular tissue upto 24 hours of contrast induction. Decrease in IL-18 at 48 hours may indicate the auto regulatory mechanism which has countered the ischaemia [Table/Fig-1,2].

[Table/Fig-2]:

Changes in serum interleukin-18 of male Wistar rats following contrast medium.

*p-values<0.05* statistically significant for paired t-test.

Contrast Induced Nephropathy (CIN) is a growing concern among nephrologists, is one of the hospital-acquired diseases on rise, 10%-15% of cases in total Acute Kidney Injury (AKI) detected a year [3]. Use of contrast media brings about vasoconstriction of renal blood vessels and causes medullary hypoxia. In a prospective cohort study, CIN occurred in 11% of patients who underwent Computed Tomography (CT) by the administration of contrast media [4]. Use of conventional biomarkers like urea and creatinine shows increased levels in serum when there is a considerable loss of kidney function or damage [5]. The novel biomarkers like NGAL, KIM 1, Cystatin C are establishing their presence in diagnosing (AKI), but all of them have failed to detect AKI early. Novel biomarkers have several potential advantages which can detect the AKI [3]. Measurement of these novel biomarkers in serum may direct the nephrologists attention to ongoing kidney damage before measurable cause of kidney dysfunction occurs. Its early detection plays an important role in discontinuation of the contrast or drugs which are associated with loss of kidney function. IL-18 is one of the promising biomarker, which is establishing its presences in identifying AKI at the earliest. Generally an increase in serum IL-18 levels indicate ischaemic renal tissue injury, injury to heart, brain, inflammation and T cell medicated immunity as reported in earlier studies [6-8] which are in agreement with the present study as the contrast given during diagnostic procedure brings about renal vascular ischaemia. In a systematic review and meta-analysis, the incidence of dialysis and death was reported in contrast exposed group [9]. Earlier studies showed that the macrophage inflammation in the interstitium of the outer medulla occurs early ischaemic AKI in rat and mouse [10,11]. Ischaemic AKI is a life-threatening illness that continues to have a big mortality rate of 50% to 80% in intensive care setups in hospitals [12]. The severity of AKI in humans has been classified according to Risk, Injury, Failure, Loss of function and end-stage kidney Disease (RIFLE) criteria [13]. Lameire N and Hoste E, Hoste postulated that there are potent vascular and tubular factors, as well as inflammatory processes are involved in the pathogenesis of renal ischaemia [14]. Earlier studies demonstrated that macrophages are the mediators of AKI in mice and rats [14-16]. In a model of macrophage depletion in using liposomal clodronate, it was demonstrated that macrophages contribute to tissue damage during acute renal allograft rejection and ischaemic Chemo Attractants Protein-1 (MCP-1) reduced macrophage infiltration and AKI [15,16]. Interleukin-18 is a mediator of ischaemic AKI in mice and a recent study demonstrated that IL-18 derived primarily from cells of bone marrow origin contributes to the renal damage observed during ischaemic AKI in mice [17]. The macrophages are the source of IL-18 in ischaemic AKI [18]. IL-18 may be activated in the proximal tubules and directly contributed to tubular injury. The role of caspase-1 and IL-18 in hypoxia induced membrane injury of freshly isolated mouse proximal tubules invitro was studied also supports the present study [19]. In a study, induced nephrotoxic AKI in mice, found IL-18 to be an indicator of tubular injury is in agreement with the present study [20]. Less difference of serum IL-18 prior and after the induction of contrast, in Group 5 (48 hours) is may be due to adaptive response of the kidney to hypoxia due to auto regulation of blood flow which may probably have caused the depletion of serum IL-18 levels by chance.

Small sample size was the limitation of the study, however, further studies are warranted with large sample size in both humans and animals.

The elevated IL-18 levels in wistar rats’ model indicate contrast induced nephropathy at the initial stages. The study depicts the role of IL-18 as an early biomarker of kidney damage, which would help to diagnose the ischaemic renal tubular injury. However further studies are warranted with large sample size in both humans and animals.