N-Acetylcysteine Attenuates Iodine Contrast Agent-Induced Nephropathy in 5/6-Nephrectomized Rats

Aims: In the present study we tested the efficacy of N-acetylcysteine (NAC) to minimize nephrotoxic effects of iodine contrast agents in intact rats as well as in 5/6-nephrectomized (5/6-Nx) rats. Methods: Rats were allocated to a group of intact rats (n = 42) and a group of 5/6-Nx rats (n = 42). After 1 month of recovery from surgery, 5/6-Nx rats and intact (sham-operated) animals received either 6 ml/kg body weight (b.w.) meglumine ioxithalamate (Telebrix 350) or 6 ml/kg b.w. iohexol (Omnipaque 350) intravenously with or without pretreatment with 100 mg/kg b.w. NAC. Plasma and urinary concentrations of creatinine, sodium and protein in 24-hour urine collections were determined prior to and on days 1, 3 and 7 after drug administration. Results: In intact animals, contrast agents caused no significant changes in kidney function throughout the duration of the experiment. In contrast, significant increases in plasma creatinine Received: November 6, 2009 Accepted: March 23, 2010 Published online: May 26, 2010 Luděk Červenka, MD, PhD Department for Experimental Medicine Institute for Clinical and Experimental Medicine 1958/9 Vídeňská, CZ–140 00 Prague 4 (Czech Republic) Tel./Fax +420 2 4172 1666, E-Mail luce @ medicon.cz © 2010 S. Karger AG, Basel 1420–4096/10/0332–0149$26.00/0 Accessible online at: www.karger.com/kbr D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 12 :1 3 P M Šochman /Peregrin /Bürgelová /Kopkan / Kramer /Červenka Kidney Blood Press Res 2010;33:149–156 150 by hypovolemia [2, 3] . Previous studies have demonstrated that a decrease in renal tissue perfusion after administration of iodinated radiographic contrast media presents a serious risk to renal tubular integrity and function due to decreasing oxygen supply, especially in the outer renal medullary region. It has been proposed that this is the trigger mechanism mainly responsible for contrastmedium-induced acute renal failure [4–6] . Moreover, it has been shown that the impaired renal tissue perfusion is associated with increased production of vasoconstrictor agents including adenosine and endothelin, whereas the counter-regulatory action of endogenous vasodilatory compounds, primarily of nitric oxide (NO) and prostaglandins, is reduced [7–11] . Thus, it is likely that multiple interactions are involved in the development of CIN. Although in the majority of cases the application of contrast media produces no significant adverse reactions, it has been noted that CIN is more likely to develop in patients with preexisting renal impairment associated with concomitant diseases such as chronic inflammatory kidney disease, diabetes mellitus, hypertension or heart failure [12, 13] . The incidence of adverse reactions varies with the type of contrast media used. Based on these observations, nephrotoxicity of different contrast media and strategies to potentially protect against CIN have been evaluated [14–17] . These include administration of saline-bicarbonate solutions, diuretics or antioxidants [18–22] . From these latter compounds, sulfhydryl donors such as N-acetylcysteine (NAC) or thiosalicylic acid were shown to decrease the level of oxygen radicals and to exhibit a protective effect against vascular dysfunction, mostly by increasing NO bioavailability [23, 24] . In the earlier studies we reported that ischemia-reperfusion injury in dogs and humans with myocardial infarction can be successfully managed with NAC [25, 26] . Furthermore, we and others have shown beneficial effects of NAC also in patients with renal insufficiency [22, 24, 27] and ischemic renal failure [28] . Thus, in the present study we tested the potential of NAC to prevent or minimize the nephrotoxic effects of contrast agents in intact rats as well as in 5/6-nephrectomized (5/6Nx) rats – a model of chronic renal insufficiency [29, 30] . Since the differences in production of adverse effects between administration of the standard high-osmolar ionic monomer sodium-meglumine-ioxi thalamate contrast medium (Telebrix) and the low-osmolar non-ionic contrast medium iohexol (Omnipaque) are still controversial in clinical practice [17, 31–33] , we evaluated whether any differences in the development of CIN can be revealed when either Telebrix or Omnipaque were employed. Animals and Methods The present study was performed in male Wistar rats (Charles River Laboratories, Germany) in accordance with guidelines and practices established by the Institute for Clinical and Experimental Medicine Animal Care and Use Committee. All animals were housed in facilities accredited by the Czech Association for Accreditation of Laboratory Animal Care. Rats [initial body weight (b.w.) 230–270 g] were randomly allocated into an intact group (n = 42) and a 5/6-Nx group (n = 42). In this latter group, 5/6-nephrectomy was performed under anesthesia (tiletamine + zolazepam, Virbac SA, Carros, France, 8 mg/kg, and xylasine, Spofa, Prague, Czech Republic, 4 mg/kg i.m.) as described previously [28] . An abdominal midline incision was performed to expose the kidneys. The right kidney and both poles of the left kidney were removed surgically in order to remove 5/6 of renal parenchyma as estimated according to kidney weight. The abdominal wall and the skin were sutured. The animals were then allowed to recover from surgery for 1 month and to adapt to the 5/6-nephrectomy. In sham-operated control rats only an abdominal midline incision was performed. After 1 month of recovery from surgery, 24-hour urine collections in metabolic cages were performed and blood samples were taken from the tail vein to determine basal levels of creatinine and sodium in plasma and urine as well as urinary protein [30, 34] . On the day before the contrast agent was administered, drinking water with the addition of furosemide (40 mg/l) and potassium chloride (500 mg/l) was given for 12 h. The animals were then deprived of water for 12 h to mimic a hypovolemic state. On the following day, in intact and 5/6-Nx rats a cannula (PE50) was inserted into the femoral vein under the usual anesthesia. The following solutions were given intravenously via the implanted femoral vein catheter: 3.5 ml of sterile saline or 3.5 ml of saline with NAC 100 mg/kg (b.w) (ACC Injekt; Hexal AG, Holzkirchen, Germany). Additional groups of intact and 5/6-Nx rats received saline or saline + NAC followed by an intravenous injection of either 6 ml/kg b.w. high-osmolar (1,860 mosm/l) ionic monomer sodiummeglumine-ioxithalamate (MIO, Telebrix 350; Guerbet, Roissy, France) or 6 ml/kg b.w. low-osmolar (780 mosm/l) non-ionic iohexol (IOH, Omnipaque 350; Amersham Health, Cork, Ireland). As iodinated contrast agent, one of the most commonly used ionic contrast medium (MIO) and one of the currently used nonionic medium (IOH) were selected. The dose of contrast medium exceeded the average routinely applied dose to possibly aggravate the renal insult. Based on the infused agents, the animals were allocated into the following six groups of intact rats: (1) control group (n = 7), (2) NAC group (n = 7), (3) MIO group (n = 7), (4) MIO + NAC group (n = 7), (5) IOH group (n = 7), and (6) IOH + NAC group (n = 7) and into the following six groups of 5/6-Nx rats: (1) control group (n = 6), (2) NAC group (n = 6), (3) MIO group (n = 8), (4) MIO + NAC group (n = 8), (5) IOH group (n = 7), and (6) IOH + NAC group (n = 7). 24-hour urine collections and blood sampling were repeatedly performed before and after drug administration on days 1, 3, and 7. The creatinine concentration in plasma and urine samples was determined by a commercial kinetic colorimetric assay (Roche Diagnostics Corp., Indianapolis, Ind., USA) and creatinine clearance was calculated to estimate glomerular filtration rate which was expressed per gram of kidney weight. Protein concentration D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /4 /2 01 7 6: 12 :1 3 P M Contrast Agent Nephropathy in Rats Kidney Blood Press Res 2010;33:149–156 151 in urine samples was measured by the Biuret method using a commercially available kit (Lachema, Brno, Czech Republic). The concentration of sodium was determined by flame photometry. Statistical Analysis Results are expressed as means 8 SEM. Statistical comparisons within groups were conducted by the use of ANOVA for repeated measurements, followed by Newman-Keuls test. Two-way ANOVA followed by post-hoc test was used for comparisons between groups for each time point of the study. Statistical significance is defined at a value of p ! 0.05.