A novel contrast-induced acute kidney injury model based on the 5/6-nephrectomy rat and nephrotoxicological evaluation of iohexol and iodixanol in vivo

Abstract

Contrast-induced acute kidney injury (CI-AKI) is a serious complication in patients after administration of iodinated contrast media. Proper animal models of CI-AKI can help understand the mechanisms involved and prevent the disorder. We used the 5/6-nephrectomized (NE) rat to develop a CI-AKI model and to evaluate differences in the toxic effects on the kidney between iohexol and iodixanol. We found that six weeks after ablative surgery was the preferred time to induce CI-AKI. We compared multiple pretreatment plans and found that dehydration for 48 hours before iodixanol (320, 10 mL/kg) administration was optimal to induce CI-AKI in the 5/6 NE rats. Compared with iodixanol, iohexol induced a significantly greater reduction in renal function, severe renal tissue damage, intrarenal hypoxia, and apoptotic tubular cells. Iohexol and iodixanol resulted in similarly marked increases in levels of inflammation and oxidative stress. In summary, the 5/6 NE rat combined with dehydration for 48 hours is a useful pretreatment to establish a novel and reliable CI-AKI model. Iohexol induced more severe CI-AKI than iodixanol in this model.

Figure 1

Study protocol.

Figure 2

BP increased gradually after 5/6 NE in rats and renal function was most stable 6 weeks after the ablative surgery. Changes in the levels of (a) BP, (b) Scr, (c) BUN, and (d) Ccr before (week 0) after (1, 2, 4, 6, 8, and 10 weeks) 5/6 NE in rats; n = 6.

Figure 3

A marked reduction in renal function was induced by dehydration for 48 hours before iodixanol administration in 5/6 NE rats. (a) BP levels were not different among the six groups before saline or ICM injection. Changes in the levels of (b) Scr, (c) BUN, and (d) Ccr before and after an intravenous injection of iodixanol or saline. Animals showed marked deterioration of renal function 24 hours after iodixanol injections in the 48 h dehydration + iodixanol group. In the saline group, the 48 h dehydration + saline group, the iodixanol group, the 24 h dehydration + iodixanol group, and the 48 h dehydration + low dose iodixanol group, there were no statistical changes of renal function between baseline and final levels (Figures 2(b)–2(d)). ^** P < 0.01 and ^* P < 0.05 end point versus baseline in every group; n = 5.

Figure 4

Renal morphologic injury was induced by combined dehydration for 48 hours with iodixanol (10 mL/kg) administration in 5/6 NE rats. ((a)–(c)) Representative photomicrographs of kidney injury from experimental rats treated with dehydration for 48 hours and iodixanol (10 mL/kg). (a) Representative photomicrographs of the most severe and pronounced alterations were observed in the renal corticomedullary boundary zone (the cortex and outer stripe of the outer medulla). (b) Representative photomicrographs of tubular dilation (black arrow), foamy degeneration (black arrow head), detachment of tubular cells (white arrow), and naked basement membranes (white arrow head) were observed. (c) Representative photomicrographs of proteinaceous casts (arrow) and inflammatory cell infiltration (arrow head) were observed. ((d), (e)) Representative photomicrographs of proliferation and hypertrophy (arrow) in tubular epithelial cells in 5/6 NE rats six weeks after the ablative surgery. (f) Representative photomicrographs of the renal morphology in a healthy rat. Original magnifications: ×100 ((a), (d)); ×400 ((b), (c), (e), (f)). Hematoxylin and eosin stain. Calibration bar = 20 μm.

Figure 5

Iohexol resulted in a more significant reduction of renal function compared with iodixanol in 5/6 NE rats. Changes in the levels of (a) BP, (b) Scr, (c) BUN, and (d) Ccr before and at 24 h, 48 h, and 72 h after an intravenous injection of saline, iohexol, or iodixanol. ^** P < 0.01 and ^* P < 0.05 versus the control group; n = 8.

Figure 6

Iohexol resulted in more severe morphological injury compared with iodixanol in 5/6 NE rats. (a) Representative photomicrographs of tubular cell injury (arrow) in rat kidney tissue sections of the control, iohexol, and iodixanol groups. (b) Quantitative analysis of histologic scoring. Original magnifications: ×100. Hematoxylin and eosin stain. Calibration bar = 20 μm. ^** P < 0.01 versus the control group; n = 8.

Figure 7

Iohexol and iodixanol resulted in similarly marked increases in inflammation and oxidative stress levels. (a) Representative photomicrographs of immunostaining for ED-1 and TNF-α in the renal sections of the control, iohexol, and iodixanol groups. Brown color indicates positive staining (arrow). (b) Quantitative analysis of ED-1-positive cells in the three groups by the percentage of ED-1-positive cells. (c) Quantitative analysis of the extent and intensity of TNF-α staining. (d) Representative photomicrographs of immunofluorescent labeling (green) for the redox product oxidized derivative of 8-OHdG in the renal sections of the control, iohexol, and iodixanol groups. Nuclei were stained with DAPI (blue). (e) Quantitative analysis of the extent and intensity of 8-OHdG-positive cells. (f) Serum TNF-α levels. (g) MDA concentrations in renal tissues. Original magnifications: ×200 (a); ×630 (d). Calibration bar = 20 μm. ^** P < 0.01 versus the control group; n = 8.

Figure 8

Urine volume, renal hypoxic conditions, and immunofluorescent labeling for TUNEL in the control, iohexol, and iodixanol groups. (a) Urine volume of rats in the three groups (recorded from the start of the bolus injection to 15 min or 30 min after the injection). The urine volume of rats in the iohexol group is more than that in the iodixanol group. (b) Quantitative analysis of the extent and intensity of renal Hypoxyprobe in the three groups. A little staining in the representative immunostaining of the control group, more staining in the representative immunostaining of the iohexol group compared with the control group, and less staining in the representative immunostaining of the iodixanol group. (c) Representative photomicrographs of Hypoxyprobe (anti-pimonidazole protein adducts antibody) immunostaining in renal sections 15 min and 30 min after a saline, iohexol, or iodixanol injection. Brown color indicates positive staining (arrow). Original magnifications: ×200. Calibration bar = 20 μm. ^** P < 0.01 versus the control group; n = 8. Data are means ± SD.

Figure 9

Immunofluorescent labeling for TUNEL in rat kidney tissue sections of the control, iohexol, and iodixanol groups. (a) Representative photomicrographs of TUNEL-positive cells in renal sections. Rat kidney tissue was stained for TUNEL (green). Nuclei were stained with DAPI (blue). Few TUNEL-positive cells in the representative immunofluorescent staining of the control group. A large number of TUNEL-positive cells in the representative immunofluorescent staining of iohexol group compared with control group, and less TUNEL-positive cells in the representative immunofluorescent staining of the iodixanol group. (b) Quantitative analysis of TUNEL-positive cells in the three groups by the percentage of TUNEL-positive cells. Original magnifications: ×630. Calibration bar = 20 μm. ^** P < 0.01 versus the control group; n = 8. Data are means ± SD.