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Am J Physiol Renal Physiol 295: F741-F748, 2008. First published June 25, 2008; doi:10.1152/ajprenal.90244.2008
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{alpha}2-Adrenergic agonists protect against radiocontrast-induced nephropathy in mice

F. T. Billings, IV,1 Sean W. C. Chen,1 Mihwa Kim,1 Sang Won Park,1 Joseph H. Song,1 Shuang Wang,2 Joseph Herman,4 Vivette D'Agati,3 and H. Thomas Lee1

Departments of 1Anesthesiology, 2Biostatistics, and 3Pathology, College of Physicians and Surgeons, Columbia University, New York, New York; and 4Division of Clinical Pharmacology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Submitted 10 April 2008 ; accepted in final form 23 June 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Radiocontrast nephropathy (RCN) is a common clinical problem for which there is no effective therapy. Utilizing a murine model, we tested the hypothesis that {alpha}2-adrenergic receptor agonists (clonidine and dexmedetomidine) protect against RCN induced with iohexol (a nonionic low-osmolar radiocontrast). C57BL/6 mice were pretreated with saline, clonidine, or dexmedetomidine before induction of RCN. Some mice were pretreated with yohimbine (a selective {alpha}2-receptor antagonist) before saline, clonidine, or dexmedetomidine administration. {alpha}2-Agonist-treated mice had reduced plasma creatinine, renal tubular necrosis, renal apoptosis, and renal cortical proximal tubule vacuolization 24 h after iohexol injection. Yohimbine reversed the protective effects of clonidine and dexmedetomidine pretreatment. Injection of iohexol resulted in a rapid (~90 min) fall of renal outer medullary blood flow. Clonidine and dexmedetomidine pretreatment significantly attenuated this perfusion decrease without changing systemic blood pressure. To determine whether proximal tubular {alpha}2-adrenergic receptors mediate the cytoprotective effects, we treated cultured human proximal tubule (HK-2) cells and rat pulmonary microvascular endothelial cells with iohexol after vehicle, clonidine, or dexmedetomidine pretreatment. Iohexol caused a direct dose-dependent reduction of HK-2 and rat pulmonary microvascular endothelial cell viability, but {alpha}2-agonists failed to preserve the viability of both cell types. We conclude that {alpha}2-adrenergic receptor agonists protect mice against RCN by preserving outer medullary renal blood flow. As {alpha}2-agonists are widely utilized during the perioperative period, our findings may have significant clinical relevance to improving outcomes following radiocontrast exposure.

acute renal failure; iohexol; clonidine; dexmedetomidine; yohimbine; HK-2 cells; medullary ischemia

ARTERIOGRAPHY, ANGIOCARDIOGRAPHY, and contrast-enhanced CT scans account for >3,000,000 iodinated radiocontrast exposures each year in the United States (15). Renal dysfunction secondary to radiocontrast administration remains prevalent and debilitating (3). Radiocontrast nephropathy (RCN) remains the third most common cause of inpatient acute renal failure, behind ischemia-reperfusion injury and nephrotoxic medication administration (23). The diagnosis of RCN confers a 5.5-fold increase in hospital mortality (18, 29), may necessitate hemodialysis (7), and is associated with an increased length of hospital stay and incidence of myocardial infarction (25).

Although the pathogenesis of RCN remains incompletely understood, tubular hypoxic injury, due to a reduction of renal medullary blood flow, and direct tubular cytotoxicity play a substantial role (8, 12, 31). The risk of developing nephropathy after radiocontrast exposure may be as high as 50%, depending on numerous risk factors (21). Preexisting renal dysfunction and dehydration are the most predictive contributors to RCN, whereas volume of contrast exposure, contrast osmolality, congestive heart failure, diabetes, anemia, and advanced age also increase risk (2, 3). Despite the exploration of numerous prophylactic regimens (N-acetylcysteine, theophylline, sodium bicarbonate, dopamine, fenoldopam, and calcium channel blockers) and attempts at developing less toxic (lower osmolar) contrast agents, isotonic intravenous hydration remains the only proven RCN prophylaxis (3, 22, 30).

{alpha}2-Adrenergic agonists have diuretic (26) and sympatholytic (20) effects. Having noted the corticomedullary ischemic pathogenesis of RCN and the decreased systemic vascular resistance and diuretic properties of {alpha}2-agonism, we hypothesized that exogenously administered {alpha}2-agonists would preserve outer medullary renal blood flow and protect against renal dysfunction after iodinated radiocontrast exposure. To test this hypothesis, we utilized in vivo and in vitro models of RCN.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Male C57BL/6 mice (25 g body wt) were purchased from Harlan Laboratories (Indianapolis, IN). The Columbia University Institutional Animal Care and Use Committee approved the animal care protocol for the experiments performed in this study.

Murine model of RCN. RCN was induced in mice as described previously (16). After overnight (16 h) water deprivation and prior inhibition of prostaglandin and nitric oxide synthesis, mice were injected subcutaneously with the low-osmolar monomeric iodinated radiocontrast medium iohexol (Omnipaque, 1.5 g iodine/kg). For inhibition of cyclooxygenase and nitric oxide synthase, mice were injected with indomethacin (10 mg/kg sc, dissolved in dimethylsulfoxide) and NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg sc, dissolved in 0.9% saline), respectively, 15 min before iohexol injection. This model reliably produces nephropathy following radiocontrast injection and has been previously validated in mice and rats (6, 16). Animals were then given access to food and water and killed 24 h later for serum creatinine (Cr) determination and kidney removal. Sham mice received subcutaneous injections of saline, instead of iohexol, after indomethacin and L-NAME injection and 16 h of water deprivation.

To determine whether {alpha}2-adrenergic receptor agonists protect against murine RCN, we assigned mice to the following treatment groups: 1) 100 µl sc bolus + 1 µl/h sc infusion of saline, 2) 5 or 10 µg/kg sc bolus of clonidine, 3) 5 or 10 µg/kg sc bolus + 5 or 10 µg·kg–1·h–1 sc infusion of clonidine, 4) 3 µg/kg sc bolus of dexmedetomidine, and 5) 3 µg/kg sc bolus + 2 or 4 µg·kg–1·h–1 sc infusion of dexmedetomidine. Subcutaneous infusion (1 µl/h) of drugs or saline was achieved by micro-osmotic pumps (model 1003D, Alzet, Cupertino, CA) implanted 16 h before iohexol injection. To determine whether blockade of {alpha}2-receptors prevents dexmedetomidine- or clonidine-mediated protection against RCN, some mice were injected with 0.1 mg/kg yohimbine (a selective {alpha}2-adrenergic receptor antagonist) after mini-osmotic pump placement and before bolus injection of saline, clonidine, or dexmedetomidine.

Assessment of nephropathy following iohexol injection. Using the colorimetric method based on the Jaffe reaction (11) and assessing kidneys for necrosis, apoptosis, and cortical vacuolization (osmotic nephrosis), we evaluated renal function 24 h after radiocontrast injection by determining plasma Cr concentration.

For histological light-microscopic preparations, we selected kidneys from six randomly selected mice from the saline RCN, clonidine (10 µg·kg–1·h–1 + 10 µg/kg) RCN, and dexmedetomidine (4 µg·kg–1·h–1 + 3 µg/kg) RCN groups 24 h after iohexol injection (after blood collection). Kidneys were bisected along their long axis and fixed in 10% formalin for 24 h. After automated dehydration through a graded alcohol series, transverse kidney specimens were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. An experienced renal pathologist, blinded to the animal treatment group, assessed proximal tubular necrosis, apoptosis, and cortical tubule vacuolization. Tubular necrosis was quantified as the number of necrotic tubules per field (x200). At least 25–30 tubules were counted in each field, and six fields were examined for each slide.

Renal tubular apoptosis was qualitatively assessed by in situ terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), which detects the DNA fragmentation characteristic of apoptosis. Fixed mouse kidney sections, obtained 24 h after iohexol injection, were deparaffinized in xylene and rehydrated through graded ethanols to water. In situ labeling of fragmented DNA was performed with TUNEL (green fluorescence) using a commercially available in situ cell death detection kit (Roche, Indianapolis, IN) according to the manufacturer's instruction. For visualization of the total number of cells in the field, kidney sections were also stained with Hoechst 33342 (blue fluorescence). For blind quantification of the sections, the labeled cells were counted in x100-magnified fields.

Quantification of plasma concentrations of clonidine and dexmedetomidine during radiocontrast injection. To measure the plasma concentration of {alpha}2-agonists (clonidine or dexmedetomidine), we collected plasma 16 h after the placement of drug infusion micro-osmotic pumps. Plasma concentrations of clonidine and dexmedetomidine were quantified by solid-phase extraction and high-pressure liquid chromatography before tandem mass spectroscopic analysis (Children's Hospital of Philadelphia), as previously described (17).

Assessment of outer medullary renal blood flow after iohexol injection. In a separate cohort of male C57BL/6 mice, outer medullary blood flow was recorded for 90 min after iohexol injection in our murine model of RCN. Systemic blood pressure was also recorded during this period by carotid artery cannulation. Outer medullary renal blood flow was quantified using laser-Doppler flow probes, as described previously (1). Laser-Doppler flow probes provide reliable measurements of relative change in regional blood flow in rodent kidney (27). Briefly, a needle flow probe (480 µm diameter; model TSD145) connected to a laser-Doppler flowmeter (Biopac Systems, Goleta, CA) measures red blood cell volume and velocity in the cubic millimeter 1 mm distal to the tip of the flow probe. Flow is derived as the product of red blood cell volume and velocity. The relative change in outer medullary blood flow before and after radiocontrast injection was measured in mice pretreated with saline, clonidine (10 µg/kg), and dexmedetomidine (3 µg/kg).

After overnight (16 h) dehydration, C57BL/6 mice were anesthetized with pentobarbital sodium (50 mg/kg ip or to effect) and placed on a heating pad to maintain body temperature at 36–38°C. Additional pentobarbital sodium (10% of the original dose) was given as needed based on tail pinch. The right carotid artery was cannulated for blood pressure measurements, and the left kidney was exposed by laparotomy. The needle tip of the flow probe was then inserted directly into the renal cortex and advanced into the outer medulla (~1.5 mm beneath the surface of the kidney). Although the insertion of the probe is invasive, blood flow is measured in the undisturbed region 1 mm beneath the tip of the optical probe. Voltage output was recorded on a computer connected to a data acquisition system (Biopac Systems) and displayed as blood perfusion units. The flow data are then represented as the percentage of the baseline blood flow for each mouse.

Baseline renal blood flow and systemic blood pressure were established before intraperitoneal administration of saline (100 µl), clonidine (10 µg/kg), or dexmedetomidine (3 µg/kg). After 15 min, indomethacin (10 mg/kg) and L-NAME (10 mg/kg) were injected intraperitoneally. Iohexol (1.5 g iodine/kg ip) was injected 15 min later. Medullary blood flow and systemic blood pressure were continuously recorded for 90 min after iohexol injection. At the end of each experiment, the mouse was killed, and the kidney was removed and bisected to confirm the position of the needle probe tip in the outer medulla. Mice with incorrectly placed probes were excluded from the study.

Cell culture. Immortalized human proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged in culture medium (50:50 mixture of low-glucose DMEM and Ham's F-12 + 5% serum) and antibiotics (100 U/ml of penicillin G, 100 µg/ml of streptomycin, and 0.25 µg/ml of amphotericin B) at 37°C in a 100% humidified atmosphere of 5% CO2-95% air. Rat pulmonary microvascular endothelial cells (RPMEC) were obtained from Dr. E. Heidi Jerome (Departments of Anesthesiology and Pediatrics, Columbia University) and grown in Ham's F-12 medium + 10% serum. Cells were plated in 24-well plates and used in the experiments described below when confluent.

In vitro models of RCN. An in vitro model of radiocontrast injury was used as described previously (13, 16). Confluent monolayers of HK-2 cells or RPMEC were pretreated with saline, clonidine (10 µM), or dexmedetomidine (10 µM) for 30 min before iohexol (0, 50, 100, or 150 mg/ml iodine) treatment. After 16 h of radiocontrast exposure, HK-2 cell viability was quantified by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Measurement of cell viability. Cell viability was assessed and quantified using an MTT cytotoxicity assay, as described previously (22). The MTT assay measures mitochondrial dehydrogenase activity, a component of the tricarboxylic acid cycle and a marker of energy production and cell viability. Spectrophotometry quantifies the formation of the dark-blue formazan product of the reduction of MTT's tetrazolium ring by mitochondrial dehydrogenase. An MTT tetrazolium salt solution was prepared fresh in serum-free medium at a final concentration of 0.5 mg MTT/ml. After removal of cell plate medium, 0.5 ml of this MTT solution was added to each well of cells pretreated with saline, clonidine, or dexmedetomidine, treated with iohexol, and incubated for 3 h. The medium was then removed. The MTT dark-blue formazen was then solubilized and extracted by addition of 0.5 ml of 0.05 M HCl in isopropanol to each well. After 15 min at room temperature, the optical densities of the formazen extracts of each well were quantified at 570 nm using a spectrophotometer, and the results are expressed as the percentage of vehicle-treated (saline-pretreated, 0 mg/ml iohexol-treated) cells.

Materials. Iohexol was obtained from Amersham Health (Princeton, NJ), clonidine and yohimbine from Tocris (Ellisville, MO), and dexmedetomidine from Hospira (Lake Forest, IL). All other reagents were obtained from Sigma (St. Louis, MO).

Statistical analysis. Continuous variables are summarized and expressed as means ± SE. For comparison of plasma Cr concentrations, necrotic tubules per high-power field, and viability of HK-2 cells, data were analyzed using Student's t-test. For preliminary analysis of outer medullary cortical renal blood flow and systemic blood pressure following radiocontrast exposure, we used Student's t-test at each time point. For better assessment of the treatment effect, we then used the linear mixed-effect model to analyze outer medullary renal blood flow and systemic blood pressure as longitudinal repeated measurements. During this comparison and assessment for differences between outer medullary renal blood flow (or systemic blood pressure) profiles in clonidine- and saline-treated (or dexmedetomidine- and saline-treated) animals, fixed effects included treatment, time from radiocontrast exposure, and treatment-by-time interaction. To account for the correlations between repeated measurements at different time points, mice were modeled using random effect. If the treatment-by-time interaction was statistically significant, the two profiles differ. That is, the treatment effect differs at different time points. No statistical significance for the treatment-by-time interaction suggests that the two profiles are parallel and the treatment effect is the same at all time points. In this case, difference in profiles is assessed using overall-effect analysis.

P < 0.05 was considered statistically significant. We used SAS version 9.1 (SAS Institute, Cary, NC) for all statistical analyses.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Development of RCN in mice. In sham C57BL/6 mice (with prostanoid and nitric oxide synthesis inhibition but without iohexol injection), plasma Cr concentration was 0.4 ± 0.1 mg/dl (n = 4). Mice treated with iohexol (1.5 g iodine/kg) after prostanoid and nitric oxide depletion developed acute renal failure (Cr = 1.5 ± 0.2 mg/dl, n = 8, P < 0.001 vs. sham mice, 24 h after iohexol injection). Our previous studies demonstrated that, without prostanoid and nitric oxide inhibition, iohexol-injected mice do not develop RCN (16). Activation of {alpha}2-adrenergic receptors with clonidine or dexmedetomidine before iohexol injection protected mice against RCN (Fig. 1, A and B). At 24 h after radiocontrast exposure, plasma Cr concentrations were lower in the 10 µg/kg bolus clonidine cohort (Cr = 1.0 ± 0.1 mg/dl, n = 15, P = 0.047), the 10 µg/kg bolus + 5 µg·kg–1·h–1 infusion clonidine cohort (Cr = 0.7 ± 0.2 mg/dl, n = 10, P = 0.010), and the 10 µg/kg bolus + 10 µg·kg–1·h–1 infusion clonidine cohort (Cr = 0.8 ± 0.1 mg/dl, n = 10, P = 0.013) than in saline-treated mice, indicating preserved glomerular filtration and less nephropathy.


Figure 1
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  		Fig. 1. A: plasma creatinine (Cr) in saline sham, clonidine (Clon, 10 µg·kg–1·h–1) sham, saline radiocontrast nephropathy (RCN), and clonidine (5 µg/kg bolus, 10 µg/kg bolus, 5 µg/kg bolus + 5 µg·kg–1·h–1 infusion, 10 µg/kg bolus + 5 µg·kg–1·h–1 infusion, and 10 µg/kg bolus + 10 µg·kg–1·h–1 infusion) RCN mice. RCN mice were injected with iohexol 24 h before treatment. B: plasma Cr in saline sham, dexmedetomidine (Dex, 4 µg·kg–1·h–1) sham, saline (100 µl) RCN, and dexmedetomidine (3 µg/kg bolus, 3 µg/kg bolus + 2 µg·kg–1·h–1 infusion, and 3 µg/kg bolus + 4 µg·kg–1·h–1 infusion) RCN mice. RCN mice were injected with iohexol 24 h before treatment. C: plasma Cr in saline sham, yohimbine (Yo) sham, saline RCN, clonidine (10 µg/kg bolus + 10 µg·kg–1·h–1 infusion) + yohimbine (0.1 mg/kg x2) RCN, and dexmedetomidine (4 µg/kg bolus + 3 µg·kg–1·h–1 infusion) + yohimbine (0.1 mg/kg x2) RCN mice. RCN mice were injected with iohexol 24 h before treatment. *P < 0.05 vs. saline RCN. #P < 0.05 vs. saline sham. Error bars represent SE.

 
Dexmedetomidine (3 µg/kg bolus + 4 µg·kg–1·h–1 infusion cohort) also reduced the rise in plasma Cr observed after iohexol injection (Cr = 0.8 ± 0.1 mg/dl, n = 10, P = 0.008). Mice treated with dexmedetomidine bolus (3 µg/kg) only were not protected from RCN (Cr = 1.5 ± 0.4 mg/dl, n = 8, P = 0.871).

After 16 h of water deprivation and prostanoid and nitric oxide synthesis inhibition, clonidine (10 µg/kg bolus), dexmedetomidine (3 µg/kg bolus + 4 µg·kg–1·h–1 infusion), or yohimbine (0.1 mg/kg bolus) administration alone (clonidine, dexmedetomidine, or yohimbine sham), without iohexol injection, did not alter plasma Cr concentrations: 0.4 ± 0.1, 0.5 ± 0.1, and 0.4 ± 0.1 mg/dl for clonidine sham, dexmedetomidine sham, and yohimbine sham, respectively (n = 4 each).

Smaller doses of clonidine and dexmedetomidine, although insignificant, revealed dose-response tendencies toward reductions in Cr rise after iohexol injection compared with saline controls: for 5 µg/kg bolus clonidine cohort, Cr = 1.1 ± 0.2 mg/dl (n = 7, P = 0.226); for 5 µg/kg bolus + 5 µg·kg–1·h–1 infusion clonidine cohort, Cr = 0.9 ± 0.2 mg/dl (n = 10, P = 0.100); and for 3 µg/kg bolus + 2 µg·kg–1·h–1 infusion dexmedetomidine cohort, Cr = 1.0 ± 0.2 mg/dl (n = 8, P = 0.109).

Yohimbine, a selective {alpha}2-antagonist, abolished the protective effects observed in clonidine- and dexmedetomidine-treated animals. The significant reductions in plasma Cr observed in the 10 µg·kg–1·h–1 + 10 µg/kg clonidine and 4 µg·kg–1·h–1 + 3 µg/kg dexmedetomidine cohorts were reversed by the injection of yohimbine (0.1 mg/kg) at the time of mini-osmotic pump placement and 15 min before clonidine or dexmedetomidine bolus injection: Cr = 1.5 ± 0.4 mg/dl for 10 µg·kg–1·h–1 + 10 µg/kg clonidine + 0.1 mg/kg yohimbine x2 vs. 10 µg·kg–1·h–1 + 10 µg/kg clonidine (n = 6, P = 0.031), and Cr = 1.5 ± 0.2 mg/dl for 4 µg·kg–1·h–1 + 3 µg/kg dexmedetomidine + 0.1 mg/kg yohimbine x2 vs. 4 µg·kg–1·h–1 + 3 µg/kg dexmedetomidine (n = 10, P = 0.002). Moreover, these plasma Cr concentrations were indistinguishable from those of saline iohexol controls: Cr = 1.5 ± 0.4 mg/dl for 10 µg·kg–1·h–1 + 10 µg/kg clonidine + 0.1 mg/kg yohimbine x2 (n = 6, P = 0.959), and Cr = 1.5 ± 0.2 mg/dl for 4 µg·kg–1·h–1 + 3 µg/kg dexmedetomidine + 0.1 mg/kg yohimbine x2 (n = 10, P = 0.890). Yohimbine did not produce nephropathy when injected without iohexol: Cr = 0.38 ± 0.1 mg/dl for yohimbine sham (n = 4, P = 1.000). Yohimbine alone also did not exacerbate RCN after iohexol injection [Cr = 1.6 ± 0.2 mg/dl (n = 5, P = 0.716)] compared with saline iohexol controls (Fig. 1C).

We performed additional in vivo studies to extend the course of RCN and examined plasma Cr beyond 24 h. Six C57BL/6 mice were subjected to RCN (iohexol injection after overnight water deprivation and indomethacin and L-NAME injection) after clonidine (10 µg/kg bolus and 5 µg·kg–1·h–1 infusion) or saline treatment. Our model of unresuscitated RCN resulted in severe renal injury with high mortality: three of six mice in the saline-vehicle group died before day 2, and an additional mouse died before day 3. In contrast, only one mouse died before day 2, and the remaining five mice survived to day 3 in the clonidine-treated group. The plasma Cr values were 0.67 ± 0.03 mg/dl (n = 3) and 0.47 ± 0.1 mg/dl (n = 2) for the saline group at days 2 and 3, respectively. Clonidine treatment led to plasma Cr of 0.58 ± 0.09 mg/dl (n = 5) and 0.45 ± 0.07 mg/dl (n = 5) at days 2 and 3, respectively.

Plasma concentrations of clonidine and dexmedetomidine. The plasma concentration of clonidine at the time of iohexol injection was 0.70 ± 0.11 ng/ml (n = 5). Dexmedetomidine plasma concentration at the time of iohexol injection was 0.51 ± 0.07 ng/ml (n = 5).

Histological findings. Histological evaluation of kidney sections from all mouse cohorts subjected to RCN revealed cortical proximal tubular necrosis, vacuolization, and apoptosis. However, proximal tubular necrosis was significantly less in animals pretreated with clonidine (10 µg/kg bolus + 10 µg·kg–1·h–1 infusion) and dexmedetomidine (3 µg/kg bolus + 4 µg·kg–1·h–1 infusion) than in RCN saline control animals: 1.6 ± 0.5 necrotic tubules/x400 field for clonidine (n = 6, P < 0.034), 0.5 ± 0.4 necrotic tubules/x400 field for dexmedetomidine (n = 6), and 5.7 ± 1.6 necrotic tubules/x400 field for saline (P < 0.01, n = 6). {alpha}2-Agonist-treated animals also developed less cortical vacuolization (Fig. 2). TUNEL revealed more apoptotic cells in renal cortices of iohexol-exposed animals treated with saline than in animals treated with clonidine or dexmedetomidine (Fig. 3) : 38 ± 10 TUNEL-positive cells/x100 field for clonidine (n = 6, P < 0.05 vs. saline), 23 ± 5 TUNEL-positive cells/x100 field for dexmedetomidine (n = 6, P < 0.01 vs. saline), and 122 ± 11 TUNEL-positive cells/x100 field for saline (n = 6).


Figure 2
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  		Fig. 2. Representative light-microscopic images of renal cortices from saline sham, saline RCN, clonidine (10 µg/kg bolus + 10 µg·kg–1·h–1 infusion) RCN, and dexmedetomidine (3 µg/kg bolus + 4 µg·kg–1·h–1 infusion) RCN mice. Sections were stained with hematoxylin and eosin; magnification x200. Saline-treated mice subjected to RCN show increased vacuolization of renal cortices (representative of 6 experiments). Clonidine sham and dexmedetomidine sham images are not shown, but they were indistinguishable from saline sham images.

 

Figure 3
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  		Fig. 3. Representative fluorescent-microscopic images of renal juxtaglomerular junction in sham, saline RCN, clonidine (10 µg/kg bolus + 10 µg·kg–1·h–1 infusion) RCN, and dexmedetomidine (4 µg/kg bolus + 3 µg·kg–1·h–1 infusion) RCN mice. Apoptosis was assessed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. Magnification x200. Saline-treated mice subjected to RCN show increased apoptosis of renal cortices (representative of 6 experiments).

 
Renal outer medullary blood flow following radiocontrast exposure. Outer medullary renal blood flow decreased rapidly and significantly in all animals after radiocontrast injection during the 120-min experiment. However, clonidine (10 µg/kg bolus) or dexmedetomidine (3 µg/kg bolus) pretreatment preserved renal outer medullary blood flow after radiocontrast exposure compared with saline pretreatment. For the clonidine cohort, the linear mixed-effect model revealed no pretreatment-by-time interaction, and overall-effect analysis demonstrated significant differences between clonidine and saline renal blood flow profiles (P = 0.020). For the dexmedetomidine cohort, the linear mixed-effect model verified significant pretreatment-by-time interaction, suggesting unique renal blood flow profiles for dexmedetomidine- and saline-treated mice (P = 0.004). Systemic blood pressure did not change significantly throughout the 90 min following iohexol injection in saline, clonidine, or dexmedetomidine cohorts (Fig. 4).


Figure 4
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  		Fig. 4. Systolic systemic blood pressure (BP, A) and outer medullary renal blood flow (RBF, B) during initiation of RCN injury and for 90 min after iohexol injection in mice pretreated with saline, clonidine (10 µg/kg), and dexmedetomidine (3 µg/kg). Indo, indomethacin; L-NAME, NG-nitro-L-arginine methyl ester. *P < 0.05 vs. saline.

 
Radiocontrast exposure and direct renal tubular toxicity in vitro. Isolated HK-2 proximal tubule cells experienced dose-dependent reductions in viability after 16 h of exposure to 50, 100, and 150 mg iodine/ml iohexol: 91 ± 9, 52 ± 7, and 24 ± 7% viability, respectively (n = 12 each). {alpha}2-Adrenergic agonist pretreatment failed to decrease the cytotoxicity secondary to iohexol exposure in HK-2 cells compared with saline pretreatment (data not shown).

Iohexol also caused dose-dependent reductions in viability after 16 h of exposure to 50, 100, and 150 mg iodine/ml iohexol: 71 ± 3, 53 ± 3, and 8 ± 2% viability, respectively (n = 6 each) in RPMEC. Again, neither of the {alpha}2-adrenergic agonist pretreatments decreased the cytotoxicity secondary to iohexol exposure in vascular endothelial cells compared with saline pretreatment (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have revealed the renal protective effects of {alpha}2-adrenergic receptor activation during radiocontrast exposure in mice. Administration of clonidine or dexmedetomidine, {alpha}2-agonists, reduced the rise in serum Cr observed 24 h after iohexol injection. Yohimbine, an {alpha}2-receptor antagonist, inhibited the protection observed in the {alpha}2-agonist-treated animals. Histological analysis of the protected {alpha}2-agonist-treated animals’ kidneys revealed decreased tubular necrosis, cellular apoptosis, and intracellular vacuolization compared with saline-treated controls. Renal medullary blood flow assessment revealed a preservation of blood flow after iohexol injection in {alpha}2-agonist-treated animals. Clonidine or dexmedetomidine pretreatment did not decrease the direct cytotoxicity of iohexol to cultured HK-2 cells or vascular endothelial cells in vitro.

The pathogenesis of RCN remains a topic of active research, inasmuch as the clinical diagnosis of RCN has significant clinical relevance. Evidence from basic research supports several mechanisms, including intense intrarenal arteriolar vasoconstriction with consequent medullary hypoxia, direct cellular toxicity, and stress-mediated oxidative renal cell injury (8, 31). A critical decrease in corticomedullary oxygen supply vs. demand characterizes clinically observed RCN.

Adrenergic receptors mediate numerous biological functions, including regulation of blood flow in all organ systems, including the kidney. {alpha}2-Adrenergic receptors are primarily associated with presynaptic neurons, where their autocrine behavior reduces neuronal release of norepinephrine and, consequently, regulates autonomic sympathetic tone (32). Reductions of regional vascular resistance, mediated by {alpha}2-adrenergic agonism, may maintain renal medullary blood flow during radiocontrast exposure. Decreases in renal outer medullary blood flow were observed in all animal cohorts exposed to radiocontrast. However, a significant reduction of this perfusion decrement was observed in {alpha}2-agonist-treated animals. This preservation of outer medullary perfusion may explain the improved markers of renal physiology, namely, lower serum Cr and decreased markers of tissue injury, specifically less tubular necrosis, intracellular vacuolization, and apoptosis, 24 h after radiocontrast injection.

With our murine model of RCN, we utilized a clinically applicable dose of radiocontrast (1.5 g iodine/kg iohexol). Although the volume of radiocontrast administered in clinical practice depends on the requirements of each examination or procedure, coronary diagnostic angiograms often require 0.3 g iodine/kg, CT scans require 0.7 g iodine/kg, peripheral vascular interventions require 0.5 g iodine/kg, and interventional coronary imaging requires 1.6 g iodine/kg. Our choice of radiocontrast agent, iohexol (a low-osmolar nonionic dimeric iodinated contrast medium), also reflects common clinical practice. We also chose in vitro concentrations of iohexol (50–150 mg iodine/ml) to mimic the renal tubular concentration achieved in clinical practice. For example, routine injection of radiocontrast medium results in plasma iodine concentrations of ~10–20 mg/ml (9). After ~60–80% of the water and solute content of the glomerular filtrate is reabsorbed into the bloodstream from the proximal renal tubule, local cellular iodine concentrations substantially rise, leading to proximal tubule iodine concentrations similar to those employed in the present study. In rats, injection of 1.6 g iodine/kg iohexol results in urinary iodine concentrations of 125–200 mg/ml (4).

We chose clonidine or dexmedetomidine to activate {alpha}2-adrenergic receptors. Both of these drugs are commonly used clinically and have proven safety profiles. Direct measurement of systemic blood pressure in mice demonstrated that the doses of clonidine and dexmedetomidine we studied do not lead to hypotension. Therefore, protective effects of clonidine and dexmedetomidine on renal corticomedullary blood flow cannot be due to the changes in systemic blood pressure.

The plasma levels of clonidine (0.7 ng/ml) and dexmedetomidine (0.51 ng/ml) demonstrated that the preservation of medullary renal blood flow, maintenance of renal function, and reduced markers of tissue injury were achieved at concentrations commonly found in humans treated with clonidine and dexmedetomidine. Specifically, during the management of hypertension, effective clonidine therapy is commonly achieved at steady-state plasma concentrations of 0.8 ng/ml (19). Dexmedetomidine's effects are achieved with a plasma concentration of 0.81 ng/ml (5, 19).

Differences in pharmacokinetics likely account for the discrepancy between the clonidine bolus-only group, which showed a significant reduction of plasma Cr rise after iohexol exposure and preserved renal medullary blood flow, and the dexmedetomidine bolus-only group, which showed no reduction of Cr rise but preserved renal medullary blood flow (Figs. 1A, 1B, and 4). Dexmedetomidine's relatively short elimination half-life of 2 h (28) renders a dexmedetomidine bolus dose eliminated, while radiocontrast exposure persists throughout the 24-h nephropathy experiment. Clonidine's elimination half-life of 12–16 h (19) accounts for extended {alpha}2-agonism and, consequently, a reduced rise in plasma Cr 24 h after exposure to radiocontrast. A continuous infusion of clonidine is not required. Dexmedetomidine-mediated renal protection requires a continuous infusion throughout the 24-h nephropathy experiment. Since the 120-min renal medullary blood flow experiments were complete before elimination of either a clonidine or a dexmedetomidine bolus, drug infusion remained unnecessary during these blood flow studies.

We did not observe protection from the cytotoxic effects of iodinated radiocontrast administration in {alpha}2-agonist-pretreated proximal tubule (HK-2) cells or vascular endothelial cells in vitro. Both of these cell types contain {alpha}2-adrenoreceptors (10, 14). Tubular toxicity, a consistent finding in isolated proximal tubule cells (8), was observed in all groups of HK-2 cells exposed to radiocontrast. This finding remains specific to iodinated radiocontrast exposure. Equivalent changes in cell media osmolality or volume, as achieved with mannitol, have not led to similar cytotoxicity (16). After exposure to escalating concentrations of radiocontrast medium, the viability of clonidine-, dexmedetomidine-, and saline-pretreated cells was equivalent. Although {alpha}2-agonists failed to protect isolated proximal tubule cells or vascular endothelial cells from direct toxicity, radiocontrast-mediated cytotoxicity is exacerbated by reperfusion injury (33). Consequently, preserved regional blood flow may limit cytotoxicity in vivo, even though cell culture experiments failed to show benefit from {alpha}2-agonists administered to isolated tubular or endothelial cells in vitro.

To better investigate the mechanisms and possible therapies for RCN, we developed a murine model of RCN. One of the limitations of the present study is that our model utilizes young and healthy mice. These mice may not comprehensively mimic the infirm patients that typically develop clinical RCN. Patients at high risk for developing RCN frequently are intravascularly dehydrated and have exhausted the cellular mechanisms that maintain medullary oxygen delivery (24). Therefore, in our murine model, we withheld water for 16 h before iohexol injection and we inhibited prostanoid and nitric oxide synthesis. With these interventions, we were able to create a reliable and consistent model of RCN, a model that has been previously described in mice and rats (6, 12, 16).

We attempted to extend the course of RCN in the present study to examine plasma Cr beyond 24 h. However, our model of unresuscitated RCN resulted in severe renal injury, leading to high mortality (50%) in <48 h for the saline-treated mice. Although the plasma Cr values were similar between saline- and clonidine-treated mice subjected to RCN at day 2, we believe that the saline-treated mice that survived did so because their renal injury was less extensive than that of the mice that died. These surviving mice, as well as their plasma Cr values, reflect a selection bias. One of the limitations of the present study is that, because of the severe nature of our RCN model, we were unable to conclusively examine whether {alpha}2-agonist-mediated renal protection persists beyond 24 h.

In summary, we have demonstrated that clonidine and dexmedetomidine attenuate the reduction of renal blood flow after radiocontrast injection and attenuate the development of subsequent nephropathy. Developing renal protective therapies remain vital to decreasing the morbidity and mortality associated with radiocontrast exposure. Reductions in renal function following radiocontrast exposure may be permanent, and 40% of patients meeting the diagnostic criteria for RCN may require hemodialysis (21). Whether {alpha}2-agonists benefit humans subjected to the diagnostic and therapeutic procedures that utilize iodinated radiocontrast medium remains to be determined. Furthermore, whether {alpha}2-adrenergic agonist-mediated renal benefits surpass those of intravascular hydration alone warrants investigation.


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This work was funded by intramural grant support from the Department of Anesthesiology, College of Physicians and Surgeons, Columbia University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. T. Lee, Dept. of Anesthesiology, Anesthesiology Research Laboratories, Columbia Univ., P & S Box 46 (PH-5), 630 West 168thSt., New York, NY 10032-3784 (e-mail: tl128{at}columbia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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