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Am J Physiol Renal Physiol 290: F1367-F1375, 2006. First published January 17, 2006; doi:10.1152/ajprenal.00347.2005
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A1 adenosine receptor knockout mice are protected against acute radiocontrast nephropathy in vivo

H. Thomas Lee, Michael Jan, Soo Chan Bae, Jin Deok Joo, Farida R. Goubaeva, Jay Yang, and Mihwa Kim

Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York

Submitted 25 August 2005 ; accepted in final form 14 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of renal A1 adenosine receptors (A1AR) in the pathogenesis of radiocontrast nephropathy is controversial. We aimed to further elucidate the role of A1AR in the pathogenesis of radiocontrast nephropathy and determine whether renal proximal tubule A1AR contribute to the radiocontrast nephropathy. To induce radiocontrast nephropathy, A1AR wild-type (WT) or knockout (KO) mice were injected with a nonionic radiocontrast (iohexol, 1.5–3 g iodine/kg). Some A1WT mice were pretreated with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a selective A1AR antagonist) before iohexol injection. A1AR contribute to the pathogenesis of radiocontrast nephropathy in vivo as the A1WT mice developed significantly worse acute renal failure, more renal cortex vacuolization, and had lower survival 24 h after iohexol treatment compared with the A1KO mice. DPCPX pretreatment also protected the A1WT mice against radiocontrast-induced acute renal failure. No differences in renal cortical apoptosis or inflammation were observed between A1WT and A1KO mice. To determine whether the proximal tubular A1AR mediate the direct renal cytotoxicity of radiocontrast, we treated proximal tubules in culture with iohexol with or without 2-chloro-N6-cyclopentyladenosine (a selective A1AR agonist) or DPCPX pretreatment. We also subjected cultured proximal tubule cells overexpressing A1AR or lacking A1AR to radiocontrast injury. Iohexol caused a direct dose-dependent reduction in proximal tubule cell viability as well as proliferation. Neither the A1AR agonist nor the antagonist treatment affected proximal tubule viability or proliferation. Moreover, overexpression or lack of A1AR failed to impact the iohexol toxicity on proximal tubule cells. Therefore, we conclude that radiocontrast causes acute renal failure via mechanisms dependent on A1AR; however, renal proximal tubule A1AR do not contribute to the direct tubular toxicity of radiocontrast.

acute renal failure; HK-2 cells; iohexol; knockout mice; LLC-PK1 cells; vasoconstriction

RADIOCONTRAST-INDUCED NEPHROPATHY is a frequent clinical problem and is a major cause of acute renal failure (4, 27). The incidence of radiocontrast nephropathy approaches 30–50% in patients with volume depletion, congestive heart failure, preexisting renal failure, or diabetes mellitus (4–6). The pathophysiology of radiocontrast nephropathy is incompletely understood. However, a combination of renal vasoconstriction with resultant renal medullary hypoxia and direct renal tubular toxicity of radiocontrast have been implicated in the pathogenesis of radiocontrast nephropathy (9, 21, 22).

Several previous studies suggested that adenosine may be a mediator, at least in part, of the pathogenesis of radiocontrast nephropathy (3, 11, 12, 43). These suggestions are initially based on the hemodynamic effects of intravenous or intrarenal adenosine injection (1, 3, 8). Adenosine, via activation of A1AR, causes direct renal arterial vasoconstriction and a reduced glomerular filtration rate, features seen during the initiation phase of radiocontrast nephropathy (35). Subsequently, administration of theophylline (a nonselective AR antagonist) or 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902; a potent and selective adenosine A1-receptor antagonist) has been shown to prevent the drop in glomerular filtration rate in response to radiocontrast media in vivo (11, 43, 44). However, two recent studies raised questions regarding an obligatory role of A1AR in generating radiocontrast nephropathy. Oldroyd et al. (36) demonstrated in isolated rat kidney that both a nonselective (theophylline) and selective A1AR antagonist (KW-3902) prevented a fall in glomerular filtration rate after radiocontrast injection. However, neither AR antagonist prevented the decrease in renal blood flow after radiocontrast injection. In addition, Liss et al. (34) recently showed that an A1AR antagonist (8-cyclopentyl-1,3-dipropylxanthine; DPCPX) failed to reverse a fall in renal medullary blood flow with radiocontrast injection. Moreover, in one study a significant reduction in creatinine (Cr) clearance remained following administration of high-osmolar contrast, despite the prophylactic use of theophylline, suggesting that there may be other mechanisms involved (10).

In addition to the renovascular effects and the effects on glomerular filtration rate, radiocontrast may cause direct tubular toxicity and induce renal dysfunction (17, 19, 25). However, no study to date has examined the role of A1AR in direct proximal tubular toxicity of radiocontrast. For example, it is unclear whether AR antagonists previously studied in radiocontrast nephropathy (KW-3902, theophylline) have direct protective effects on renal tubules after radiocontrast injection. Therefore, in this study, we tested the hypothesis that mice with deletionally lacking A1AR (A1KO mice) would have improved renal function compared with A1AR wild-type (A1WT) mice when subjected to radiocontrast nephropathy. We also questioned whether the direct proximal tubular cytotoxicity of radiocontrast iohexol is modulated by A1AR. For our in vitro studies, we used primary cultures of proximal tubules from A1WT as well as A1KO mice. In addition, we used immortalized cultures of human (HK-2) and porcine (LLC-PK1) proximal tubules that either expressed normal or increased density of A1AR.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice. Breeder pairs of A1AR heterozygous mice were donated by Dr. J. Schnermann (National Institutes of Health). The generation and initial characterization of the A1WT and A1KO mice with a C57BL/6 background have been described previously (33, 39). The animal care protocol for the experiments performed in this study was approved by the Columbia University Institutional Animal Care and Use Committee.

Model of radiocontrast-induced nephropathy in mice. We used a model of radiocontrast nephropathy combining injection of the radiocontrast iohexol (Omnipaque) with prior inhibition of prostanoids and nitric oxide synthesis as previously described in rats (12, 13). A1WT and A1KO mice were injected with a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester, 10 mg/kg) and an inhibitor of prostaglandin synthesis (indomethacin, 10 mg/kg) intraperitoneally (ip) before iohexol (350 mg iodine/ml, 1.5–3 g iodine/kg ip) injection. This model creates a reproducible acute renal failure following radiocontrast injection. Sham mice received ip injections of saline instead of iohexol after indomethacin and NG-nitro-L-arginine methyl ester injection. Some A1WT mice were pretreated with 1 mg/kg DPCPX ip, a selective adenosine A1AR antagonist, 15 min before the radiocontrast injection. To exclude any nonspecific effects of DPCPX, some A1KO mice were injected with DPCPX before the radiocontrast injection. To account for the increased osmolality of radiocontrast injection, some mice were injected with an equiosmolar concentration of mannitol after indomethacin and NG-nitro-L-arginine methyl ester injection.

Assessment of renal function after iohexol injection. Renal function was assessed 1) by measuring plasma Cr 24 h after radiocontrast injection by a colorimetric method based on the Jaffé reaction (18) and 2) by estimating glomerular filtration rate 4 h after radiocontrast injection. The glomerular filtration rate was estimated with fluorescein isothiocyanate (FITC-inulin) clearance technique as described by Qi et al. (38).

Assessment of renal tubular necrosis and apoptosis. For histological preparations, explanted mice kidneys were bisected along the long axis and fixed in 10% formalin solution overnight. Following automated dehydration through a graded alcohol series, transverse kidney slices were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin-eosin. Morphological assessment was performed by an experienced renal pathologist who was blinded to the treatment for each animal. A grading scale of 0–4, as outlined by Jablonski et al. (26), was used for the histopathological assessment of radiocontrast nephropathy-induced damage of the proximal tubules.

We utilized in situ terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining to detect DNA fragmentation in apoptosis. Fixed mouse kidney sections obtained at 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) according to the manufacturer’s instruction. To visualize the total number of cells in the field, kidney sections were also stained with Hoechst 33342 (blue fluorescence). We also performed DNA laddering on kidney samples obtained 24 h following radiocontrast injection, as previously described (14).

Semiquantitative RT-PCR for proinflammatory cytokines. Twenty-four hours after iohexol injection, renal cortexes were dissected and total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) reagent. RT-PCR reactions for proinflammatory mRNAs (ICAM-1, monocyte chemoattractant protein 1, interferon-induced protein 10, TNF-{alpha}, keratinocyte-derived chemokine, and macrophage inflammatory protein 2) were performed with a one-step RT-PCR kit as described previously (Access RT-PCR System, Promega, Madison, WI) (30). We used primers designed to recognize mouse sequences based on sequences of proinflammatory genes (Sigma Genosys, Woodlands, TX) (Table 1). They were chosen to yield expected PCR products of 200–600 bp, have a 50–60% GC content, and all span an intron to distinguish from genomic DNA contamination. Cycle numbers for each primer set were chosen so as to yield a linear increase in product. Five microliters of the RT-PCR product were analyzed on a 6% acrylamide gel stained with syber green for analysis with a FlourS Multi Imager (Bio-Rad, Hercules, CA). Semiquantification analysis of mRNA expression gene was accomplished by obtaining the ratio of the band density of the gene product of interest to that of GAPDH (a housekeeping gene) from the same sample. Results are expressed as the ratio of the band density of the gene product of interest to the band density of GAPDH obtained from the same sample.


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  		Table 1. Primers used to amplify mRNAs encoding proinflammatory cytokines based on published GenBank sequences for mice

 
Quantification of renal cortical and medullary vacuolization after radiocontrast nephropathy. We noted that radiocontrast injection in mice caused extensive renal cortical vacuolization, as described in rat kidneys previously (20). The number of cortical and medullary vacuoles was counted per x400 field on kidney sections stained by hematoxylin-eosin using the colony counter feature of LabWorks Software. Labworks Software counted and displayed the number of vacuoles within the correct color range, size, and roundness for each image. Images from randomly chosen microscopic fields (magnification, x400) were examined: four images from the medulla and four images from the cortex from each kidney section were quantified and results were averaged for each mouse.

Cell culture. HK-2 and LLC-PK1 cells (immortalized human proximal tubular cell line and porcine renal tubule cell line, respectively, American Type Culture Collection, Manassas, VA) were grown and passaged in culture medium (50:50 mixture of low-glucose DMEM and F-12 plus 5% serum for HK-2 and medium 199 plus 5% serum for LLC-PK1) 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. They were plated in 6- or 24-well plates when 80% confluent and used in the experiments described below when confluent.

To test the effects of A1AR overexpression and A1AR deletion on an in vitro model of radiocontrast nephropathy, we generated two separate proximal tubule cell lines that overexpress A1AR (HK-2 and LLC-PK1) as well as utilized primary cultures of proximal tubules that do not express A1AR (A1KO mice proximal tubule primary culture) as described below.

Generation of A1AR-overexpressing proximal tubule cells in vitro. We generated two independent proximal tubule cell lines that overexpress A1AR. LLC-PK1 cells (porcine renal tubule cell line) were transfected with A1AR-enhanced green fluorescence protein (EGFP) or an EGFP-expressing plasmid (kindly provided by Dr. Ray Penn, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA), and G418-resistant colonies overexpressing A1AR-EGFP or EGFP alone were isolated and propagated. HK-2 cells (human proximal tubule cell line) were infected with A1AR-expressing lentivirus, and cells stably overexpressing A1AR were sorted for propagation. Lentivirus encoding A1AR was generated by subcloning A1AR-EGFP plasmid into a shuttle vector (pLL3.7) and transfecting HEK293-FT cells with pVSVG (Invitrogen) and p{Delta}8.9 (from Dr. Van Parjs, MIT, Cambridge, MA) utilizing opti-MEM and Lipofectamine 2000.

Primary cultures of A1AR WT and KO proximal tubules. A1WT or A1KO mice were killed with an overdose of pentobarbital sodium. Kidneys were rapidly removed in a sterile fashion. Proximal tubules were enriched using the methods of Vinay et al. (40a). In brief, renal cortexes were minced and suspended in sterile buffer A containing (in mM) 105 NaCl, 24 NaHCO3, 5 KCl, 1.5 CaCl2, 1 MgSO4, 2.0 NaH2PO4, and 10 HEPES as well as 0.2% BSA, pH 7.4, with the following additions (in mM): 8.3 glucose and 1 alanine, plus 0.03% collagenase (type I, Sigma) and 0.01% soybean trypsin inhibitor (Sigma), gassed with 95% O2-5% CO2 at 37°C. The cortical suspension was incubated for 45 min with gentle agitation. The suspension was strained through a large sieve, centrifuged at 50 g for 10 min, resuspended in oxygenated buffer A, and washed three times. The resulting pellet was mixed with oxygenated and chilled 40% Percoll solution with the identical ionic composition as buffer A. The Percoll solution was centrifuged at 12,200 g for 30 min at 4°C. The lowermost band of four, enriched in proximal tubules, was washed three times in buffer A and cultured in a 50:50 mix of high-glucose DMEM and F-12 plus 5% serum. The A1WT and A1KO mice proximal tubule cells were plated, and primary cultures were used to study the cytotoxicity of iohexol in vitro.

Radioligand binding assays for A1AR. Saturation radioligand binding assays for A1AR in HK-2 and LLC-PK1 cells were performed according to the general procedure as previously described using 1,3-[3H]-dipropyl-8-cyclopentylxanthine (a highly selective A1AR antagonist) as a radioligand (31, 32).

In vitro models of radiocontrast nephropathy. An in vitro model of radiocontrast injury was adapted from the method of Hizoh et al. (23, 24). Confluent monolayers of proximal tubule cells in culture were incubated with iohexol (omnipaque, 350 mg iodine/ml, stock osmolality = 844 mosmol/kgH2O) yielding a final iodine concentration of 50 (final osmolality = 372 mosmol/kgH2O) and 100 mg/ml (osmolality = 451 mosmol/kgH2O) for 30 min or overnight. To control for the dilution of cell culture media as well as increased osmolality after iohexol, some cells were incubated with equal volumes of saline (isosmolar, 294 mosmol/kgH2O) or mannitol (to mimic the osmolality of 50 and 100 mg/ml iodine iohexol). Some cells were pretreated with 1–10 µM 2-chloro-N6-cyclopentyl-adenosine (CCPA; a selective A1AR agonist) or 1–10 µM DPCPX (a selective A1AR antagonist) 30 min before iohexol addition to determine the effects of A1AR modulation of in vitro radiocontrast toxicity.

Measurement of cell viability. Proximal tubule cell viability was tested with a 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphery]tetradium bromide (MTT) cytotoxicity assay (a measure of mitochondrial Krebs cycle activity). The MTT cytotoxicity assay evaluates mitochondrial dehydrogenase function and cell viability by quantifying the formation of a dark blue formazan product formed by the reduction of the tetrazolium ring of MTT. An MTT tetrazolium salt solution was prepared fresh in serum-free medium at a final concentration of 0.5 mg MTT/ml. After iohexol treatment, 0.5 ml MTT containing medium was added to each well and incubated at 37°C for 3 h. At the end of the incubation period, the unreduced MTT-containing medium was removed. The MTT formazan was solubilized and extracted by adding 0.5 ml of 0.05 M HCl in isopropanol to each well. After 15 min at room temperature, the optical density of formazan extracts was quantified at 570 nm using a spectrophotometer, and results are expressed as the percentage of vehicle-treated cells.

Cell proliferation assay. Cell proliferation was quantified by the incorporation of [3H]thymidine into DNA. HK-2 cells were serum deprived for 24 h before treatment with iohexol and [3H]thymidine (0.1 µCi/ml) was added for the last 4 h. The medium was removed, and cells were washed twice with 500 µl of cold PBS. Following two washes in 500 µl of ice-cold 10% trichloroacetic acid and a rinse with 500 µl of 70% ethanol, the cells were solubilized in 250 µl of 0.2 N NaOH at room temperature for 15 min. This method allows for the recovery of [3H]thymidine incorporated into the DNA as opposed to measuring the total cellular uptake of [3H]thymidine and is therefore a more sensitive method of cellular (i.e., DNA) proliferation. Incorporated [3H]thymidine was quantified by scintillation counting, and the results are expressed as means ± SE.

Materials. Iohexol was obtained from Amersham Health (Princeton, NJ). CCPA and DPCPX (Sigma, St. Louis, MO) were dissolved in 10% DMSO in saline. The final concentration of DMSO in cell culture media was <0.5%. Control groups were nevertheless treated with appropriate vehicle (diluted DMSO).

Statistical analysis. The data were analyzed using Student’s t-test when means between two groups were compared or with one-way analysis of variance plus Tukey’s post hoc multiple comparison test to compare mean values across multiple treatment groups. The ordinal values of the Jablonski scale were analyzed by the Kruskal-Wallis nonparametric test with Dunn’s posttest comparison between groups. In all cases, a probability statistic <0.05 was taken to indicate significance. All data are expressed throughout the text as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Development of radiocontrast nephropathy in A1WT and A1KO mice. As previously described in rats (12), without prostanoid and nitric oxide inhibition, iohexol-injected mice did not develop radiocontrast nephropathy (Cr = 0.47 ± 0.1 mg/dl, n = 6, and 0.44 ± 0.1, n = 5, for A1WT and A1KO mice, respectively). Injection of saline after prostanoid and nitric oxide inhibition failed to increase plasma Cr in A1WT (Cr = 0.49 ± 0.1 mg/dl, n = 4) and A1KO mice (Cr = 0.51 ± 0.1 mg/dl, n = 4). Both A1WT and A1KO mice treated with iohexol (1.5 g iodine/kg) after prostanoid and nitric oxide depletion developed acute renal failure 24 h after injection. However, the degree of acute renal failure was significantly less for A1KO mice (Cr = 0.88 ± 0.11 mg/dl, n = 12) compared with A1WT mice (Cr = 1.55 ± 0.11 mg/dl, n = 9, P < 0.001, Fig. 1). A1WT mice also had significantly higher mortality at 24 h (55.6% or 11/20) compared with A1KO mice (25% or 4/16). A1AR heterozygous (HZ) mice developed an intermediate degree of acute renal failure [Cr = 1.22 ± 0.21 mg/dl, n = 12 vs. A1HZ sham mice (Cr = 0.47 ± 0.05 mg/dl, n = 8) and mortality (39% or 7/19)]. Injection of a higher dose of iohexol (3 g iodine/kg) resulted in more severe acute renal failure in both A1WT (Cr = 1.83 ± 0.31 mg/dl, n = 4) and A1KO (Cr = 1.81 ± 0.43 mg/dl, n = 3) mice; however, the degree of acute renal failure was equivalent between A1WT and A1KO mice. Treatment with a selective A1AR antagonist (DPCPX) before radiocontrast injection significantly protected against radiocontrast nephropathy in A1WT mice (Cr = 0.71 ± 0.05 mg/dl, n = 10, P < 0.0001 vs. A1WT mice injected with radiocontrast, P < 0.05 vs. A1WT sham mice and P = 0.29 vs. A1KO mice injected with 1 radiocontrast, Fig. 1). DPCPX pretreatment did not alter plasma Cr in A1KO mice injected with radiocontrast (Cr = 0.91 ± 0.1 mg/dl, n = 4). In addition, an equiosmolar mannitol injection did not significantly increase plasma Cr (Cr = 0.56 ± 0.1 mg/dl, n = 5) compared with saline-injected mice.


Figure 1
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  		Fig. 1. Plasma creatinine (Cr) values of A1 adenosine receptor (A1AR) wild-type (WT; n = 9), A1AR heterozygous (HZ; n = 9), and A1AR knockout (KO; n = 12) mice injected with iohexol 24 h earlier. Control mice received saline (Sham; n = 6 for WT, HZ, and KO). Some A1WT mice (n = 10) or A1KO (n = 4) were pretreated with DPCPX (a selective A1AR antagonist) before iohexol injection. Some A1WT mice were injected with an equiosmolar concentration of mannitol (n = 5). Error bars represent SE. *P < 0.05 vs. A1WT sham. #P < 0.05 vs. A1WT iohexol.

 
We measured the glomerular filtration rate in mice with the FITC-inulin clearance technique as described by Qi et al. (38). The glomerular filtration rate in saline-injected A1WT and A1KO mice was similar [0.96 ± 0.08, n = 3 vs. 0.98 ± 0.12 ml·min–1·100 g body wt (BW)–1, n = 3]. Radiocontrast injection significantly reduced the glomerular filtration rate in A1WT mice in 4 h (0.28 ± 0.06 ml·min–1·100 g BW–1, n = 6). Reductions in glomerular filtration rate were significantly less in A1KO (0.79 ± 0.2 ml·min–1·100 g BW–1, n = 6, P < 0.05 vs. A1WT mice injected with radiocontrast) and A1HZ (0.51 ± 0.06 ml·min–1·100 g BW–1, n = 6, P < 0.05 vs. A1WT mice injected with radiocontrast) mice injected with radiocontrast. Pretreatment of A1WT mice with DPCPX significantly reduced the fall in glomerular filtration rate after radiocontrast injection (0.52 ± 0.05 ml·min–1·100 g BW–1, n = 6, P < 0.05 vs. A1WT mice injected with radiocontrast).

Histological evaluation for necrosis. Little corticomedullary necrosis occurred in either A1WT (Jablonski score = 0.6 ± 0.3, n = 4) or A1KO mice (Jablonski score = 0.8 ± 3, n = 5) injected with iohexol after prostanoid and nitric oxide inhibition. Saline-injected sham mice after prostanoid and nitric oxide inhibition had a Jablonski score of 0 in both groups. Histological evaluation of kidney sections from mice subjected to radiocontrast nephropathy show vacuolization of the renal cortex [specifically, the proximal portion of the proximal convoluted tubules (S1 segment) with near complete sparing of the distal portions of the proximal tubules (S3 segment)]. The degree of renal cortical vacuolization was significantly greater for A1WT mice subjected to radiocontrast nephropathy compared with A1KO mice (Fig. 2). Renal cortical vacuolization was significantly reduced in A1WT mice subjected to radiocontrast nephropathy after treatment with an A1AR antagonist (DPCPX, Fig. 2). Medullary vacuolization was significantly less and was equivalent between A1WT and A1KO mice after radiocontrast injection (Fig. 2). These morphological changes are similar to the changes in human radiocontrast nephropathy (6, 20).


Figure 2
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  		Fig. 2. A: representative light microscopic photographs of renal cortexes from A1WT and A1KO control mice (sham) and A1WT and A1KO mice injected with iohexol 24 h earlier. Some A1WT mice were pretreated with DPCPX (a selective A1AR antagonist) before iohexol injection (hematoxylin and eosin staining, magnification x400). B: quantification of vacuoles in renal cortex and medulla from A1WT and A1KO mice subjected to saline injection (sham; n = 6 and 6 for A1WT and KO mice, respectively), to iohexol injection 24 h earlier (n = 8 and 12 for A1WT and KO mice, respectively) or from A1WT mice pretreated with DPCPX before iohexol injection (n = 6). Error bars represent SE. *P ≤ 0.05 vs. A1WT medulla sham. #P ≤ 0.05 vs. A1WT cortex sham. $P ≤ 0.05 vs. A1WT cortex iohexol.

 
Renal apoptosis and inflammation with radiocontrast nephropathy. We assessed renal tubular apoptosis with two independent methods; TUNEL staining and DNA laddering. Little apoptosis occurred after radiocontrast nephropathy with either methods (data not shown), and there were no differences between A1WT and A1KO mice subjected to radiocontrast nephropathy.

We assessed renal proximal tubule inflammation by measuring proinflammatory mRNA expression. Radiocontrast-induced acute renal failure was associated with significantly increased proinflammatory mRNA expression (ICAM-1, monocyte chemoattractant protein 1, interferon-induced protein 10, TNF-{alpha}, keratinocyte-derived chemokine, and macrophage inflammatory protein 2). However, there were no differences between A1KO and A1WT mice for proinflammatory mRNA expression (Fig. 3). Moreover, DPCPX pretreatment did not change proinflammatory mRNA expression in A1WT mice. Proinflammatory mRNA expression did not differ between A1KO and A1WT mice pretreated with DPCPX and subjected to radiocontrast nephropathy.


Figure 3
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  		Fig. 3. Densitometric quantifications of relative band intensities from RT-PCR reactions for proinflammatory mRNAs. Error bars represent SE. *P < 0.05 vs. A1WT sham.

 
A1AR radioligand binding. We tested A1AR expression with radioligand binding assays (with [3H]DPCPX), which demonstrated robust expression of A1AR in A1-EGFP-LLC-PK1 cells compared with EGFP-transfected cells (GFP-LLC-PK1). The Bmax of A1AR for the A1-EGFP-LLC-PK1 was ~118-fold higher (Bmax = 2,002 ± 195 fmol/mg protein, n = 3) than the Bmax in EGFP vector-transfected controls (Bmax = 17 ± 1 fmol/mg protein, n = 3, P < 0.01). The number of A1AR for the A1-EGFP-HK-2 was even higher (Bmax = 14,400 ± 1,100 fmol/mg protein, n = 3) compared with that of EGFP vector-transfected controls (Bmax = 25 ± 2 fmol/mg protein, n = 3, P < 0.01). The Kd values were not significantly different, indicating that the receptor affinity for the antagonist radioligand was unaffected.

Radiocontrast produces direct renal tubular toxicity in vitro. Iohexol treatment caused direct time- and dose-dependent toxicity {reduced MTT conversion (viability) and [3H]thymidine incorporation (proliferation)} in all three cell lines of proximal tubules (HK-2, LLC-PK1, and mouse proximal tubule primary culture, Table 2). For example, overnight treatment with 100 mg iodine/ml iohexol reduced MTT conversion to 79.5 ± 1.6% (n = 9, P < 0.05), 56.8 ± 2.6% (n = 6, P < 0.05), and 73.1 ± 6.2% (n = 6, P < 0.05) of untreated A1WT proximal tubule, HK-2, and LLC-PK1 controls, respectively. Similarly, overnight treatment with 100 mg iodine/ml iohexol reduced proximal tubule cell proliferation to 13.0 ± 1.7% (n = 9, P < 0.05), 65.9 ± 1.9% (n = 6, P < 0.05), and 20.8 ± 1.0% (n = 6, P < 0.05) of untreated A1WT proximal tubule, HK-2, and LLC-PK1 controls, respectively. Thirty-minute or overnight treatment with equal volumes of saline had no appreciable effect on cell viability or proliferation in all three cell lines (data nor shown). Thirty-minute treatment with an equiosmolar concentration of mannitol also had no effect on cell viability or proliferation in any cell lines. Overnight treatment with an equiosmolar concentration of mannitol had a small reduction in proliferation in all three cell lines and reduction of viability in HK-2 cells (Table 2).


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  		Table 2. Iohexol-induced changes in cell viability (MTT assay) and proliferation ([3H]thymidine incorporation) in primary cultures of proximal tubules from A1 wild-type and A1 knockout mice, HK-2 cells overexpressing the A1 adenosine receptor and control HK-2 cells, and LLC-PK1 cells overexpressing the adenosine receptor A1 and control LLC-PK1 cells

 
There were no differences in proliferation ([3H]thymidine uptake) or cell viability (MTT assay) in A1WT or A1KO proximal tubules in culture in response to iohexol (Table 2). Overexpression of A1AR had no impact on cytotoxicity of iohexol in HK-2 or LLC-PK1 cells (Table 2). In addition, the A1AR antagonist or agonist failed to alter the cellular injury response to iohexol (Table 2). Similar to the finding in vivo, little apoptosis occurred in vitro HK-2 cells and LLC-PK1 cells by TUNEL staining (data not shown) and DNA laddering (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this study are that the A1WT mice developed significantly worse acute renal failure compared with the A1KO mice after a nonionic radiocontrast (iohexol) injection, indicating that the A1AR contribute to the pathogenesis of radiocontrast nephropathy. Pretreatment with a selective A1AR antagonist (DPCPX) also protected against radiocontrast nephropathy. However, because A1KO and A1WT mice pretreated with DPCPX also developed acute renal failure after radiocontrast injection, A1AR activation cannot solely be responsible for the pathogenesis of radiocontrast nephropathy. In vitro, iohexol produced dose- and time-dependent renal tubular cell toxicity that is not modulated by A1AR, as neither a selective A1AR agonist nor antagonist affected renal tubular cell death. In addition, renal proximal tubular cells from A1WT and A1KO mice showed a similar degree of iohexol cytotoxicity. Proximal tubule cells overexpressing A1AR did not show worse renal cell death with radiocontrast in vitro. Therefore, we conclude that radiocontrast produces renal dysfunction via direct renal tubular toxicity that is not mediated by the A1AR as well as nonrenal proximal tubular effects (perhaps vascular effects and subsequent renal hypoxia) that are mediated by the A1AR.

Radiocontrast nephropathy remains an important clinical problem despite the use of modern contrast media with lower osmolality. Patients with preexisting renal insufficiency, diabetes, or volume depletion are particularly at high risk of developing radiocontrast nephropathy (4–6). The pathogenesis of radiocontrast nephropathy appears to be multifactorial and includes a deleterious reduction of renal arteriolar blood flow and glomerular filtration rate as well as the direct renal tubular toxicity caused by the radiocontrast agents. Indeed, radiocontrast injection in vivo reduces renal blood flow, medullary PO2, and glomerular filtration rate (6, 21). These effects could be due to modulation of the intrarenal synthesis and release of vasoactive mediator(s) after radiocontrast injection. Adenosine has been proposed as one such mediator of radiocontrast nephropathy (10, 12, 13, 43).

Radiocontrast injection increases renal oxygen consumption and ATP breakdown due to their osmotic load. Increased osmotic load increases renal tubular Na+-K+-ATPase activity, which is the driving force for transepithelial electrolyte and water transport (21). Radiocontrast injection increases urinary adenosine (3). Increased adenosine levels as a result of enhanced ATP breakdown have been proposed to induce or potentiate the physiological changes associated with radiocontrast application and mediate acute renal failure. Specifically, it has been proposed that the release of adenosine and activation of A1AR produce the physiological changes observed in radiocontrast nephropathy: the fall in glomerular filtration rate, renal blood flow, and urine output. Intrarenal or intravenous injection of A1AR agonists produces similar physiological changes, including vasoconstriction of renal arterioles and reduction in glomerular filtration rate (2, 3, 11, 13, 34, 43). We demonstrate in this study that radiocontrast injection after prostanoid and nitric oxide inhibition resulted in greater reduction in glomerular filtration rate in A1WT mice compared with A1KO mice. Moreover, a selective A1AR antagonist (DPCPX) significantly reduced the fall in glomerular filtration rate in A1WT mice. These data are consistent with the hypothesis that the increased release of adenosine after radiocontrast injection mediates the reduction in glomerular filtration rate and potentiates radiocontrast nephropathy via activation of A1AR.

Several adenosine-receptor antagonists have been shown to prevent radiocontrast nephropathy (11, 34, 36, 43). Injection of selective as well as nonselective A1AR antagonists in vivo and in isolated kidneys reversed the reduction in renal blood flow and glomerular filtration rate (11, 34, 43). In preclinical studies, radiocontrast-mediated renal failure is attenuated with theophylline (nonselective A1AR antagonist) as well as with selective A1AR antagonists (2, 11). In clinical studies, patients treated with theophylline had a lower incidence of acute renal failure (37). In this study, we also confirmed that a selective A1AR antagonist (DPCPX) reduced radiocontrast-induced acute renal failure in A1WT mice.

Our study is the first to use A1KO mice to study the role of A1AR in in vivo radiocontrast nephropathy. Previous studies used pharmacological agonists and antagonists in rats (12, 13, 21, 43). These studies are limited by the selectivity of the adenosinergic agents used. We therefore utilized mice lacking A1AR to confirm the pharmacological studies (with DPCPX) that A1AR participates in the pathogenesis of radiocontrast nephropathy.

Our study verifies an important role of endogenous A1AR in contributing to radiocontrast nephropathy. DPCPX (an A1AR antagonist) attenuated the acute renal failure induced by iohexol, and the A1KO mice had better preserved renal function compared with A1WT mice after radiocontrast injection. However, A1KO mice did develop renal dysfunction after radiocontrast injection. Therefore, radiocontrast nephropathy is mediated by both A1AR-dependent and A1AR-independent mechanisms. The pathogenic role of A1AR in radiocontrast nephropathy is in stark contrast to their renal protective role in other forms of acute renal failure, including ischemia-reperfusion injury and sepsis, indicating that fundamental differences exist in the mechanisms of pathogenesis of acute renal failure due to radiocontrast and ischemia-reperfusion (15, 28, 33).

Both prostanoid as well as nitric oxide cause compensatory vasodilation after radiocontrast injection, which may attenuate the progression into acute renal failure (20–22). Inhibition of nitric oxide and prostanoids unmasks the renal hemodynamic responses to contrast media. In this study, we used a model of radiocontrast nephropathy after prostanoid and nitric oxide depletion. The combined acute inhibition of both nitric oxide synthesis and prostaglandin generation predisposed mice to consistent acute renal failure from radiocontrast injection, as previously described in rats (20–22). We confirm several previous findings that without inhibition of prostanoid and nitric oxide inhibition, mice did not develop radiocontrast nephropathy.

We have demonstrated that with ischemia-reperfusion injury in rats and mice, there is severe necrosis in proximal tubules, with the most predominant injury occurring in the distal S3 segments of the proximal tubules (28, 29). In contrast, we detected a very different pattern of renal tubular injury after radiocontrast injection. There was significant proximal tubular vacuolization, with the most severe injury occurring in the most proximal part of proximal tubules (the S1 segment) and near-complete sparing of the S3 segment. Moreover, little or no gross proximal tubular necrosis occurred, unlike after ischemia-reperfusion injury. In addition, our model of radiocontrast nephropathy was associated with profound vacuolization of the renal cortex, with relative sparing of the medulla. This vacuole formation seen in radiocontrast nephropathy was significantly worse for the A1WT mice compared with the A1KO mice. This profound vacuole formation after iohexol administration in mice resembles what has been described in human radiocontrast-induced acute renal failure as osmotic nephrosis (20).

We show that expression of proinflammatory mRNAs were similar between A1WT and A1KO mice cortexes after iohexol injection. Moreover, little renal cortical apoptosis was observed in both A1WT and A1KO mice after iohexol injection. Therefore, inflammation or apoptosis does not play a role in A1AR-mediated exacerbation of radiocontrast nephropathy in A1WT mice.

As mentioned, radiocontrast nephropathy has a multifactorial pathogenesis. Some pathogenic mechanisms like alterations in renal blood flow or intrarenal oxygen content can only be studied in vivo. In contrast, the direct chemical toxicity of radiocontrast (perhaps explaining the renotoxic effects of iohexol in A1KO mice) may be better studied in vitro to exclude the confounding variables of in vivo studies. Therefore, in this study we used proximal tubules in culture to test the direct in vitro renal tubular toxicity of radiocontrast and its modulation by the A1AR. Previous studies described the direct toxic effects of radiocontrast in LLC-PK1 proximal tubule cells (19, 41, 42). For example, several types of radiocontrast produced dose- and time-dependent reductions in viability, proliferation, and apoptosis in LLC-PK1 cells (19). The proposed mechanisms of renal tubular toxicity after radiocontrast application in vitro are not fully elucidated; however, osmolar toxicity, free radical generation, severe ATP depletion, a disruption of calcium homeostasis, a disturbance of tubular cell polarity, and programmed cell death (apoptosis) have been proposed (16). We also confirm dose- and time-dependent reductions in viability (MTT assay) and proliferation (thymidine incorporation assay) in HK-2 cells, LLC-PK1 cells, as well as in primary cultures of mouse proximal tubules.

No previous study examined the direct effects of A1AR modulation on the toxicity of radiocontrast in proximal tubules. We used HK-2 cells, which are immortalized human proximal tubules, in culture as well as LLC-PK1 cells and primary cultures of mouse proximal tubules isolated from A1WT and A1KO mice. Use of several proximal tubular cell lines strengthens the conclusion derived from our studies that the cytotoxic renal tubular effect of iohexol is not specific to a single cell line. We show in this study that unlike our murine model of radiocontrast nephropathy, A1AR does not appear to play a role in modulating the toxicity of radiocontrast in cultured proximal tubules. Our study shows that neither the lack nor overexpression of A1AR had an effect on renal tubular damage from radiocontrast. Moreover, a selective A1AR agonist (CCPA) or an antagonist (DPCPX) failed to modulate the cytotoxicity of radiocontrast. Collectively, these data indicate that the direct tubular toxicity of radiocontrast is independent of A1AR. We used an equiosmolar concentration of mannitol to control for cytotoxicity of increased osmolarity and found that iohexol toxicity cannot solely be explained by high osmolarity. However, unlike previous studies (23, 24, 42), we were unable to observe significant apoptosis (DNA laddering and TUNEL staining) with radiocontrast injury of proximal tubules. The reason for differences in these results is only speculative, including differences in cell culture conditions and the specific radiocontrast (iohexol) used.

We utilized both in vitro and in vivo models of radiocontrast nephropathy. In this study, injection of mice with a clinically applicable radiocontrast dose of 1.5 g iodine/kg produced reproducible renal dysfunction. This dose of radiocontrast injection is routinely used in angiographic practice as well as several previous studies of radiocontrast nephropathy in rats as well as mice (5, 6). We also chose the in vitro concentrations of iohexol to induce proximal tubular cell injury (50–100 mg iodine/ml) to mimic the renal tubular concentration achieved in clinical practice. For example, the routine injection volume of radiocontrast media results in plasma concentrations of ~10–20 mg iodine/ml (17). Proximal tubule concentrations, however, are significantly higher, as ~60–80% of the water and solute content of the glomerular filtrate is reabsorbed in this portion of the renal tubule (40). In previous studies, urinary iodine concentration ranged from 125 to 200 mg iodine/ml in rats after injection with clinical doses of iohexol (1.6 g iodine/kg) (7). However, when a "physiological" or "clinical" dose of radiocontrast is being predicted, the renal tubular effects may differ depending on whether the action is intraluminal or interstitial, even at an equimolar concentration of radiocontrast.

In summary, the current study demonstrates that endogenous A1AR activation participates in the pathogenesis of radiocontrast-induced acute renal failure in vivo. Mice lacking the A1AR are protected from acute renal failure following radiocontrast injection. However, direct tubular toxicity of radiocontrast is not modulated by A1AR. Therefore, A1AR is partially responsible for the pathogenesis of acute renal dysfunction after radiocontrast injection. Application of selective A1AR antagonist may benefit patients highly susceptible to acute renal failure following radiocontrast injection.


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


   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 W. 168th St., 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.


   REFERENCES
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