Am J Physiol Renal Physiol 306: F864–F872, 2014. First published January 15, 2014; doi:10.1152/ajprenal.00302.2013.

Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback Zhi Zhao Liu,1 Kristin Schmerbach,1 Yuan Lu,2 Andrea Perlewitz,1 Tatiana Nikitina,1 Kathleen Cantow,1 Erdmann Seeliger,1 Pontus B. Persson,1 Andreas Patzak,1 Ruisheng Liu,2 and Mauricio M. Sendeski1 1

Institut für Vegetative Physiologie, Charité, Universitätsmedizin Berlin, Berlin, Germany; and 2Department of Physiology and Biophysics, Division of Nephrology, University of Mississippi Medical Center, Jackson, Mississippi Submitted 28 May 2013; accepted in final form 14 January 2014

Liu ZZ, Schmerbach K, Lu Y, Perlewitz A, Nikitina T, Cantow K, Seeliger E, Persson PB, Patzak A, Liu R, Sendeski MM. Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback. Am J Physiol Renal Physiol 306: F864 –F872, 2014. First published January 15, 2014; doi:10.1152/ajprenal.00302.2013.—Iodinated contrast media (CM) have adverse effects that may result in contrastinduced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CMinduced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CMperfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle’s loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration. acute kidney injury; cell damage; iodinated contrast media; thick ascending limb; tubuloglomerular feedback

(CM) can cause acute kidney injury (AKI). More than 75 million doses of CM are administered worldwide each year, while the frequency of contrast-induced AKI (CIAKI) is ⬃7% (3). CIAKI may reach up to 10% of all hospital-acquired cases of AKI diagnosed in Europe and USA (4), and it is associated with prolonged hospital stays and increased short- and long-term mortality (9, 10). Although the incidence of CIAKI in healthy patients is low, factors like volume depletion, previous chronic kidney disease, diabetes mellitus, use of other nephrotoxic drugs, and pre- and periprocedural hemodynamic instability may increase the incidence of IODINATED CONTRAST MEDIA

Address for reprint requests and other correspondence: A. Patzak, Institut für Vegetative Physiologie, Charité, Universitätsmedizin Berlin, Berlin, Germany (e-mail: [email protected]). F864

CIAKI up to 50% (28). CIAKI has currently no specific prophylaxis or treatment; hydration and keeping the load of CM as low as possible—which are the only widely recommended preventive measures for CIAKI—are nonspecific, and only diminish the risk, but cannot totally prevent CIAKI (1, 42). The mechanisms of CIAKI are complex and not completely understood. They involve a combination of 1) renal hypoperfusion and hypoxia, specially of the renal medulla (16, 35), 2) auto- and paracrine derangements (e.g., increased release of endothelin and adenosine, lower bioavailability of nitric oxide, and increased local oxidative stress in the kidney) (15, 45), and 3) CM-induced direct cell damage (36). CM-induced direct cell damage has been demonstrated firstly and primarily in cell culture studies, and it is caused by all types of CM. Although the mechanism of CM-induced direct cell damage is still unknown, it has been demonstrated in the absence of hypoxia, oxidative stress, and extremes of CM osmolarity and/or viscosity (36). It has been shown that medullary hypoxia and hypoperfusion in CIAKI may be largely dependent on CMinduced direct cell damage, leading to endothelial dysfunction, and consequent constriction of vasa recta (37–39). The renal medulla has a unique circulatory anatomy (20), which causes medullary thick ascending limbs of the Henle’s loop (TAL) to perform energetically demanding ion transport in a situation of relative hypoxia compared with the renal cortex (8). Furthermore, in vivo studies show that extremes of viscosity may worsen the adverse effects of some CM on urine formation and renal perfusion (35) along with prolonged CM retention in the kidneys, thus keeping kidney cells exposed to CM direct toxic effects for a longer period of time (18). Endothelial lesion of vasa recta and other renal vessels could explain why levels of endothelin increase and nitric oxide levels decrease in the kidney. Since the renal medulla sustains a more pronounced worsening in perfusion than other areas of the kidney, it is assumed that a circulatory component plays a role in the cell damage by CM observed in TAL (16, 35). It has been proposed that a mismatch between the metabolic demands of TAL and the medullary blood supply could cause a surplus of superoxide (O⫺ 2 ), consequently inducing TAL damage due to oxidative stress superimposed on ischemic damage (15). On the other hand, proximal and distal convoluted tubules, which are not so compromised by hypoxia and hypoperfusion in CIAKI as the renal medulla, also show cell damage after use of CM (6, 43, 44). This suggests that oxidative stress caused by hypoperfusion and hypoxia is not the only factor playing a role in tissue lesion in CIAKI in vivo. Thus, we hypothesize that 1)

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CM causes direct cell damage of TAL in vitro, independently of hypoxia or oxidative stress, 2) increased O⫺ 2 and oxidative stress in TAL in vitro might be a consequence rather than a cause of cell damage by CM, and 3) increased O⫺ 2 and oxidative stress in macula densa cells may increase tubuloglomerular feedback (TGF) in concentrations not high enough to cause cell death. To test our hypotheses, we investigated functional parameters as well as signs of cell damage and oxidative stress in isolated, in vitro perfused TAL, and the TGF response in isolated juxtaglomerular apparatus (21) (JGA; the macula densa along with the corresponding distal tubule, glomerulus, and afferent arteriole)—in an environment where tissue hypoxia is absent and therefore does not contribute to cell damage and oxidative stress. Furthermore, we also investigated whether acute administration of CM in vivo could influence the expression of genes related to oxidative stress and the activity of the O⫺ 2 -dismutase (SOD). MATERIALS AND METHODS

Dissection and perfusion of isolated TAL and JGA. SpragueDawley rats (male, 120 –200 g) were used for experiments with TAL, and New Zealand white rabbits (male, 1.5–2.0 kg) were used for experiments with JGA. The animals were sedated (50 mg/kg ip ketamine/xylazine), killed, and the kidneys were removed, sliced along the corticomedullary axis, and then placed in ice-cold buffered solution. TAL and JGA were manually isolated using sharpened micro-forceps under magnification and transferred into a thermoregulated chamber filled with buffered bath solution on the stage of an inverted microscope. The solution used for dissection, perfusion, and bath solution of TAL consisted of (in mM) 130 NaCl, 2.5 NaH2PO4, 4 KCl, 1.2 MgSO4, 6 L-alanine, 1 Na3C6H5O7, 5.5 glucose, 2 C6H10CaO6, and 10 HEPES. The solution for preparation of JGA was MEM (GIBCO, Grand Island, NY) containing 5% BSA (Sigma, St. Louis, MO). All solutions were adjusted to pH 7.4 at 37°C. The procedures involving animals were approved by the appropriate control organ of our state and were in accordance with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” (7) and with the Guide for the Care and Use of Laboratory Animals (29) adopted by the National Institutes of Health. Microperfusion procedure. TAL and the afferent arteriole as well as the distal tubule of the JGA preparation were perfused using a system of concentric pipettes intended for holding and perfusing them as described previously (11, 26). Briefly, TAL, as well as the afferent arteriole and the end of distal tubule, were mounted on concentric pipettes, cannulated at one end, and held at the other end in a manner that permits free flow into the collection pipette and administration of CM from the luminal side. The afferent arteriole of JGA preparations was perfused with DMEM at a pressure of 60 mmHg, and the distal tubule was perfused with physiological saline comprising (in mM) 10 HEPES, 1 CaCO3, 0.5 K2HPO4, 4 KHCO3, 1.2 MgSO4, 5.5 glucose, 0.5 Na acetate, 0.5 NaC3H5O3, and either 80 or 10 NaCl at a rate of 40 nl/min controlled by a pump. The bath for JGA experiments consisted of MEM exchanged continuously into the chamber at a rate of 1 ml/min. Dissection and cannulation were achieved within 40 min, and after achieving stable perfusion of TAL and JGA, temperature at the chamber was kept at 37°C. The CM used to perfuse TAL and the distal tubule of JGA preparations was iodixanol, a dimeric, nonionic, iso-osmotic CM (GE Healthcare, Munich, Germany). The concentrations used were 11 and 23 mg iodine/ml. Iodixanol at the concentration of 23 mg iodine/ml has been shown to cause endothelial cell damage and functional disturbances with ultimate contraction of human and rat descending vasa recta in vitro (39).

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Quantification of O⫺ 2 and nitric oxide in single isolated, perfused TAL. Before perfusion with CM, perfused TAL were loaded with either dihydroethidium (DHE) or 4-amino-5 methylamino-2=,7=-difluorofluorescein (DAF-FM) diacetate in the bathing solution. DHE is oxidized within cells by O⫺ 2 to fluorescent products that are intercalated into DNA. Images for O⫺ 2 quantification were obtained through excitation from xenon arc lamps at 365 and 490 nm, and emissions were monitored with band pass filters at 400 – 450 and 520 – 600 nm. The proportion between the measured fluorescence in these two wavelength windows is considered as indicative of O⫺ 2 production. DAF-FM diacetate is deacetylated within cells by action of intracellular esterases, reacting then with nitric oxide and producing fluorescent benzotriazol. DAF-FM emissions were measured after excitation at 490 and emissions at 535 nm. Before NO measurements, perfused TAL were treated for 25 min with 50 ␮M L-arginine—at this concentration, L-alanine supports but does not stimulate nitric oxide production (30). Serial computerized fluorescence images were acquired and analyzed for serial measurements of changes in the light emission, signifying higher or lower concentration of O⫺ 2 and nitric oxide. These techniques are well-established for measurements of O⫺ 2 and nitric oxide in similar experimental settings using TAL (12, 13, 30). Imaging of propidium iodide fluorescence for assessment of cell damage in isolated, perfused TAL. Only cells that lose integrity of cell membrane due to cell death become permeable to propidium iodide, so it can be used to quantify cell damage in tubular cells (22). After a stable perfusion was achieved, 5 ⫻ 10⫺3 mol/l propidium iodide was added to the bath solution for 5 min. Excitation from xenon arc lamps was done at 490 nm, and emissions with band pass filters were monitored at 520 – 600 nm at 30-s intervals. After the first minute of recording, the perfusate was exchanged to contain either vehicle or CM (23 mg iodine/ml). Propidium iodide was also added to the perfusate. After exchange of perfusate, the pressure in the pressure head was set to 10 mmHg for the whole experimental period. TAL were imaged by a ⫻40 oil immersion objective. Acquisitions were analyzed over time using the software VisiView (from Visitron Systems, Munich, Germany). Figure 1 shows representative samples of an experiment using propidium iodide. In some experiments, tempol (10⫺4 mol/l) was used in the bath solution and perfusate. Staining with Trypan blue solution. After the fluorescence protocols were finished with propidium iodide, 50% Typan blue (Sigma, Munich, Germany) was applied for 5 min into the chamber. Pictures of TAL were then taken after Trypan blue was washed out, and the amount of Trypan blue uptake by TAL was estimated as follows: a “region of interest” (ROI) following the contours of each TAL was drawn using the program ImageJ (National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/), and the amount of transmitted light on the area of TAL was measured in it. Afterwards, the background light of a neighboring area using the same ROI obtained from TAL was then subtracted from the initial measurement, to equalize possible changes in environment light. The amount of Trypan blue absorbed by TAL was considered to be the difference between the light transmitted through TAL between the final and initial measurements, and is depicted in relative values. They represent roughly how much “less light” travels through the TAL as a result of Trypan blue absorption ¨ C or “how dark” the TAL become. Figure 2 shows an example of Trypan blue absorption of an isolated, perfused TAL that received CM (Fig. 2A: before CM application; Fig. 2B: after CM application). Measurement of TGF. After a 30-min equilibration period at 37°C, the perfusate of the distal tubule was switched from 10 to 80 mM NaCl, and luminal diameter of the afferent arteriole was measured for 5 min. The average change in diameter of the afferent arteriole was taken as a control TGF response. To study the effect of CM, CM (11 or 23 mg iodine/ml) was added in the tubule for 10 min and present during the whole experiment, while the afferent arteriole was still perfused by MEM. Then, the TGF response was measured again. In

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magnets. Tubular structures were separated from the supernatant by centrifugation (4,000 rpm for 5 min at 4°C). Total RNA extraction, reverse transcription, and RNA microarray. The total RNA from medullary tubules was extracted using RNA-Bee RNA Isolation Reagent (AMS Biotechnology Limited, Abingdon, UK). RNA concentrations were measured using the absorption at 260 nm according to the manufacturer’s protocol (NanoDrop 2000, Thermo Fisher Scientific, Bremen, Germany) and all samples had parity of RNA ratios at 260/280 nm above 2.0. An equal amount of RNA (2 ␮g) was taken for all samples and all probes belonging to one of the four groups were pooled which finally resulted in three probes per group. Reverse transcription (RT2 First Strand Kit, Qiagen, Hilden, Germany) was followed by the measurement of relative gene expression of 84 genes potentially involved in oxidative stress, as described in the manufacturer’s protocol, using the commercial kit RT2 Profiler PCR Array Rat Oxidative Stress and Antioxidant Defense (Qiagen) on a StepOne Real-Time PCR System (Applied Biosystems). Thermocycler conditions were 90°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Afterwards, specific PCR products were evaluated using the melting curves (95°C for 15 s, 60°C for 1 min, and 95°C for 15 s). Relative changes in gene expression were calculated using the comparative quantification cycle (⌬⫺⌬Cq) method with five reference genes (Rplp1, Hprt1, Rpl13a, Ldha, Actb) (2, 33). SOD activity assay. The determination of SOD activity was independently performed two times by using the commercial kit OxiSelect O⫺ 2 Dismutase Activity Assay (Cell Biolabs, San Diego, CA). The assay principle is based on the ability of a xanthine/xanthine oxidase (XOD) system to generate O⫺ 2 anions that are detected with a chromagen solution. In the presence of SOD, these O⫺ 2 anion concentrations are reduced, yielding lower colorimetric signal. One unit (U) of SOD activity was defined as the amount that reduced the absorbance at 490 nm (46). Statistics and analysis. Results of experiments were expressed as means ⫾ SE. The size of each treatment group is given together with the group’s results. Data sets of microperfusion experiments with TAL and JGA were compared using Brunner’s test (a nonparametric ANOVA-like test for repeated measurements and multiple compari-

Fig. 1. Representative pseudocolor microscopic presentation of light intensity in rat thick ascending limb (TAL) stained with propidium iodide, a fluorescent dye used for identification of dead cells. Light intensity (represented as a shift from blue to red in light spektrum) increases with propidium iodide uptake by nonviable cells. A: at beginning of an experiment. B: at 20th min of perfusion with iodixanol. C: after 4 h of perfusion with iodixanol.

some experiments, tempol (10⫺4 mol/l) was used in the bath solution and perfusate. Isolation of medullary tubular structures from medullary vessels using iron oxide. Male Sprague-Dawley rats were anesthetized with urethane (6 mg/kg ip), and they received either vehicle or CM solution (a bolus of 1.5 ml of iodixanol 320 into the thoracic aorta). N was 10 for each experimental group. The animals were killed 2 h after application of CM. The circulatory bed of the kidneys was isolated by tying the abdominal aorta rostrally and caudally to the renal arteries, opening the aorta, and inserting a catheter into the aortic lumen. Both kidneys were then rinsed with PBS and perfused with a solution of PBS plus 1% iron oxide and 1% albumin. The medulla of perfused kidneys was separated, chopped on a plate over cold ice for 4 min, and collagenase 1A (0.45 mg/ml) was added for 20 min at 37°C. Mechanical separation of tubular and vascular structures was then accomplished via passages through needles gauge 23 (5 passages) and 24 (5 passages), and vascular structures, which are filled with magnetized iron particles, were isolated from tubules using high-performance

Fig. 2. Representative example of Trypan blue uptake in a microscopic transmitted light presentation of rat TAL. A: before perfusion with iodixanol: the typical aspect of TAL. B: after iodixanol perfusion: cell darkening due to Trypan blue uptake indicates loss of cell membrane permeability due to cell death.

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Fig. 3. Relative changes of DHE-ratio fluorescence over time in rat isolated TAL perfused with vehicle solution (control group, n ⫽ 8), 11 mg iodine/ml iodixanol (n ⫽ 8), 23 mg iodine/ml iodixanol (n ⫽ 7), and 23 mg iodine/ml iodixanol⫹tempol (n ⫽ 6). *P ⬍ 0.05.

sons) (5). For testing differences between groups in RNA microarray and O⫺ 2 dismutase activity assay measurements, the Student’s t-test was performed. Statistical differences were evaluated by comparison of CM treatment to control. P ⬍ 0.05 was used to reject the null hypothesis. RESULTS

TAL perfused with CM show impairment of functional responses to flow: Increased production of O⫺ 2 in TAL perfused with CM. As shown in Fig. 3, O⫺ 2 production increased in TAL perfused with CM. Perfusion with CM at 23 mg iodine/ml over 4 min increased O⫺ 2 concentration by 9.58 ⫾ 1.43%, significantly more than in the control group (1.72 ⫾ 1.01%; P ⫽ 0.001). CM at 11 mg iodine/ml did not differ from controls (0.22 ⫾ 1.88%; P ⫽ 0.468). On the other hand, the difference between CM at 23 and 11 mg iodine/ml was statistically significant (P ⫽ 0.003). Tempol, a O⫺ 2 dismutase mimetic, prevented the O⫺ 2 increase caused by CM at 23 mg iodine/ml (0.35 ⫾ 1.33%, P ⫽ 0.044). Decreased nitric oxide bioavailability in TAL perfused with CM. We used the fluorescent dye DAF-FM diacetate to quantify the bioavailability of nitric oxide in isolated TAL perfused with CM (Fig. 4). In the control group, nitric oxide bioavailability after 12 min was increased to 1.63 ⫾ 0.34% of baseline. CM at 23 mg iodine/ml decreased nitric oxide bioavailability to ⫺0.55 ⫾ 0.64% of baseline after 12 min (P ⫽ 0.005 compared with control group). L-NAME, a nonselective inhibitor of the nitric oxide synthase, had an effect similar to CM—nitric oxide bioavailability was decreased to ⫺0.29 ⫾ 0.76% from baseline after 12 min (P ⫽ 0.004 compared with control group). CM causes cell death in isolated, perfused TAL. To discern whether low nitric oxide and high O⫺ 2 in isolated TAL during perfusion with CM at 23 mg iodine/ml were due to a reversible, functional impairment of TAL cells, or whether this could be due to cell damage by CM, we used two disctinct techniques to detect loss of cell membrane integrity.

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Fig. 4. Relative changes of DAF-FM fluorescence over time in rat isolated TAL perfused with vehicle solution (control group, n ⫽ 7), 23 mg iodine/ml iodixanol (n ⫽ 7), and L-NAME (n ⫽ 8). *P ⬍ 0.05.

Increased propidium iodide fluorescence in CM-perfused TAL. Figure 5 shows the propidium iodide fluorescence curves of TAL perfused with CM, vehicle, and CM plus tempol over a period of 20 min. Compared with the control group, there was a significant increase in propidium iodide fluorescence in the group receiving CM at 23 mg iodine/ml (P ⫽ 0.022), indicating a higher rate of cell death. The group receiving CM at 23 mg iodine/ml and treated with tempol was not significantly different from CM alone at the same concentration, but differed significantly from controls (Fig. 5). Increased uptake of Trypan blue in CM-perfused TAL. As Fig. 6 shows, isolated TAL perfused with CM showed a

Fig. 5. Relative changes of propidium iodide (PI) fluorescence over time in rat isolated TAL perfused with vehicle solution (control group, n ⫽ 5), and 23 mg iodine/ml iodixanol with (n ⫽ 6) and without tempol 10⫺4 mol/l (n ⫽ 8). *P ⬍ 0.05.

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American College of Radiology or by the European Society of Urogenital Radiology (1, 42). A closer look at the assumptions made when developing anti-oxidant strategies for CIAKI shows that the mechanisms of CM-induced increased oxidative stress in CIAKI have been until now only hypothesized, and not actually identified. We believe that our experiments provide new insights into the pathophysiology of oxidative stress in CIAKI, and they help to explain why anti-oxidative therapy does not improve outcomes in the clinical setting. A possible explanation proposed for the signs of oxidative stress in CIAKI is that the renal hypoperfu-

Fig. 6. Absorption of transmitted light by cellular uptake of Trypan blue as an indicator of cell membrane lesion in isolated, perfused TAL after perfusion with 23 mg iodine/ml iodixanol (n ⫽ 7) and vehicle solution (n ⫽ 3). *P ⬍ 0.05.

significantly higher cell uptake of Trypan blue over time, what corresponds to a higher rate of cell death. CM increased TGF in isolated JGA. TGF was determined by the changes of afferent arteriole diameter in response to increasing the NaCl concentration from 10 to 80 mM in isolated, perfused JGA preparations as shown in Fig. 7. CM at 11 mg iodine/ml significantly enhanced the TGF response of our isolated JGA compared with controls (4.5 ⫾ 0.4 and 2.8 ⫾ 0.4 ␮m, respectively). Effects of CM at 23 mg iodine/ml on TGF response (2.3 ⫾ 0.3 ␮m) did not significantly differ from controls. JGA treated with tempol and receiving CM (11 mg iodine/ml) had no increase in the TGF response and showed a similar TGF response to JGA receiving either vehicle or tempol alone (Fig. 8). Quantification of expression of genes related to oxidative stress and SOD activity ex vivo. Due to a decrease in medullary blood flow and oxygenation, as well as CM-induced direct cell damage, O⫺ 2 may increase in the renal medulla. To investigate whether CM might acutely influence the expression of genes that are related to reactive oxygen species, we used a quantitative RT-PCR array to compare the expression of 84 oxidative stress and antioxidant defense genes in tubular structures isolated from the medulla of rats 2 h after intra-aortic administration of either CM or vehicle. We found differences between groups only for two genes (Fig. 9): a diminished expression of thioredoxin-interacting protein (TXNIP) as well as O⫺ 2 dismutase 3 (SOD3). Because enzyme activity could be influenced in vivo by CM independently of changes in gene expression, we used the material from the same set of probes used for RT-PCR measurements (i.e., tubular structures isolated from the medulla of rats treated with either CM or vehicle) to investigate the SOD activity. The result is shown in Fig. 9, and there were no significant differences between groups. DISCUSSION

Evidence of oxidative stress in experimental models of CIAKI led to several attempts to use anti-oxidant agents as a prophylaxis for CIAKI (15). However, anti-oxidant therapy did not consistently reduce incidence of CIAKI in the clinical setting, and it is currently not recommended either by the

Fig. 7. Effects of iodixanol at the concentration of 11 (n ⫽ 3) and 23 mg iodine/ml (n ⫽ 4) on isolated, perfused juxtaglomerular apparatuses (JGA) in which the distal tubules were perfused first with 10 mM NaCl, and then 80 mM, compared with controls (n ⫽ 4). A: absolute diameter of the perfused afferent arterioles of our isolated JGA. B: changes in the tubuloglomerular feedback response (TGF; defined as the net diameter change of afferent arterioles in response to changes in the concentration of NaCl). The significant differences between groups are indicated (*P ⬍ 0.05).

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spheric air conditions, with higher oxygen partial pressure than in the medullary milieu in vivo. This rules out hypoxia as a cause for increased oxidative stress in our setting, indicating that the increased O⫺ 2 concentration found in our experiments with perfused TAL must have a different cause than the hypoxia observed in vivo. Our experiments show that O⫺ 2 concentration is significantly increased by CM in TAL perfused in vitro. While in vivo models of CIAKI may provide an approximation of what

Fig. 8. Effects of 11 mg iodine/ml iodixanol (n ⫽ 3, as in Fig. 7) and 10⫺4 mol/l tempol (n ⫽ 6) either alone or in combination (n ⫽ 6) on isolated, perfused JGA in which the distal tubules were perfused first with 10 mM NaCl, and then 80 mM, compared with controls. A: absolute diameter of the perfused afferent arterioles of our isolated JGA. B: changes in the TGF response. The significant differences between groups are indicated (*P ⬍ 0.05).

sion observed in several models, combined with local hypoxia and occurring mainly in the renal medulla, leads to a shift in mitochondrial oxidative reactions toward increased free radical production [for a review on the interaction between hypoxia and oxidative stress in CIAKI, see the review of Heyman et al. (15)]. However, indications of increased oxidative stress following exposition to CM may also be found in experimental models using cell culture models, where oxygen partial pressure can be controlled during experiments, and tissue hypoxia does not occur (23, 47). In accordance, our models of microperfusion using TAL and JGA are performed in atmo-

Fig. 9. A: effects of iodixanol on the relative expression of genes involved in reactive oxidative stress and antioxidant defense of medullary tubular structures quantified through microarray analysis. Depicted are the fold regulation vs. controls of the 5 most up- and downregulated genes (*P ⬍ 0.05). B: effects of iodixanol on superoxide dismutase (SOD) activity of medullary tubular structures (n ⫽ 8 –9). There was no statistically significant difference between groups.

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happens in humans, they are not adequate to differentiate to which extent factors such as hypoxia, CM-induced direct cell damage, and oxidative stress contribute to tissue damage seen in kidneys—to make this differentiation, in vitro experiments are more appropriate. As an example, although hypoperfusion and relative hypoxia of the renal medulla have long been recognized as a hallmark of CIAKI in vivo, only recently it has been possible to demonstrate, using in vitro studies, that all contemporary types of CM, independent of their properties, lead to a marked constriction of outer descending medullary vasa recta (38). Conversely, increased medullary O⫺ 2 production may, independently of its cause or sources, have important consequences in vivo, because O⫺ 2 stimulates NaCl absorption by TAL (31), enhances Na⫹ transport and Na⫹-K⫹-2Cl⫺ cotransporter activity (19, 40), and decreases nitric oxide bioavailability (30). Importantly, increased O⫺ 2 in TAL may be a consequence of factors like increased flow (17) or mechanical stress on TAL cells (12). Such increases in local O⫺ 2 might be specially important in the case of types of CM that may become extremely viscous when concentrated inside tubules (34). The repercussions of all these functional derangements in TAL are hard to separate from each other in vivo, and most of them could potentially worsen oxidative stress in vivo. In our experiments, increased O⫺ 2 goes along with signs of concomitant cell death detected both by Trypan blue dye and propidium iodide. However, perfused TAL which received CM at 23 mg iodine/ml together with tempol showed a rate of cell death similar to TAL which received only CM—indicating that TAL cell damage also takes place when increases in local O⫺ 2 along with oxidative stress are prevented. Because of the strict control of the milieu surrounding TAL in our setting, we are able to exclude that TAL cell damage could have been caused by factors that cannot be controlled in vivo, such as hypoxia, extremes of CM viscosity and/or osmolarity, and metabolic overload due to changes Na⫹ load in the tubular fluid. Importantly, several in vitro studies showed that all types of CM directly cause cell damage in the absence of other identifiable causal factors, and our findings of TAL epithelial cell damage by CM are consistent with such studies (36). Furthermore, our results help understand why neither anti-oxidative therapy nor drugs directed to improve renal medullary perfusion can prevent CIAKI: since CM-induced direct cell damage may be the cause of both oxidative stress as well as medullary hypoperfusion, the real trigger factor of CIAKI is not actually being targeted. We also show that the production of nitric oxide in TAL as a response to flow and changes in intraluminal pressure is significantly disrupted by CM. This might have important functional consequences for TAL in vivo, since nitric oxide 1) inhibits NaCl absorption via reduction of Na⫹-K⫹-2Cl⫺ cotransport activity, 2) inhibits absorption of bicarbonate via reduction of Na⫹/H⫹ exchange activity, and 3) enhances the activity of luminal K⫹ channels (14). Furthermore, O⫺ 2 reacts with nitric oxide, thus decreasing its bioavailability in TAL (30). This explains why our findings of decreased nitric oxide bioavailability in TAL perfused with CM also fit our measurements of increased O⫺ 2 in a similar setting. Production of O⫺ 2 is important for the tubulovascular “crosstalk” taking place between TAL and descending vasa recta (DVR) (8), and in the macula densa as part of the signaling of the TGF (24 –26). Consistently, in our experiments with iso-

lated, perfused JGA, the TGF response was increased by CM at 11 mg iodine/ml, and this increase was prevented by tempol. Although our TAL which received CM showed significant increases in O⫺ 2 using fluorescent techniques only at concentrations higher than 11 mg iodine/ml, we believe that our TAL and JGA experiments are in agreement because 1) JGA and medullary TAL are surrounded by different levels of O⫺ 2 in vivo, with medullary TAL experiencing relative hypoxia compared with JGA. This might differently influence the production of mitochondrial O⫺ 2 in JGA and TAL when exposed to similar levels of oxygen as in our setting, and it precludes a direct comparison of data from our JGA with our medullary TAL; 2) O⫺ 2 has distinct physiological roles in JGA and medullary TAL, and it is possible that functional repercussions of increased O⫺ 2 due to exposure to CM show effects earlier in TGF response of isolated JGA than what can be measured with fluorescence in isolated TAL. Up to our knowledge, our experiments are the first direct evidence that increased TGF might contribute to the decreases in medullary perfusion due to CM observed in vivo—in addition to other factors such as 1) direct DVR constriction by CM due to endothelial damage and dysfunction (37–39), 2) a more marked constrictive effect of CM in afferent compared with efferent arterioles (27), and 3) properties of CM such as high viscosity or extremes of osmolarity (35). Importantly, in our setting the concentration of CM reaching the macula densa is kept constant. This prevents the influence of CM on the composition of the tubular fluid that happens in vivo (35), and this is meaningful because tubular fluid composition directly influences the signaling between macula densa and the afferent arteriole (32, 41), independently of a possible effect on O⫺ 2 production by either CM and/or hypoxia. Interestingly, a higher concentration of CM (23 mg iodine/ml) did not increase the TGF response. Since this concentration significantly increases cell death rate in our TAL experiments, it is possible that JGA perfused with CM at 23 mg iodine/ml were already rendered dysfunctional due to CMinduced direct cell damage—while in JGA perfused with CM at 11 mg iodine/ml, cell damage is still incipient, but it increases O⫺ 2 already enough to influence macula densa signaling and increase TGF. Taken together, our data show that JGA and medullary TAL may react to increasing CM concentrations in manners that are neither exactly similar nor technically comparable, but which stand overall in agreement with the diverse anatomical location and physiological roles of JGA and medullary TAL. Furthermore, the effects of CM on the TGF response may contribute to the worsening of kidney perfusion in CIAKI, and these effects vary according to the concentration of CM in each structure. It has been shown that CIAKI may be linked to increased oxidative stress in the renal medulla, and our experiments show increased local O⫺ 2 production in TAL perfused with CM. Since it is not possible to recover perfused TAL in sufficient abundance to perform protein expression studies, we used RT-PCR to quantify the expression of 84 genes potentially related to free radical metabolism in our samples of tubular structures from the renal medulla of rats that received intraarterial CM. Only two of the 84 genes investigated showed significant changes: SOD3 and TXNIP (which may be involved in the initiation of local inflammation) were less expressed compared with controls. Interestingly, our SOD activity assays were performed using samples of the same probes

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which provided material for the RT-PCR measurements, and they show that the lower SOD3 expression does not go along with significant decreases in SOD activity. Combined, both techniques indicate that both oxidative stress-related gene expression as well as SOD activity are not significantly altered in our setting. While such results may at first seem to be in conflict with previous evidence of increased oxidative stress in CIAKI, it is possible that the interval of 2 h we used between CM administration and kidney removal is too short for significant changes in expression to be detected in most genes. Longer periods of time, however, would diminish the comparability of this setting to our acute in vitro experiments. In this context, our RT-PCR and SOD activity studies provide evidence that the acute increases in O⫺ 2 production observed in our CM-perfused TAL are not related to significant increases in the expression of the genes related to oxidative stress, and they agree with our finding that tempol—a SOD mimetic— does not decrease the rate of CM-induced direct cell damage in CMperfused TAL. In conclusion, our results show that CM-induced direct cell damage leads to oxidative stress and low-nitric oxide levels in TAL also in absence of tissue hypoxia and hypoperfusion. Increased O⫺ 2 at the macula densa also increases TGF, what might aggravate renal hypoperfusion in vivo. Strategies for prevention of CIAKI in the long run should therefore concentrate on diminishing CM-induced direct cell damage, instead of focusing on preventing oxidative stress and renal hypoperfusion— given that, in optimal clinical conditions of hydration as well as CM osmolarity and viscosity, measures directed to prevent oxidative stress and improve medullary perfusion do not diminish the incidence of CIAKI. Induction of genes related to oxidative stress and changes of SOD activity do not seem to play a role in our setting. ACKNOWLEDGMENTS The excellent technical assistance of Jeannette Werner, Ariane Anger, and Andrea Gerhardt is greatly appreciated. GRANTS This work was supported by grants from the Else Kröner-Fresenius-Stiftung (P40/09//A29/09) and the Deutsche Forschungsgemeinschaft (PA 479/10-1). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). P. B. Persson is a member of the speakers bureau of Bayer HealthCare Pharmaceuticals (Berlin, Germany). AUTHOR CONTRIBUTIONS Author contributions: M.M.S., R.L., and A.P.: conception and design of research; Z.Z.L, M.M.S., K.S., Y.L., A.P., T.N., and K.C.: performed experiments; M.M.S., Z.Z.L., Y.L., A.P., T.N., and K.S.: data analysis; M.M.S., R.L., Y.L., A.P., and Z.Z.L.: interpretation of results; M.M.S., Z.Z.L., Y.L., and K.S.: prepared figures; M.M.S. and Z.Z.L.: drafted manuscript; M.M.S., R.L., E.S., P.B.P., and A.P. manuscript edition and review. REFERENCES 1. American College of Radiology (ACR) Committee on Drugs and Contrast Media. ACR Manual on Contrast Media, Version 8. ACR Committee on Drugs and Contrast Media, Reston, VA, 2012. 2. Baqai FP, Gridley DS, Slater JM, Luo-Owen X, Stodieck LS, Ferguson V, Chapes SK, Pecaut MJ. Effects of spaceflight on innate immune function and antioxidant gene expression. J Appl Physiol 106: 1935–1942, 2009.

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