Contributed equally
The aim of the current study was to investigate the cytotoxic effects of hypotonic (iopamidol) and isotonic (iodixanol) contract media (CMs)
Iodinated contrast medium (CM) is widely used in percutaneous cardiac interventions. The use of CMs in cardiac interventions may lead to a type of kidney dysfunction called contrast-induced nephropathy (CIN). As the number of interventional cardiac procedures is steadily increasing, CIN is now associated with the third highest number of hospital acquired nephropathy diagnoses (
CM-induced nephrotoxicity is closely related with the permeability, viscosity, ionization degree and dosage of selected CMs (
A total of 60 Wistar rats were included in the current study. The rats were randomly divided into three groups (20 rats/group) and injected with iodixanol (4 g iodine/kg body weight), iopamidol (4 g iodine/kg body weight) or an equal volume of normal saline via tail vein. The rats were then randomly divided into 4 subgroups according to the time points following injection and were sacrificed at 0.5, 2, 12 or 24 h following injection, respectively. Blood samples, collected from the abdominal aorta for each animal, were treated with heparin to prevent coagulation and centrifuged at 670.8 × g for 10 min at 4°C. Plasma samples were then immediately transferred to −80°C for future tests. All animal experiments were conducted according to the ethical guidelines.
HUVEC cells were cultured in F12K medium containing 10% fetal bovine serum (FBS), 0.1 mg/ml heparin and 0.03–0.05 mg/ml endothelial cell growth supplement. H5V cells (mouse endothelial cells) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS. All of these cells were maintained in an atmosphere of 5% CO2 at 37°C.
Cells were seeded into 96-well plates (5×104/well) and cultured for 24 h before use. Cells were then treated with 10 mg iodine/ml iodixanol or iopamidol for 30 min, 2, 12 or 24 h, respectively. Cell viability was determined by using CCK-8 agents following the manufacturer's manual (Dojindo Molecular Technologies, Inc., Kumamoto, Japan).
Cells were lysed with a protein extraction buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40 and 0.5% sodium deoxycholate], then quantified using a bicinchoninic acid kit (cat no. orb219872; Wuhan Booute Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Proteins (100 µg/well) were then loaded onto 10% SDS-PAGE gels for electrophoresis. Protein was blotted onto nitrocellulose membrane and blocked with 5% skimmed milk at room temperature for 2 h. Then, the membrane was incubated at 4°C for 2 h with antibodies against ABCG1 (cat no. PL0402507; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (cat no. A01020; 1:10,000; Abbkine Scientific Co., Ltd., Wuhan, China). Following washing with TBS containing 0.1% Tween-20 for 4 times, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (cat no. B1254; 1:1,000; Beijing Yonghui Biological Technology Co., Ltd., Beijing, China) at 4°C for 2 h. Specific bands were measured by using SuperSignal West Femto Maximum Sensitivity substrate (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). The specific band intensities were quantified with Quantity One software (version, 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Abdominal aortae from each subgroup were washed with PBS and fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin and cut into 4 µm thick sections. The sections were de-paraffinized with xylene and subsequently rehydrated. For routine histological analysis, the sections were stained with hematoxylin and eosin. For immunohistochemistry staining, the sections were treated with 3% H2O2 in PBS for 30 min to block the endogenous peroxidase and heat-repaired in 0. 01 M citrate buffer for 15 min. The sections were further blocked by normal goat serum for 30 min at room temperature prior to primary anti-endothelial nitric oxide synthase antibody (cat no. 4231; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) incubation at 4°C overnight. On the next day, the sections were incubated with biotin-conjugated goat anti-rabbit secondary antibody (cat no. sc-2007; 1:1,000; Santa Cruz Biotechnology, Inc.) for 2 h at 37°C followed by incubation in S-A/horseradish peroxidase (Santa Cruz Biotechnology, Inc.) for 15 min at room temperature. Following this, the sections were counterstained with hematoxylin, dehydrated, mounted with neutral gum and observed using Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan).
Plasma ET, von Willebrand factor (vWF), tissue type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI), D-Dimer, fibrinogen (FG), anti-thrombin III (AT-III), plasminogen (PLG) and NOS from cell culture medium were measured by using ELISA kits [Beijing Yonghui Biological Technology Co, Ltd.; Plasma ET (cat no. GD-VN9950), vWF, t-PA, PAI, D-Dimer and FG (cat no. QY-VN3291), AT-III (cat no. E04298), PLG (cat no. E00403) and NOS (cat no. JK-(a)-1609)]. Briefly, samples were applied to a 96-well plate (1×105/well) with pre-coated capture antibodies for 2 h at 37°C. Then, the plates were washed and incubated with biotinylated antibodies (cat no. 500-P39Bt; 1:1,000; PeproTech, Inc., Rocky Hill, NJ, USA) at 37°C for 2 h. The plates were then washed five times and incubated with streptavidin-horseradish peroxidase (Beijing Yonghui Biological Technology Co, Ltd.; cat. no. 554066; 1:1,000) at 37°C for 30 min. The plates were washed again for five times and horseradish peroxidase substrates were added. The reactions were stopped after 30 min. Optical density was measured at 450 nm by using a microplate reader (Thermo Fisher Scientific, Inc.).
All values were presented as means ± standard deviation. Two-tailed Student's t-test was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) to determine the statistical significance of the results between two groups. P<0.05 was considered to indicate a statistically significant difference.
To examine the effect of CMs on endothelial cells
To further investigate whether CMs induce endothelial cell injury
To measure the levels of ET, NOS and ABCG1 in CM-treated cells
To compare whether iopamidol and iodixanol have similar effects on thrombin and fibrinolytic system, plasma levels of vWF, t-PA, PAI-1, D-Dimer, FG, AT-III and PLG were measured using ELISA. The data indicated that plasma vWF level was upregulated following iopamidol and iodixanol treatments at 0.5, 2 and 12 h after injection. However, plasma vWF returned to a normal level at 24 h in iodixanol group, but remained high in iopamidol group (
In the present study, the toxicity of two commonly used CMs on endothelial cells was investigated
Endothelial cells synthesize and secrete numerous vasoactive substances (
ABCG1 is a member of ABC transporter that is broadly expressed in various tissues (
Dysfunction of endothelial cells may cause thrombin and fibrinolytic system disorder. A previous study reported that CMs upregulate plasma vWF level (
The present study was supported by the Capital Health Research and Development Special Foundation of China (grant no. 2014-4-2082), and Basic Clinical Research Cooperation Project of Capital Medical University (grant no. 14JL87).
Effect of contrast media on endothelia
Effect of contrast media on endothelin in (A) HUVEC cells, (B) H5V cells and (C) rat plasma. The cells were incubated with 10 mg iodine/ml iopamidol or iodixanol for the indicated time, and ELISA assays were performed to measure endothelin levels. Wistar rats were injected with 4 g iodine/kg iopamidol, iodixanol or equal volume of normal saline (as control) for the indicated time, and plasma ET was detected by using ELISA assay (n=5). All data were obtained from at least three independent experiments and are expressed as means ± standard deviation. *P<0.05, **P<0.01 vs. control group. ET, endothelin.
Effect of contrast media on NOS in (A) HUVEC cells, (B) H5V cells, and (C) rat plasma (magnification, ×100). The cells were incubated with 10 mg iodine/ml iopamidol or iodixanol for the indicated time, and ELISA assays were performed to detect the expression of NOS. Wistar rats were injected with 4 g iodine/kg iopamidol, iodixanol or equal volume of normal saline (as control) for the indicated time. eNOS in endothelia of abdominal aorta was detected by immunohistochemistry. Representative graphs are shown. All data were obtained from at least three independent experiments and are expressed as means ± standard deviation. **P<0.01 vs. control group. NOS, nitric oxide synthase; eNOS, endothelium-derived NOS.
Effect of contrast media on the expression of ABCG1 in (A and B) HUVEC cells and (C and D) H5V cells. The cells were incubated with 10 mg iodine/ml IOP or IOD for the indicated time, and western blotting was performed to detect the expression of ABCG1 in the cells. (B and D) Data were obtained from three independent experiments and are expressed as means ± standard deviation. (A and C) The band intensity was normalized to GAPDH. **P<0.01 vs. control group. ABCG1, ATP-binding cassette subfamily G member 1; IOD, iodixanol; IOP, iopamidol.
Effect of contrast media on the levels of plasma thrombin and fibrinolytic biomarkers. (A-G) Wistar rats were injected with 4 g iodine/kg iopamidol, iodixanol or equal volume of normal saline (as control) for the indicated time. (A) Plasma von Willebrand factor, (B) t-PA, (C) PAI, (D) D-Dimer, (E) FG, (F) AT-III and (G) PLG were measured by using ELISA assays. Data were obtained from at least three independent experiments and are expressed as means ± standard deviation. *P<0.05, **P<0.01 vs. control group; #P<0.05 vs. iopamidol group. vWF, von Willebrand factor; t-PA, tissue type plasminogen activator; PAI, plasminogen activator inhibitor; FG, fibrinogen; AT-III, anti-thrombin III; PLG, plasminogen.