TITLE: A rat model of contrast-induced acute kidney injury following intra-arterial contrast administration
AUTHORS: Zhiqing Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Contrast-induced acute kidney injury (CI-AKI) is an iatrogenic complication frequently developed after cardiac catheterization procedures with the administration of </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">iodinated</span></a><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">contrast med</span></a><span>ia. However, the precise pathological mechanisms remain obscure. Thus, the establishment of a reproducible rat model following </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">intra-arterial contrast medium (CM) injection</span></a><span> to simulate </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">the process of arteriography</span></a><span> is of great importance. Here, a detailed protocol for establishing a CI-AKI model was describe. Three-month-old male Sprague-Dawley rats, weighing approximately 300~400g at the start of the experiment, were used. The rats were pretreated with water dehydration for 48 hours, plus indomethacin and N-ω nitro-L-arginine methyl ester (L-NAME) injection, before intra-arterial contrast medium (CM) administration. Levels of serum creatinine (SCr) were measured and histopathological assessment of the kidney tissue by H&E staining were conducted. As expected, the administration of CM, following the pretreatment by indomethacin and L-NAME, induced remarkable renal dysfunction at 12hours </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">post-procedure</span></a><span> in comparing with controls. The pronounced histopathological alterations in the tubular epithelial cells of the inner medulla and outer medulla were also observed in the CI-AKI rats. Thus, this rat model was reliable as a valuable tool for exploring the pathogenesis of CI-AKI.</span></div></div>

[STEPS]
?. [Surgical Procedure]
Rats were deprived of water for 48 hours。
?. Body weight of each rat was measure.
?. Deeply anaesthetized was conducted by pentobarbital sodium (40 mg/kg, i.p.).
?. The right femoral vein and carotid artery were both cannulated by short peripheral catheters which were widely used in the nursing practice (24 G×21 mm).
?. A baseline blood sample was drawn (1 ml) from the carotid artery for determination of SCr.
?. Indomethacin was dissolved in phosphate buffer (pH 8.4) at a concentration of 5mg/ml, then administrated intravenously at the dose of 10 mg/kg.
?. After 15 min, L-NAME (dissolved in 0.9% normal saline at a concentration of 10mg/ml) was administrated intravenously at the dose of 10 mg/kg.
?. After another 15 min, these rats were randomized to receive iopromide (CI-AKI group) or normal saline (Control group) at the dose of 7.8 ml/kg via the carotid artery cannulation over a time course of 5 min.
?. The rats were then allowed to recover in individual cages with free access to tap water and standard chow.
?. [Sample Collection]
At 12 hours post CM injection, the rats were deeply anaesthetized by pentobarbital sodium (40 mg/kg, i.p.).
?. [Sample Collection]
A second set of arterial blood sample was obtained from the ventral aorta.
?. [Sample Collection]
The blood cells were removed by perfusing with 0.9% normal saline through the bilateral renal arteries.
?. [Sample Collection]
Both kidneys were harvested and bisected longitudinally. The right kidney, containing cortex and medulla, was fixed in 10 % neutral formalin liquid for histological analysis.
?. [Sample Collection]
The rats were euthanized with an overdose of pentobarbital anesthesia (200 mg/kg, i.p.).
?. [Measurement of SCr level]
Blood samples were centrifuged within 2 hours at 1000 × g for 15 min.
?. [Measurement of SCr level]
The levels of SCr were measured at the hospital clinical laboratory by a standard method.
?. [Histopathological assessment of the kidney tissue by H&E staining]
The histological samples were embedded in paraffin and sectioned at 3 to 4 μm thick.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Deparaffinize each slide with xylene (repeated 3 times, 2 min each).
?. [Histopathological assessment of the kidney tissue by H&E staining]
Hydrate tissue sections with gradient ethanol (100% ethanol repeated 3 times, 1 min each; 80% ethanol for 1 min) to distilled water in a fume hood.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Incubate the slides with hematoxylin solution in a staining jar for 4 min, and then rinse with tap water for 2 min to remove the unbound dye from slides.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Transfer the slides to 0.1% hydrochloric acid in alcohol for 30 sec. Then rinse with tap water for 1 min.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Transfer the slides to 0.5% ammonia solution for 1 min until the background turns light blue. Then rinse with tap water for 1 min.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Transfer the slides to 80% ethanol for 1 min.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Incubate the slides with 1% eosin in a staining jar for 50 sec, and then rinse with tap water to remove the unbound dye from slides.
?. [Histopathological assessment of the kidney tissue by H&E staining]
Dehydrate with 100% ethanol (repeated 3 times, 1 min each) and clear in xylene (repeated 3 times, 1 min each).
?. [Histopathological assessment of the kidney tissue by H&E staining]
The slides were then mounted with resinous mounting medium and stored at room temperature.
?. [Histopathological assessment of the kidney tissue by H&E staining]
After being visualized by hematoxylin and eosin (H&E) staining, the slides of each sample were reviewed by lightmicroscope.
?. [Histopathological assessment of the kidney tissue by H&E staining]
FTubular injury was graded by the Paller score, a semi-quantitative evaluation. For each kidney, 100 tubules from 10 highmagnification (×200) fields of the medulla and the outer medulla were scored.
As expected, the administration of CM, following the pretreatment by indomethacin and L-NAME, induced remarkable renal dysfunction at 12 hours post-procedure. The average elevation of SCr was (59.9±23.0) % over baseline in the CI-AKI rats, comparing to (-10.5±17.1) % in the control rats injected with normal saline (Fig 1, p The pronounced histopathological alterations in the tubular epithelial cells of the inner medulla and outer medulla were observed in the CI-AKI rats, including vacuole-like denaturation, nucleus pycnosis and apoptotic body formation(Fig 2A). Additionally, the mean Paller score for the contrast treated kidneys was 8.2 ± 0.9, whereas that for controls was 3.6 ± 1.2 (Fig 2B, p 5), indicating of a greater extent of tubular injury after the intervention of contrast.