Forty infant Wistar albino rats were housed with their mothers until they reached four weeks old; then, they were housed in rat cages with free access to a standard rodent diet and tap water under a 12:12-hr artificial light cycle, with a mean temperature of 21 ± 2 °C and a mean humidity of 55 ± 2%. All animals were randomized to four groups and treated as follows:
Group 1 (n = 10): Control, normal saline alone, injected with saline (10 ml/kg) i.p.
Group 2 (n = 10): L-Carnitine alone, injected with L-Carnitine (300 mg/kg) i.p.
Group 3 (n = 10): Irradiation alone (RT), injected with saline (10 ml/kg) i.p. 30 minutes before irradiation.
Group 4 (n = 10): L-Carnitine before irradiation (L-Carnitine + RT), injected with LC (300 mg/kg) i.p. 30 minutes before irradiation.
All experimental procedures were performed on anesthetized rats. Anesthesia was maintained with ketamine and xylazine (35 mg/kg body weight (BW) and 3 mg/kg BW intramuscular injection for infant rats and 50 mg/kg BW and 5 mg/kg BW intramuscular injection for adults) during irradiation and scintigraphic evaluation.
Rats were followed up for three months after the experimental procedures. During the follow-up period, all rats received veterinary care. Before euthanasia, the rats received anesthesia using a combination of ketamine and xylazine. Euthanasia was performed by decapitation, and a histopathologic assessment of the kidney was performed. All animal experiments were conducted according to the guidelines from the Institutional Animal Ethics Committee (TUHDYEK 2012/13).
The rats in the RT and L-Carnitine + RT groups were irradiated individually with a single dose of 8 Gy (6 MV photon) at a depth of 2.25 cm through an anterior 2.5 × 2 cm single portal (with a 1.5 cm bolus) covering the left kidney in its entirety. A linear accelerator treatment unit (Varian 2100 C/D, Varian Inc., Palo Alto, CA) was used at a source skin distance of 100 cm. The dose rate was 600 MU/min. The rats were anesthetized and then fixed on a 20×30 cm blue Styrofoam treatment couch (Med-Tec, Orange City, IA) in a prone position. The position of the kidney was determined in an X-ray scope image by measuring the distance between the tip of the nose and the middle of the kidney hilus. The distance information was used to position the center of the irradiation field to the middle of the kidney hilus in the anesthetized rat. Correct positioning of the fields was confirmed for each individual rat using a therapy simulator (Mecaserto-Simics, Paris, France). Special dosimetry was performed for the irregular radiotherapy fields. The dose homogeneity across the radiotherapy field was ± 3%.
All animals underwent both Tc99m DTPA dynamic renal imaging and Tc99m DMSA static renal imaging three months after radiotherapy.
For Tc99m DMSA imaging, a commercially available DMSA kit (Renocis, France) was prepared according to the manufacturer’s recommendations. 99mTc-DMSA was administered intravenously through the 24F cannula in the tail vein at a regular dose of 37-50 MBq (in 0.2 ml). Posteroanterior views of the abdomen of the rats were taken in the supine position (at least 300 counts/view) using a single-head gamma camera (Philips, Eindhoven, the Netherlands) with a low-energy, high-resolution pinhole and parallel collimators in a 256 × 256 matrix format at 3 hours after the injection of Tc-99m DMSA. The relative uptake of the kidneys was quantitatively calculated by drawing regions of interest for kidney and background areas on the computed anterior and posterior images. 99mTc-DMSA uptakes (mean value/pixel) of renal tissue and background tissue were calculated from images with the drawn region of interest. The total renal function percentage for the left kidney (LF %) was calculated using the following formulas:
Eq. 1: LF%= Left kidney uptake/(Left kidney uptake + Right kidney uptake) ×100.
For 99mTc-DTPA imaging, a commercially available DTPA kit (CIS, France) was prepared according to the manufacturer’s recommendations. Dynamic scans were acquired for 20 minutes on a single-head gamma camera (Philips, Eindhoven, the Netherlands) with low-energy, high resolution, and parallel collimators in a 64×64 matrix format after the injection of 99mTc-DTPA. Regions of interest (ROIs) were placed around each kidney. Curves of the 30-second frame rate were generated from both ROIs over the 20-minute study. The time to peak count (Tmax) and time from peak count to a half count (T1/2) was calculated from the curve data.
The left kidneys were dissected and decapsulated after imaging and then prefixed in formaldehyde for 24 h for further histopathological evaluation. The kidneys were divided into halves with a central transverse section. After formalin fixation, they were processed into paraffin wax tissue blocks, and thin tissue sections (4 nm) were produced using a microtome. All sections were then stained with hematoxylin and eosin (H&E) and evaluated under a light microscope. A certified pathologist who was blinded to the experimental protocol assessed the tissue sections.
To evaluate RIN, we evaluated proximal tubular degeneration, proximal tubular atrophy, interstitial fibrosis, and glomerular damage microscopically. Cytoplasmic eosinophilia, apical blebbing, loss of intercellular adhesions, cytoplasmic vacuolization, karyorrhexis, and karyolysis were defined as proximal tubular degeneration. Capillary loop collapse and dilatation of Bowman’s capsule were defined as glomerular damage. Proximal tubular degeneration, proximal tubular atrophy, interstitial fibrosis, and glomerular damage were scored as follows: 0 (no abnormality), 1 (weak lesions affecting <25% of the kidney samples), 2 (moderate lesions affecting 25-50% of the kidney samples), and 3 (marked lesions affecting >50% of the kidney samples).
Scintigraphy data are presented as the mean (±) standard deviation (SD), and histopathological data are presented as the median (min-max). Differences in the scored parameters among the four groups were analyzed with ANOVA. Intergroup comparisons were tested by post hoc Bonferroni tests. These differences were considered significant when the probability was less than 0.05.
The results are expressed as the median (interquartile range). The normality distribution of the variables was tested by a one-sample Kolmogorov–Smirnov test. The Kruskal–Wallis test was used to assess the statistical significance of comparisons, and then the Dunn test was used for multiple comparisons when significant results were obtained. Statistics were performed with Statistica version 7.1 (Statsoft Inc., Tulsa, OK, USA). Statistical differences were considered significant when the p-value was smaller than 0.05 with two-sided probability.