Adult beagle dogs of both genders, weighing 10-13 kg (Marshal Farms, USA) were used in this study. The animals were acclimatized and maintained on a standard diet, a routine lighting cycle and room temperature for 6 months, and had a normal renal function before the surgical procedure. The dogs were assigned randomly into a control group (n = 4) and ascorbic acid treatment group (n = 4).

The left kidney was freed from the perirenal tissue and fat, and the left renal artery and vein were then clamped using an atraumatic vascular clamp. The ligation and resection of the vessels was performed at the aorta and caudal vena cava, and moved to a tray filled with ice.

After the left kidney had been removed, all the arteries in the control group were perfused with cold normal saline. Cold normal saline and ascorbic acid (100 mg/kg; Dai Han Pharm, Korea) were perfused in the treatment group followed by autotransplantation.

After an overnight fast, the animals are premedicated with atropine sulfate (0.04 mg/kg, SC; Huons, Korea), and an antibiotic prophylaxis with cefazolin sodium (20 mg/kg, IV; Chong Kun Dang, Korea) and an analgesic with meloxicam (0.2 mg/kg, IV; Boehringer Ingelheim, Korea) were administered at induction. The animals were induced with thiopental sodium (12.5 mg/kg, IV; Dai Han Pharm, Korea) and maintained with isoflurane 2% and a 100% oxygen supply during the procedure. All the dogs were administered a balanced electrolyte solution (10 ml/kg/h, IV). The dogs were administered mannitol (1 g/kg, IV; Dai Han Pharm, Korea) 20 min before the nephrectomies. The kidneys were exposed through a midline incision, and the left renal artery, vein, and ureter were mobilized for transplantation. The kidneys were harvested, flushed with a cold, heparinized saline solution, and placed in a cold, sterile saline solution before the anastomosis. The renal vein was anastomosis to the caudal vena cava in an end-to-side manner using a simple continuous suture pattern of 6-0 polypropylene. The renal artery was anastomosed to the external iliac artery in an end-to-end manner using 7-0 polypropylene in a simple interrupted pattern. If 2 renal arteries were present, both arteries were managed with a bridging of the renal arteries using a modification of the technique reported by Sarin et al. [20]. The ureter was sutured to the bladder mucosa in a modified ureteroneocystostomy technique using 5-0 polyglyconate in a simple interrupted pattern. A nephropexy was created by suturing the renal capsule to the abdominal wall with simple interrupted sutures made from 4-0 polyglycolic acid. After implanting the autograft, contralateral kidneys were removed. Postoperatively, all the dogs were allowed access to water and food ad libitum.

Three days after surgery, vitamin C (30 mg/kg) was injected intravenously in treatment group with the same amount of vehicle (physiological saline solution) being injected in the control group.

The blood urea nitrogen (BUN) and creatinine levels were determined from the serum samples taken on days 0, 1, 3, 5, and 7 before and after the procedure from the jugular vein, using a commercially available kit (VetTest; IDEXX, Japan). The results are expressed in milligrams per deciliter.

The invasive blood pressure was measured using Pulscan-Component (Scionic, USA) and a monitoring-set was used for the arterial blood pressure measurement (B/Braun, Germany) at the renal and external iliac arteries. The blood pressure was measured at pre-anastomosis, 10 and 60 min after the anastomosis through a direct invasion using a 24 G scalp vein set (Korea Vaccine, Korea) in the lumen of the artery.

Blood samples were collected using an anticoagulant as EDTA, and centrifuged at 700~1,000 × g for 10 min at 4℃. The samples were then pipetted off the top yellow plasma layer without disturbing the buff layer, and the plasma samples were stored on ice until assayed or were freeze at -80℃. The superoxide dismutase (SOD) activity was determined using a commercial SOD assay kit (Cayman, USA) for measuring the SOD activity from the plasma. The activity was recorded spectrophotometrically at 450 nm. The enzyme activity was calculated as U/ml. The glutathione peroxidase (GSHPx) activity was measured using a commercial GSHPx assay kit (Cayman, USA). The activity was recorded spectrophotometrically at 340 nm. The catalase (CAT) activity was measured spectrophotometrically at 540 nm using a commercial CAT assay kit (Cayman, USA).

The study protocol called for euthanizing the surviving canines after the third post-transplant week if the serum creatinine values had normalized to <1.8 mg/dl. The tissue samples from the left kidney were taken for a histology examination after euthanasia on postoperative day 21. The tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned in 4 µm slices, and mounted on slides. After deparaffinizing, each specimen was stained with hematoxylin and eosin for an optical microscopy examination. The morphological characteristics of the kidneys were determined using a blinded histology examination.

All the values are expressed as a means ± SD of the determinations for all the dogs in the group. The data was analyzed using an analysis of the variance followed by 2-way repeated measures analysis (ANOVA) and then by a Student's t-test. A p value <0.05 or 0.01 was considered significant.

The serum creatinine levels, which are an index of the kidney function, increased significantly to 6.73 ± 2.12 mg/dl in the control group (baseline levels; 0.72 ± 0.07) after 5 days of reperfusion and then decreased gradually. On the other hand, the serum creatinine levels in the treatment group (baseline levels; 0.87 ± 0.06) increased to 2.71 ± 1.32 mg/dl after 3 days reperfusion and then decreased gradually by 7 days. The BUN was also measured as a second index of the kidney function. Similar to the serum creatinine level, the BUN levels in the control group (baseline levels; 11.7 ± 6.68) increased significantly to 176.6 ± 49.32 mg/dl by 5 days after reperfusion. The BUN levels in the treatment group (baseline levels; 14.4 ± 1.26) increased to 59.87 ± 21.04 mg/dl after 7 days reperfusion.

The blood pressure was measured directly at the external iliac artery. The systolic blood pressure in the control group increased from 86.5 ± 19.67 to 98.5 ± 11.32 mmHg while in the treatment group, these levels decreased from 86 ± 12.67 to 84.75 ± 15.32 mmHg. In the control group, the diastolic blood pressure also increased from 77.75 ± 18.24 to 88 ± 11.51 mmHg while these levels decreased from 78.5 ± 12.23 to 77 ± 13.83 mmHg in the treatment group. In the control group, the mean blood pressure increased from 80.5 ± 18.92 to 91.75 ± 11.14 mmHg by 60 min after the anastomosis. In contrast, in the treatment group, the mean blood pressure increased to 85.5 ± 15.69 mmHg by 10min and then decreased gradually thereafter, and was almost normalized to 79.5 ± 14.20 mmHg by 60 min. However, there was no significant difference between the groups.

The antioxidant enzyme activity in the plasma after the renal autotransplantation was evaluated. The specific SOD, GSHPx and CAT activities before surgery and 72 h after reperfusion are expressed as the mean ± SD. The activity of SOD increased slightly from 2.14 ± 0.22 to 2.31 ± 0.17 nmol/min/ml in the control group. In contrast, the activity of SOD in the treatment group increased from 2.37 ± 0.21 to 2.69 ± 0.25 nmol/min/ml by 72 h of reperfusion (p = 0.037, Fig. 1). There was no significant change in the GSHPx activity in the control group. On the other hand, in the treatment group, the level of activity increased significantly from 109.16 ± 19.51 to 132.47 ± 15.50 nmol/min/ml by 72 h of reperfusion (p = 0.032, Fig. 2). There was no significant change in the CAT activity in the control group whereas the CAT activity in the treatment group increased from 2.73 ± 0.30 to 3.74 ± 0.37 nmol/min/ml by 72 h of reperfusion (p = 0.038, Fig. 3).

At the autopsy 21 days after the transplant, the control kidneys in which a normal saline had been administered demonstrated moderate cystic dilation of the tubules along with inflammatory cell infiltration, regeneration of the tubules and congestion (Fig. 4A & B). In contrast, the kidneys in the treatment group, which had been administered ascorbic acid after the renal transplantation showed slight damage to the tubules. The tubular epithelium was essentially normal, even though there was occasional, mild cystic dilation of tubules with mild peritubular inflammatory cell infiltration and regeneration of the tubules (Fig. 4C & D).