80 male and female Wistar rats (weighting 200 ± 20g) were kept at a constant temperature of 23-25 ºC and 12h light/12h dark cycle. The rats were fed with rat chow and water ad libitum. The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the US guidelines (NIH publication #85-23, revised in 1985). The rats were purchased from Animal House of Hormozgan University of Medical Sciences, Bandar Abbas, Iran. The protocol of experiment was approved in advance by the Hormozgan University of Medical Sciences Ethics Committee (HUMS.REC.1396.138).
The animals were randomly assigned into 8 experimental groups, n=10 in each groups, (4 groups of male and 4 groups of female: Group1 (sham): male rats were exposed to surgery without ischemia process. Group 3, 5 and 7 (named ISC3hr, ISC24hr, and ISC48hr): male rats were subjected to renal ischemia with 3 hr, 24 hr or 48 reperfusions respectively. Groups 2, 4, 6 and 8 were subjected to same procedure of the groups 1, 3, 5 and 7 respectively except female rats used instead of male. To obtain renal ischemia, the animals body weights (BW) were measured and they were anesthetized with chloral hydrate injection (450 mg/kg; ip), the kidneys were carefully excised. Special care was done to avoid damage to the organs. The both kidney arteries and veins were occluded for 45 min by clamping.
The occurrence of ischemia was visually confirmed by the observation of renal blanching. After 45 min, the clamps were removed with care to initiate kidney reperfusion. The kidneys were observed for about 10 min to ensure reperfusion. The animals that didn’t have adequate restoration of blood flow into kidneys were excluded from the study. Then, the skin and tissue were sutured and the animals were kept in the animal room under direct observation for 3 hours, 24 hours and 48 hours as assigned.
Collection of blood sample by cardiac puncture
At the end of reperfusion times, the animals were anesthetized again, with chloral hydrate injection (450 mg/kg; ip, and the blood samples were obtained via heart puncture with 22G needle and 5 ml syringe. The animals were sacrificed humanly with over dose administration of anesthetic drug. An incision was made in the chest and abdomen with a surgical blade to obtain kidney and lung tissues.
Blood samples were centrifuged at 2500 g for 10 minutes to obtain serum samples for measuring the serum level of blood urea nitrogen (BUN), creatinine (Cr), nitrite, and malondialdehyde (MDA). Serum samples were sorted at -80 ºC until measurement.
Preparation of kidney and lung tissue
The kidneys and lung tissues were also removed and weighted immediately. The kidney weight (KW) was normalized to the BW, and reported as tissue weight (kW)/100 g of BW. The right kidney and lung tissues were fixed in 10%formalin for histopathological investigation. The removed left kidney was transferred into liquid nitrogen very quickly and then was stored at -80 ºC refrigerator until measurement. In addition, a small part of kidney and lung tissues were weighted and homogenized and centrifuged at 15,000 g for 2 min, and the supernatant was used for MDA and nitrite measurements.
To evaluate the effects of IRI on lung histology, the right bronchi was tied with 3 -0 silk and one needle was placed in the right bronchi. Then, 1 ml of 10% formalin was instilled into the right lung to achieve inflation ex-vivo. Finally, it was removed and again fixed in 10% formalin for pathological examinations. To determine lung water content (index of edema), a sample of left lung tissue was taken and was immediately weighed as lung wet weight.
Measurement of pulmonary water content
Pulmonary edema was measured by determination of lung water content. The left lung was dried in the oven under 100°C for at least 48 hours until constant weight was obtained. The percentage of lung water content was calculated by the following formula:
Water content% = [(Lung wet weight – Lung dry weight)/ Lung wet weight] × 100
The serum levels Cr and BUN were determined using quantitative diagnostic kits (Pars Azmoon, Iran). Serum and kidney nitrite levels (stable metabolite of NO) were measured by using an assay kit (Promega Corporation, USA) The nitrite concentration of samples was determined by comparison with the nitrite standard reference curve. The serum and kidney levels of MDA were quantified according to the thiobarbituric acid (TBA) method .
The kidney and lung tissue were fixed in 10% formalin solution, and embedded in paraffin for histopathological staining. The Hematoxylin and Eosin stain was applied, and to determine the kidney damage, presence of tubular atrophy, ischemic necrosis, inflammation, vessels congestion, hyaline casts, vacuolization, and debris were evaluated. Based on the damage intensity and damage percentage, the samples were scored as 1-4 while score zero was assigned to normal tissue.
To determine the lung tissue damage, presence of congestion, inflammation, and fibrosis were evaluated and graded.
The data are presented as Mean ± SEM. Differences among groups in serum levels of BUN, Cr, NO, and MDA; and kidney levels of MDA and NO, kidney weight, lung water content were compared with each other by tow-way ANOVA followed by the Tukey post hoc test. Due to the qualitative nature of scoring, Kruskal-Wallis tests with Mann-Whitney were used to compare the pathological damage score of the groups. Values of P< 0.05 were considered statistically significant.