Prevention of renal ischemia / reperfusion injury in rats using acetylcysteine after anesthesia with isoflurane

PURPOSE: To evaluate the effect of N-acetylcysteine, as a renoprotective agent, when administered early after anesthesia induction, against ischemia/reperfusion injury in rats anesthetized with isoflurane. METHODS: Eighteen male Wistar rats weighing > 300g were anesthetized with isoflurane. The internal jugular vein and the left carotid artery were dissected and cannulated. The animals were randomly divided into GAcetyl, receiving intravenous N-acetylcysteine, 300mg/kg, and GIsot, isotonic saline. After 30 minutes, right nephrectomy was performed and the left renal artery was clamped during 45 minutes. The animals were sacrificed after 48 hours and blood samples were taken after anesthetic induction and upon sacrificing of the animals to evaluate blood creatinine. The kidneys were sent for histological analysis. RESULTS: The variation in serum creatinine was 2.33mg/dL ± 2.21 in GAcetyl and 4.38mg/dL ± 2.13 in GIsot (p=0.074). Two animals presented intense tubular necrosis in GAcetyl, compared to 5 in GIsot. Only GAcetyl presented animals free of tubular necrosis (two) and tubular degeneration (one). CONCLUSION: After renal ischemia/reperfusion, the rats which were given N-acetylcysteine presented less variation in serum creatinine and milder kidney injuries than the control group.


Introduction
Acute renal failure (ARF) has high incidence in intensive care units, representing an isolated prognostic factor in patients with multiple organ and system disorder¹.
Reactive oxygen species are normally produced during cell metabolism.Free radicals may affect cell vitality by several mechanisms, interfering in the cell growth and proliferation, regeneration and tissue repair, inflammatory response and immune process.The main compounds are the superoxide anion, hydrogen peroxide and the hydroxyl radical.Nitric oxide and the peroxynitrite radical also present the capacity to induce oxidation damage 2 .Kidneys are responsible for 10% of the body oxygen consumption.They are physiological sources of free radicals, especially stemming from the tubular metabolism³.This production increases during the tissue insult process and may contribute towards the installation and progression of the kidney injury 4 .
ARF incidence during the postoperative period varies in accordance with the series studied and the established definition.
Kidney dysfunction is associated with worst case prognoses, especially when dialysis is need.A review conducted on patients who underwent vascular surgery revealed an incidence ranging between 1.7% and 25% 5 .The risk factors can be related to the patient, such as pre-existing kidney or heart dysfunction, diabetes, sepsis, liver failure and exposure to nephrotoxins, or to the surgical procedure, such as procedures involving extracorporeal circulation or aortic clamping, surgeries that result in an increase in intra-abdominal pressure, kidney or liver transplants and use of intravenous contrasts 6 .
Several drugs have been used, without success, in the attempt to prevent ARF in risk situations, such as atrial natriuretic peptide 7 , calcium channel blockers 8 , dopamine 9 , loop diuretics 10 , mannitol 11 , among others.The studies will therefore continue in order to find agents that benefit more serious situations.
N-acetylcysteine is a thiol with mucolytic properties that is broadly used in clinical practice with few side effects.It has direct and indirect antioxidant properties, a precursor for L-cysteine and glutathione 12 , a source of sulfhydryl groups and free radical scanner 13 .It has been studied in prophylaxis of nephropathy induced by contrast 14 and in kidney 15,16 , lung 17 and intestinal reperfusion injury 18 , still with conflicting results.It has been administered to animals by intra-peritoneal injection, before ischemia and in different doses, as 200mg/kg, 24 and 12 hours before 15 , 200mg/kg, 24, 12 and two hours before 16 , and 40 or 300mg/kg, 24 hours and 15 minutes before 19 , usually with 45 minutes of arterial clamping.
Recent studies revealed kidney reperfusion injuries with only 25 minutes of clamping 20 .It has yet to be determined whether it will have beneficial effect if administered at the beginning of the anesthetic-surgical procedure.
The aim of this study was to evaluate the effect of N-acetylcysteine as a renoprotective agent, when administered early after induction, against ischemia/reperfusion injury in rats anesthetized with isoflurane.

Methods
This study was approved by the Committee for Ethics in Animal Experimentation at the Botucatu School of Medicine, UNESP.Eighteen male Wistar rats weighing more than 300g were anesthetized with isoflurane in an appropriate campanula.After anesthesia induction, they were intubated with an orotracheal cannula and maintained in controlled mechanical ventilation (Harvard Rodent Ventilator, USA).Anesthesia was maintained with isoflurane in 0.3 FIO 2 (Ohmeda vaporizer, USA).The right internal jugular and the left carotid artery were dissected and cannulated with 24G venocath.At that moment, 1mL of blood was collected for the serum creatinine determination, which was replaced with 2mL of isotonic saline solution.An isotonic saline solution infusion was maintained at 5mL/kg/h with an infusion pump (Anne Pump, Abbott, USA).Rectal temperature was maintained between 36 o C and 38 o C using a thermal bag under and on the animal.Mean arterial pressure (MAP1, before clamping, and MAP2, immediately after clamping the left renal artery), O 2 saturation (SatO 2 ) (sensor on the animal's tongue), end expired CO 2 (ET CO2 ) and the expired isoflurane fraction (ET iso ) (recorded every 15 minutes until the end of the experiment) were monitored by the Datex Engstron, Finland.The animals were divided into two groups using randomizing computer software: group 1 (GAcetyl) (n=9) receiving intravenous N-acetylcysteine (Zambon, Italy), 300mg/kg 19 , and group 2 (GIsot) (n=9) receiving an isotonic saline solution at the same volume.After 30 minutes, a laparotomy was performed, followed by right nephrectomy and left renal artery clamping for 45 minutes using a non-traumatic vascular clamp.After unclamping the renal artery, the venocaths from vein and artery were removed and the abdominal and cervical wall was sutured using non-absorbable thread on a single plane.The isoflurane was interrupted and the animals remained with the tracheal tube and 0.3 FIO 2 until complete recovery from anesthesia.For postoperative analgesia, the surgical wound was infiltrated with 0.25% bupivacaine.The rats were sent to a shelter and kept in a warm environment with water and food.After two days, the animals were anesthetized again with isoflurane and after performing left nephrectomy and collecting 1 mL of blood for serum creatinine determination, they were sacrificed with an overdose of intra-cardiac sodium pentobarbital.The dry chemistry method was utilized for serum creatinine determination.The kidneys were stored in a DuBoscq Brasil solution (formaldehyde, 6 mL, acetic acid, 15 mL, absolute alcohol, 12 mL and picric acid, 8 mL) for up to 36 hours and, subsequently, in alcohol 70%.
Histological analysis was conducted by a single pathologist, who did not have knowledge which group the animals were from, observing the following parameters: tubular degeneration, regeneration and necrosis, and inflammation.The injuries were graded as absent (grade zero), mild (grade I), moderate (grade II), and intense (grade III).
For statistical analysis, the Student t test was used for the categorical variables and the Mann-Whitney test for the noncategorical variables.A value of p<0.05 was adapted to statistical significance.
However, for the kidneys that suffered ischemia, tubular necrosis was not observed in two GAcetyl kidneys but it was observed in grade II in GAcetyl (4 kidneys) and GIsot (3 kidneys) and grade III in GAcetyl (2 kidneys) and GIsot (5 kidneys) (p = 0.18).Only one GAcetyl kidney did not present tubular degeneration, but there was this change in grade I in GAcetyl (4 kidneys) and GIsot (3 kidneys), grade II in GAcetyl (one kidney) and GIsot (5 kidneys) and grade III only in GAcetyl (3 kidneys) (p = 0.12).No tubular regeneration was observed in four GAcetyl kidneys and in one GIsot kidney, but there was grade I in GAcetyl (2 kidneys) and GIsot (one kidney), grade II in GAcetyl (one kidney) and GIsot (4 kidneys) and grade III in GAcetyl (one kidney) and GIsot ( 2kidneys) (p = 0.23).Tubular inflammation was not observed in three GAcetyl kidneys and one GIsot kidney, but it appeared in grade I in GAcetyl (4 kidneys) and GIsot (7 kidneys) and grade II only in GAcetyl (one kidney) (p = 0.24) (Table 1, Figure 2, A, B,   C, and D).

FIGURE 1 -
FIGURE 1 -Mean ± standard deviation of variation of serum creatinine (mg/dL) between two time points from administration of acetylcysteine early after anesthesia induction: before ischemia and 48 hours after reperfusion.

FIGURE 2 -
FIGURE 2 -A.Left kidney with tubular necrosis (grade 1, group acetylcysteine): few anucleated (necrotic) tubular eosinophilic cell (HE 400x).B. Left kidney with tubular necrosis grade 2 commitment: ischemic tubular necrosis, with early regeneration of tubular cells evidenced by mitotic figures (arrow -added detail) (group acetylcysteine) (HE 400x).C. Left kidney with grade 3 necrosis.Most tubules with eosinophilic cells in karyolysis (detail), some still with pyknotic nuclei (group isotonic saline) (HE 400x).D. Ischemic left kidney with tubular hyalin casts in the medullary some with focal areas of calcification in detail in blue (group isotonic saline) (HE 200x).

TABLE 1 -
Number of rat kidneys with histological changes in GAcetyl (group acetylcysteine) and GIsot (group isotonic saline solution) after ischemia/reperfusion. Grade zerolack of change, I -mild change, II -moderate, III -intense.