Ischemia-Reperfusion Dexmedetomidine and S ( + )-ketamine in ischemia and reperfusion injury in the rat kidney

Purpose: To investigate blood creatinine and renal histology in rats anesthetized with S(+)-ketamine (keta) or dexmedetomidine (dex) and submitted to kidney ischemia/reperfusion injury (IRI). Methods: Under intraperitoneal (ip) S(+)-ketamine, 20 male Wistar rats were divided into two groups (n=10): maintenance with iv S(+)-ketamine or dex (keta and dex groups), and submitted to right (R) nephrectomy and left (L) renal artery clamping for 45 min. Blood creatinine was measured before ischemia (T1) and 48h after reperfusion (T2), when L nephrectomy was performed. Histological analysis was performed in all kidneys. Results: Blood creatinine was significantly higher at T2 in both groups, but dex group results were lower than those of keta group. Histological changes: between groups, R kidneys did not differ; there were significant high scores for vascular dilation: keta L kidneys; for vascular congestion, tubular dilation, and necrosis: L kidneys from both groups; for tubular degeneration: keta R kidneys. Conclusion: S(+)-ketamine plus IRI were aggressive to rat kidneys, according to histological changes, and dexmedetomidine may have not totally protected the kidneys from these injuries, despite the better results of blood creatinine.


Introduction
The protective effect of brief periods of artery occlusion and reperfusion against a subsequent prolonged exposure to ischemia and reperfusion (ischemic preconditioning -IPC) has been long demonstrated in the heart and kidney 1,2 .Some intravenous anesthetics have distinctive actions on mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels and these channels play a pivotal role in mediating cardiac preconditioning 3 .The intravenous agent dexmedetomidine is a potent and highly selective α 2 -adrenoceptor agonist that facilitates analgesia and anesthesia in humans.However, it has been demonstrated in a rat model of focal cerebral ischemia that, rather than mimicking IPC protecting the kidney, it reduces total ischemic volume in the cortex 4 .Because IRI may be an event in the perioperative period, and the role of intravenous anesthetics in the course of IRI has not been fully established, research on this protection is welcome.According to the literature, S(+)-ketamine did not affect mitoK(ATP) channel activity 3,5 , so the purpose of this study was to assess creatinine level and renal histology in rats anesthetized with S(+)-ketamine which received dexmedetomidine and were submitted to kidney IRI.

Methods
This study was approved by the Institution's Animal Experimentation Ethics Committee.Under S(+)-ketamine anesthesia (100 mg/kg-ip), 20 male Wistar rats remained in spontaneous ventilation using a mask with O 2 (1L/min).In order to establish the experimental model, a 24G catheter was inserted into the right internal jugular vein and the left carotid artery.
Ringer's lactate solution (5mL/kg/h) was administered and body temperature was kept at 36 o C -38 o C by placing the rats on a heating pad.
The animals were assigned to two groups (n=10 animals each) according to anesthesia maintenance: keta group, intravenous (iv) intermittent S(+)-ketamine (2mg/kg); dex group, dexmedetomidine, 1.0μg/kg iv, during 10min, and 1.0μg/kg/h (Abbott infusion pump Anne, USA).After 20 min with anesthesia maintenance, all animals underwent median laparotomy, rightsided nephrectomy and clamping of the left renal artery for 45 min.Mean arterial pressure and heart rate were monitored through the catheter in the carotid artery (Datex Engstrom, Finland).After renal artery declamping, the abdomen and neck were closed and ropivacaine 0.5% was injected into the incisions.The animals were allowed to recover from anesthesia, remaining 48 hours in a controlled-environment room with food and water freely available.Serum creatinine was measured immediately before ischemia (time point T1) and 48 hours after reperfusion (time point T2), under sodium pentobarbital anesthesia (50mg/kg, ip) and immediately before left nephrectomy and sacrifice.Full crosssection of the right and left kidneys were processed, the organs were fixed in a Duboscq-Brazil solution (120 mL formaldehyde, 30 mL acetic acid and 2 g picric acid), for a minimum of 12 hours and a maximum of 36 hours; they were then transferred to 70% ethanol and sectioned.The sections were stained with hematoxylin and eosin, and identified by a study number not known to the research pathologist who scored the histological changes observed (0=none, 1=mild, 2=moderate, 3=severe).

Statistical analysis
Statistical analysis was performed with the help of Stata/

Results
Weight (g) (mean ± SD) in the keta and dex groups was 436 ± 35 and 375 ± 43, respectively (p=0.002).Blood creatinine (mg/dL) values at T1 and T2 were, respectively: 0.4 ± 0.1 and 5.1 ± 3.1 in the keta group (p=0.005);0.3 ± 0.1 and 3.2 ± 2.8 in the dex group (p=0.005).In T2, the means are not different (p=0.16)(Figure 1).Vascular dilation (Table 1) was observed in right and left kidneys of all groups, but in the keta group the scores were higher in the left than in the right kidneys (p<0.05).
TABLE 1 -Scores of vascular dilation (VD) and vascular congestion (VC) in the right (R) and left (L) kidney according to group, keta or dex.
Data are reported as median (25 th and 75 th percentile).* median value of left (L) and right ® kidney significantly different (p<0.05);# median value significantly lower among groups (p<0.05).
The keta and dex groups had the same degree of vascular congestion (Table 1) and the left kidneys of both groups had higher scores than right kidneys (p<0.05).Tubular dilation (Table 2) scores in the keta and dex groups were higher in the left than in the right kidney (p<0.05), and there was no significant difference between the results of these groups.Although the keta group showed less tubular degeneration (Table 2) in the left than in the right kidney, there were no statistical differences between groups.
In the keta and dex groups, left kidneys had equally significant higher scores of necrosis (p>0.05)(Figure 3).

Discussion
Renal injury associated with ischemia-reperfusion results from a dynamic process involving the vasculature and tubules in a complex interaction whereby events modulating the vasculature will alter oxygen and nutrient delivery to the epithelial cell, and the injured epithelial cell will respond by producing autocrine factors that will affect its own survival and paracrine factors that will affect the vasculature.There is a complex activation of signaling cascades resulting in hemodynamic changes, leukocyte accumulation and cytoskeletal breakdown, loss of cell polarity, apoptosis and necrosis, desquamation of viable and necrotic cells, tubular obstruction and backleak 6 .
Serum creatinine is not a reliable biomarker during acute changes in kidney function.Indeed, it does not accurately depict kidney function until steady-state equilibrium has been reached, which may require several days 7  an abrupt decrease in glomerular filtration rate is followed by a slow rise in creatinine over 48 to 72 hours.The limitations to the use of blood creatinine as a marker of kidney function are due to differences in age, sex, dietary intake, muscle mass, and clearance by tubular secretion that can result in significant variation in baseline serum creatinine values 9,10 .In addition, certain factors can reduce the accuracy of serum creatinine assays, and lead to artifactual increases in serum creatinine levels 9 .Assuming that our experimental model is homogeneous, dex group had most reduced serum creatinine levels two days after reperfusion than keta group.
Delbridge et al. 11 reported that the temperature of the animal during the ischemic period of IRI significantly affects injury severity, and that the use of a homeothermic heating blanket led to an increase in creatinine level by day three.In our study, the animals were kept between normothermia-mild hypothermia, which explains the increased serum creatinine.
Jolkkonen et al. 4 administered dexmedetomidine to rats as an intravenous bolus of 3µg/kg over 5 min followed by a 120 min infusion of 3µg/kg/h.After occlusion of the middle cerebral artery, they observed that dexmedetomidine decreased total ischemic volume in the cortex, and was associated with some minor degree we infused dexmedetomidine continuously, as in clinical practice, and after ip S(+)-ketamine only, and we studied the kidneys after 48h of reperfusion.So, our results are from kidneys with more accentuated alterations and they are not totally comparable to those of Kocoglu et al. 14 .
In this study, S(+)-ketamine was the induction anesthetic of choice because it could facilitate the administration of dexmedetomidine without interfering with study results 15 .In a renal histology study, after acute hemorrhage of 30% of volemia, it was found that rats receiving sodium pentobarbital or S(+)ketamine had hypotension from hemorrhage with S(+)-ketamine, and that the score of histological changes was higher in the S(+)ketamine group 16 .Vascular dilation and congestion, as well as tubular dilation and degeneration in the right kidney were observed in all our study groups.The fact that these changes were seen even in keta group suggests that S(+)-ketamine caused these alterations.
The association of IRI with S(+)-ketamine and dexmedetomidine also provoked vessel changes.Tubular degeneration was also caused by S(+)-ketamine as indicated by the high scores seen in the right kidneys that did not undergo ischemia.Our results demonstrate that S(+)-ketamine plus the experimental model of IRI used here was aggressive to rat kidneys, and dexmedetomidine did not protect the kidneys from these aggressions.

Conclusion
Our results demonstrate that S(+)-ketamine plus the experimental model of IRI used here was aggressive to rat kidneys, according to histological changes, and dexmedetomidine may have not totally protected the kidneys from these injuries, despite the better results of blood creatinine.However, further studies should be conducted to elucidate the role of venous anesthetic agents in IRI.

SE 9 .
0 for Windows (Stata Corporation, College Station, Texas, USA).Creatinine values and body weight were expressed by the mean ± standard deviation.Student's T test was used to compare creatinine values between groups at the same time point.Mann-Whitney test was used in the comparison between groups at the same time point for histological scores.The Wilcoxon test was used to compare variables between the two studied time points (T1 and T2).Significance level was set at p < 0.05.

*
In all groups, histological analysis (Figure2) revealed no statistical difference among the right kidneys.

TABLE 2 -
Scores of tubular dilation (TD) and tubular degeneration (TDe) in the right ® .01andleft (L) kidney according to group.Data are reported as median (25 th and 75 th percentile).* median value of left (L) and right (R) kidney significantly different (p<0.05);# median value significantly lower among groups (p<0.05).
. According to Moran et al.8 , of hyperglycemia and hypotension besides being safer regarding kidney toxicity.The protein kinase C (PKC) is important for IPC as it activates intracellular signaling pathways providing organ protection -the opening of sarcolemal and mitochondrial ATPsensitive potassium channels (sarcK ATP and mitoK ATP , respectively) -and yet through genetic transcription, promoting the protective cellular protein synthesis.An investigation of alternative signaltransduction pathways has demonstrated that α 2B -adrenoceptor stimulates PKC activity and inositol triphosphate production in kidney distal convoluted tubule cells.This fact could be responsible for the same cell protection conferred by IPC12,13 .Kocoglu et al.14 studied the role of dexmedetomidine on renal histological alterations after IRI.They found normal glomeruli and slight edema of the tubular cells, and according to them dexmedetomidine could reduce the renal injury caused by IRI of the kidney.The α 2 -adrenoceptor agonist was administered ip, in single dose of 100 µg/kg, at the starting time of reperfusion, and the rats had been anesthetized previously with ketamine, 50mg/kg, and xylazine, 20mg/kg, ip.Time of ischemia was 60 minutes and the rats were sacrificed after 45 minutes of reperfusion.Differently,