Kidney ischemia and reperfunsion syndrome: effect of lidocaine and local postconditioning

A síndrome de isquemia e reperfusão contribui com 60-70% da morbimortalidade relacionada à lesão renal aguda presente em várias situações clínicas, como o transplante renal e a embolia de artéria renal1-5. Embora a reperfusão arterial imediata seja a melhor conduta para eliminar o processo isquêmico6, a reoxigenação celular está associada a um aumento na peroxidação lipídica, no dano celular e na piora da função7,8. A recente descrição das técnicas de “condicionamento tecidual”, que consistem em breves ciclos alternados de isquemia e reperfusão, é uma abordagem promissora para o controle dos danos causados pela isquemia prolongada e pela lesão de reperfusão6. Em 2003, Zhao et al.9 desenvolveram o conceito do pós-condicionamento isquêmico (POS), que consiste em pequenos ciclos de isquemia e reperfusão antes da reperfusão livre em um tecido previamente isquêmico. Esta técnica pode ser facilmente aplicada em situações inesperadas de isquemia, principalmente quando comparada ao pré-condicionamento, com estudos que comprovam seus efeitos benéficos em seres humanos10,11. Os mecanismos envolvidos no efeito protetor do POS contra a síndrome de isquemia e reperfusão são pouco compreendidos. Sabe-se que este procedimento atua na modulação dos canais de potássio da mitocôndria, nos poros de transição de permeabilidade mitocondriais e na via de sinalização p13-kinase-pAKt12. Os exatos mecanismos intracelulares do POS ainda não foram totalmente determinados. Um mecanismo sugerido por alguns estudos seria a modulação do tônus vascular13,14, em que o pós-condicionamento aumentaria o número e duração da contração das arteríolas impedindo um grande influxo de oxigênio e consequentemente a geração de espécies reativas de oxigênio. Contudo, na revisão de literatura realizada, não foi identificado nenhum estudo que avaliou diretamente esta via. A lidocaína é um anestésico local muito utilizado para a realização de pequenos procedimentos ci-


INTRODUCTION
T he ischemia and reperfusion syndrome contributes to 60-70% of the morbidity and mortality related to the Cute Kidney Injury (AKI) present in various clinical situations, such as renal transplantation and renal artery embolism [1][2][3][4][5] .Although the immediate arterial blood reperfusion is the best approach to eliminate the ischemic process 6 , cell reoxygenation is associated with an increase in lipid peroxidation, in cellular damage and in deterioration of the function 7,8 .
The recent description of "tissue conditioning" techniques, consisting of alternate cycles of brief ischemia and reperfusion, is a promising approach for controlling damage by prolonged ischemia and reperfusion injury 6 .
In 2003, Zhao et al. 9 developed the concept of ischemic postconditioning (POS), which consists of small ischemia and reperfusion cycles before free reperfusion in a previously ischemic tissue.This technique can be easily applied in unexpected ischemias, especially when compared with preconditioning, with studies showing beneficial effects in humans 10,11 .
The mechanisms involved in the protective effect of POS against ischemia and reperfusion injury are poorly understood.It is known that this procedure works in modulation of mitochondrial potassium channels, the mitochondrial permeability transition pore and via p13-kinase-pAkt signaling 12 .
The exact POS intracellular mechanisms have not been fully determined.A mechanism suggested by some studies would be the vascular tone modulation 13,14 , wherein the postconditioning would increase the number and duration of contraction of the arterioles, preventing a large influx of oxygen and therefore the generation of reactive oxygen species.However, in the literature review, we identified no study directly assessing this route.

Kidney ischemia and reperfunsion syndrome: effect of lidocaine and local postconditioning
Lidocaine is a local anesthetic, often used to perform minor surgical procedures.It acts by blocking sodium channels, preventing the nerve impulse and the painful feeling 15,16 .However, when administered systemically, it has a vasodilatory effect, which could be used to block the vascular tone modulation 17 .
Thus, this study aims to assess the effects of blocking the regulation of vascular tone in ischemia and reperfusion syndrome in rats, through the use of lidocaine in the ischemic postconditioning technique.

METHODS
We used thirty-five male Wistar rats (Rattus We randomly divided the animals into seven groups of five animals each: 1) Control group (CON), in which renal ischemia was not induced; 2) Group ischemia and reperfusion (IR), which underwent renal ischemia for 30 minutes followed by reperfusion without any conditioning technique; 3) Group ischemia and saline reperfusion (IRS), which underwent renal ischemia for 30 minutes followed by saline infusion in the renal artery at a rate of two drops per minute (Figure 1); 4) Ischemic postconditioning group (POS), subjected to 30 minutes of renal ischemia followed by 30 minutes of local postconditioning (three cycles of five minutes of perfusion interspersed by five minutes of ischemia) 13 ; 5) Ischemic postconditioning and saline group (POSS), submitted 30 minutes of renal ischemia followed by 30 minutes of the local postconditioning (3 cycles of 5 minutes of renal perfusion interspersed with five min of renal ischemia) 11 and instillation of saline in the renal artery at a rate of two drops per minute; 6) Lidocaine group (LIDO), subjected to 30 minutes of renal ischemia followed by 30 minutes of lidocaine instillation into the renal artery at a rate of two drops per minute; 7) Postconditioning and lidocaine group (POLI), submitted 30 minutes of renal ischemia followed by 30 minutes of the local postconditioning and instillation of lidocaine in the renal artery.
We performed all procedures under anesthesia with ketamine 70mg/kg and xylazine 10mg/kg intraperitoneally.In all groups, we performed right nephrectomy and dissected the left renal artery with the aid of a microsurgical microscope.We induced renal ischemia by applying a microsurgical clamp on the left renal artery for 30 minutes.We maintained the temperature of the animals at 37°C using a thermal blanket throughout the procedure.We performed postoperative rehydration through saline injection (10ml/kg) subcutaneously.
During the observation period, the animals received analgesia with dipyrone 30mg/kg every eight hours and water and food ad libitum.The animals were individual in cages postoperatively.We evaluated the amount of food and water consumed before and after surgery.
Twenty-four hours after surgery, animals were again anesthetized.We obtained blood samples from the vena cava and immediately sent them to biochemical analysis.Serum urea and creatinine were measured using the colorimetric assay in a Selectra-E device using specific Labtest® kits.We removed the left kidney and fixed it in 10% buffered formalin.The pieces were stained with hematoxylin and eosin.We analyzed the presence of tubular necrosis, medullary congestion and retraction of the glomerular tuft, graduating it as 0 -absent; 1 -mild; 2 -moderate; and 3 -intense.We euthanized the animals by anesthesia overdose.

Kidney ischemia and reperfunsion syndrome: effect of lidocaine and local postconditioning
We expressed the results as mean ± standard deviation.We used the BioEstat® 5.4 software (Belém, PA, Brazil) for statistical analysis.We used the ANOVA test followed by Tukey's post-test to compare the levels of urea and creatinine between groups, and the Kruskal-Wallis test to compare the histopathological scores.We adopted a value less than 0.05 or 5% to reject the null hypothesis.

RESULTS
During the entire study period, no animal died or needed resuscitation maneuvers.There was no change in the feeding patterns of animals during pre and postoperative periods (18.63 g/day vs 17.44 g/day, p<0.05).We noticed that the IR group had higher urea levels that CON, POS and POLI groups (p<0.01),showing no significant difference when compared with the LIDO group (p=0.62).The POLI group achieved better serum levels than the POS group (p=0.045), and was the only group that showed urea levels similar to the CON group (p=0.32).
With respect to creatinine, the IR group showed the highest levels when compared with all other groups (p<0.001); the POLI group achieved better serum levels than the POS group (p=0.043) and was the only group that showed creatinine levels statistically similar to the CON group (p=0.57).The LIDO group showed higher creatinine levels than POS and POLI groups (p<0.01).Table 1 shows the serum levels of urea and creatinine per group.
Table 2 shows the results of the histopathological analysis.We identified a lesser degree of glomerular injury in all treated groups (p<0.05),but only in the postconditioning groups there was a reduction in the level tubular injury (p<0.05).

DISCUSSION
The exact mechanism of ischemic postconditioning is poorly understood.It is known that its effects are mediated through potassium channels 16,18 , modulated by neural and humoral mechanisms that critically depend on the time at which the conditioning is applied.When performed immediately after the main ischemia, the neuronal pathway is dominant, through a parasympathetic action 19 .However, when applied shortly after the main ischemia, the humoral mechanism is predominant, through the RISK and SAFE pathways 8 .
One of the parasympathetic actions is the regulation of vascular tone that, when activated, causes an increase in blood flow by vasodilatation 20 .
The tone of the renal artery is regulated primarily by the sympathetic pathway, which promotes dilation when inhibited 21 .Our hypothesis with lidocaine instillation in the renal artery is that it should generate a parasympathetic effect through of the sympathetic-type blocking of the vascular tone, resulting in a sustained vasodilation with blocking of the postconditioning neurogenic pathway.
The blood markers of renal function were decreased in the group subjected to the postconditioning, as identified in several other studies.However, lidocaine-treated animals also showed a beneficial effect, particularly in relation to creatinine, displaying an additional glomerular protection 22 .We did not observed this difference regarding urea levels, demonstrating a higher tubular compromise since, under normal conditions, the loop of Henle is responsible for 60% of urea urinary secretion 23 .
It is important to note that the groups treated with saline had no effect similar to the lidocaine group, indicating that the reduction of markers was due to the pharmacological properties of lidocaine and not to a possible volume overload of the instilled lidocaine.
One possible mechanism involved in the lidocaine protective effect of is the blockade of NaV 1.9 channels, which are closely related to the pathophysiology of ischemia and reperfusion syndrome 16 .
When this channel is blocked, there is a decrease in the intracellular influx of sodium and less reperfusion injury.
However, Lee et al. 24 demonstrated that the continuous infusion of lidocaine in rats' subcutaneous tissue had deleterious effects on the ischemia and reperfusion syndrome, suggesting that there is still need for greater understanding of this drug effects and the influence of the route of administration.
Histologically, no significant differences were evident when comparing the histological grade of glomerular and tubular injury, with results similar to those identified in the analysis of serum urea and creatinine and confirming the initial conclusions.We observed no further lesion by the use of lidocaine, which some studies proved to be toxic to the renal epithelium 24,25 .
Further studies are required before the clinical use of lidocaine, especially using other analysis parameters such as oxidative stress and cell viability, not tested in this initial work 26 .Furthermore, there is controversy in the literature on whether the ischemic postconditioning is influenced by the number of cycles or by the conditioning time 27 .Five-minute cycles may not have been the appropriate model to determine the maximum protective effect on ischemia and reperfusion.Thus, if we had applied an ideal cycle, lidocaine solution could not have presented the described effects.
In conclusion, lidocaine did not block the postconditioning effects on the ischemia and reperfusion syndrome, but determined a better result in glomerular protection when applied in conjunction with this technique.

R E S U M O
norvegicus) aged 15 to 20 weeks, weighing 250-300 g study.We kept the animals in a vivarium of the Experimental Surgery Laboratory of the Pará State University, in a controlled environment, with water and food offered ad libitum.This research followed the Brazilian law of animal experimentation (11,794/08).The research project was approved by the Ethics in Research Committee of the Pará State University (43/12)

Figure 1 .
Figure 1.Technique of instillation in the left renal artery.