Ischaemia or severe hypotension causes acute renal failure which is associated with high morbidity and mortality in patients . Renal failure is a common clinical problem associated with some surgical procedures like kidney transplantation or abdominal aortic aneurysm surgery that induce ischaemia/reperfusion injury of the kidney. In the latter group of patients, postoperative kidney failure occurs in 8–40% of patients  and little is known about the modifying aspect of anaesthetic drugs in these situations.
In the heart, a protective effect by anaesthetic preconditioning has been confirmed in patients [3–5] and may be relevant for patient outcome. Increased evidence suggests that an endogenous protective cascade can be activated in other organs by preconditioning with short ischaemic episodes or by administering an anaesthetic [6–8].
Data on preconditioning of the kidney, however, are equivocal to date [9–12]. Pharmacological preconditioning with either morphine  or local anaesthetics  was not effective in protecting the rat kidney against ischaemia/reperfusion injury, and the effects of preconditioning with volatile anaesthetics are still in the investigative stage .
The aim of the present study was to evaluate and compare the effect of sevoflurane and ischaemia-induced preconditioning on renal function and morphological cell damage after 45 min of renal ischaemia and 3 days of reperfusion in the rat in vivo.
The study was performed in accordance with the regulations of the German Animal Protection Law and was approved by the Bioethics Committee of the District of Duesseldorf.
To prevent compensation by the contra-lateral kidney, right-sided nephrectomy was performed 14 days prior to the ischaemia/reperfusion experiments. Nephrectomy was carried out under general anaesthesia with S-ketamine (100 mg kg−1 intraperitoneal (i.p.); Abbott Laboratories) in spontaneously breathing male Wistar rats (body weight 301 ± 31 g, mean ± SD). For the ischaemia/reperfusion experiments, S-ketamine anaesthetized animals (100 mg kg−1) were ventilated after tracheal intubation (small animal ventilator, Type 10 mL, Class 34931; Rhema-Labortechnik, Germany) with a FiO2 of 0.4 and a positive end-expiratory pressure of 3–5 cmH2O. Arterial pH and PCO2 were maintained within the normal physiological range by adjusting the ventilation rate. Throughout the experiments, body temperature was maintained at 37°C by placing the rats on a heating pad. During the ischaemia/reperfusion experiment all animals received 3–5 mL of saline. For repeated blood withdrawal during the reperfusion period (3 days), a central venous catheter (CVC) was inserted into the right external jugular vein. Renal ischaemia was induced by exposing the left renal pedicle (through a flank approach) and tightening a ligature snare around the renal artery for 45 min. Successful reperfusion was determined by the disappearance of surface cyanosis. The abdominal wall was subsequently closed with 4.0 polypropylene sutures through muscle and skin layers.
The nephrectomized rats were anaesthetized with a bolus of S-ketamine (100 mg kg−1 i.p.). In five rats (SHAM) preparation was performed in the same manner as in the other groups apart from renal ischaemia. After 70 min of S-ketamine anaesthesia (0.5 mg kg−1 min−1 intravenous (i.v.)), rats were allowed to recover and were housed for measurement of renal function in metabolic cages for 3 days.
All other rats were randomly assigned to one of the following groups (Fig. 1):
- Control group (CON; n = 9): After the end of preparation, rats received 21 mg kg−1S-ketamine through the CVC and 25 min later the renal artery was occluded for 45 min. After the occluder had been removed, the animals were allowed to recover from anaesthesia. During occlusion, anaesthesia was maintained by intermittent i.v. administration of S-ketamine (in total 15 mg kg−1).
- Ischaemic preconditioning group (IPC; n = 8): We used the protocol described by Yamashita and colleagues  consisting of three episodes of 2 min renal artery occlusion followed by 5 min of reflow 10 min prior to renal artery occlusion. Successful reperfusion was verified by the disappearance of renal surface cyanosis. During the preconditioning procedures and renal artery occlusion, anaesthesia was maintained by intermittent i.v. administration of S-ketamine (in total 36 mg kg−1).
- Sevoflurane preconditioning group (SPC; n = 8): After preparation was completed rats received sevoflurane (2.4 vol% corresponding to one minimal alveolar concentration, MAC, for 15 min ) followed by 10 min of washout prior to renal ischaemia. Expiratory sevoflurane concentration was measured at the tip of the endotracheal tube (Datex Capnomac Ultima, Division of Instrumentarium Corp., Helsinki, Finland) at a sampling rate of 200 mL min−1. By using a high rate of inspiratory gas flow (12 L min−1), changes in sevoflurane concentration could be achieved within 15 s. During sevoflurane washout and renal artery occlusion, anaesthesia was maintained by intermittent i.v. administration of S-ketamine (in total 28 mg kg−1).
All animals were housed in a controlled-environment room (22 ± 2°C, illuminated from 07.00–18.00 h) with food pellets and water freely available. After the ischaemia/reperfusion experiments rats recovered quickly from anaesthesia and were transferred to metabolic cages for measurement of fluid intake and urine output during 3 days of reperfusion. To assess renal function, urine was collected and quantified separate from fecal pellets or drinking water. Blood samples (1.3 mL) were taken after 24, 48 and 72 h of reperfusion. Blood creatinine (mg dL−1; Reflotron®, Nr 886874; Roche Diagnostics GmbH, Mannheim, Germany) and blood urea nitrogen (BUN, mg dL−1; Reflotron®, Nr 1200666; Roche Diagnostics GmbH, Mannheim, Germany) were measured. Sodium, potassium and pH were measured in blood and in urine (EML510/ABL710, Radiometer; Copenhagen, Denmark).
At the end of the reperfusion period, the kidney was harvested by a mid-abdominal approach under S-ketamine anaesthesia (100 mg kg−1 i.p.). Transverse slices of tissue were fixed in Bouin's solution, dehydrated in a graded alcohol series, embedded in paraffin and further processed for histological examination. Four micrometer sections were stained by periodic acid-Schiff (PAS) and morphologically assessed for injury by two observers blinded for treatment. Loss of brush border and portions of cytoplasm leading to flattening of tubular cells, dilation of tubules and tubular obstruction with protein casts as well as the appearance of mitoses and the location of the area involved were used as indicators of the degree of ischaemia/reperfusion injury. The morphological criteria for assessing cell damage were slightly modified from those introduced by Jablonski and colleagues . Micrographs were acquired with a digital camera (Leica DC 300 F; Leica Microsystems, Wetzlar, Germany) on a light microscope (Leica DM LB; Leica Microsystems, Wetzlar, Germany) with the 40× objective.
Results are expressed as mean values ± SD. The statistical analysis was performed by a two-way analysis of variance (ANOVA) for time and treatment. Time effects (changes from baseline value) during the 3 days following ischaemia were analysed by applying Dunnett's post hoc test. If an overall significance between groups was found, comparison was made for each time point using one-way ANOVA followed by Tukey's post hoc test where appropriate. pH-values were expressed as negative logarithm (base 10) of the mean values and SD of the calculated proton concentration.
Experiments were performed in a total of 35 animals. During the 3 days of reperfusion two animals of the sevoflurane group died shortly before the third blood sampling due to pulmonary oedema. Two animals died after right-sided nephrectomy (one in the CON and in the IPC group, respectively). One animal of the IPC group was not evaluated because the kidney showed gross signs of malformation not related to ischaemia; its creatinine concentration was the highest of any animal. For all other rats complete data sets were collected.
Both glomerular and tubular function were decreased in all groups subjected to renal ischaemia: whereas blood concentration of creatinine and urea nitrogen (BUN) remained stable in the SHAM-operated group (creatinine: 0.7 ± 0.3 mg dL−1; BUN: 72 ± 13 mg dL−1; values after 3 days of reperfusion), creatinine concentration increased more in the preconditioned groups (SPC: 4.0 ± 1.1 mg dL−1; IPC: 3.3 ± 1.2 mg dL−1) than in the CON group (both P < 0.05; a similar increase was observed in BUN, Figs 2 and 3).
As variables of impaired tubular function, the concentration of electrolytes in the urine decreased in all groups subjected to ischaemia and reperfusion followed by an increase of serum potassium content (K: 3.4 ± 0.4 vs. 4.8 ± 1.2 mmol L−1, P < 0.05 vs. baseline, Table 1). Potassium excretion was mostly impaired after ischaemic preconditioning (K: 6.1 ± 1.5 mmol L−1; P < 0.05 vs. all other groups, Table 2).
Histological examination in PAS-stained sections
Four levels of injury to the proximal convoluted tubules were characterized:
- Many proximal straight tubules (S3 segments) had portions of shed apical cytoplasm including brush border floating in the lumen; the basal cytoplasm with the nucleus was generally intact. Numerous mitoses were seen in damaged areas. The S1 and S2 segments of proximal tubules and the corpuscles appeared intact. This level of injury was concentrated in the outer medullary stripe and extended only slightly into the medullary rays (Figs 4a and b).
- A greater number of proximal straight tubules were injured with a greater loss of apical cytoplasm than in level 1 but the epithelium was nevertheless usually continuous; numerous tubules at the inner medullary stripe were congested with cell debris; most proximal tubules of medullary rays were injured; some mitoses were found in damaged areas; convoluted tubules next to corpuscles were slightly distended (Figs 4c and d).
- Most proximal straight tubules at the outer medulla and in medullary rays were practically denuded of epithelial cells; cell debris congested many tubules at the inner medulla. Mitoses were rare. The S1 and S2 segments next to corpuscles were generally intact, although often distended (Figs 4e and f).
- There was a loss of cells in almost all proximal tubule segments, including convoluted ones in the outer cortex, amorphous debris congesting them. Distal tubules were generally intact, even when filled with PAS-positive material (Figs 4g and h).
These levels of tissue injury were not evenly distributed in the sections. Individual animals were classified to a certain level of injury if that level was predominant. No animals of the SHAM group showed lesions as described above. With the exception of two animals, all control rats had level 1 damage. These two exceptions also had the highest increase of creatinine within that group. Five rats of the IPC group had level 3 damage, two had level 2. The levels of injury among the SPC were spread between 2 and 4. The scatter plot relates the levels of tubular injury to the increases in serum creatinine for each animal (Fig. 5).
The present study compares the function and morphological integrity of the rat kidney after renal ischaemia in animals preconditioned with sevoflurane or preconditioned with short episodes of ischaemia. An untreated group served as control. Our main finding was that neither sevoflurane nor ischaemic preconditioning protected the rat kidney against ischaemia/ reperfusion injury after a 45-min renal artery occlusion.
Whereas different anaesthetics used as preconditioning agents are known to reduce the size of myocardial infarcts [18–20], to date little has been reported on anaesthetic preconditioning of the kidney. We used a 45-min period of unilateral renal artery occlusion to determine whether sevoflurane or ischaemic preconditioning protects the kidney from ischaemia/reperfusion injury. The 45-min period is known to produce severe but not fatal injury and has been used in numerous earlier studies on ischaemia/ reperfusion in the rat kidney [14,15,21].
Reports on the use of S-ketamine in heart ischaemia experiments  show that it does not interfere with ischaemic or anaesthetic preconditioning. Since all of our animals were anaesthetized with S-ketamine during preconditioning and ischaemia, S-ketamine cannot account for the differences between experimental groups. Sham-operated animals did not have any histologically detectable lesions. We found a lower increase of blood creatinine and urea nitrogen in the control group (no preconditioning) than in both the ischaemia preconditioned and the sevoflurane preconditioned groups. These functional variables were mirrored by higher level of histological injury in both preconditioned groups than in the controls (Fig. 5).
The pattern of tubular necrosis seen in our animals resembles in many respects that described in earlier reports on ischaemia-induced injury. The most common similarity is the finding that the proximal straight tubule shows the highest degree of susceptibility [23–25]. One of the major differences between our protocol and that of numerous other studies is the unilateral nephrectomy 2 weeks prior to ischaemia. The single kidney may be more susceptible to injury than when an unaffected kidney compensates . Islam and colleagues  did not find any differences between injury in ischaemic preconditioned and injury in non-preconditioned kidneys in animals which had one unaffected kidney. In experiments with a different animal species (pig), Behrends and colleagues found that ischaemic preconditioning even enhanced the level of injury . Another study which used a highly sensitive method able to detect damage even in the sham group, showed that ischaemic preconditioning protected the kidney. However, that study used a shorter period of ischaemia, a model without nephrectomy and female rats . It is well known that male rats are more susceptible to ischaemia/reperfusion injury . From all these findings, one could speculate that with the relatively severe ischaemic injury in our study, ‘preconditioning’ might not have a protective effect.
In our study S-ketamine was the anaesthetic used during preparation and ischaemia and it cannot be excluded that sevoflurane, in combination with S-ketamine, increases damage where it would not do so used alone. In this respect our observations are very similar to those of Lee and colleagues  who found that ischaemia/reperfusion under sevoflurane anaesthesia alone led to a much lower degree of injury than if the animals had received sevoflurane followed by ischaemia/reperfusion under pentobarbital.
The reduction of renal function by sevoflurane pretreatment raises the question of whether the drug caused the cell damage by itself. Kharasch and colleagues investigated the nephrotoxicity of sevoflurane in many studies . They found that prolonged administration of sevoflurane to isolated rat proximal tubular cells caused cell damage . Sevoflurane is degraded in anaesthesia machines by CO2 absorbers to different compounds  and the hepatic enzyme system of the rat produces nephrotoxic metabolites [32,33]. Studies investigating the effect of sevoflurane anaesthesia on patients with unimpaired renal function or in patients with renal dysfunction could not find any postoperative deterioration of renal function [34–37]. It seems unlikely that the specific sensitivity of the rat to sevoflurane is responsible for the high level of injury seen in several of our sevoflurane preconditioned animals. We did not use a CO2 absorber and both dosage, and duration of preconditioning are not comparable with the higher concentration or longer administration time used in the earlier studies.
In the present study rats underwent preconditioning without measurement of renal blood flow. It is well known from previous studies on myocardial reperfusion injury that the depression of global haemodynamics by sevoflurane even after myocardial ischaemia is marginal in this species [38,39]. Therefore it seems unlikely that alterations of renal blood flow or perfusion pressure might have influenced the effect of ischaemic or sevoflurane preconditioning.
In cardio-surgical patients Julier and colleagues reported an increased renal blood flow and a glomerular filtration rate (determined by cystatin c levels) after sevoflurane preconditioning prior to cardiopulmonary bypass . Unfortunately, these authors were not able to determine whether this improvement was the result of a specific effect on the kidney or was caused by a greater cardiac output in the patients who had received sevoflurane prior to bypass surgery. These positive effects are not yet evidence, but may suggest that anaesthetic preconditioning may have a positive effect on the human kidney.
Our results show that preconditioning with sevoflurane, in contrast to its effects on heart muscle, does not protect the rat kidney from ischaemia/reperfusion injury. Further research with different experimental protocols and species might be necessary before reaching a definite conclusion regarding a pharmacological preconditioning effect of sevoflurane and its importance for clinical situations in which renal ischaemia/ reperfusion injury may occur.
The technical assistance during the experiments of Maximiliane Kratz and Stefan C. Ley is gratefully acknowledged. Furthermore, we thank Gabriele Berthold (Dept. of Anatomy II) for her skilled help with the histological preparation of the renal tissue and the staining procedures. This work is a part of the MD thesis of Saskia Dettwiler.
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