Propofol is a lipophilic hypnotic drug widely used in anesthesia and intensive care. The protective effects of propofol have been described in models of ischemia-reperfusion (I-R) injury in several organs.1–5 Renal I-R injury is a serious condition associated with high mortality in humans. It may result from hemorrhagic, traumatic, or septic shock, graft rejection after renal transplantation, or certain surgical conditions, including aortic clamping.6 Both apoptosis and necrosis have been reported during renal I-R injury, although it is difficult to evaluate the contribution of each one to overall cell death.7 Several studies reported that renal ischemic preconditioning may protect the kidneys against I-R injury, improving renal morphology and function by reducing cellular necrosis8 and diminishing the severity of postischemic dysfunction of the kidneys.9
One of the mechanisms involved in ischemic preconditioning in different tissues is the activation of adenosine triphosphate-sensitive potassium (KATP) channels, located in mitochondria, plasma membrane, and/or sarcolemma, although the mechanisms by which these channels contribute to the observed effects are still not clear.10–12
Propofol has been shown to diminish renal I-R-induced injury in both rats and piglets.5,13 In humans, it has been shown that propofol attenuated I-R-induced oxidative injury in renal transplantation, when compared with thiopental.14 Although the mechanisms involved in the renal effects of propofol are yet to be elucidated, several studies have shown propofol's effects on KATP channels in cells of other organs.15–19 Because propofol may induce a pharmacologic preconditioning in other organs,19,20 this study was performed to evaluate the possible preconditioning properties of propofol in a cellular model of renal I-R.
Propofol and its lipidic vehicle were generously supplied by Cristália Produtos Químicos e Farmacêuticos Ltda, Brazil, without any charge or commitment.
Dulbecco's modified Eagle medium (DMEM), antimycin A, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), annexin V–fluorescein isothiocyanate, diazoxide, glibenclamide, 5-hydroxidecanoic acid (5HD), l-glutamine, and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Sodium bicarbonate was purchased from Quimibrás Indústrias Químicas, Brazil. Penicillin/streptomycin solution, trypsin-ethylenediaminetetraacetic acid, and fetal bovine serum (FBS) were purchased from Invitrogen, Brazil.
LLC-PK1 cells were cultured in plastic bottles (TPP, Switzerland) containing DMEM with 10% FBS at 37°C. For each experiment, the cells were plated in 24 (105 cells/well, for trypan blue viability assay) or 96 wells (104 cells/well, for MTT assay) and incubated for 24 h before the experiments, for complete cell adhesion.
On the day of the experiment, the cells were washed 3 times with warmed phosphate-buffered saline (PBS) and incubated with prewarmed solution (125 mM NaCl; 5 mM K2PO4; 2 mM MgSO4; 1.5 mM CaCl2; 12.6 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid without glucose, amino acids, and serum) and with 0.1 μM antimycin A (an inhibitor of oxidative phosphorylation) for 45 min for depletion of ATP. After chemical anoxia, the cells were washed with PBS and allowed to recover for 2 h in DMEM.
Treatment with Propofol
Propofol (25 μM) and the correspondent amount of the vehicle solution (10% emulsion, phospholipid stabilized soybean oil—intralipid) were added to the culture medium at different periods of time: 1 or 24 h before I-R, only during ischemia, or only during reperfusion. The other substances, 10 μM diazoxide (D), 10 μM glibenclamide (G), or 100 μM 5HD, were added to the culture medium 1 h before I-R in the presence or absence of propofol or its vehicle.
Cell Viability Assays
The cellular viability was measured either by trypan blue exclusion assay or by MTT reduction test.
Trypan Blue Exclusion
The cells were plated onto 24-microwell plates (105 cells/well) and incubated for 24 h for complete cell adhesion. After I-R, the medium was aspirated and centrifuged to recover the cells detached from the monolayer due to the treatment. The cells still attached were trypsinized with 2% trypsin-ethylenediaminetetraacetic acid and centrifuged at 10,000 g for 5 min. Cell pellets from adherent cells and detached cells were suspended together in PBS, incubated in equal volumes with trypan blue (1%) for 10 min at room temperature, and then counted on a hemocytometer.
The cells were plated on 96-microwell plates (104 cells/well) and incubated for 24 h for complete cell adhesion. The next day, the I-R was performed as described above, with 10 μM diazoxide, 10 μM glibenclamide, or 100 μM 5HD added to the culture medium 1 h before I-R in the presence or absence of propofol or its vehicle. Four independent experiments were performed in triplicate. After the end of I-R, 20 μL of a 0.5% MTT solution diluted in PBS was added to each microwell. After 3 h of incubation with the reagent, the supernatant was removed and replaced with 200 μL dimethyl sulfoxide. The optical density (OD) of each well sample was measured in a microplate spectrophotometer reader (BIO-TEK, Benchmark, BioRad) at 570 nm with background subtraction at 630 nm.
Analysis of Cell Necrosis and Apoptosis by Immunofluorescence of Cells Stained with Hoescht 33358 and Propidium Iodide
Fluorescence microscopy was used to distinguish apoptosis and necrosis. For immunofluorescence studies, 1 × 105 cells were plated on round glass coverslips in 24-well plates and incubated for 24 h for complete cell adhesion. After I-R performed as described above, the cells were stained with Hoescht 33258 to identify apoptosis by nuclear condensation and fragmentation or with propidium iodide (PI) to observe necrotic cells. The cells were then observed under a Zeiss Axiovert 100 microscope, using a 40× objective.
Evaluation of Apoptosis by Flow Cytometry
Apoptosis was also evaluated by flow cytometry. The cells were plated on 24-well plates at a concentration of 105 cells/mL, and the experiments were performed 24 h after seeding. The cells were preincubated (or not) with propofol for 1 h and I-R was performed as described earlier. The cells were then washed with PBS, harvested, and labeled with annexin V–fluorescein isothiocyanate according to the manufacturer's instructions. The cell fluorescence, a measure of apoptosis, was then analyzed in a FACSCalibur flow cytometer (BD, San Jose, CA) equipped with an air-cooled argon laser tuned to emit 15 mW in 488 nm. The fluorescence was measured through a 530-nm long-pass filter.
All values are expressed as means ± se. Statistical analysis was performed by one-way analysis of variance followed by Dunnett or Tukey multiple-comparison tests. Differences among groups were considered as significant at P values <0.05.
Propofol Attenuated I-R-Induced LLC-PK1 Cell Death
Figure 1 shows the survival of cells pretreated with propofol (25 μM) before being submitted to I-R, as evidenced by trypan blue exclusion assay. This concentration was used to mimic the blood propofol concentration in major surgeries.21–23 Treatment of cells with I-R induced approximately 70% of cell death, and this was partially reversed by preincubation of cells with propofol for 1 or 24 h before the I-R (approximately 30% of cell death). There was no statistical difference between propofol 1 or 24 h. The vehicle solution had no effect on cell viability, demonstrating that the effect observed was indeed due to propofol. Similar results were observed when MTT was used as a measure of cellular viability (data not shown). Propofol and vehicle alone had no effect on cell viability (data not shown).
To assess whether propofol could also give a protection during or even after the I-R, propofol was added only during the ischemia or only during the reperfusion periods. Figure 2 shows that no increase in survival was observed when propofol was added only during ischemia (Fig. 2A); however, the addition of propofol during the reperfusion period promoted a slight but significant improvement in cell survival (Fig. 2B). In both situations, the vehicle showed no effect. For the next experiments, propofol was added only 1 h before I-R.
Propofol Reduces Both Necrosis and Apoptosis Induced by I-R
To evaluate whether propofol was protecting cells against necrosis and/or apoptosis, after I-R, the cells were stained with PI, an intercalating agent used to observe necrotic cells, or with Hoescht 33358, an apoptotic marker that detected apoptotic nuclei with condensed and/or fragmented DNA. Virtually no PI staining was seen in the control cells (Fig. 3, A and B). After I-R (Fig. 3, C and D), a few cells presented nuclear PI fluorescence, a marker of cell necrosis, but propofol conferred protection against necrosis (Fig. 3, E and F). Similarly, in Figure 4, it is shown that control cells presented no apoptotic nuclei (Fig. 4, A and B), which was seen only in cells submitted to I-R (Fig. 4, C and D, arrows). Again, pretreatment with propofol protected cells against apoptosis (Fig. 4, E and F). This result was further confirmed by flow cytometry, using annexin V. As can be seen in Figure 5, propofol greatly reduced the percentage of cells undergoing apoptosis, visualized as a diminished labeling of cells in region M2.
Effect of Diazoxide, Glibenclamide, or 5HD on Propofol Preconditioning
The effects of diazoxide (a selective opener of ATP-sensitive K+ channel), glibenclamide (a sarcolemmal ATP-dependent K+ channel blocker), and 5HD (a mitochondrial ATP-dependent K+ channel blocker) on propofol preconditioning were evaluated. Because trypan blue exclusion and MTT produced similar results in the evaluation of cell death induced by I-R, MTT was used in the next experiments because of its simplicity and reproducibility. Figure 6 shows that I-R led to a 50% decrease in OD, a result consistent with that obtained with trypan blue exclusion (Fig. 1). Propofol pretreatment significantly diminished I-R-induced cell death, restoring it to approximately 75% of control values (Fig. 6A), and the vehicle solution showed no significant effect (Fig. 6B). However, propofol's protection was abolished by either glibenclamide or 5HD, whereas diazoxide had no effect on the protective effect of propofol against I-R injury (Fig. 6A). Glibenclamide and 5HD alone did not alter the cell death induced by I-R. Preincubation with diazoxide, however, promoted a small but significant improvement on cell viability (Fig. 6C).
Renal ischemic preconditioning can protect against I-R injury, reducing the severity of postischemic dysfunction of the kidneys.24–26 In this study, the protective effect of propofol against renal I-R injury was evaluated in a well established in vitro experimental model of chemically induced I-R. This model causes a rapid, reversible, reproducible, and significant depletion of ATP and has been widely used to study the signaling pathways of apoptosis and necrosis in LLC-PK1 cells. These cells are commonly used as a model of renal proximal tubular cells and for I-R studies.27,28 The cells were incubated with 0.1 μM antimycin A for 45 min, because higher concentrations of antimycin A or longer periods of incubation lead to intense necrosis (data not shown). The concentration of propofol used was chosen based on studies showing that the blood propofol concentration at which 95% of patients did not respond to verbal command was 5.4 μg/mL,21 which corresponds to approximately 30 μM, and that plasma concentrations typically ranged from 10 to 20 μM during anesthetic maintenance.22,23
By using this experimental model, we observed that propofol significantly attenuated I-R-induced cell necrosis and apoptosis when given before I-R. We also observed a slight but significant protection against cell death when propofol was added only during the reperfusion period, but this protective effect could not be demonstrated when propofol was added only during ischemia. Because protein binding of propofol exceeds 95%, free fractions of propofol were probably smaller in the experiments with preincubation and postincubation, which were performed in DMEM with 10% FBS, than that in which propofol was added during ischemia, performed in a serum-free solution. Therefore, our results are suggestive of a preconditioning-like effect of propofol in the kidneys.
Our results are in agreement with those obtained from other groups showing that propofol reduced renal I-R injury in vivo. Propofol reduced the increase of blood urea nitrogen and serum creatinine levels induced by renal I-R in rats.5 Propofol also minimized renal I-R injury after aortic cross-clamp in pigs, compared with sevoflurane anesthesia.29 However, these studies were unable to present a possible mechanism of action of propofol in renal cells. By using cultured cells, we were able to study such mechanisms, showing that propofol protection is, at least in part, mediated by the activation of ATP-sensitive K+ channels.
Although KATP channels play a critical role in I-R preconditioning in myocardium and endothelium,30–35 and propofol has been shown to act by activating KATP channels in other organs,17,18 the role of KATP channels in renal ischemic preconditioning remains unclear. It has been shown that sodium reabsorption was improved by KATP activation with diazoxide and that this effect was reversed by glibenclamide in a model of renal I-R.12 KATP channels have also been shown to exist in kidney mitochondria.36 More recently, it was demonstrated that treatment with diazoxide before ischemia reduced the deleterious effects associated with renal I-R injury.37
We showed that diazoxide had no effect on I-R-induced LLC-PK1 cell death, but both glibenclamide and 5HD reduced the protection induced by propofol. Moreover, diazoxide alone protected LLC-PK1 cells from cell death. Therefore, our results suggest that the activation of KATP channels, probably the same opened by diazoxide, is involved in propofol preconditioning of renal cells against I-R injury.
In this study, no difference between preconditioning was observed for 1 or 24 h of preincubation with propofol. In a previous study, Al-Jahdari et al.38 showed that no significant changes of propofol concentration in culture media of chick neurons were found at zero, 2, and 24 h, indicating that neither dilution nor metabolism of propofol had occurred. On the other hand, Chen et al.39 observed that the remaining concentrations of propofol in macrophages and culture medium were one-half and one-third after 1 and 6 h of its administration, respectively, and that propofol was undetectable after 24 h of incubation in cells and culture medium. Taken together, these 2 studies suggest that the propofol concentration in the culture medium and in the cells depends on the cell type.
Because in our conditions propofol was withdrawn after 1 and 24 h of preincubation and the experiments of I-R were performed in the absence of propofol, our results suggest that, at least in renal cells, propofol may trigger a signaling pathway that, once activated, no longer needs its presence. This pathway is probably mediated by KATP activation.
Finally, although I-R is a complex issue, involving both tubular necrosis and altered hemodynamics, the use of simpler systems, such as cultured tubular cells, is necessary to gain direct mechanistic insights into the cellular events underlying ischemic injury to renal tubular cells. Therefore, this is the first study showing propofol's preconditioning protection against renal tubular cell I-R injury and relating this effect to the activation of KATP channels. Although it has been shown that propofol diminished renal I-R-induced injury in both rats and piglets5,13,40 and that propofol attenuated I-R-induced oxidative injury in renal transplantation in humans,14 these renal effects of propofol have not been studied in detail. Therefore, its mechanisms of action in renal tissue are unknown. We showed here that propofol has a direct effect in tubular cells, attenuating I-R-induced cell necrosis and apoptosis in an established cellular model of renal I-R,27,28 and that this direct protection is related to the activation of KATP channels. Further studies are necessary to fully understand the mechanisms of propofol protection against renal I-R injury.
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