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Am J Physiol Renal Physiol 294: F371-F378, 2008. First published December 5, 2007; doi:10.1152/ajprenal.00277.2007
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Sevoflurane-mediated TGF-β1 signaling in renal proximal tubule cells

H. Thomas Lee, Mihwa Kim, Joseph H. Song, Sean W. C. Chen, Gina Gubitosa, and Charles W. Emala

Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York

Submitted 18 June 2007 ; accepted in final form 26 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several volatile anesthetics, including sevoflurane, protect against renal ischemia-reperfusion injury in vivo by reducing necrosis and inflammation. Furthermore, in cultured renal tubule cells, sevoflurane directly induced the phosphorylation of the cytoprotective kinases (ERK and Akt), upregulated 70-kDa heat shock protein (HSP70), and attenuated nuclear translocation of the proinflammatory transcription factor NF-{kappa}B. It has been shown that sevoflurane increases the release of transforming growth factor-β1 (TGF-β1) in human proximal tubule (HK-2) cells via externalization of plasma membrane phosphatidylserine (PS), and this increase in TGF-β1 protected HK-2 cells against hydrogen peroxide-mediated necrosis. In this study, we aimed to determine whether the sevoflurane-mediated phosphorylation of ERK and Akt, induction of HSP70, and reduction in NF-{kappa}B activation are due to TGF-β1 receptor-mediated signaling after PS externalization in HK-2 cells. Exogenous TGF-β1 and a liposome mixture containing PS mimicked sevoflurane-mediated ERK and Akt phosphorylation and HSP70 induction in HK-2 cells. Sevoflurane and TGF-β1 caused the nuclear translocation of the SMAD3 transcription factor in HK-2 cells. Furthermore, a neutralizing TGF-β1 antibody or exogenous annexin V to bind PS prevented sevoflurane-induced ERK and Akt phosphorylation and HSP70 induction in HK-2 cells. Finally, a TGF-β1 antibody and annexin V attenuated the reduction in nuclear translocation of NF-{kappa}B by sevoflurane. Therefore, we demonstrate in this study that sevoflurane-mediated cytoprotective and anti-inflammatory effects in HK-2 cells are at least partially due to the externalization of PS and activation of TGF-β1 signaling pathways.

acute renal failure; Akt (protein kinase B); extracellular signal-regulated kinase; heat shock protein 70; volatile anesthetic

PERIOPERATIVE ACUTE RENAL failure (ARF) is a serious and frequent complication without effective therapy (1, 10, 22, 26). Development of perioperative ARF implies a poor prognosis with high mortality and is frequently complicated by many other life-threatening conditions, including respiratory failure, sepsis, and a multiorgan dysfunction syndrome (1, 10, 22, 23). Unfortunately, the mortality and morbidity rates from perioperative ARF have changed little over the past 50 years (10).

Nearly all patients subjected to general anesthesia are administered volatile anesthetics. Volatile anesthetics, in addition to providing anesthesia, analgesia, and amnesia, have nonanesthetic properties in many organ systems, including the kidney. Previously, our group (13) demonstrated that volatile anesthetics, including sevoflurane, protected against renal ischemia-reperfusion injury in vivo by reducing necrosis and inflammation. Our group also demonstrated that sevoflurane produced antinecrotic and anti-inflammatory effects in in vitro cultures of proximal tubules and that the mechanisms involved in this protection included the activation of the cytoprotective kinases ERK and Akt, the induction of 70-kDa heat shock protein (HSP70), and the attenuation of the nuclear translocation of a proinflammatory transcription factor (NF-{kappa}B) (11). Subsequently, we showed that volatile anesthetics including sevoflurane increased the release of transforming growth factor-β1 (TGF-β1) in human proximal tubule (HK-2) cells via externalization of plasma membrane phosphatidylserine (PS) and that this increase in TGF-β1 protected HK-2 cells against hydrogen peroxide (H2O2)-mediated necrosis (12).

In this study, we further probed the mechanisms of sevoflurane-mediated renal tubular protection in vitro by testing the hypothesis that sevoflurane-induced release of TGF-β1 mediates ERK and Akt phosphorylation, the induction of HSP70, and the reduction in NF-{kappa}B nuclear translocation. We therefore determined whether exogenous TGF-β1 or a liposome mixture containing PS mimics and whether a TGF-β1-neutralizing antibody prevents sevoflurane-induced activation of ERK and Akt, upregulation of HSP70, and attenuation of nuclear translocation of NF-{kappa}B and whether sevoflurane causes nuclear translocation of a key TGF-β1 transcription factor, SMAD3 (mothers against decapentaplegic homolog 3).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human renal proximal tubule cell culture. HK-2 cells (immortalized human proximal tubular cell line; American Type Culture Collection, Manassas, VA) were grown and passaged in 75-cm2 cell culture flasks containing culture medium (50:50 mixture of DMEM-F-12 with 10% FBS; Invitrogen, Carlsbad, CA) and antibiotics [100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (Invitrogen)] at 37°C in a 100% humidified atmosphere of 5% CO2-95% air. This cell line has been characterized extensively and retains the phenotypic and functional characteristics of proximal tubule cells in culture (21). Cells were plated in six-well plates when 80% confluent and used in the experiments described below when confluent after 24 h of serum deprivation.

Exposure of HK-2 cells to sevoflurane. For sevoflurane treatment, HK-2 cells were placed in an air-tight, 37°C, humidified modular incubator chamber (Billups-Rothenberg, Del Mar, CA) with inflow and outflow connectors. The inlet port was connected to an in-line agent-specific calibrated vaporizer (Datex-Ohmeda) to deliver sevoflurane mixed with 95% air and 5% CO2 (carrier gas) at 10 l/min. The outlet port was connected to a Datex-Ohmeda 5250 RGM gas analyzer that measured sevoflurane concentrations. Exposure to sevoflurane lasted 0–16 h. Control cells were exposed to carrier gas in a modular incubator chamber.

Exposure of HK-2 cells to recombinant TNF-{alpha}, recombinant TGF-β1, TGF-β1 neutralizing antibody, or PS liposome. To induce inflammation, we treated some HK-2 cells with human recombinant TNF-{alpha} (10 ng/ml for 6 h; Sigma, St Louis, MO). We previously demonstrated that treatment of HK-2 cells with volatile anesthetics including sevoflurane led to the release of TGF-β1 via externalization of PS to the outer leaflet of the cell membrane. Some HK-2 cells were treated with recombinant TGF-β1 (1–10 ng/ml, 0–16 h; R&D Systems, Minneapolis, MN) in lieu of sevoflurane treatment. To mimic PS exposure after sevoflurane treatment, small unilamellar liposomes containing a 50-to-50 molar ratio of PS (derived from brain) (Avanti Polar Lipids, Alabaster, AL) to phosphatidylcholine (PC; derived from bovine liver) (Avanti Polar Lipids) were made as described by Fadok et al. (6, 7, 9, 27). Briefly, the individual phospholipids, stored in chloroform-methanol (90:10), were added to glass tubes and dried under nitrogen. PBS was added, and the lipid mix was vortexed and then sonicated for 3 min. The PC-PS liposomes were used at a concentration of 10 µM total lipids per well and treated for 0–22 h. To block the effects of TGF-β1 or PS exposure, some HK-2 cells were pretreated with neutralizing TGF-β1 antibody (1 or 10 µg/ml; R&D Systems) or 10 nM annexin V for 30 min before sevoflurane treatment, respectively. We also used nonneutralizing control isotype antibody to test the specificity of the neutralizing TGF-β1 antibody (BD Biosciences, San Jose, CA). Moreover, we also tested the nonspecific effects of control liposomal preparations to establish the specificity of PS and to exclude nonspecific alterations on plasmalemmal integrity, structure, and/or fluidity. We prepared PC liposome (from bovine liver; Avanti Polar Lipids) and purchased the commercial liposome N-(2,3-dioleoyloxy-1-propyl)trimethylammonium methyl sulfate (DOTAP; Sigma) and treated HK-2 cells for 0–22 h.

Immunoblot analyses. We measured nuclear translocation of SMAD3 transcription factor via immunoblotting of nuclear fractions (see below) after sevoflurane treatment. We also measured the activation (via phosphorylation) of ERK and Akt (protein kinase B) as well as HSP70 induction by immunoblotting of whole cell lysates after treatment with 1–10 ng/ml TGF-β1 as described previously (13). The primary antibodies for SMAD3, phospho-ERK1/2, total ERK1, and HSP70 (inducible and total) were from Santa Cruz Biotechnologies (Santa Cruz, CA). The antibody for phospho-Akt was from Cell Signaling Technologies (Danvers, MA). The antibody for β-actin was from Sigma.

EMSA for NF-{kappa}B and SMAD3. EMSA was performed with the Gel Shift assay system (Promega, Madison, WI) as described previously (11). The oligonucleotides for NF-{kappa}B and SMAD3 (Promega) consensus sequences were end labeled with 10 µCi of [{gamma}32-P]ATP (Perkin-Elmer Life Technology, Waltham, MA) and purified with a G-25 spin column (Amersham Biosciences/GE Healthcare, Piscataway, NJ). HK-2 cells were scraped in 500 µl of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT, protease inhibitor cocktail; Roche, Indianapolis, IN) for 10 min at 4°C. The cells were homogenized with a Polytron homogenizer for 5 s to release the nuclei into solution and centrifuged at 18,000 g for 5 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 50 µl of buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.5 mM EDTA, 25% glycerol, 0.1% Triton X-100, 0.2 mM PMSF, 0.5 mM DTT, protease inhibitor cocktail) and incubated for 1 h at 4°C with occasional swirling to extract nuclear protein. The solubilized pellet was centrifuged at 16,000 g for 15 min, and the supernatant containing nuclear proteins was used for EMSAs. Nuclear extract (10 µg) was incubated with 1 µl of the labeled probe for 20 min at room temperature and electrophoresed on a 4% polyacrylamide gel (200 V at 4°C). Two micrograms of HeLa nuclear extract (Promega, Madison, WI) were used for positive control. The gel was then dried to blotting paper and exposed to X-ray film or scanned with a PhosphorImager (Molecular Dynamics/GE Healthcare, Piscataway, NJ).

Protein determination. Protein content was determined with the Pierce Chemical (Rockford, IL) bicinchoninic acid protein assay reagent with BSA as a standard.

Statistical analysis. The data were analyzed with Student's t-test when comparing means between two groups or with one-way ANOVA plus Tukey's post hoc multiple comparison test to compare mean values across multiple treatment groups. In all cases, a probability statistic of <0.05 was taken to indicate significance. All data are expressed throughout the text as means ± SE.

Materials. All drugs were in saline. Unless otherwise specified, all chemicals were obtained from Sigma.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Recombinant TGF-β1 mimics, whereas neutralizing TGF-β1 antibody blocks, sevoflurane-mediated ERK and Akt phosphorylation and HSP70 induction. We have previously demonstrated that sevoflurane-induced HSP70 and phosphorylated ERK and Akt in HK-2 cells (11). Similar to sevoflurane, recombinant TGF-β1 (1 ng/ml) treatment caused a time-dependent increase in HSP70 induction (Fig. 1; n = 6 experiments, 0–16 h of treatment) and ERK/Akt phosphorylation (data not shown) in HK-2 cells. Moreover, neutralizing TGF-β1 antibody (1 or 10 µg/ml, 30-min pretreatment) inhibited sevoflurane-mediated induction of the expression of the inducible form of HSP70 (Fig. 2, A and B; 2.2% for 16 h, n = 6) and the phosphorylation of ERK (Fig. 3; 2.2% sevoflurane for 4 h, n = 3) and Akt (data not shown) in HK-2 cells. Nonneutralizing control antibody did not inhibit the sevoflurane-mediated induction of the expression of the inducible form of HSP70 (Fig. 2C) and the phosphorylation of ERK and Akt (data not shown).


Figure 1
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  		Fig. 1. Transforming growth factor-β1 (TGF-β1; 1 ng/ml) causes time-dependent increases in the expression of inducible 70-kDa heat shock protein (HSP70i) in human proximal tubule (HK-2) cells. Representative immunoblot image (top) and band intensity quantifications (bottom) are shown for HSP70i; n = 6 experiments. *P < 0.05 vs. 0 h.

 

Figure 2
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  		Fig. 2. TGF-β1 antibody (1 or 10 µg/ml) prevents sevoflurane (Sevo)-mediated induction of HSP70 in HK-2 cells (n = 6). HK-2 cells were treated with 2.2% Sevo for 16 h. TGF-β1 antibody (AB) was given 30 min before Sevo treatment. A: representative immunoblot images. B: band intensity quantifications, expressed as fold increases in HSP70i or total HSP70 (HSP70t) compared with vehicle-treated cells; n = 6. #P < 0.05 vs. carrier gas + vehicle. *P < 0.05 vs. Sevo + vehicle. C: nonspecific mouse IgG antibody (10 µg/ml) had no effect on Sevo-mediated induction of HSP70 (representative images from 3 experiments). HK-2 cells were treated with 2.2% Sevo for 16 h. Mouse antibody was given 30 min before Sevo treatment.

 

Figure 3
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  		Fig. 3. TGF-β1 (10 ng/ml) causes and TGF-β1 antibody (1 µg/ml) attenuates ERK phosphorylation (pERK2) by 2.2% Sevo (4-h treatment). Top: representative immunoblot images of phosphorylated p42 and p44 ERK. Bottom: band intensity quantifications; n = 3. #P < 0.05 vs. Sevo. *P < 0.05 vs. vehicle.

 
Sevoflurane or TGF-β1 translocates SMAD3 transcription factor to the nucleus. We measured nuclear translocation of SMAD3, a key transcription factor of TGF-β1 signaling, by two independent methods. We performed EMSA with SMAD3 consensus sequence oligonucleotides and immunoblotting assays of nuclear protein extracts with SMAD3 antibody. Four-hour treatment of HK-2 cells with recombinant TGF-β1 (0.1–10 ng/ml) or 4 or 8 h of treatment with 2.2% sevoflurane induced increased nuclear translocation of the SMAD3 transcription factor determined by both EMSAs (Fig. 4; n = 4) and immunoblotting (Fig. 5; n = 4).


Figure 4
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  		Fig. 4. Sevo (2.2% for 4 or 8 h) or TGF-β1 (0.1–10 ng/ml, 4 h) causes nuclear translocation of SMAD3 protein, as measured by EMSA of SMAD3 protein in nuclear extracts. Left: representative gel images. Right: band intensity quantifications; n = 4. *P < 0.05 vs. carrier gas + vehicle.

 

Figure 5
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  		Fig. 5. 2.2% Sevo (2.2% for 4 h) or TGF-β1 (10 ng/ml for 4 h) causes nuclear translocation of SMAD3 protein, as measured by immunoblotting of SMAD3 protein in nuclear extracts. Top: representative immunoblot images. Bottom: band intensity quantifications; n = 4. *P < 0.05 vs. carrier gas + vehicle.

 
TGF-β1 mimics and TGF-β1 neutralizing antibody or annexin V prevents sevoflurane-mediated attenuation of nuclear translocation of NF-{kappa}B. Six hours of TNF-{alpha} (10 ng/ml) treatment demonstrated the expected increase in NF-{kappa}B nuclear translocation. Sevoflurane treatment significantly reduced the TNF-{alpha}-induced translocation of NF-{kappa}B in HK-2 cells (Fig. 6, A and B; n = 4). TGF-β1 neutralizing antibody (30-min pretreatment) reversed the sevoflurane-induced decrease in NF-{kappa}B binding activity in HK-2 cells (Fig. 6, A and B). Moreover, PC-PS liposome and recombinant TGF-β1 mimicked, whereas annexin V prevented, the sevoflurane-induced reduction in nuclear translocation of NF-{kappa}B (Fig. 6C). TGF-β1, PC-PS, or annexin V alone had no effect on NF-{kappa}B binding compared with vehicle or TNF-{alpha}-treated HK-2 cells (Fig. 6C; n = 4).


Figure 6
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  		Fig. 6. A: representative EMSA image. B: quantification of NF-{kappa}B binding activities of HK-2 cells treated with vehicle (n = 4), 25 ng/ml TNF-{alpha} for 16 h (n = 4), TNF-{alpha} plus 2.2% Sevo (n = 4), or TNF-{alpha} plus 2.2% Sevo plus TGF-β1-neutralizing monoclonal antibody (1 or 10 µg/ml; n = 4 each). C: quantification of NF-{kappa}B binding activities of HK-2 cells treated with vehicle (n = 4), 25 ng/ml TNF-{alpha} (n = 4), 2.2% Sevo + vehicle (n = 4), TNF-{alpha} plus 2.2% Sevo (n = 4), 1 ng/ml TGF-β1 (n = 4), TNF-{alpha} plus 1 ng/ml TGF-β1 (N = 4), 10 µM phosphatidylcholine (PC)-phosphatidylserine (PS) liposome, TNF-{alpha} plus PC-PS liposome, 2.2% Sevo plus 10 nM annexin V (n = 4), or TNF-{alpha} plus 2.2% sevoflurane plus annexin V (n = 4). HeLa cell nuclear extract acted as positive (Pos) control. Supershift, supershift with NF-{kappa}B p65 antibody. *P < 0.05 vs. vehicle, #P < 0.05 vs. TNF-{alpha}. $P < 0.05 vs. Sevo + TNF.

 
PC-PS liposome mixture mimics sevoflurane-mediated phosphorylation of ERK and induction of HSP70. We demonstrated previously that sevoflurane treatment externalized PS in HK-2 cells (12). Figure 7, A and B, shows that a mixture of PC-PS (10 µM, 0–22 h) induced the phosphorylation of ERK and induced the expression of inducible HSP70 in a time-dependent manner in HK-2 cells (n = 4) . Control PC liposomes and DOTAP liposomes failed to induce phosphorylation of ERK or Akt or upregulate HSP70 (Fig. 7C).


Figure 7
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  		Fig. 7. PC-PS liposome (Lipo) mixture (10 µM) causes phosphorylation of ERK and induces HSP70. A: representative immnoblot images. B: band intensity quantifications. Band intensities of HSP70i were expressed as a ratio of β-actin and phosphorylated ERK (pERK1) was expressed as a ratio of total ERK1; n = 4. *P < 0.05 vs. vehicle. C: in contrast, PC liposome (10 µM) failed to cause phosphorylation of ERK and Akt and induction of HSP70. Representative immnoblot images from 3 experiments are shown.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The key findings of this study are that, in cultured human proximal tubule cells, 1) TGF-β1 mimicked cytoprotective signaling changes induced by sevoflurane treatment (inducible HSP70 induction and phosphorylation of ERK and Akt), 2) neutralization of TGF-β1 prevented ERK and Akt phosphorylation and HSP70 induction after sevoflurane treatment, 3) blocking PS externalization or TGF-β1 neutralization attenuated the sevoflurane-mediated reduction in NF-{kappa}B nuclear translocation after TNF-{alpha}, 4) mimicking PS externalization with PS liposomes reduced the nuclear translocation of NF-{kappa}B, phosphorylated ERK/Akt, and induced HSP70 similar to the sevoflurane's effects, and 5) sevoflurane treatment led to SMAD3 nuclear translocation. These data are consistent with our hypothesis that sevoflurane's anti-inflammatory and cytoprotective effects are mediated by PS externalization and TGF-β1 release.

Sevoflurane [2,2,2-trifluoro-1-(trifluoromethyl)ethyl fluoro methyl ether] is one of the most commonly used volatile anesthetics for induction and maintenance of general anesthesia in the United States. In our previous studies, we showed that clinically relevant concentrations of volatile anesthetics including sevoflurane protected against renal ischemia-reperfusion injury in vivo (13). Subsequently, we showed that sevoflurane produced dose- and time-dependent anti-necrotic and anti-inflammatory effects in in vitro cultures of proximal tubules and that the mechanisms involved in this protection included activation of the cytoprotective kinases ERK and Akt and the induction of HSP70 (11). Externalization of PS, ligation of PS receptors of neighboring cells, and subsequent TGF-β1 release have been described in macrophages undergoing apoptosis (6–9, 27). PS externalization and TGF-β1 release have been implicated to promote the anti-inflammatory phenotype of macrophages and shown to help to protect the surrounding tissue from inflammatory tissue damage. These events result in inhibition of inflammation and necrotic cell death after tissue or cell injury and serve as potent cytoprotective mechanisms. Our group (12) previously showed that volatile anesthetics, including sevoflurane, increased the release of TGF-β1 in human proximal tubule (HK-2) cells via externalization of plasma membrane PS and that this increase in TGF-β1 protected HK-2 cells against H2O2-mediated necrosis. In this study, we mechanistically linked the externalization of PS and TGF-β1 release to the intracellular cytoprotective signaling cascades of sevoflurane in HK-2 cells including ERK/Akt phosphorylation, HSP70 induction, and SMAD3 nuclear translocation. Additionally, TGF-β1 released by sevoflurane treatment reduced the proinflammatory NF-{kappa}B activation as either PS-PC liposome or exogenous TGF-β1 mimicked whereas annexin V prevented sevoflurane-mediated reduction in NF-{kappa}B translocation. Use of control liposomes without PS (PC liposomes and DOTAP liposomes) failed to induce ERK activation and upregulate HSP70. Therefore, we can establish the specificity of PS for the signaling changes observed and exclude any nonspecific alterations on plasmalemmal integrity, structure, and/or fluidity from liposome treatment.

TGF-β1 is a well-known anti-inflammatory molecule and has been shown to produce both anti-inflammatory and antinecrotic effects in vivo as well as in vitro (9, 25). We demonstrate in this study that TGF-β1, similar to sevoflurane, induced the phosphorylation of ERK and Akt in HK-2 cells. Moreover, blockade of TGF-β1 via neutralizing TGF-β1 antibody prevented phosphorylation of ERK and Akt. ERK and Akt are well-known kinases that produce cytoprotection and reduce necrosis in several organs (5, 17, 24). Volatile anesthetics are known to phosphorylate these kinases in the heart and in the kidney (3, 15, 16, 24). Our in vitro studies showed that sevoflurane induced phosphorylation of prosurvival kinases ERK and Akt in proximal tubules in a time-dependent manner, and inhibition of ERK or Akt prevented protection against H2O2-induced necrosis (11). However, we noticed that the peak phosphorylation of ERK and Akt occurred between 2 and 4 h after the initiation of sevoflurane treatment. This was a surprising finding because ERK and Akt phosphorylation (e.g., via G-protein-coupled receptor activation) usually occurred much more rapidly (within minutes) and suggested that perhaps indirect mechanisms via the release of autocrine/paracrine molecule(s) may be involved. In this study, we show that sevoflurane achieves this by the release of TGF-β1. This may explain the longer than expected kinetic profile of sevoflurane-mediated phosphorylation of ERK and Akt.

The HSP70 proteins are a family of ubiquitously expressed proteins with powerful cytoprotective functions (2, 18). HSP70 can reduce necrosis and inflammation, as well as apoptosis, in many organs both in vivo and in vitro (4, 28). Our group has previously demonstrated that volatile anesthetics induced both mRNA and protein expression of inducible HSP70 in renal proximal tubular cells (11). In this study, we demonstrate that TGF-β1 can mimic inducible HSP70 induction and that neutralizing TGF-β1 can block the sevoflurane-mediated inducible form of HSP70. These data are consistent with the hypothesis that sevoflurane-mediated TGF-β1 release increases the synthesis of HSP70. HSP70 proteins were originally described to be upregulated in response to cellular stresses, including heat, heavy metals, and toxic chemicals. Therefore, it is surprising that volatile anesthetics can upregulate HSP70 in HK-2 cells. It is possible that cell membrane perturbations and the externalization of PS can be perceived by the cell as a stress and that the cell responds with HSP70 induction.

In this study, we demonstrate that sevoflurane treatment led to nuclear translocation of SMAD3, a key transcription factor involved in TGF-β1 signaling. SMAD proteins constitute the basic components of the core intracellular signaling cascade carrying TGF-β1 signals from the cell surface directly to the nucleus (14, 19). The activated heteromeric complex of TGF-β type I and type II transmembrane serine/threonine kinase receptors induces phosphorylation of SMAD3. The complexes then translocate to the nucleus and regulate transcriptional responses together with DNA binding cofactors. In addition, TGF-β receptor tyrosine kinase may phosphorylate enzymes involved in cytoprotective signaling such as ERK or Akt (20). It remains to be determined with future studies whether SMAD3 activation is absolutely required for HSP70 induction, ERK and Akt activation, or cytoprotection.

It should be noted that sevoflurane is only half as effective as TGF-β1 in activating ERK (Fig. 3) but is twice as effective at increasing SMAD3 DNA binding capacity, suggesting that these paths do not share complete identity. The fundamental difference in relative potency for SMAD3 DNA binding (sevoflurane > TGF-β1) and MAPK activation (TGF-β1 > sevoflurane) cannot be explained by differences in SMAD3 nuclear translocation (Fig. 5) and suggests inputs additional to TGF-β1 receptor activation, as depicted in Fig. 8. SMAD receptors such as SMAD3 may be either positive or negative effectors of cell function, with target gene specificity presumably conferred via interaction with a variety of factors that include, but are not restricted to, co-SMADs. As such, SMAD3 binding may reflect cross talk with other signaling pathways.


Figure 8
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  		Fig. 8. Schematic of proposed cellular mechanisms of volatile anesthetic-mediated protection in renal tubule cells. PSR, phosphatidylserine receptor; Akt, protein kinase B. It remains to be determined whether a serial activation of ERK and Akt occur in response to TGF-β1 generation, whether ERK and Akt activation occur upstream, as well as downstream, of TGF-β1, and what the role is of SMAD3 phosphorylation/activation in renal tubule protection.

 
We previously demonstrated that sevoflurane pretreatment attenuated H2O2-induced necrotic cell death in HK-2 cells (11). Sevoflurane also reduced ATP depletion (with calcium ionophore, antimycin A, and 2-deoxyglucose mixture)-induced HK-2 cell necrosis (unpublished observations). Zager et al. (29) have also demonstrated that a volatile anesthetic isoflurane protected against HK-2 cell death induced by severe ATP depletion. Therefore, both isoflurane and sevoflurane reduce necrotic cell death in HK-2 cells after severe ATP-depletion.

The mechanism of sevoflurane-mediated externalization of PS to the outer plasma membrane remains to be determined. Zager et al. (29) showed that isoflurane caused a direct inhibition of aminophospholipid translocase activity in HK-2 cells. Inhibition of aminophosphatidyl-translocase function, which is critical for maintenance of normal transmembrane phospholipid asymmetry, would promote PS exposure to the outer leaflet of the plasma membrane. Whether sevoflurane also has similar inhibitory effects on aminophosphatidyl-translocase activity remains an intriguing but likely hypothesis and will require future testing.

In this study, we identified some of the signaling pathways of sevoflurane-mediated renal protection inducing necrosis and inflammation. Clinical significance is high as inhalational anesthetics such as sevoflurane are the center piece of general anesthetic regimens in the United States. Identification of distal signaling pathways of sevoflurane- and other volatile anesthetic-mediated renal tubular effects could lead to the development of drugs that could mimic sevoflurane's renal protective effects without having to deal with the systemic hemodynamic effects of inhalational anesthetics (e.g., hypotension, negative inotropic effects). Our group previously showed that, in vitro, sevoflurane's protection against H2O2-mediated necrosis required at least 4 h of pretreatment (11). This relatively prolonged time course of sevoflurane administration required to achieve cytoprotection (4–16 h) could be a concern in sevoflurane's immediate clinical applicability. Identification of the specific distal effectors may provide more practical therapeutic targets requiring less pretreatment time.

In conclusion, we demonstrate in this study that mimicking externalization of PS or exogenous TGF-β1 activates similar cytoprotective signaling cascades as observed with sevoflurane treatment. Moreover, neutralizing PS externalization or TGF-β1 attenuated sevoflurane's effects of ERK/Akt, HSP70, and NF-{kappa}B (Fig. 8). Because volatile anesthetics such as sevoflurane are critical components of perioperative medicine, harnessing the organ-protective mechanisms would have significant clinical implications. Future work are needed to determine 1) whether a serial activation of ERK and Akt occur in response to TGF-β1 generation, 2) whether ERK and Akt activation occur upstream, as well as downstream, of TGF-β1, and 3) the role of SMAD3 phospharylation/activation in renal tubule protection.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was funded by the intramural grant support from the Department of Anesthesiology, Columbia University College of Physicians and Surgeons, and by National Institutes of Health Grant R01 GM-067081.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. T. Lee, Dept. of Anesthesiology, Anesthesiology Research Laboratories, Columbia Univ., P&S Box 46 (PH-5), 630 West 168th St., New York, NY 10032-3784 (e-mail: tl128{at}columbia.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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