Critical Role of Interleukin-11 in Isoflurane-mediated Protection against Ischemic Acute Kidney Injury in Mice

ACUTE kidney injury (AKI) remains a major perioperative complication and results in extremely high mortality and morbidity costing more than 10 billion dollars per year in the United States.^1,2  Furthermore, AKI is frequently associated with other life-threatening complications including remote multiorgan injury and sepsis.^2–5  Renal ischemia–reperfusion (I/R) injury or ischemic AKI is a frequent complication for patients subjected to major cardiac, hepatobiliary, or transplant surgery.^3,6  Unfortunately, there is no effective clinically proven therapy for ischemic AKI. Furthermore, patients who survive initial renal injury frequently suffer from long-term chronic kidney disease.

Volatile halogenated anesthetics are one of the most widely used drugs during the perioperative period.⁷  Our previous studies demonstrated that clinically used volatile halogenated anesthetics including isoflurane at clinically relevant concentrations (approximately 1–2 minimum alveolar concentration) protect against ischemic AKI by attenuating renal tubular necrosis and by retarding renal tubular inflammation with reduction in influx of proinflammatory leukocytes.^8,9  We also demonstrated that volatile halogenated anesthetics produce direct antiinflammatory and antinecrotic effects in cultured human kidney proximal tubule (HK-2) cells.^10,11  Subsequently, we discovered that volatile halogenated anesthetics protect against renal tubular necrosis and inflammation by direct renal tubular production of transforming growth factor-β1 (TGF-β1).^11–13  However, the downstream signaling mechanisms of volatile halogenated anesthetic-mediated renal protection generated by TGF-β1 remain incompletely understood. Moreover, isoflurane therapy for critically ill patients may be limited by its anesthetic and cardiovascular effects. One way to mitigate this is to use the distal signaling molecules synthesized with isoflurane treatment devoid of systemic hemodynamic and anesthetic effects.

Interleukin (IL)-11 is a 20 kDa member of the IL-6-type cytokine family. IL-11 promotes megakaryocyte maturation and is already clinically approved to increase platelet counts in patients receiving chemotherapy.¹⁴  In addition to its hematopoietic effects, IL-11 protects against intestinal, cardiomyocyte, and endothelial cell death.¹⁵  We recently showed that recombinant human IL-11 treatment before or after renal ischemia attenuated ischemic AKI in mice.¹⁶  Specifically, IL-11 administration significantly attenuated necrosis, inflammation, and apoptosis after ischemic AKI closely mimicking the renal protective effects of volatile halogenated anesthetics. This IL-11–mediated protection against ischemic AKI requires the downstream induction of another cytoprotective protein sphingosine kinase-1. Interestingly, we also showed that isoflurane-mediated protection against ischemic AKI also requires induction of sphingosine kinase-1.¹⁷  Finally, previous studies suggest that TGF-β1 induces IL-11 in lung epithelial cells and fibroblasts.^18,19  Therefore, in this study, we tested the hypothesis that isoflurane induces TGF-β1–mediated renal proximal tubular IL-11 synthesis. We also tested whether IL-11 plays a critical role in isoflurane-mediated renal protection.

Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco Modified Eagle Media/F12 with 10% fetal bovine serum (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% carbon dioxide–95% air. This cell line has been characterized extensively and retains the phenotypic and functional characteristics of proximal tubule cells in culture.²⁰  We also cultured mouse kidney proximal tubule cells. Mouse kidneys were removed, minced, and digested in collagenase A (1 mg/ml; Sigma, St. Louis, MO) at 37°C for 45 min with occasional agitation. The cellular digest was filtered through a nylon mesh, centrifuged at 600g for 10 min, and washed twice. Mouse kidney proximal tubules were isolated according to the method by Vinay et al.²¹  with the use of Percoll density gradient separation. Cells were used in the experiments described below when confluent after 24-h serum deprivation.

HK-2 cells or mouse proximal tubules in culture were placed in an air tight, 37°C, humidified modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA) with inflow and outflow ports. The inlet port was connected to a vaporizer (Datex-Ohmeda, GE Healthcare, Oklahoma City, OK) to deliver isoflurane (Abbott Laboratories, North Chicago, IL) mixed with 95% air and 5% carbon dioxide (carrier gas) at 10 l/min. The outlet port was connected to a Datex-Ohmeda 5250 RGM gas analyzer that measured isoflurane concentrations. Exposure to isoflurane (1.25–2.5%) lasted 0–16 h. Control cells were exposed to carrier gas in an identical modular incubator chamber. To block the effects of TGF-β1 generated by isoflurane, some HK-2 cells were pretreated with neutralizing TGF-β1 antibody (10 µg/ml; R&D Systems, Minneapolis, MN) 30 min before isoflurane treatment, respectively. We also used nonneutralizing control isotype antibody to test the specificity of the neutralizing TGF-β1 antibody (BD Biosciences, San Jose, CA).

By using reverse transcription polymerase chain reaction, we measured messenger RNAs (mRNAs)-encoding human (HK-2 cells) or mouse IL-11 as described.²²  Amplification of the human IL-11 complementary DNA was performed using the following primers: forward primer, 5′-CTG AAG ACT CGG CTG TGA CC-3′ and reverse primer, 5′-CAG GGC AGA AGT CTG TGG AC-3′ at an annealing temperature of 66°C resulting in a 300 base pair product. Amplification of the mouse IL-11 complementary DNA was performed using the following primers: forward primer, 5′-AAC TGT GTT TGT CGC CTG GT-3′ and reverse primer, 5′-AAG CTG CAA AGA TCC CAA TG-3′ at an annealing temperature of 68°C resulting in a 267 base pair product. Glyceraldehyde-3-phosphate dehydrogenase complementary DNA amplification was performed to control for lane loading: forward primer, 5′-ACC ACA GTC CAT GCC ATC AC-3′ and reverse primer, 5′-CAC CAC CCT GTT GCT GTA GCC-3′ at an annealing temperature of 65°C resulting in a 450 base pair product.

HK-2 cell supernatant or mouse kidney cortex lysate IL-11 protein expression was measured using human- or mouse-specific sandwich IL-11 enzyme-linked immunosorbent assay kit (R&D Systems), respectively.

After approval from the Columbia University Institutional Animal Care and Use Committee (New York, New York), we used adult male IL-11 receptor–deficient (IL-11R knockout) mice or wild-type (WT) litter mates (IL-11R WT) on a C57BL/6 background (B6.129S1-Il11ra1^tm1Wehi/J; Jackson Labs, Bar Harbor, ME). Mice were initially anesthetized with intraperitoneal pentobarbital (Henry Schein Veterinary Co., Indianapolis, IN; 50 mg/kg body weight, or to effect) and subjected to right nephrectomy and 30 min of left renal ischemia or to sham-operation (laparotomy, right nephrectomy without renal ischemia).^8,9  After closure of the abdomen in two layers, the mice were then exposed to an additional 4 h of equipotent doses of either pentobarbital or 1.2% isoflurane as described previously.²³  The mice were placed on a heating pad under a warming light to maintain body temperature approximately 36°–38°C. To neutralize IL-11 in vivo, some IL-11R WT mice were injected intravenously with 1 mg/kg monoclonal anti-IL-11 (MAB418; R&D Systems) 20 min before reperfusion of ischemic kidney or sham-operation. We collected kidney (cortex and corticomedullary junction) and plasma 6–24 h after I/R injury to examine the severity of renal dysfunction (plasma creatinine, renal histology, apoptosis, and neutrophil infiltration) and IL-11 mRNA and protein detection. Plasma creatinine was measured as described with an enzymatic creatinine reagent kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA).²⁴  Unlike the Jaffe method, this method of creatinine measurement largely eliminates the interferences from mouse plasma chromagens.

Some IL-11R WT mice were anesthetized with pentobarbital or with 1.2% isoflurane without being subjected to renal I/R injury. To test the critical role of TGF-β1 signaling in isoflurane-mediated IL-11 induction in vivo, some IL-11 WT mice were injected intravenously with 5 mg/kg monoclonal anti-TGF-β1 (MAB240; R&D Systems) or control isotype antibody 30 min before anesthesia with isoflurane or pentobarbital.

Morphological assessment of kidney hematoxylin and eosin stain staining was performed by renal pathologist (Dr. D’Agati) who was unaware of the treatment that each animal had received. An established grading scale of necrotic injury (0–4, renal injury score) to the proximal tubules was used for the histopathological assessment of I/R-induced damage as outlined by Jablonski et al.²⁵  and as described previously in our studies.^26,27  We detected kidney apoptosis with terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick end-labeling staining as described elsewhere²⁸  using a commercially available in situ cell death detection kit (Roche, Indianapolis, IN) according to the instructions provided by the manufacturer. Apoptotic terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick end-labeling–positive cells were quantified in five to seven randomly chosen ×100 microscope image fields in the corticomedullary junction, and results were expressed as apoptotic cells counted per ×100 field. Immunohistochemistry for neutrophils was performed as described previously²⁹  with a rat anti-mouse Ly6B monoclonal antibody against polymorphonuclear leukocytes (clone 7/4; AbD Serotec, Raleigh, NC). A primary antibody that recognized IgG_2a (MCA1212; AbD Serotec) was used as a negative isotype control in all experiments. Neutrophils infiltrating the kidney were quantified in five to seven randomly chosen ×200 microscope image fields in the corticomedullary junction, and results were expressed as neutrophils counted per ×200 field.

Immunohistochemistry detected mouse kidney IL-11 protein expression and localization 4 h after anesthesia with pentobarbital or 1.2% isoflurane with rat anti-IL-11 antibody (MAB418; 1:50 dilution; R&D Systems) and biotin-conjugated anti-rat IgG (1:100 dilution; Vector Laboratories, Burlingame, CA). Normal rat IgG2a (Vector Laboratories) was used at the same concentration as the primary antibody as a negative isotype control. Kidney IL-11 immunohistochemistry was quantified as described by Matkowskyj et al.³⁰  with some modifications. Integrated image densities of five to seven randomly selected renal tubule areas from each slide were averaged, and background measured from isotype control slides was subtracted. Renal tubular IL-11 intensity was expressed as fold increase over pentobarbital-anesthetized mice.

The data were analyzed with two-tailed Student t est when comparing means between two groups or one-way or two-way ANOVA plus Tukey post hoc multiple comparison test when comparing multiple groups. The ordinal values of the renal injury scores were analyzed by the Mann–Whitney nonparametric test. We used Graphad InStat and Graphad Prism for statistical analyses (GraphPad Software, Inc., La Jolla, CA). In all cases, a probability statistic P value less than 0.05 was taken to indicate significance. All data are expressed throughout the text as means ± SD.

Figure 1A shows a concentration-dependent (0–2.5%) induction of IL-11 mRNA in HK-2 cells after isoflurane treatment (N = 6). Figure 1B shows that 2.5% isoflurane significantly increased IL-11 mRNA after 3- or 6-h treatment. Isoflurane treatment (2.5%) in HK-2 cells for 6–16 h also induced IL-11 protein (released into cell culture media; fig. 1C) in HK-2 cells compared with carrier gas–treated cells (N = 6).

HK-2 cells pretreated with control isotype antibody (mouse IgG) demonstrated a significant induction of IL-11 mRNA (fig. 2A) and protein (fig. 2B) expression (N = 6) after isoflurane exposure (2.5% for 6 h). We determined that TGF-β1–neutralizing antibody (10 µg/ml) significantly attenuated the up-regulation of IL-11 mRNA as well as protein expression after isoflurane treatment.

Figure 3 shows that primary cultures of mouse kidney proximal tubule cells pretreated with control isotype mouse IgG had significant IL-11 mRNA induction after 6-h treatment with 2.5% isoflurane (N = 4). TGF-β1 neutralization significantly attenuated the up-regulation of IL-11 mRNA in isoflurane-treated mouse proximal tubule cells. Therefore, our studies show that isoflurane-induced TGF-β1 directly promotes the synthesis of IL-11 in both immortalized and primary cultures of renal proximal tubule cells.

Figure 4 shows that anesthesia with isoflurane (1.2% for 4 h) significantly induced IL-11 mRNA (fig. 4A) and protein expression (fig. 4B) measured in mouse kidneys compared with mice anesthetized with pentobarbital for 4 h (N = 4). We also determined that TGF-β1–neutralizing antibody (5 mg/kg monoclonal anti-TGF-β1, MAB240) prevented the induction of kidney IL-11 mRNA and protein expression after anesthesia with isoflurane in mice (fig. 4; N = 4).

Figure 5A shows diffuse renal tubular IL-11 staining in mice anesthetized with pentobarbital and increased IL-11 staining in the kidneys of mice anesthetized with 1.2% isoflurane for 4 h (representative of four experiments, ×100 and ×400 images shown). Quantification of immunohistochemical staining confirmed significant increase in IL-11 immunoreactivity in mice anesthetized with isoflurane (fig. 5B; N = 4). This increase in IL-11 immunoreactivity during anesthesia with isoflurane was again attenuated by treating mice with TGF-β1–neutralizing antibody before anesthesia with isoflurane. Therefore, from these experiments, we concluded that isoflurane induces IL-11 and synthesis in vivo via TGF-β1 signaling. IL-11 staining was not visible in the kidneys stained with negative isotype control antibody.

IL-11R WT mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h had similar plasma creatinine values after sham-operation (fig. 6). Similarly, IL-11R WT mice pretreated with IL-11–neutralizing antibody or IL-11R knockout mice had similar plasma creatinine values after sham-operation during anesthesia with pentobarbital. Plasma creatinine significantly increased in IL-11R WT mice subjected to 30 min of renal ischemia and 2-h reperfusion compared with sham-operated mice (fig. 6; N = 6). However, IL-11R WT mice anesthetized with 1.2% isoflurane for 4 h after renal ischemia (isoflurane postconditioning) had significantly decreased plasma creatinine 24 h after injury compared with mice anesthetized with pentobarbital after renal ischemia. Supporting a critical role of IL-11 in isoflurane-mediated protection against ischemic AKI, IL-11R knockout or IL-11R WT mice pretreated with IL-11–neutralizing antibody before renal ischemia were not protected against ischemic AKI during anesthesia with isoflurane after renal I/R (fig. 6). IL-11R knockout mice and IL-11R WT mice pretreated with IL-11–neutralizing antibody had similar degree of renal injury after I/R during anesthesia with pentobarbital. Collectively, these studies suggest that IL-11 induction by isoflurane is required to trigger in vivo renal protection.

Figure 7A demonstrates severe necrotic renal injury in IL-11R WT mice subjected to renal I/R during anesthesia with pentobarbital. Compared with sham-operated mice (not shown), the kidneys of mice subjected to renal I/R showed significant tubular necrosis, cast formation, and congestion. In contrast, consistent with the plasma creatinine data, anesthesia with isoflurane reduced renal tubular necrosis 24 h after I/R injury (fig. 7A). Supporting a critical role of IL-11 in isoflurane-mediated renal protection against I/R, isoflurane failed to reduce renal tubular necrosis in IL-11R WT mice pretreated with a neutralizing IL-11 antibody or in IL-11R knockout mice. The Jablonski scale²⁵  renal injury score was used to grade renal tubular necrosis 24 h after renal I/R (fig. 7B). Thirty minutes of renal ischemia and 24 h of reperfusion resulted in severe acute tubular necrosis in IL-11R WT mice anesthetized with pentobarbital after renal I/R injury. Consistent with the renal histology data, isoflurane significantly reduced the renal injury score in IL-11R WT mice but not in IL-11R knockout mice or in IL-11R WT mice pretreated with IL-11–neutralizing antibody.

Figure 8A shows representative images (from four to six experiments) of neutrophil immunohistochemistry in kidneys (magnification ×200) of mice subjected to 30 min of renal ischemia and 24-h reperfusion. In sham-operated mice, we were unable to detect any neutrophils in the kidney (data not shown). There was heavy neutrophil infiltration (dark brown) in the kidneys of pentobarbital-anesthetized IL-11R WT mice subjected to renal I/R. In contrast, IL-11R WT mice anesthetized with isoflurane for 4 h after renal ischemia had significantly reduced neutrophil infiltration in the kidney 24 h after I/R (fig. 8B). Again, isoflurane failed to reduce kidney neutrophil infiltration in IL-11R WT mice treated with IL-11–neutralizing antibody and in IL-11R knockout mice.

Terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick end-labeling staining (from four to five experiments) detected apoptotic renal cells in the kidneys of mice subjected to renal I/R resulting in severe proximal tubule cell apoptosis (fig. 9A, ×100). Renal ischemia and 24 h of reperfusion resulted in significant apoptosis in the kidneys of pentobarbital-anesthetized IL-11R WT mice. However, IL-11R WT mice anesthetized with isoflurane for 4 h after renal ischemia had significantly reduced number of apoptotic terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick end-labeling–positive cells in the kidney 24 h after I/R (fig. 9B). Again, isoflurane failed to reduce renal tubular apoptosis in IL-11R WT mice treated with IL-11–neutralizing antibody and in IL-11R knockout mice.

We have shown in this study that isoflurane increases renal tubular IL-11 mRNA and protein synthesis via TGF-β1–dependent signaling in vivo as well as in vitro. TGF-β1 is a powerful antiinflammatory cytokine that regulates multiple cellular processes including immune modulation, cellular differentiation and proliferation, and oncogenesis.^31,32  We previously demonstrated that volatile halogenated anesthetics including isoflurane cause translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane (membrane externalization) resulting in the release of antiinflammatory TGF-β1.^11,12  Furthermore, volatile halogenated anesthetics-mediated reduction in renal tubular necrosis and inflammation was dependent on the release of renal tubular TGF-β1. Finally, volatile halogenated anesthetic-mediated renal protection was abolished in mice deficient in TGF-β1 or WT mice treated with neutralizing TGF-β1 antibody.³³ 

We also recently demonstrated that exogenous human recombinant IL-11 attenuated ischemic AKI in mice and reduced necrosis and apoptosis in human kidney (HK-2) proximal tubule cells.¹⁶  In mice, recombinant IL-11 treatment attenuated renal tubular necrosis and apoptosis as well as the influx of proinflammatory neutrophils after renal I/R. We further demonstrated in this study that isoflurane-mediated protection against renal tubular necrosis (Jablonski score), inflammation (neutrophil infiltration), and apoptosis (terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick end-labeling staining) is directly mediated by the induction of IL-11 as isoflurane failed to reduce these critical indices of renal injury in IL-11R–deficient mice or mice treated with IL-11–neutralized antibody. Taken together, as blockade or genetic deletion of either TGF-β1 or IL-11 abolished renal protective effects of isoflurane, our current and previous studies suggest that isoflurane induces nonredundant proximal (TGF-β1) and distal (IL-11) cytoprotective signaling molecules to protect against ischemic AKI.

The most exciting aspect of isoflurane-mediated induction of IL-11 leading to renal protection is that recombinant IL-11 (Oprelvekin; Wyeth Pharmaceuticals, Philadelphia, PA) is already clinically approved to treat chemotherapy-induced thrombocytopenia. IL-11 is a member of the IL-6-type cytokine family isolated from bone marrow–derived stromal cells.³⁴  IL-11 receptor activation potently regulates hematopoiesis by promoting megakaryocyte maturation.³⁵  However, in addition to its effects on platelets, recent studies show a cytoprotective role of IL-11 as exogenous IL-11 administration attenuates cell death in several cell types and organs.^16,36–38  In the heart, intestine, and endothelial cells, IL-11 directly reduces necrotic as well as apoptotic cell death via mitogen-activated protein kinase and the Janus kinase/signal transducers and activators of transcription signaling.¹⁵  In addition to its antiapoptotic and antinecrotic properties, IL-11 receptor activation attenuates lipopolysaccharide-induced systemic inflammation in mice, toxic nephritis, and leukocyte-mediated liver injury.^39–42  Therefore, we propose that isoflurane-mediated induction of IL-11 and renal tubular IL-11 receptor activation produce powerful protection against ischemic AKI by targeting all three pathways of cell death: necrosis, apoptosis, and inflammation. Furthermore, as IL-11 as well as IL-11 receptors are expressed in many tissues and cell types,¹⁵  it remains to be determined whether isoflurane can induce IL-11 in nonrenal cells (e.g., hepatocytes, intestinal epithelial cells, and endothelial cells) and confer protection in these organs against AKI-induced remote organ injury.

The signal transduction mechanisms of IL-11–induced cytoprotection have been investigated in other cell types. IL-11 ligand and receptor complex interacts with a common receptor subunit, glycoprotein 130 (gp130), leading to gp130-associated kinase-mediated tyrosine phosphorylation.⁴³  In cardiac myocytes, IL-11 reduces injury and fibrosis by the Janus kinase/signal transducers and activators of transcription 3 activation.^38,43,44  In vascular endothelial and intestinal epithelial cells, IL-11 protects against oxidant-induced necrosis and apoptosis via mechanisms involving extracellular signal-regulated kinase, mitogen-activated protein kinase, protein kinase B, and/or induction of heat shock protein 25.^37,45,46 

Isolfurane-induced IL-11 generation may also produce antiinflammatory effects after renal I/R via modulating the nuclear factor-κB activity. Indeed, nuclear factor-κB is one of the most important proinflammatory transcription factors.⁴⁷  IL-11 has been shown to attenuate transcription factor NF-kB in several cell lines and in mouse models of kidney inflammation.^40,48,49  Furthermore, IL-11 treatment in vivo decreases glomerular nuclear factor-κB activity and reduces renal injury in experimental glomerulonephritis.⁵⁰ 

We previously showed that IL-11 produces renal protection by direct induction of sphingosine kinase-1 via nuclear translocation of hypoxia-inducible factor-1α.¹⁶  Sphingosine kinase-1 produces sphingosine 1-phosphate—a well-known antiinflammatory immune modulator.⁵¹  Based on our previous^9,52  and current experimental data, we propose that isoflurane may directly induce several antiinflammatory signaling molecules (e.g., IL-11, sphingosine 1-phosphate) to protect against hyperinflammatory response after ischemic AKI. Interestingly, several of the IL-11–mediated cytoprotective signal transduction proteins (e.g., extracellular signal-regulated kinase, mitogen-activated protein kinase, protein kinase B, sphingosine kinase) are also activated with volatile halogenated anesthetics as we demonstrated previously.^12,13,17 

We have shown here that isoflurane postconditioning produces potent renal protection against ischemic AKI via IL-11–mediated reduction in kidney tubule necrosis, apoptosis, and renal inflammation. We propose that after renal I/R injury, necrotic tubules cells release proinflammatory cytokines (e.g., monocyte chemotactic protein-1 release) that can further aggravate inflammation by stimulating resident kidney macrophages and dendritic cells.⁴  These cells in turn release additional proinflammatory cytokines (e.g., macrophage inflammatory protein 2α, keratinocyte-derived cytokine) to promote cytotoxic T-lymphocyte, neutrophil, and macrophage infiltration into the kidney interstitial space. Infiltrating proinflammatory leukocytes release additional cytotoxic proinflammatory and proapoptotic cytokines (e.g., tumor necrosis factor-α) which will exacerbate renal tubular cell necrosis as well as apoptosis. Figure 10 summarizes the potential mechanisms of isoflurane-mediated renal protection involving renal tubular IL-11 synthesis via TGF-β1 signaling.

We believe that potential for clinical use of recombinant IL-11 therapy would be far superior to therapy against ischemic AKI with recombinant TGF-β1. Active form of TGF-β1 has a very short plasma half-life (2–3 min) as nonlatent TGF-β1 gets rapidly taken up by the liver, kidneys, lungs, and spleen and degraded.⁵³  This is in contrast to the more prolonged half-life of recombinant IL-11 (approximately 7 h).³⁴  Furthermore, prolonged exposure to TGF-β1 may promote kidney fibrosis.⁵⁴  Therefore, prolonged TGF-β1 therapy may transiently reduce inflammation and necrosis but may cause increased renal tubular fibrosis that may paradoxically prolong renal dysfunction after I/R. Unlike TGF-β1, prolonged and high-dose IL-11 therapy does not induce tissue fibrosis.

We recently showed that isoflurane via TGF-β1 induces ecto-5′-nucleotidase (CD73) to generate cytoprotective adenosine in renal proximal tubule cells.⁵⁵  CD73 induction and CD73-mediated adenosine generation were critical for isoflurane-mediated renal protection against ischemic AKI. However, the downstream target of isoflurane-mediated adenosine generation as well as the specific adenosine receptor subtype(s) responsible for renal protection against ischemic AKI remains unclear. We hypothesize that isoflurane-mediated induction of CD73 activity and adenosine generation directly stimulates renal tubular IL-11 synthesis to protect against ischemic AKI. Consistent with this hypothesis, we recently also showed that a specific A₁ adenosine receptor agonist 2-chloro cyclopentyladenosine also induces IL-11 in proximal tubule cells.⁵⁶  It remains to be tested in future studies whether isoflurane-mediated adenosine generation results in the activation of renal tubular A₁ adenosine receptors to protect against renal ischemia and reperfusion injury.

There are several limitations to our study. Our results may not completely translate to clinical setting as differences in pathophysiology between human and mouse ischemic AKI exist. Furthermore, our studies focused on renal tubular synthesis of IL-11 and protection whereas clinical AKI results in both impairment of glomerular filtration and renal tubular dysfunction. In addition, our in vitro studies have limitation as HK-2 cells used in this study are immortalized and primary culture of proximal tubule cells may undergo rapid phenotypic changes ex vivo. Finally, complete isoflurane concentration–response curves were not generated in most of our experiments.

In summary, we demonstrated that a widely used volatile halogenated anesthetic isoflurane protects against renal tubular necrosis and inflammation after renal I/R by inducing cytoprotective IL-11 generation.

The authors appreciate the surgical, analytical, and technical assistance provided by Sang Won Park, Ph.D. (Assistant Professor of Pharmacology, Gyeongnam National University, Jin Ju, South Korea).