Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A and B. IL-11 mRNA measured with reverse transcription polymerase chain reaction (RTPCR) in HK-2 cells treated with 0-2.5% isoflurane for 6 h (A. N = 6) or 2.5% isoflurane for 0-6 h (B, N = 6). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was quantified to normalize lane loading. Data are presented as means ± SD. * P < 0.05 vs. IL-11 mRNA measured after 0% isoflurane-treatment (A) or at 0 h (B). C. Isoflurane increases IL-11 protein (pg/ml) in cell culture media from HK-2 cells (N = 6). HK-2 cells were treated with 2.5% isoflurane or with carrier gas for 6 h or 16 h. * P < 0.05 vs. carrier gas treated group.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 messenger ribonucleic acid (mRNA) (detected with reverse transcription polymerase chain reaction (RTPCR)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas plus immunoglobulin G (IgG) isotype antibody treated controls. B. IL-11 protein (detected with enzyme-linked immunosorbent assay (ELISA)) expression in HK-2 cells treated with 2.5% isoflurane for 6 h (N = 6). * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in human proximal tubule cells.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in primary culture of mouse proximal tubule cells treated with 2.5% isoflurane for 6 h (N = 4). Representative images (top) and band intensity quantifications (bottom) expressed as fold increases in IL-11 expression over carrier gas and immunoglobulin G (IgG) isotype antibody treated controls. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was also quantified to normalize lane loading. * P < 0.05 vs. carrier gas group treated with IgG isotype antibody. # P < 0.05 vs. isoflurane group treated with IgG isotype antibody. Error bars represent 1 SD. TGF-β1 antibody (10 μg/ml) prevents isoflurane-mediated induction of IL-11 mRNA and protein expression in mouse proximal tubule cells.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Naïve IL-11 wild-type (WT) mice were exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h. A. Representative bands for IL-11 mRNA (reverse transcription polymerase chain reaction (RTPCR)) expression in mouse kidney (N = 4). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal loading control. B. Kidney lysate IL-11 protein (detected by enzyme-linked immunosorbent assay (ELISA)) in IL-11 naïve WT mice exposed to pentobarbital (PB) or 1.2% isoflurane (ISO) for 4 h (N = 4). To neutralize TGF-β1 in vivo, some IL-11 WT mice were injected with 5 mg/kg monoclonal anti-TGF-β1 (MAB240) antibody intravenous injection. TGF-β1 neutralization prevented the induction of IL-11 after isoflurane anesthesia. * P < 0.05 vs. pentobarbital anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with IgG isotype antibody. Error bars represent 1 SD. Isoflurane anesthesia significantly increased kidney IL-11 mRNA and protein expression in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Enzyme-linked Immunosorbent Assay, In Vivo, Injection, Neutralization

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. IL-11 immunohistochemistry (400× shown, N = 4) in kidneys from IL-11 receptor wild type (IL-11R WT) mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. IL-11 staining was darker in kidneys of mice anesthetized with isoflurane. TGF-β1 neutralizing antibody attenuated isoflurane-mediated increase in IL-11 immunoreactivity. Finally, IL-11 staining was not visible in kidneys from mice stained with negative isotype control antibodies (representative of four experiments). B. Quantifications of renal tubular IL-11 staining in mice anesthetized with pentobarbital or with 1.2% isoflurane for 4 h. Kidney IL-11 immunoreactivity significantly increased in mice anesthetized with isoflurane and attenuated with TGF-β1 neutralizing antibody. # P < 0.05 vs. isoflurane anesthetized mice treated with immunoglobulin G (IgG) isotype antibody. * P < 0.05 vs. pentobarbital-anesthetized mice. Error bars represent 1 SD.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Immunohistochemistry, Staining, Control

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Plasma creatinine levels from IL-11 wild-type (WT) or IL-11 deficient (KO) mice subjected to 30 min renal ischemia and 24 h reperfusion (N = 4-6 per group). After renal ischemia reperfusion (RIR), mice were further anesthetized with 1.2% isoflurane (ISO) or with equi-anesthetic dose of pentobarbital (PB). Some IL-11 receptor (IL-11R) WT mice were pretreated with an IL-11 neutralizing antibody (1 mg/kg, intravenous injection) 20 min before reperfusion or sham-operation. Isoflurane postconditioning significantly reduced plasma creatinine after RIR injury in IL-11R WT mice. However, IL-11R deficiency or IL-11 neutralizing antibody prevented the renal protective effects of isoflurane post-conditioning. * P < 0.05 vs. respective sham-operated mice. # P < 0.05 vs. pentobarbital anesthetized mice subjected to RIR. Data are from 6 mice per group and represented as mean ± SD.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Clinical Proteomics, Injection

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of five to six experiments for hematoxylin and eosin staining (magnification 200×) of kidneys of IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min renal ischemia and 24-h reperfusion (I/R). B. Summary of Jablonski scale renal injury scores (N = 4, graded from hematoxylin and eosin staining, scale 0-4) for mice subjected to renal I/R. * P < 0.05 vs. pentobarbital-anesthetized IL-11R WT mice subjected to renal I/R. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed severe renal tubular necrosis. Isoflurane post-conditioning significantly attenuated renal tubular necrosis and renal injury scores after renal IR. IL-11R deficiency (IL-11R KO) or IL-11 neutralization prevented renal protection with isoflurane postconditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Staining, Neutralization

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of four to six experiments for immunohistochemistry (brown staining) for neutrophil infiltration (200×) from kidneys IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min. renal ischemia and 24-h reperfusion. B. Quantifications of infiltrated neutrophils per 200× field in the kidneys of mice after renal ischemia reperfusion (RIR). * P < 0.05 vs. vehicle-treated pentobarbital anesthetized mice subjected to RIR. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed heavy neutrophil infiltration. Isoflurane postconditioning significantly attenuated renal tubular neutrophil infiltration after RIR. IL-11 deficiency (IL-11R KO) or IL-11 neutralization attenuated these reductions in renal neutrophil infiltration with isoflurane post-conditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Immunohistochemistry, Staining, Neutralization

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: A. Representative photomicrographs of four to six experiments for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (representing apoptotic nuclei, magnification 100×) from kidneys IL-11 receptor wild-type (IL-11R WT) mice, IL-11 receptor deficient (IL-11R KO [knockout]) mice and IL-11R WT mice pretreated with IL-11 neutralizing antibody and subjected to 30 min renal ischemia and 24-h reperfusion. B. Quantifications of apoptotic cells per 100× field in the kidneys of mice after renal ischemia reperfusion (RIR). * P < 0.05 vs. vehicle-treated pentobarbital anesthetized mice subjected to RIR. Error bars represent 1 SD. IL-11R WT mice anesthetized with pentobarbital after renal ischemia showed numerous TUNEL positive cells. Isoflurane postconditioning significantly attenuated renal tubular apoptosis after RIR. IL-11 deficiency (IL-11R KO) or IL-11 neutralization attenuated these reductions in renal tubular apoptosis with isoflurane postconditioning in mice.

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: TUNEL Assay, Staining, Knock-Out, Neutralization

Journal: Anesthesiology

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

doi: 10.1097/ALN.0b013e3182a950da

Figure Lengend Snippet: Collectively, our data suggest that isoflurane anesthesia increases interleukin (IL)-11 messenger RNA (mRNA) and protein synthesis via TGF-β1 signaling. We propose that IL-11 synthesized then subsequently activates IL-11R in neighboring renal tubules, or endothelial cells to induce cytoprotective signaling. Since previous studies have shown that IL-11 reduces the activity of a well-known proinflammatory transcription factor NF-kB49;50, it is highly possible that IL-11 generated with isoflurane treatment may also attenuate NF-kB activity to protect against renal inflammation and injury after acute kidney injury. SMADs are intracellular proteins that transduce extracellular signals from TGF-β1 to the nucleus to initiate downstream gene transcription. Hypothetical pathways (e.g., NF-kB inhibition) leading to cytoprotection are shown in dashed lines. We previous showed that IL-11 produces renal protection by direct induction of sphingosine kinase-1 via nuclear translocation of HIF-1α.14

Article Snippet: Human and mouse proximal tubule cell culture and exposure to isoflurane Immortalized human renal proximal tubule (HK-2) cells (American Type Culture Collection, Manassas, VA) were grown and passaged with 50:50 mixture of Dulbecco’s 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.

Techniques: Synthesized, Activity Assay, Generated, Inhibition, Translocation Assay