Journal: Journal of Clinical Medicine

Article Title: A Specific Tubular ApoA-I Distribution Is Associated to FSGS Recurrence after Kidney Transplantation

doi: 10.3390/jcm10102174

Figure Lengend Snippet: Localization of ApoA-I and megalin, a specific marker of the proximal tubular brush border, in recurrent FSGS and non-FSGS kidney allograft biopsies. ApoA-I and megalin were detected in kidney allograft biopsies by immunofluorescence using specific antibodies and nuclei were counterstained with Hoechst 33342. The figure shows a representative image of the megalin (in green), ApoA-I (in red) and Hoechst (in blue) staining obtained in recurrent-FSGS and non-FSGS patients; as well as the merged image. Megalin staining (in green) localized at the brush border of the proximal tubular cells in both recurrent FSGS and non-FSGS patients. Regarding ApoA-I, relapsing FSGS patients showed a strong ApoA-I-megalin colocalization, which was not observed in non-FSGS patients (see details of each case in the merged image zoom box).

Article Snippet: The sections were then blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with antibodies against ApoA-I (dilution 1:1000 of rabbit polyclonal PAB8546, Abnova, Jhongli, Taiwan) and megalin (dilution 1:50 of mouse monoclonal MAB9578, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Marker, Immunofluorescence, Staining

Journal: Frontiers in Molecular Biosciences

Article Title: SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice

doi: 10.3389/fmolb.2021.725319

Figure Lengend Snippet: Six1 is upregulated in the proximal tubular epithelial cells (TECs) after unilateral ischemia/reperfusion injury (IRI). (A) Serum creatinine (SCr) and blood urea nitrogen (BUN) levels in I/R injury. (B) Histological changes (H and E staining). Bar = 100 μm. (C) mRNA expression of Six1 in the I/R injured kidney was assessed by qRT-PCR. (D) Western blotting analysis of Six1 expression in the I/R injured kidney. (E) Immunofluorescence analysis of Six1 in IRI 2 days kidneys or sham. Bar = 50 μm. (F) Immunohistochemistry analysis of Six1 in IRI 2 days kidneys or sham. Bar = 50 μm. Data are mean ± SD for groups of six mice. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 versus sham ( t -test); # p <0.05 versus IRI 2 days (Bonferroni correction, two comparisons were made). IRI 1d, 1 day after IRI; IRI 2d, 2 days after IRI; IRI 3d, 3 days after IRI; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

Article Snippet: Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Immunohistochemistry

Journal: Frontiers in Molecular Biosciences

Article Title: SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice

doi: 10.3389/fmolb.2021.725319

Figure Lengend Snippet: Mouse Six1 suppresses monocyte chemotactic protein-1 ( MCP-1 ) expression by inhibiting NF-κB activation. (A) pNFκB-TA-luc reporter containing four NFκB binding sites (top). pNFκB-TA-luc co-transfected with pRL-SV40-N (internal control reporter plasmid) in mouse Six1 overexpression HK2 cells (HK2-Six1 6#) and the control 1#. Cells were harvested 24 h after transfection or treatment with 25 ng/ml TNFα for another 24 h, and luciferase activity was measured (bottom). (B) Plasmids (pNFκB-TA-luc and pRL-SV40-N) were co-transfected into the mouse renal tubular epithelial cells Six1 knockout (TCMK1-Six1 −/− 5#) and the Control 1#. Luciferase activity was measured. The relative luciferase activity was quantified by adjusting it to the renilla luciferase activity and untreated control samples were normalized to 1.0. (C) mRNA expression of mouse Six1 , human NFκB subunit RELA , human cofactors genes of RELA ( AES and FUS ) were assessed by qRT-PCR in the HK2-Six1 6# and the Control 1# cell lines. (D, E) Western blotting was applied to SIX1, RELA, p -RELA, AES, and FUS in HK2-Six1 cell lines (4# and 6#) and the Control cell lines (1# and 2#). (F) SIX1 DNA binding compatible motif (ACCTGA) in AES and FUS gene promoter regions and chromatin immunoprecipitation (ChIP) analysis of SIX1 occupancy of the AES and FUS genes from HK2-Six1 6# and the control 1# cell lines. The location of each primer set compared to the transcription start site (TSS) is shown (left). IgG control samples were normalized to 1.0. (G, H) After treatment by 25 ng/ml TNFα for 24 h or no treatment, mRNA expression of human MCP-1 and mouse Mcp-1 assessed by qRT-PCR in HK2-Six1 6# and Control 1#, TCMK1-Six1 −/− 5# and Control 1#, respectively. Data are expressed as mean ± SD. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 versus the Control group ( t -test). GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

Article Snippet: Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Binding Assay, Transfection, Control, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Knock-Out, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation

Journal: Frontiers in Molecular Biosciences

Article Title: SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice

doi: 10.3389/fmolb.2021.725319

Figure Lengend Snippet: Schematic depicting the effects of Six1 activation in renal tubular epithelial cells (TECs) on cell proliferation/migration and anti-inflammation and underlying mechanism.

Article Snippet: Mouse renal tubular epithelial cells (TCMK1) (iCell Bioscience Inc., Shanghai, China) were transfected with 8 μg Six1-Cas9/sgRNA plasmids with 2 μg pCMV-tdTomato vector (Clontech, Mountain View, CA) using Lipofectamine 3000 according to the manufacturer’s protocol.

Techniques: Activation Assay, Migration

Journal: Oncotarget

Article Title: A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

doi: 10.18632/oncotarget.18540

Figure Lengend Snippet: Diabetes was induced in mice by intraperitoneal injection of STZ (50 mg/kg). Then APX-115 (60 mg/kg/day) or losartan (1.5 mg/kg/day) was administered orally for 12 weeks to diabetic mice. After 12 weeks, urine and blood samples were collected for analysis of ( A ) urinary albumin excretion, ( B ) albumin/creatinine ratio, ( C ) creatinine clearance rate, and ( D ) plasma cystatin C. ( E ) Kidneys were fixed in paraffin and cut into 3 μm sections that were subsequently stained with PAS reagent. Scale bar: 10 μm; original magnification: 630×. After PAS staining, ( F ) glomerular volume, ( G ) mesangial area, and ( H ) tuft area were analyzed using Image-Pro Plus 4.5.1. DM, STZ-induced diabetic mice. Data are presented as means ± SE of 10–12 mice/group; * p < 0.05 vs. control, † p < 0.05 vs. DM.

Article Snippet: A murine mesangial cell line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Injection, Clinical Proteomics, Staining, Control

Journal: Oncotarget

Article Title: A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis

doi: 10.18632/oncotarget.18540

Figure Lengend Snippet: ( A ) Plasma LPO, ( B ) urinary LPO, ( C ) kidney tissue LPO, ( D ) Nox1, ( E ) Nox2, and ( F ) Nox4 mRNA expression levels in kidneys were measured using real-time PCR. ( G and H ) Frozen kidney sections were stained with DHE at 5 µM (original magnification: 400×; scale bar: 20 μm). (A–H) Data are presented as means ± SE of 10–12 mice/group; * p < 0.05 vs. control, † p < 0.05 vs. DM. ( I ) Mesangial cells were incubated with or without APX-115 (1 µM) for 30 min and stimulated with or without 30 mM high glucose (HG) for 24 h followed by angII for 30 min. After that cells were incubated with 10 µM DCF-DA for 10 min and the fluorescence intensity was measured with a Zeiss vision system. Data are presented as means ± SE of at least 2 independent experiments; * p < 0.05 vs. control, † p < 0.05 vs. angII or angII+HG in DMSO.

Article Snippet: A murine mesangial cell line (MES-13, cloned from mice transgenic for the early region of SV-40 virus, passage 25) was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Clinical Proteomics, Expressing, Real-time Polymerase Chain Reaction, Staining, Control, Incubation, Fluorescence

Journal: Journal of medical virology

Article Title: Swine pseudorabies virus attenuated vaccine reprograms the kidney cancer tumor microenvironment and synergizes with PD-1 blockade.

doi: 10.1002/jmv.29568

Figure Lengend Snippet: FIGURE 1 The oncolytic activity of Pseudorabies virus live attenuated vaccine(PRV‐LAV) against kidney cancer in vitro. (A) Cell morphology of A498, Caki‐1, 786‐0, and Renca cells 48 h postinfection with PRV‐LAV. Phase‐contrast micrographs were captured using a Thermo EVOS M7000. Scale bars, 100 μm. (B) Cell viability assays were performed on A498, Caki‐1, 786‐0, and Renca cells 72 h after exposure to PRV‐LAV. Data are presented as the mean ± s.d. values (n = 5). A t‐test was used to determine the significance of differences between the PRV‐LAV infection group and the Mock group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) The expression of PRV gB in the infected cells was detected by immunofluorescence assay. Graphs were captured by Leica DMI8. Scale bars, 250 μm. (D) The expression of PRV gB was analyzed by western blot analysis after cancer cells were infected with PRV‐LAV (MOI = 1) and cultured for 0, 12, 24, 36, and 48 h. GAPDH was used as a loading control. Virus titer in the culture supernatant of A498 (E), Caki‐1 (F), 786‐0 (G), and Renca (H) cells post‐PRV‐LAV infection. Data are presented as the mean ± s.d. values (n = 4). PRV‐LAV, Pseudorabies virus live attenuated vaccine.

Article Snippet: Human kidney cancer cell line A498 (HTB‐44) and mouse kidney cancer cell line Renca (CRL‐2947) were purchased from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Virus, In Vitro, Infection, Expressing, Immunofluorescence, Western Blot, Cell Culture, Control

Journal: Journal of medical virology

Article Title: Swine pseudorabies virus attenuated vaccine reprograms the kidney cancer tumor microenvironment and synergizes with PD-1 blockade.

doi: 10.1002/jmv.29568

Figure Lengend Snippet: FIGURE 2 The safety and therapeutic activity of Pseudorabies virus live attenuated vaccine (PRV‐LAV) against kidney cancer in vivo. (A) Timeline of the experimental setup for the experiments in the BALB/c mouse model. Weight curve (B) and survival rate curve (C) of mice after intravenous injection of vehicle or PRV‐LAV (4 × 107 PFUs) into the tail vein. Data are presented as the mean ± s.d. values (n = 6). (D) Timeline of the experimental setup for the experiments in the BALB/c nude mouse model. (E) Tumor volume curves of mice bearing A498 tumors treated with vehicle or PRV‐LAV (1 × 107 PFUs, intratumorally). Data are presented as the mean ± s.d. values (n = 6). (F) Timeline of the experimental setup for the experiments in the Renca syngeneic models. (G) Tumor volume curves of mice bearing Renca tumors treated with vehicle or PRV‐LAV (1 × 107 PFUs, intratumorally) with an initial tumor volume of approximately 100mm3. Data are presented as the mean ± s.d. values (n = 5). (H) Timeline of the experimental setup for the experiments in the Renca syngeneic models. (I) Tumor volume curves of mice bearing Renca tumors treated with vehicle or PRV‐LAV (1 × 107 PFUs, intratumorally) with an initial tumor volume of approximately 200 mm3. Data are presented as the mean ± s.d. values (n = 5). In (E, G, I), comparisons were performed by AUC analysis. Statistical analysis was performed by t‐test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. PRV‐LAV, Pseudorabies virus live attenuated vaccine.

Article Snippet: Human kidney cancer cell line A498 (HTB‐44) and mouse kidney cancer cell line Renca (CRL‐2947) were purchased from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Virus, In Vivo, 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 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