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1.  Nox2 is a mediator of chronic CsA nephrotoxicity 
We hypothesized that Nox2, the classical phagocytic NADPH oxidase, plays an important role in calcineurin inhibitor (CNI)-induced renal fibrosis. We tested this hypothesis in vitro, in animal and in human studies. Cyclosporine A (CsA) and tacrolimus (TAC) were associated with greater levels of Nox2 mRNA and epithelial to mesenchymal transition (EMT) in NRK52E cells. CsA increased Nox2, α-SMA and phosphorylated-p38MAPK, Smad3, and NFκB proteins. Nox2 upregulation and EMT were inhibited in TGF-β1 knockout cells suggesting that TGF-β1 is required for Nox2 activation. Fisher344 rats treated with high dose CsA showed increased Nox2 in the tubulointerstitium and greater Nox2, α-SMA, phosphorylated Smad3 and nitrotyrosine by immunoblot analyses. Inhibition of Nox2 by coadministration of apocynin or diphenyleneiodonium was associated with reduced fibrogenesis. We validated these findings by treating wild type and Nox2 null (B6.129S-CybbTm1Din/J) mice with high dose CsA. Western blot analyses confirmed the absence of Nox2 and significantly lower levels of α-SMA and 4-hydroxynonenal in CsA-treated knockout mice. These findings were clinically relevant since Nox2 and α-SMA were increased in the tubulointerstitium of kidneys from 15 liver transplant recipients with biopsy-confirmed chronic CsA or TAC nephrotoxicity. In conclusion, specific Nox2 inhibition strategies may improve chronic CNI nephrotoxicity in solid organ transplantation.
doi:10.1111/j.1600-6143.2012.04081.x
PMCID: PMC3409317  PMID: 22568654
CsA; Nox2; Fibrosis; EMT; Oxidative Stress
2.  Tubular expression of heat shock protein 27 inhibits fibrogenesis in obstructive nephropathy 
Kidney international  2012;83(1):84-92.
Morphological changes that occur during kidney injury involve actin skeleton remodeling. Here we tested whether heat shock protein 27 (HSP27), a small stress response protein involved in cytoskeletal remodeling, protects the kidney from tubulointerstitial fibrosis in obstructive nephropathy. Tubular cell HSP27 immunostaining was significantly increased in human kidneys with ureteropelvic junction obstruction; supporting the clinical relevance of our studies. To develop an animal model for mechanistic studies we generated transgenic mice that specifically overexpress human HSP27 in renal tubules, under the kidney androgen-regulated protein promoter, and determined the effects of HSP27 overexpression on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis following unilateral ureteral obstruction. This was associated with decreased fibrogenesis as evidenced by significant declines in phosphorylated p38MAPK, collagen III, α-smooth muscle actin, 4-hydroxynonenal, and reduced trichrome staining following obstruction. Notably, E-cadherin and β-catenin remained at the cell membrane of tubular cells in transgenic mice with an obstructed ureter. Monocyte/macrophage infiltration, however, was not significantly affected in these transgenic mice. Thus, tubular HSP27 inhibits fibrogenesis in obstructive nephropathy. Further studies are needed to determine pathways regulating the interactions between HSP27 and the E-cadherin-β-catenin complex.
doi:10.1038/ki.2012.336
PMCID: PMC3525804  PMID: 22971995
3.  An Extracellular Signal–Regulated Kinase 2 Survival Pathway Mediates Resistance of Human Mesothelioma Cells to Asbestos-Induced Injury 
We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)–linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal–regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death–related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag–immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.
doi:10.1165/rcmb.2010-0282OC
PMCID: PMC3262687  PMID: 21454801
mesothelioma; asbestos; toxicity; epidermal growth factor receptor; protein kinase B/AKT
4.  Coadministration of DNA Encoding Interleukin-6 and Hemagglutinin Confers Protection from Influenza Virus Challenge in Mice 
Journal of Virology  1998;72(2):1704-1708.
This study was conducted to investigate whether Accell gene gun coadministration of DNA encoding human interleukin-6 (IL-6) would enhance protective immune responses in mice to an equine influenza A virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone exhibited accelerated clearance of homologous challenge virus but were not protected from infection. In contrast, mice that received both HA and IL-6 DNA had no detectable virus in their lungs after challenge. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.
PMCID: PMC124660  PMID: 9445082
5.  Immunization of Pigs with a Particle-Mediated DNA Vaccine to Influenza A Virus Protects against Challenge with Homologous Virus 
Journal of Virology  1998;72(2):1491-1496.
Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.
PMCID: PMC124630  PMID: 9445052
7.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(311):655-660.
PMCID: PMC2310453  PMID: 20744657
8.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(313):713-715.
PMCID: PMC2310424  PMID: 20744665
9.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(312):688-690.
PMCID: PMC2310412  PMID: 20744663
10.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(310):627-629.
PMCID: PMC2310392  PMID: 20744655
11.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(307):541-544.
PMCID: PMC2310366  PMID: 20744648
12.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(308):567-570.
PMCID: PMC2310344  PMID: 20744652
13.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(305):489-493.
PMCID: PMC2310338  PMID: 20744643
14.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(299):321-323.
PMCID: PMC2310313  PMID: 20744623
15.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(301):373-377.
PMCID: PMC2310298  PMID: 20744632
16.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(300):347-350.
PMCID: PMC2310284  PMID: 20744627
17.  On Diseased Conditions of the Knee-Joint 
British Medical Journal  1866;2(303):427-431.
PMCID: PMC2310243  PMID: 20744636

Results 1-17 (17)