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      Apocynin-Treatment Reverses Hyperoxaluria Induced Changes in NADPH Oxidase System Expression in Rat Kidneys: A Transcriptional Study

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          Abstract

          Purpose

          We have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition.

          Approach

          Age-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H 2O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes.

          Results

          Genes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with p21rac and Rap1a, were coordinately up-regulated significantly in both renal medulla and cortex tissues in the HLP-fed rats compared to normal healthy untreated controls. Activation of NADPH oxidase appears to occur via the angiotensin-II/angiotensin-II receptor-2 pathway, although the DAG-PKC pathway of neutrophils may also contribute. Immuno histochemical staining confirmed up-regulated gene expressions. Simultaneously, genes encoding ROS scavenger proteins were down-regulated. HLP-fed rats receiving Apocynin had a complete reversal in the differential-expression of the NADPH oxidase system genes, despite showing similar levels of hyperoxaluria.

          Conclusions

          A strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.

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          Most cited references58

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          Identification of renox, an NAD(P)H oxidase in kidney.

          Oxygen sensing is essential for homeostasis in all aerobic organisms, but its mechanism is poorly understood. Data suggest that a phagocytic-like NAD(P)H oxidase producing reactive oxygen species serves as a primary sensor for oxygen. We have characterized a source of superoxide anions in the kidney that we refer to as a renal NAD(P)H oxidase or Renox. Renox is homologous to gp91(phox) (91-kDa subunit of the phagocyte oxidase), the electron-transporting subunit of phagocytic NADPH oxidase, and contains all of the structural motifs considered essential for binding of heme, flavin, and nucleotide. In situ RNA hybridization revealed that renox is highly expressed at the site of erythropoietin production in the renal cortex, showing the greatest accumulation of renox mRNA in proximal convoluted tubule epithelial cells. NIH 3T3 fibroblasts overexpressing transfected Renox show increased production of superoxide and develop signs of cellular senescence. Our data suggest that Renox, as a renal source of reactive oxygen species, is a likely candidate for the oxygen sensor function regulating oxygen-dependent gene expression and may also have a role in the development of inflammatory processes in the kidney.
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            Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5.

            gp91phox is the catalytic subunit of the respiratory burst oxidase, an NADPH-dependent, superoxide generating enzyme present in phagocytes. In phagocytes, the enzyme functions in host defense, but reactive oxygen generation has also been described in a variety of non-phagocytic cells, including cancer cells. We previously reported the cloning of Nox1 (NADPH oxidase1), a homolog of gp91phox, its expression in colon and vascular smooth muscle, and its oncogenic properties when overexpressed [Suh et al. (1999). Nature 401, 79-82]. Herein, we report the cloning and tissue expression of three additional homologs of gp91phox, termed Nox3, Nox4 and Nox5, members of a growing family of gp91phox homologs. All are predicted to encode proteins of around 65 kDa, and like gp91phox, all show 5-6 conserved predicted transmembrane alpha-helices containing putative heme binding regions as well as a flavoprotein homology domain containing predicted binding sites for both FAD and NADPH. Nox3 is expressed primarily in fetal tissues, and Nox4 is expressed in not only fetal tissues, but also kidney, placenta and glioblastoma cells. Nox5 is expressed in a variety of fetal tissues as well as in adult spleen and uterus. Nox isoforms are aberrantly expressed in several cells derived from human cancers, with Nox4 being the isoform most frequently expressed in the tumor cells investigated. Thus, expression of Nox family members is likely to account for some of the reactive oxygen generation seen in non-phagocytic cells.
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              Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells.

              Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91phox, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                16 October 2012
                : 7
                : 10
                : e47738
                Affiliations
                [1 ]Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, United States of America
                [2 ]Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida, United States of America
                [3 ]Department of Infectious Diseases and Pathology, University of Florida College of Veterinary Medicine, Gainesville, Florida, United States of America
                Emory University, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SRK ABP. Performed the experiments: SJ BTS WW. Analyzed the data: SJ ABP. Contributed reagents/materials/analysis tools: SJ BTS WW ABP. Wrote the paper: SJ ABP SRK. Prepared figures: SJ ABP.

                Article
                PONE-D-12-22095
                10.1371/journal.pone.0047738
                3473023
                23091645
                f5083f02-67b1-4e10-b485-b7095a33ed1e
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 July 2012
                : 14 September 2012
                Page count
                Pages: 11
                Funding
                Supported by National Institutes of Health grant #RO1-DK078602 and University of Florida Center for the Study of Lithiasis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Model Organisms
                Animal Models
                Rat
                Medicine
                Metabolic Disorders
                Nephrology
                Bladder and Ureteric Disorders
                Chronic Kidney Disease
                Hypertension
                Mineral Metabolism and the Kidney
                Tubulointerstitial Disease
                Urology
                Endourology
                Kidney Stones

                Uncategorized
                Uncategorized

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