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NADPH oxidase 4 mediates flow-induced superoxide production in thick ascending limbs.

ABSTRACT: We previously showed that luminal flow stimulates thick ascending limb (TAL) superoxide (O(2)(-)) production by stretching epithelial cells and increasing NaCl transport, and reported that the major source of flow-induced O(2)(-) is NADPH oxidase (Nox). However, the specific Nox isoform involved is unknown. Of the three isoforms expressed in the kidney-Nox1, Nox2, and Nox4-we hypothesized that Nox4 is responsible for flow-induced O(2)(-) production in TALs. Measurable flow-induced O(2)(-) production at physiological flow rates of 0, 5, 10, and 20 nl/min was 5 ± 1, 9 ± 2, 36 ± 6, and 66 ± 8 AU/s, respectively. RT-PCR detected mRNA for all three Nox isoforms in the TAL. The order of RNA abundance was Nox2 > Nox4 >>> Nox1. Since all three isoforms are expressed in TALs and pharmacological inhibitors are not selective, we used rats transduced with siRNA and knockout mice. Nox4 siRNA knocked down Nox4 mRNA expression by 63 ± 7% but did not reduce Nox1 or Nox2 mRNA. Flow-induced O(2)(-) was 18 ± 9 AU/s in TALs transduced with Nox4 siRNA compared with 77 ± 9 AU/s in tubules transduced with scrambled siRNA. Flow-induced O(2)(-) was 81 ± 5 AU/s in Nox2 knockout mice compared with 83 ± 13 AU/s in wild-type mice. In TALs transduced with Nox1 siRNA, flow-induced O(2)(-) was 82 ± 7 AU/s. We conclude that Nox4 mediates flow-induced O(2)(-) production in TALs.

SUBMITTER: Hong NJ

PROVIDER: S-EPMC3469675 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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NADPH oxidase 4 mediates flow-induced superoxide production in thick ascending limbs.

Hong Nancy J NJ Garvin Jeffrey L JL

American journal of physiology. Renal physiology 20120815 8

We previously showed that luminal flow stimulates thick ascending limb (TAL) superoxide (O(2)(-)) production by stretching epithelial cells and increasing NaCl transport, and reported that the major source of flow-induced O(2)(-) is NADPH oxidase (Nox). However, the specific Nox isoform involved is unknown. Of the three isoforms expressed in the kidney-Nox1, Nox2, and Nox4-we hypothesized that Nox4 is responsible for flow-induced O(2)(-) production in TALs. Measurable flow-induced O(2)(-) produc ...[more]

PMID: 22896039

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Project description:Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.

Project description:Superoxide (O(2) (.-)) may function as a second messenger or regulator of signal transduction when produced at low concentrations in the proper locations within cells. The purpose of these studies was to determine whether human corneal stromal (HCS) fibroblasts are capable of producing O(2) (.-) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, a family of protein complexes believed to be responsible for the localized and limited production of O(2) (.-) with regulatory activity.HCS cells, grown as primary and low-passage cultures of fibroblasts, were used as the sources of RNA for reverse transcriptase PCR, with primers specific for mRNAs encoding the proteins that comprise NADPH oxidases. Small interfering (si)RNAs were used to knockdown specific NOX mRNAs. Proteins composing the NADPH oxidase complexes were identified using western blots. The production of O(2) (.-) by whole cells and cell-free preparations was assessed by measurement of NADPH-dependent superoxide dismutase-inhibitable cytochrome c reduction.Whole cells and cell-free extracts of corneal stromal fibroblasts produced O(2) (.-) in an NADPH-dependent manner. These fibroblasts constitutively produced mRNAs encoding eight proteins known to comprise NADPH oxidase complexes. mRNAs encoding NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac were expressed. Treatment of HCS fibroblasts with siRNA pools specific for each of these three NOXs significantly reduced the steady state levels of the respective mRNAs. Western blots confirmed the existence of all the proteins required for O(2) (.-) production. Rac 1, a regulator of the activity of some forms of NADPH complexes was present in membranous cell fractions containing the oxidase proteins.HCS fibroblasts produced O(2) (.-) in a NADPH-dependent manner via at least three isoforms of NADPH oxidase. These cells expressed NOX1, NOX4, NOX5, p22 phox, p47 phox, p67 phox, and p40 phox as well as Rac. SiRNAs directed against each of the three putative isoforms of NOX significantly reduced the steady state levels of the appropriate NOX mRNA pools, thus confirming the existence of the three isoforms. The O(2) (.-) produced by the NADPH oxidases in HCS fibroblasts is a potential contributor to signal transduction pathways and a regulator of gene expression as well as a potential participant in processes that occur during inflammation.

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NADPH oxidase 4 mediates flow-induced superoxide production in thick ascending limbs.

Publications

NADPH oxidase 4 mediates flow-induced superoxide production in thick ascending limbs.

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