NADPH oxidase 4 mediates ROS production in radiation-induced senescent cells and promotes migration of inflammatory cells

Excessive DNA damage induced by ionising radiation (IR) to normal tissue cells is known to trigger cellular senescence, a process termed stress-induced premature senescence (SIPS). SIPS is often accompanied by the production of reactive oxygen species (ROS), and this is reported to be important for the initiation and maintenance of SIPS. However, the source of ROS during SIPS after IR and their significance in radiation-induced normal tissue damage remain elusive. In the present study, we tested the hypothesis that the NADPH oxidase (NOX) family of proteins mediates ROS production in SIPS-induced cells after IR and plays a role in SIPS-associated biological events. X-irradiation of primary mouse embryonic fibroblasts (MEFs) resulted in cellular senescence and the concomitant increase of intracellular ROS. Among all six murine NOX isoforms (NOX1–4 and DUOX1/2), only NOX4 was detectable under basal conditions and was upregulated following IR. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. Meanwhile, NOX4 deficiency did not affect the induction of cellular senescence after IR. Furthermore, the migration of human monocytic U937 cells to the culture medium collected from irradiated MEFs was significantly reduced by NOX4 inhibition, suggesting that NOX4 promotes the recruitment of inflammatory cells. Collectively, our findings imply that NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

NADPH氧化酶与ROS信号区域化

最近有关活性氧物质 (ROS)的研究取得了突飞猛进的进展,尤其是其作为第二信使介导了许多生理性与病理性细胞事件,包括细胞分化、过度生长、增殖及凋亡.为了避免ROS的毒性产生特异性的信号转导,ROS的产生与代谢必须被严格调控;其具体的调控机制一直是人们关注的焦点. 最近有关ROS区域化观点的提出解决了这一问题. NADPH是生成ROS的主要来源. 研究发现,NADPH氧化酶及其衍生的ROS存在于机体的多种组织内,且在细胞中呈区域化分布,对细胞内信号的精确调控具有至关重要的作用. NADPH一方面通过小窝/脂筏组装成功能型复合物,从而产生ROS区域化;另一方面,NADPH通过其不同亚细胞定位亚基与各种靶蛋白之间的相互作用,产生ROS特异性. 本文系统综述了NADPH衍生的ROS信号区域化,为进一步理解ROS信号在各种生理或病理过程的分子调控机制提供理论依据.     

A novel danshensu derivative ameliorates experimental colitis by modulating NADPH oxidase 4‐dependent NLRP3 inflammasome activation

We have previously reported a novel compound [4‐(2‐acetoxy‐3‐((R)‐3‐(benzylthio)‐1‐methoxy‐1‐oxopropan‐2‐ylamino)‐3‐oxopropyl)‐1,2‐phenylene diacetate (DSC)], derived from danshensu, exhibits cytoprotective activities in vitro. Here, we investigated the effects and underlying mechanisms of DSC on dextran sodium sulphate (DSS)‐induced experimental colitis. We found that DSC treatment afforded significant protection against the development of colitis, evidencing by suppressed inflammatory responses and enhanced barrier integrity. Intriguingly, DSC specifically down‐regulated DSS‐induced colonic NADPH oxidase 4 (Nox4) expression, accompanied by a balanced redox status, suppressed nuclear factor‐κB (NF‐κB) and NLRP3 inflammasome activation and up‐regulated nuclear factor (erythroid‐derived 2)‐like 2 and haeme oxygenase‐1 expression. In vitro study also demonstrated DSC also markedly decreased Nox4 expression and activity associated with inhibiting reactive oxygen species generation, NF‐κB activation and NLRP3 inflammasome activation in bone marrow‐derived macrophages. Either lentiviral Nox4 shRNA‐mediated Nox4 knockdown or Nox4‐specific small‐interfering RNA mimicked effects of DSC by suppressing NLPR3 inflammasome activation to alleviate experimental colitis or inflammatory macrophage response. Collectively, our results provide the first evidence that DSC ameliorates experimental colitis partly through modulating Nox4‐mediated NLRP3 inflammasome activation.     

Brain-derived neurotrophic factor can act as a pronecrotic factor through transcriptional and translational activation of NADPH oxidase

Several lines of evidence suggest that neurotrophins (NTs) potentiate or cause neuronal injury under various pathological conditions. Since NTs enhance survival and differentiation of cultured neurons in serum or defined media containing antioxidants, we set out experiments to delineate the patterns and underlying mechanisms of brain-derived neurotrophic factor (BDNF)-induced neuronal injury in mixed cortical cell cultures containing glia and neurons in serum-free media without antioxidants, where the three major routes of neuronal cell death, oxidative stress, excitotoxicity, and apoptosis, have been extensively studied. Rat cortical cell cultures, after prolonged exposure to NTs, underwent widespread neuronal necrosis. BDNF-induced neuronal necrosis was accompanied by reactive oxygen species (ROS) production and was dependent on the macromolecular synthesis. cDNA microarray analysis revealed that BDNF increased the expression of cytochrome b558, the plasma membrane-spanning subunit of NADPH oxidase. The expression and activation of NADPH oxidase were increased after exposure to BDNF. The selective inhibitors of NADPH oxidase prevented BDNF-induced ROS production and neuronal death without blocking antiapoptosis action of BDNF. The present study suggests that BDNF-induced expression and activation of NADPH oxidase cause oxidative neuronal necrosis and that the neurotrophic effects of NTs can be maximized under blockade of the pronecrotic action.     

NADPH oxidase produces reactive oxygen species and maintains survival of rat astrocytes

Reactive oxygen species (ROS) produced by activated astrocytes have been considered to be involved in the pathogenesis of neurodegenerative diseases, while NADPH oxidase is an essential enzyme involved in ROS-mediated signal transduction. The goal of the present study was to determine whether NADPH oxidase plays a role in ROS generation and cell survival in rat astrocytes. We found that the release of ROS in rat astrocytes was significantly increased by stimulation with calcium ionophore or opsonized zymosan, which are known to trigger a respiration burst in phagocytes by the NADPH oxidase pathway. Further study indicated that diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, significantly suppressed the increase of ROS release caused by the calcium ionophore or opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI dose- and time-dependently decreased the viability of normal astrocytes, whereas exogenous supplementation of H2O2 can reverse the survival of DPI-treated astrocytes. For the first time, our results suggest that NADPH oxidase is an important enzyme for the generation of ROS in astrocytes, and the ROS generated by NADPH oxidase play an essential role in astrocyte survival.     

Lipid microdomain polarization is required for NADPH oxidase-dependent ROS signaling in Picea meyeri pollen tube tip growth

The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca²⁺ gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.     

Evasion of Legionella pneumophila from the bactericidal system by reactive oxygen species (ROS) in macrophages

We demonstrated that the production of reactive oxygen species (ROS) by U937 macrophage-like cells was suppressed upon infection with a wild type Legionella pneumophila strain, whereas such suppression was not observed in the case of infection with intracellular growth-deficient mutants. This was supported not only by measuring ROS released into the supernatants of cell cultures by chemiluminescence assaying but also by detecting intracellular ROS with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), under a confocal laser scanning microscope. Furthermore, more than 60% of the phagosomes containing intracellular growth-deficient mutants were colocalized with p47(phox), which is the cytosolic subunit of NADPH oxidase, consistently throughout the observation period in an early stage of bacterial infection. In contrast, the colocalization of p47(phox) was suppressed after infection with the wild type strain. These results suggest that the interference with ROS production by U937 cells infected with wild type L. pneumophila is due to a failure of NADPH oxidase activation through inhibition of p47(phox) recruitment to phagosomes harboring bacteria. The results also highlighted the difference in the nature of phagosomes between ones harboring the wild type and ones the intracellular growth-deficient strains.     

SUMO1 attenuates stress-induced ROS generation by inhibiting NADPH oxidase 2

Small ubiquitin-like modifier 1 (SUMO1) is a member of the superfamily of ubiquitin-like proteins. Despite its structural similarity with ubiquitin, SUMO1 does not seem to play any role in protein degradation and its precise biological function is poorly understood. During our studies on heat-shock responses, we found that heat-shock stress increased SUMO1 conjugation in a dose-dependent manner. Intriguingly, SUMO1 conjugation resulted in decrease of intracellular ROS generation and protection cells from death under heat-shock stress. We showed that NADPH oxidase 2 (NOX2) is a target protein of sumoylation by SUMO1 using immunoprecipitation and is colocalized with SUMO1 at plasma membrane. Additionally, we demonstrated that the attenuation in intracellular ROS generation resulted from inhibition of NADPH oxidase complex (NOX) activity. These results suggested that SUMO1 plays an important role in modulation of NOX activity required for ROS generation.     

Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk desensitizes EGF-induced ROS generation

Accumulating evidence indicates that protein phosphorylation regulates Nox activity. In this report, we show that serine282 residue of Nox activator 1 (NoxA1) is phosphorylated by Erk in response to EGF resulting in desensitization of Nox1 activity. Specifically, murine NoxA1 is detected as two independent protein bands in SDS PAGE, and the form of protein with higher mobility shifted to and merged with the one with lower mobility in response to EGF treatment. Pretreatment with PD98059 resulted in inhibition of NoxA1 migration in response to EGF indicating that Erk was involved in the process. Site-directed mutagenesis showed that S282A mutant but not S239A mutant failed to respond to EGF, demonstrating that serine282 is the target amino acid of Erk. Expression of S282A mutant of NoxA1 in these cells led to increased superoxide anion production in response to EGF compared to expression of the wild type, whereas the expression of S282E, a phosphomimetic mutant, resulted in significantly decreased superoxide anion generation. We also tested whether the phosphorylation of serine282 of NoxA1 affects Rac activation. Expression of S282A mutant NoxA1 up-regulated the Rac activity, whereas expression of S282E mutant led to the abrogation of Rac activation. Taken together, these results demonstrate that phosphorylation of NoxA1 is a part of the feedback mechanism that functions through activation of Rac with a net outcome of negative modulation of Nox1 activity.     

Engineering stability in NADPH oxidases: A common strategy for enzyme production

NADPH oxidases (NOXs) are membrane enzymes whose sole function is the generation of reactive oxygen species. Humans have seven NOX isoenzymes that feature distinct functions in immune response and cell signaling but share the same catalytic core comprising a FAD-binding dehydrogenase domain and a heme-binding transmembrane domain. We previously described a mutation that stabilizes the dehydrogenase domain of a prokaryotic homolog of human NOX5. The thermostable mutant exhibited a large 19?°C increase in the apparent melting temperature (app T_m) and a much tighter binding of the FAD cofactor, which allowed the crystallization and structure determination of the domain holo-form. Here, we analyze the transferability of this mutation onto prokaryotic and eukaryotic full-length NOX enzymes. We found that the mutation exerts a significative stabilizing effect on the full-length NOX5 from both Cylindrospermum stagnale (app T_m increase of 8?°C) and Homo sapiens (app ΔT_m of 2?°C). Enhanced thermal stability resulted in more homogeneous preparations of the bacterial NOX5 with less aggregation problems. Moreover, we also found that the mutation increases the overall expression of recombinant human NOX4 and NOX5 in mammalian cells. Such a 2–5-fold increase is mainly due to the lowered cell toxicity, which leads to higher biomasses. Because of the high sequence identity of the catalytic core within this family of enzymes, this strategy can be a general tool to boost the production of all NOXs.     

Essential role of Rac1/NADPH oxidase in nerve growth factor induction of TRPV1 expression

Nerve growth factor (NGF) regulates the nociceptive properties of a subset of small diameter sensory neurons by increasing the expression of the heat-sensing transient receptor potential (TRP) channel, TRPV1. This action involves activation of the tyrosine kinase receptor (Trk) A/p38 MAPK pathway. Recent studies indicate that activation of TrkA promotes superoxide generation via NADPH oxidase. In this study, we determined whether the NADPH oxidase pathway is involved in NGF-stimulated TRPV1 expression using a rat pheochromocytoma 12 line and rat dorsal root ganglion neurons. Treatment of these cells with NGF (100 ng/mL) increased TRPV1 protein expression (approx. twofold) but not mRNA. This increase was mimicked by H(2)O(2) and attenuated by catalase and inhibitors of NADPH oxidase. NGF stimulated NADPH oxidase activity, while 24 h exposure further increased expression of the Rac1 and gp91(phox) subunits of the holoenzyme. Inhibition of NADPH oxidase by transient transfection of a dominant negative Rac1 mutant (RacN17) plasmid blocked NGF-stimulated TRPV1 protein expression, while expression of a constitutively active Rac1 increased basal and NGF-stimulated TRPV1 levels. Inhibition of NADPH oxidase activity also attenuated NGF-dependent p38 MAPK activation. We conclude that the Rac1/NADPH oxidase pathway regulates p38 activation and TRPV1 expression which aids in the maintenance of peripheral neuron integrity and pain perception.     

Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a d‐[ ³ H] aspartate (glutamate) release assay to explore the ROS‐dependent regulation of glutamate‐permeable volume‐regulated anion channels (VRACs). Exposure of rat microglia to hypo‐osmotic media stimulated Cl^? currents and d ‐[³H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H₂O₂ potently increased swelling‐activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan‐stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin‐insensitive NOX4. Using RT‐PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.     

NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a collection of diseases in which rod photoreceptors die from a variety of mutations. After rods die, the level of tissue oxygen in the outer retina becomes elevated and there is progressive oxidative damage to cones that ultimately triggers apoptosis. In this study, we investigated the hypothesis that NADPH oxidase (Nox) and/or xanthine oxidase serve as critical intermediaries between increased tissue oxygen and the generation of excessive reactive oxygen species that cause oxidative damage to cones. Apocynin, a blocker of Nox, but not allopurinol, a blocker of xanthine oxidase, markedly reduced the superoxide radicals visualized by hydroethidine in the outer retina in the retinal degeneration-1 ( rd1 ^+/+ ) model of RP. Compared to rd1 ^+/+ mice treated with vehicle, those treated with apocynin, but not those treated with allopurinol, had significantly less oxidative damage in the retina measured by ELISA for carbonyl adducts. Apocynin-treated, but not allopurinol-treated, rd1 ^+/+ mice had preservation of cone cell density, increased mRNA levels for m- and s-cone opsin, and increased mean photopic b-wave amplitude. In Q344ter mice, a model of dominant RP in which mutant rhodopsin is expressed, apocynin treatment preserved photopic electroretinogram b-wave amplitude compared to vehicle-treated controls. These data indicate that Nox, but not xanthine oxidase, plays a critical role in generation of the oxidative stress that leads to cone cell death in RP and inhibition of Nox provides a new treatment strategy.     

质膜上的活性氧制造者--NOX家族

NADPH氧化酶特异存在于吞噬细胞质膜,能生成用于清除病原微生物的活性氧（reactive oxygen species,ROS）。NOX是NADPH氧化酶催化亚基gp91^phox的同源物,存在于多种非吞噬细胞。目前发现的NOX有NOX1、NOX3、NOX4及NOX5,虽然它们有一定的组织特异性,但与NADPH氧化酶一样均有催化生成ROS的能力。与吞噬细胞中NADPH氧化酶所制造的ROS不同,NOX所产生的ROS并不主要起细胞防御功能,而是作为第二信使,参与细胞增殖、分化、凋亡的调节。此外,NOX对血管生成及骨吸收也有一定的影响,同时还可作为氧感受器调节促红细胞生成素（EPO）的产生。     

NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro

Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1α and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1α and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling.     

RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.     

NADPH氧化酶在电磁辐射生物效应中的作用

电磁辐射是一种复合电磁波,而人体生命活动包含一系列的生物电活动,这些生物电对环境的电磁波敏感,因此,电磁辐射可对人体造成危害.关于电磁辐射诱导的生物效应研究虽多,然而其具体机制尚不清楚.近年来,发现烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH氧化酶),又称呼吸爆发氧化酶(respiratory burst oxidase homologue,Rboh)在电磁辐射产生的生物学效应中扮演重要角色,电磁辐射可直接或间接活化NADPH氧化酶复合体,然后NADPH氧化酶可以将细胞内NADPH的电子转移而形成活性氧,或者通过一系列炎症因子和相关基质金属蛋白酶等参与炎症、防御以及组织修复等生命过程.本文对NADPH氧化酶在电磁生物学效应中的作用进行了综述.     

NADPH氧化酶抑制剂apocynin对力竭运动大鼠运动性蛋白尿的影响

目的：研究NADPH氧化酶抑制剂apocynin对力竭运动大鼠运动性蛋白尿产生的影响及其机制。方法：32只SD雄性大鼠随机分为安静对照组（C组）、对照+药物组（CA组）、力竭运动组（E组）、力竭运动+药物组（EA组）。药物注射按10 mg/kg体重,每天一次,连续3 d,并在末次药物注射1 h后进行一次性跑台力竭运动。测定运动后尿UP、血液BUN水平、肾脏ROS浓度、NOS活性、NOS与3-NT蛋白含量。结果：结果显示,E组UP、肾脏ROS、iNOS含量及活性、3-NT明显升高,而EA组的这些指标与C组相比无显著性差异。结论：力竭运动可明显增加肾组织NADPH氧化酶活性,从而产生大量的ROS,后者可迅速地与由肾脏iNOS催化生成的NO反应,产生过量的ONOO^-,诱发运动性蛋白尿的生成。