| IntroductionActivation of the intrarenal renin-angiotensin system(RAS) is a characteristic feature in the development and progression of chronic kidney disease.AngiotensinⅡ(AngⅡ),the main effector of RAS,is implicated in the pathogenesis of renal diseases. AngⅡinitiates its effects by interaction with at lease two pharmacologically distinct subtypes of cell-surface receptors,AT1 and AT2,The major functions of AngⅡin cardiovascular system are mediated through AT1 receptor.An intriguing concept has emerged that AngⅡnot only mediates intraglomerular hypertension but also behaves as a pro-inflammatory and growth-stimulating factor contributing to the renal hypertension and sclerosis.AngⅡhas growth-stimulating properties on different renal cell types.However,possible inflammatory effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvascularture have not formally been investigated.Glomerular endothelial cells(GECs),being exposed to the bloodstream,are the target of many different factors,that may alter the functional state of these cells and induce them to release inflammatory cytokines.Inflammatory processes involve both the synthesis of inflammatory cytokines, such as monocyte chemoattractant protein-1(MCP-1),and the activation of their distinct signaling cascades.Recent findings suggest that AngⅡactivates intracellular signaling processes,including the polyol pathway and generation of reactive oxygen species(ROS).In cellular systems,a major source of ROS derives from the membrane-bound NAD(P)H oxidase system.Activation of NAD(P)H oxidase system requires the participation of p47phox,one of the NAD(P)H oxidase subunits,which plays a central role in the scenario of NAD(P)H oxidase activation.10Activation of nuclear factor -ΚB(NF-ΚB) is involved in the expression of pro-inflammatory genes.11 In this study,we investigated wether AngⅡactivates the NF-ΚB cascade by ROS and whether the ROS-dependent mechanism may be involved in AngⅡ-induced MCP-1 formation.Further,we tested whether telmisartan,an AT1 receptor blocker,and DPI,an inhibitor of NAD(P)H oxidase,modulate endothelial inflammation and oxidative cell damage induced by AngⅡ-dependent stimuli in cultured rat glomerular endothelial cells.MethodsWe isolated and characterized primary cultures in rat glomerular endothelial cells(GECs).We found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B (NF-ΚB).Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AngⅡon rat GECs.We study the effect of AngⅡon the propagation and apoptosis of RGECs by the methods of MTT and AV/PI.ResultsWe found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B(NF-ΚB).And the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AugⅡon rat GECs.DiscussionThe present study demonstrates that AngⅡ-induced ROS generation in rat GECs depends on the P47phox subunit of NADPH Oxidase.ROS are additionally required for AngⅡ-induced activation of NF-ΚB.Blockade of the NADPH Oxidase by its inhibitor DPI significantly abolished AngⅡ-induced MCP-1 formation,indicating that NF-ΚB when activated by ROS,participates in the AngⅡ-induced MCP-1 production.It's now well accepted that AngⅡmay function as a potent pro-inflammatory mediator and be implicated in the pathogenesis of chronic renal disease.13,14GECs are at the interface between blood and adjacent cell population and play a crucial role in preserving the kidney function.The monocyte-endothelium interaction induced by AngⅡmay contribute to the initiation of vascular inflammation.15 MCP-1,when expressed at the plasma membrane of endothelial cells,can mediate the initial capture of monocytes.16 Our results suggest that AngⅡcan induce MCP-1 formation in rat GECs and promote the monocyte adhesion to endothelial cells.AngⅡacts via AT1 and AT2 receptors.Both receptor subtypes have been found in rat GECs.17 AngⅡexerts most of its already well-defined physiologic and pathophysiologic actions through AT1 receptors.18 Consistent with this concept,our results showed that the AngⅡeffect on MCP-1 formation was inhibited by the selective AT1 receptor antagonist telmisartan.In mammalian cells,a major source of ROS derives from the membrane-bound NADPH Oxidase system,which exists in nonphagocytic cells of the vascular wall,for example,fibroblast,vascular smooth muscle cells and endothelial cells.19 Increased ROS generation can induce cell inflammation.20,21 The present study isolated rat glomerular endothelial cells and demonstrated that the expression of p47phox protein, one of the NADPH Oxidase subunits,was up-regulated by AngⅡtreatment in accordance with the increased ROS generation.Moreover,blockade of NADPH Oxidase by DPI reduced the P47phox protein synthesis and ROS generation.Nuclear factor-ΚB normally exists in the cytoplasm in an inactive form bound to the inhibitory protein IΚB.Upon cellular activation,IΚB is rapidly degraded prior to the translocation of NF-ΚB into the nucleus and its subsequent activation,resulting in the transcriptional regulation of target genes encoding pro-inflammatory cytokines.NF-ΚB consists of two subunits,p50 and p65,with p65 containing a transcription domain.11,22 We here studied the possible involvement of NF-ΚB activation in AngⅡ-induced MCP-1 production in rat GECs.In the present study,AngⅡstimulation increased the translocation of NF-ΚB P65 subunit to the nucleus,which was reduced by DPI.The observation suggests that AngⅡ-induced NF-ΚB activation is partially mediated by ROS.In summary,we demonstrated that AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.Our finding may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with the chronic renal disease.Conclusions1 AngⅡincreases the expression of MCP- 1 and AT1mRNA;2 AngⅡincreases the expression of p47phox in a certain ranges showing a time-dependent manner.3 AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.4 the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.
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