Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin–angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of α actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of α actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Angiotensin II type 2 receptors facilitate reinnervation of phenol-lesioned vascular calcitonin gene-related peptide-containing nerves in rat mesenteric arteries

The present study was designed to investigate involvement of angiotensin II (Ang II) type 2 receptors (AT2 receptors) in restoration of perivascular nerve innervation injured by topical phenol treatment. Male Wistar rats underwent in vivo topical application of 10% phenol around the superior mesenteric artery. After phenol treatment, animals were subjected to immunohistochemistry of the third branch of small arteries, Western blot analysis of AT2 receptor protein expression in dorsal root ganglia (DRG) and studies of mesenteric neurogenic vasoresponsiveness. Ang II (750 ng/kg/day), nerve growth factor (NGF; 20 microg/kg/day) and PD123,319 (AT2 receptor antagonist; 10 mg/kg/day) were intraperitoneally administered for 7 days using osmotic mini-pumps immediately after topical phenol treatment. Losartan (AT1 receptor antagonist) was administered in drinking water (0.025%). Phenol treatment markedly reduced densities of both calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and neuropeptide Y (NPY)-LI-containing fibers. NGF restored densities of both nerve fibers to the sham control level. Coadministration of Ang II and losartan significantly increased the density of CGRP-LI-fibers but not NPY-LI-fibers compared with saline control. The increase of the density of CGRP-LI-fibers by coadministration of Ang II and losartan was suppressed by adding PD123,319. Coadministration of Ang II and losartan ameliorated reduction of CGRP nerve-mediated vasodilation of perfused mesenteric arteries caused by phenol treatment. The AT2 receptor protein expression detected in DRG was markedly increased by NGF. These results suggest that selective stimulation of AT2 receptors by Ang II facilitates reinnervation of mesenteric perivascular CGRP-containing nerves injured by topical phenol application in the rat.     

Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.     

Dual repressive effect of angiotensin II-type 1 receptor blocker telmisartan on angiotensin II-induced and estradiol-induced uterine leiomyoma cell proliferation

BACKGROUND: Although uterine leiomyomas or fibroids are the most common gynecological benign tumor and greatly affect reproductive health and well-being, the pathophysiology and epidemiology of uterine leiomyomas are poorly understood. Elevated blood pressure has an independent, positive association with risk for clinically detected uterine leiomyoma. Angiotensin II (Ang II) is a key biological peptide in the renin-angiotensin system that regulates blood pressure. METHODS: In this study, we investigated the potential role of Ang II (1-1000 nM) in the proliferation of rat ELT-3 leiomyoma cells in vitro. RT-PCR and western blot analysis with cell proliferation and DNA transfection assays were performed to determine the mechanism of action of Ang II. RESULTS: Ang II induced ELT-3 leiomyoma cell proliferation (P < 0.01) and the expression of Ang II type 1 receptor (AT(1)R) and AT(2)R mRNA and protein was confirmed. Regarding the intracellular signaling pathway, the Ang II-induced cell proliferation was AT(1)R-, epidermal growth factor receptor-, extracellular-regulated kinase- and protein kinase C-dependent but was not dependent on the AT(2)R or phosphatidylinositol-3 kinase or JAK kinase. The AT(1)R blocker telmisartan, effectively repressed Ang II-induced and estradiol-induced cell proliferation (P < 0.01). AT(1)R, but not AT(2)R, plays a role in Ang II-induced ELT-3 cell proliferation. CONCLUSIONS: These experimental findings in vitro highlight the potential role of Ang II in the proliferation of leiomyoma cells.     

血管紧张素II及其受体在血管平滑肌细胞迁移中的作用

目的:探讨血管紧张素II(AngII)及其受体(ATRs)在局部血管损伤后血管平滑肌细胞(VSMC)迁移中的作用及其机制。方法:以体外培养VSMC为基础,采用细胞化学和改良Boyden'schamber的方法,观察AngⅡ干预VSMC后AngII受体的表达、VSMC迁移能力的变化、肌动蛋白纤维丝的动态组装变化,并探讨AT₁R拮抗剂、AT₂R拮抗剂对上述观测指标的影响。结果:AngII10^-7mol/L可以刺激VSMC发生迁移,该作用是通过影响VSMC内应力纤维动态组装而实现的;AngII干预VSMC后可使AT₁R表达上调,随着作用时间延长AT₁R表达水平下降。AT₁R拮抗剂可下调AT₁R表达。AngII通过AT₁R的介导发挥其影响VSMC迁移能力的生物学效应。AT₂R对此无明显影响。结论:AngII通过AT₁R介导来调节VSMC内肌动蛋白微丝的动态组装,进而改变VSMC的迁移能力,从而发挥其介导VSMC迁移的生物学效应。     

Dynamic expression of the angiotensin II type 2 receptor and duodenal mucosal alkaline secretion in the Sprague-Dawley rat

Activation of angiotensin II type 2 receptors (AT2R) has been shown to stimulate duodenal mucosal alkaline secretion (DMAS) in Sprague-Dawley rats (S-D). This finding could not be confirmed in another line of S-D, and the present study investigates whether the level of AT2R expression determines the response to the AT2R agonist CGP42112A. DMAS was measured in anaesthetized rats using in situ pH-stat titration. Real-time PCR and Western blot were used to assess AT1R and AT2R RNA and protein expression, respectively. CGP42112A (0.1 microg kg(-1)min(-1) I.V.) elicited a 45% net increase in DMAS in the previous S-D line studied, whereas no change occurred in the new S-D line. Luminal administration of prostaglandin E2 (10(-5) M) increased DMAS similarly in both S-D lines. AT2R protein expression was significantly higher in tissue from the previous line compared to the new line. Individual AT1R to AT2R ratios (RNA and protein) were significantly higher in the new line compared to the previous S-D line. In the new S-D line intravenous infusion of angiotensin II (Ang II; 10 microg kg(-1) h(-1)) over 120 min significantly lowered the duodenal AT1aR to AT2R RNA ratio. Prolonged Ang II infusion over 240 min increased AT2R protein expression and evoked a 42% stimulatory response in DMAS to CGP42112A. The level of local AT2R expression determines the effect of the AT2R agonist CGP42112A on rat duodenal mucosal alkaline secretion. AT2R expression should be confirmed before interpreting the experimental effects of pharmacological interferences with this receptor.     

肾内血管紧张素系统在高血压性肾损害中的作用

目的:探讨血管紧张素II(AngII)及血管紧张素II受体(AT₁R)在缺血性肾损害时肾脏局部的作用。方法:采用放射免疫法及逆转录-聚合酶链反应(RT-PCR)技术,检测双肾动脉狭窄时大鼠血浆和肾脏的AngII含量和AT₁RmRNA的表达。结果:缺血性肾损害时大鼠血浆及肾组织AngII水平均高于对照组,P <0.0 5,而肾组织AT₁RmRNA的表达也较对照组高,P <0.0 1。结论:在双肾动脉狭窄缺血性肾损害时存在AngII及其AT₁R的异常,它们可参与肾脏损害作用.     

Angiotensin II maintains the structure and function of glomerular mesangium via type 1a receptor

 The angiotensin II type 1a (AT1a) receptor is the major receptor effecting the multiple actions of angiotensin II on the cardiovascular system. It is expressed abundantly in the glomerular mesangial cells of the kidney. We investigated glomerular changes in null mutant mice minus the AT1a receptor gene to gain an understanding of the in vivo action of angiotensin II via AT1a on the mesangium. Morphological observations and morphometric analysis revealed that the glomerular volume was greatly increased owing to the expansion of the mesangial area, which contained fluid-filled spaces with a small amount of fibrillar components. The mesangial cells lost contact with each other and with the perimesangial area of the glomerular basement membrane (GBM), so that the glomerular capillary neck was greatly widened. These findings suggest a defect of the anchoring function of mesangial cells resulting from some abnormality in mesangial matrix formation. We conclude that angiotensin II has an important role in the structural and functional maintenance of the mesangium via the AT1a receptor, especially by reinforcing the connection between mesangial cells and GBM via the mesangial matrix. Received: 11 December 1997 / Accepted: 30 March 1998     

A possible anti-inflammatory role of angiotensin II type 2 receptor in immune-mediated glomerulonephritis during type 1 receptor blockade

We previously reported that angiotensin II type 1 receptor (AT1R) blockade attenuates renal inflammation/fibrogenesis in immune-mediated glomerulonephritis via angiotensin II type 2 receptor (AT2R). In the present study, further in vivo experiments revealed that AT2R was expressed in tubular epithelial cells of nephritic kidneys in mice, and feedback activation of the renin-angiotensin system during AT1R blockade significantly reduced p-ERK, but not intranuclear nuclear factor-kappaB, levels via AT2R. This led to reduction in mRNA levels of the proinflammatory mediator monocyte chemoattractant protein-1 and overall interstitial inflammation and subsequent fibrogenesis. Specific blockade of ERK expression in tubular epithelium by anti-sense oligodeoxynucleotides also attenuated interstitial inflammation, mimicking the anti-inflammatory action of AT2R in nephritic kidneys. Alternatively, we succeeded in confirming such an AT(2)R function by demonstrating that AT1R blockade did not confer renoprotection in nephritic, AT2R gene-deficient mice. Additional in vitro experiments revealed that AT2R activation did not affect nuclear factor-kappaB activation by tumor necrosis factor-alpha in cultured tubular epithelial cells, although it inhibited ERK phosphorylation, which reduced monocyte chemoattractant protein-1 mRNA levels. These results suggest that feedback activation of AT2Rs in tubular epithelium of nephritic kidneys plays an important role in attenuating interstitial inflammation.     

Regulation of alveolar fluid clearance and ENaC expression in lung by exogenous angiotensin II

Angiotensin II (Ang II) has been demonstrated as a pro-inflammatory effect in acute lung injury, but studies of the effect of Ang II on the formation of pulmonary edema and alveolar filling remains unclear. Therefore, in this study the regulation of alveolar fluid clearance (AFC) and the expression of epithelial sodium channel (ENaC) by exogenous Ang II was verified. SD rats were anesthetized and were given Ang II with increasing doses (1, 10 and 100 μg/kg per min) via osmotic minipumps, whereas control rats received only saline vehicle. AT1 receptor antagonist ZD7155 (10 mg/kg) and inhibitor of cAMP degeneration rolipram (1 mg/kg) were injected intraperitoneally 30 min before administration of Ang II. The lungs were isolated for measurement of alveolar fluid clearance. The mRNA and protein expression of ENaC were detected by RT-PCR and Western blot. Exposure to higher doses of Ang II reduced AFC in a dose-dependent manner and resulted in a non-coordinate regulation of α-ENaC vs. the regulation of β- and γ-ENaC, however Ang II type 1 (AT1) receptor antagonist ZD7155 prevented the Ang II-induced inhibition of fluid clearance and dysregulation of ENaC expression. In addition, exposure to inhibitor of cAMP degradation rolipram blunted the Ang II-induced inhibition of fluid clearance. These results indicate that through activation of AT(1) receptor, exogenous Ang II promotes pulmonary edema and alveolar filling by inhibition of alveolar fluid clearance via downregulation of cAMP level and dysregulation of ENaC expression.     

Angiotensin II induced contraction of rat and human small intestinal wall musculature in vitro

Background: Angiotensin II (Ang II) is a well‐known activator of smooth muscle in the vasculature but has been little explored with regard to intestinal wall muscular activity. This study investigates pharmacological properties of Ang II and expression of its receptors in small‐intestinal smooth muscle from rats and humans. Methods: Isometric recordings were performed in vitro on small intestinal longitudinal muscle strips. Protein expressions of Ang II typ 1 (AT1R) and typ 2 (AT2R) receptors were assessed by Western blot. Results: Ang II elicited concentration‐dependent contractions of rat jejunal and ileal muscle preparations. The concentration–response curve (rat ileum, EC₅₀: 1.5 ± 0.9 × 10^?8 m ) was shifted to the right by the AT1R receptor antagonist losartan (10^?7 m ) but was unaffected by the AT2R antagonist PD123319 (10^?7 m ) as well as by the adrenolytic guanethidine (3 × 10^?6 m ) and the anticholinergic atropine (10^?6 m ). Human duodenal, jejunal and ileal longitudinal muscle preparations all contracted concentration‐dependently in response to Ang II. The concentration–response curve (human jejunum, EC₅₀: 1.5 ± 0.8 × 10^?8 m ) was shifted to the right by losartan (10^?7 m ) but was unaffected by PD123319 (10^?7 m ). Both AT1R and AT2R were detected in all segments of the rat small intestinal wall musculature, whereas only AT1R was readily detectable in the human samples. Conclusion: Ang II elicits contractions of small‐intestinal longitudinal muscle preparations from the small intestine of rats and man. The pharmacological pattern and protein expression analyses indicate mediation via the AT1R.     

Regulator of G protein signalling 2 ameliorates angiotensin II-induced hypertension in mice

Angiotensin II (Ang II) activates signalling pathways predominantly through the G-protein-coupled Ang II type 1 receptor (AT(1)R). The regulator of G protein signalling 2 (RGS2) is a negative G protein regulator. We hypothesized that RGS2 deletion changes blood pressure regulation by increasing the response to Ang II. To address this issue, we infused Ang II (0.5 mg kg(-1) day(-1)) chronically into conscious RGS2-deleted (RGS2(-/-)) and wild-type (RGS2(+/+)) mice, measured mean arterial blood pressure and heart rate (HR) with telemetry and assessed vasoreactivity and gene expression of AT(1A), AT(1B) and AT(2) receptors. Angiotensin II infusion increased blood pressure more in RGS2(-/-) than in RGS2(+/+) mice, while HR was not different between the groups, indicating a resetting of the baroreceptor reflex. Urinary catecholamine excretion was similar in Ang II-infused RGS2(-/-) and RGS2(+/+) mice, indicating a minor role of sympathetic tone for blood pressure differences. Myogenic tone and vasoreactivity in response to Ang II, endothelin-1 and phenylephrine were increased in isolated renal interlobar arterioles of RGS2(-/-) mice compared with RGS2(+/+) mice. The AT(1A), AT(1B) and AT(2) receptor gene expression was not different between RGS2(-/-) and RGS2(+/+) mice. Our findings suggest that RGS2 deletion promotes Ang II-dependent hypertension primarily through an increase of myogenic tone and vasoreactivity, probably by sensitization of AT(1) receptors.     

Effects of the differential expression of ZO‐1 and ZO‐2 on podocyte structure and function

Glomerular podocytes in the kidney originate from columnar epithelial cells possessing tight junctions. During podocyte differentiation, tight junctions are replaced by slit diaphragms, which are formed between foot processes and function as a blood filtration barrier. Although the expression of most tight junction components is suppressed during podocyte differentiation, several components, including ZO‐1 and ZO‐2, are consistently expressed. We recently showed that podocyte‐specific deletion of ZO‐1 gene impaired slit diaphragm formation, leading to proteinuria and glomerular sclerosis. Here, we address the relevance of ZO‐2, whose sequence is highly similar to ZO‐1, in the maintenance of the structure and function of podocytes. In glomerular development, the spatiotemporal expression of ZO‐2 was similar to that of ZO‐1 until the capillary loop stage. Subsequently, the distribution patterns of ZO‐1 and ZO‐2 diverged at the maturation stage, when slit diaphragms are formed. This divergence could partly rely on the ability of ZO‐2 to interact with the slit diaphragm membrane proteins. Podocyte‐specific deletion of the ZO‐2 gene did not cause overt defects; however, double knockout of ZO‐1 and ZO‐2 genes accelerated the defects observed in ZO‐1 knockout mice. These results suggest that ZO‐2 plays supportive roles in the ZO‐1‐dependent regulation of podocyte filtration barrier.     

Angiotensin II stimulates proliferation of adventitial fibroblasts cultured from rat aortic explants

It has been proposed that the local renin-angiotensin system is activated in the adventitia after vascular injury. However, the physiological role of Angiotensin II (Ang II) in the adventitia has not been studied at a cellular level. This study was designed to assess the role of Ang II in the growth response of cultured adventitial fibroblasts (AFs). Adventitial explants of the rat thoracic aorta showed outgrowth of AFs within 5-7 days. Ang II caused hyperplastic response of AF cultures. The Ang II-induced mitogenic response of AFs was mediated primarily by the AT1 receptor. Ang II caused a rapid induction of immediate early genes (c-fos, c-myc and jun B). Induction of c-fos expression was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. Epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) induced DNA synthesis in AFs. Co-stimulation of AFs with the growth factors and Ang II potentiated the incorporation of 3H-thymidine into DNA. Results from this study indicate that Ang II causes mitogenesis of AFs via AT1 receptor stimulation and potentiates the responses to other mitogens. These data suggest that the Ang II may play an important role in regulating AF function during vascular remodeling following arterial injury.     

Nephrin promotes cell-cell adhesion through homophilic interactions

Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+)concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction.