Renal Arterial Smooth Muscle Cells Research Articles

Effect and mechanism of ubiquitin-like protein FAT10 on AngⅡ induced endothelial cell inflammation

Objective: To investigate the role and related mechanism of ubiquitin-like protein FAT10 in the angiotensin Ⅱ (AngⅡ)-induced endothelial cell inflammatory responses. Methods: The Western blot was used to detect the protein expression of FAT10 in 16-weeks old WKY rat carotid artery, thoracic aorta artery, renal artery and vascular smooth muscle cells (VSMC), human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MDA-MB-231). The optimal concentration and stimulation time of AngⅡ on inducing the highest FAT10 in HUVEC were determined. The following plasmids were constructed: control plasmid, overexpression FAT10 plasmid (Flag-FAT10), invalid interference plasmid, and interference FAT10 plasmid (sh-FAT10). These plasmids were then transfected into HUVEC cells and divided into following groups: control group, Flag-FAT10 group, invalid interference group, and sh-FAT10 group. After culturing with 100 nmol/L AngⅡ for 36 h, the control group and the Flag-FAT10 group were treated with reactive oxygen species scavenger N-acetyl-L-cysteine ​​(NAC), the protein expression levels of the inflammatory factor monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were measured. Laser confocal microscopy was used to detect the generation levels of reactive oxygen species in the cells of vrious groups. Results: FAT10 was expressed in carotid artery, thoracic aorta, and renal artery of normal blood pressure rats and expressed in HUVEC, VSMC, MDA-MB-231. The expression level of FAT10 gradually increased in proportion to the increase of the time and concentration of AngⅡ stimulation in HUVEC, and the expression level of FAT10 was the highest when the HUVEC was treated with 100 nmol/L AngⅡ for 36 h (P<0.01). The protein expression level of MCP-1 (P<0.001) and TNF-α (P<0.01) was higher in AngⅡ treated HUVEC with FAT10 overexpression, while the expression level of MCP-1 and TNF-α protein was lower in AngⅡ treated HUVEC with FAT10 knockdown (all P<0.01). The level of intracellular reactive oxygen species (ROS) production was significantly increased with FAT10 overexpression (P<0.001), and the level of ROS was decreased when the expression of FAT10 was interfered (P<0.05). The increased level of MCP-1 and TNF-α proteins in FAT10 overexpressed HUVEC was reversed by NAC (all P<0.05). Conclusion: FAT10 promotes the release of inflammatory factors induced by AngⅡ in endothelial cells by increasing the level of intracellular ROS production.

G protein-coupled estrogen receptor activation improves contractile and diastolic functions in rat renal interlobular artery to protect against renal ischemia reperfusion injury.

This work aimed to investigate whether G protein-coupled estrogen receptor (GPER) can improve the renal interlobular artery vascular function by increasing the NO content, thereby protecting against renal ischemia-reperfusion (IR) injury. This study classified ovariectomised (OVX) female Sprague-Dawley rats into OVX, OVX + IR, OVX + IR + G1 (the GPER agonist G1), OVX + IR + G1+G15 (GPER blocker) and OVX + IR + G1+L-NAME (eNOS blocker) groups. Enzyme-linked immunosorbent assay was performed to detect the estrogen levels in the body and eliminate interference from endogenous estrogens. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) and HE staining, renal function test and Paller scoring were performed to identify the successful model and detect the degree of renal and renal interlobular arteries injury. The in vitro microvascular pressure diameter measurement technique was used to detect the contraction and diastolic activities of the renal interlobular arteries in each group. Immunofluorescence technique was used to observe the localisation and expression levels of GPER and eNOS in renal interlobular arteries. The GPER and eNOS protein expression levels in each group were detected by Western blot. The NO content in the serum of each group was detected by the nitrate reductase method. After OVX, the estrogen level in the body decreased significantly (P < 0.01), and TUNEL staining showed a significant increase in the degree of renal tubular epithelial cell apoptosis in the IR group. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were significantly increased in the IR group (P < 0.01), and the Paller score showed significantly increased kidney damage. When performing drug treatment, the G1 intervention group significantly decreased serum BUN and SCr levels after IR injury (P < 0.01). The Paller score showed significantly decreased the degree of renal injury (P < 0.01). After IR, the renal interlobular artery contraction rate and systolic velocity of blood vessels were significantly decreased (P < 0.01). The G1 intervention group significantly restored contraction rate and systolic velocity of blood vessels (P < 0.01), and G15 and L-NAME partially reversed this effect (P < 0.01). Immunofluorescence technique showed that GPER was expressed in renal interlobular artery smooth muscle and endothelial cells. After IR injury, the GPER protein expression increased, and the eNOS protein expression decreased significantly (P < 0.01). Western blot showed that after IR injury, the GPER protein expression increased, and the eNOS protein expression decreased significantly. After G1 intervention, the GPER content did not change, and the eNOS content increased significantly (P < 0.01). After ischemia and reperfusion, the serum NO content decreased significantly, but it increased after G1 intervention. G15 and L-NAME reversed the effects of G1 to varying degrees (both at P < 0.01). GPER may improve the renal interlobular artery vascular function by increasing the NO content, thereby protecting against renal IR injury.

IL-26 Confers Proinflammatory Properties to Extracellular DNA.

In physiological conditions, self-DNA released by dying cells is not detected by intracellular DNA sensors. In chronic inflammatory disorders, unabated inflammation has been associated with a break in innate immune tolerance to self-DNA. However, extracellular DNA has to complex with DNA-binding molecules to gain access to intracellular DNA sensors. IL-26 is a member of the IL-10 cytokine family, overexpressed in numerous chronic inflammatory diseases, in which biological activity remains unclear. We demonstrate in this study that IL-26 binds to genomic DNA, mitochondrial DNA, and neutrophil extracellular traps, and shuttles them in the cytosol of human myeloid cells. As a consequence, IL-26 allows extracellular DNA to trigger proinflammatory cytokine secretion by monocytes, in a STING- and inflammasome-dependent manner. Supporting these biological properties, IL-10-based modeling predicts two DNA-binding domains, two amphipathic helices, and an in-plane membrane anchor in IL-26, which are structural features of cationic amphipathic cell-penetrating peptides. In line with these properties, patients with active autoantibody-associated vasculitis, a chronic relapsing autoimmune inflammatory disease associated with extensive cell death, exhibit high levels of both circulating IL-26 and IL-26-DNA complexes. Moreover, in patients with crescentic glomerulonephritis, IL-26 is expressed by renal arterial smooth muscle cells and deposits in necrotizing lesions. Accordingly, human primary smooth cells secrete IL-26 in response to proinflammatory cytokines. In conclusion, IL-26 is a unique cationic protein more similar to a soluble pattern recognition receptor than to conventional cytokines. IL-26 expressed in inflammatory lesions confers proinflammatory properties to DNA released by dying cells, setting up a positive amplification loop between extensive cell death and unabated inflammation.

Downregulation of Thromboxane A2 Receptor Occurs Mainly via Nuclear Factor-KappaB Signaling Pathway in Rat Renal Artery.

Thromboxane A2 (TXA2) acts on TXA2 receptors (TP) to regulate renal artery blood flow and subsequently contributes to the pathogenesis of renal ischemia. The present study was designed to examine if nuclear factor-kappaB (NF-κB) signaling pathway is involved in the downregulation of TP receptors in rat renal artery. Rat renal artery segments were organ cultured for 6 or 24 h. Downregulation of TP receptors was monitored using myograph (contractile function), real-time PCR (receptor mRNA), and immunohistochemistry (receptor protein). Specific inhibitors (MG-132 and BMS345541) for NF-κB signaling pathway were used to dissect the underlying molecular mechanisms involved. Compared to fresh (noncultured) segments, organ culture of the renal artery segments for 24 h induced a significant rightward shift of U46619 (TP receptor agonist) contractile response curves (pEC50: 6.89 ± 0.06 versus 6.48 ± 0.04, P < 0.001). This decreased contractile response to U46619 was paralleled with decreased TP receptor mRNA and protein expressions in the renal artery smooth muscle cells. Specific inhibitors (MG-132 and BMS345541) for NF-κB signaling pathway significantly abolished the decreased TP protein expression and receptor-mediated contractions. In conclusion, downregulation of TP receptors in the renal artery smooth muscle cells occurs mainly via the NF-κB signaling pathway.

10 - Hydrogen Peroxide and Redox Control of Hypoxic Pulmonary Vasoconstriction: Identifying the Mitochondrial Redox Oxygen Sensor

The ability of the body to respond to changes in oxygen (O 2 ) is an integral part of both fetal development and adaptation to environmental hypoxia. Oxygen sensing is mediated by the homeostatic oxygen sensing system, which includes structures such as the resistance pulmonary arteries (PA), ductus arteriosus, carotid bodies and neuroepithelial bodies that function together to improve oxygen uptake and delivery. Within the PA, hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation, whereas systemic arteries dilate during hypoxia. It is hypothesized that subunit(s) of the mitochondrial electron transport chain (ETC) serve as a redox-sensitive O 2 sensor, and modulate vascular tone via release of reactive oxygen species (ROS). However, the identity of the specific subunits within the megacomplexes that accounts for O 2 sensing remains elusive. Hence, the ETC composition and function of mitochondria from rat pulmonary and renal artery (RA) smooth muscle cells (SMC) were compared. Relative to RASMC, PASMC exhibit increased Complex I activity under both normoxic and hypoxic conditions. Mass spectrometry analysis and gene expression array have identified seven candidate proteins/subunits within the mitochondrion and electron transport chain that are differentially expressed between PASMC and RASMC, including iron-sulfur subunits of Complexes I and III, and uncoupling proteins UCP2 and UCP3. We also measured hydrogen peroxide (H 2 O 2 ) flux in PASMC during the acute hypoxic response using a pH-sensitive, H 2 O 2 -specific vector under flow conditions, with increased intracellular calcium as a marker of vasoconstriction. Upon exposure to hypoxia, H 2 O 2 levels decrease immediately prior to the increase in intracellular calcium, supporting the theory that a decrease in mitochondrial ROS, and specifically H 2 O 2 , during hypoxia is the cellular signal mediating HPV. Further experiments aim to confirm the identity of the mitochondrial oxygen sensor using gene knockdown/silencing of candidate proteins. Identification of specific components of the oxygen sensing system may identify therapeutic targets for diseases in which oxygen-sensing is disordered, such as pulmonary arterial hypertension, chronic altitude sickness and chronic obstructive pulmonary disease.

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein βγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gβγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

Intracellular Acid-Extruding Regulators and the Effect of Lipopolysaccharide in Cultured Human Renal Artery Smooth Muscle Cells

Homeostasis of the intracellular pH (pHi) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na+-H+ exchanger (NHE) and the Na+-HCO3 − co-transporter (NBC) have been confirmed in many mammalian cells as major acid extruders. However, the role of acid-extruding regulators in human renal artery smooth muscle cells (HRASMCs) remains unclear. It has been demonstrated that lipopolysaccharide (LPS)-induced vascular occlusion is associated with the apoptosis, activating calpain and increased [Ca2+]i that are related to NHE1 activity in endothelia cells. This study determines the acid-extruding mechanisms and the effect of LPS on the resting pHi and active acid extruders in cultured HRASMCs. The mechanism of pHi recovery from intracellular acidosis (induced by NH4Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pHi is 7.19±0.03 and 7.10±0.02 for HEPES- and CO2/HCO3 −- buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na+-coupled HCO3 − transporter i.e. NBC, functionally co-exists to achieve acid-equivalent extrusion; (c) three different isoforms of NBC: NBCn1 (SLC4A7; electroneutral), NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5), are detected in protein/mRNA level; and (d) pHi and NHE protein expression/activity are significantly increased by LPS, in both a dose- and time- dependent manner, but NBCs protein expression is not. In conclusion, it is demonstrated, for the first time, that four pHi acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pHi and NHE in a dose- and time-dependent manner.

TRPM4 channels in smooth muscle function

The melastatin (M) transient receptor potential (TRP) channel TRPM4 is selective for monovalent cations and is activated by high levels of intracellular Ca(2+). TRPM4 is broadly distributed and may be involved in numerous functions, including electrical conduction in the heart, respiratory rhythm, immune response, and secretion of insulin by pancreatic β-cells. The significance of TRPM4 in smooth muscle cell function is reviewed here. Several studies indicate that TRPM4 channels are critically important for pressure-induced cerebral arterial myocyte depolarization and myogenic vasoconstriction as well as autoregulation of cerebral blood flow. Regulation of TRPM4 activity in arterial smooth muscle cells is complex and involves release of Ca(2+) from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and translocation of TRPM4 channels to the plasma membrane in response to protein kinase Cδ. TRPM4 is also present in colonic, urinary bladder, aortic, interlobar pulmonary and renal artery, airway, and corpus cavernosum smooth muscle cells, but its significance and regulation in these tissues is less well characterized.

Angiotensin II Type 1 receptor Regulates BK Channels Independent of G-Protein Activation in Rat Renal Arterial Smooth Muscle Cells

In vascular smooth muscles cells (SMCs),Angiotensin II (Ang II) is known to be involved in numerous biological events, including cell contraction, proliferation, migration, and senescence, through the activation of canonical G-protein-dependent signaling pathways. We previously identified a physical interaction between expressed angiotensin II type 1 receptor (AT1R) and large-conductance calcium-activated potassium (BK) channels. In the present study, we assessed the contribution of AT1R and G-protein activation in Ang II-mediated inhibition of BK currents using losartan, an antagonist of AT1R and GDPβS, an inhibitor of G-protein activation. First, we tested the effectiveness of losartan in preventing AT1R activation in HEK293T cells by measuring ERK1/2 phosphorylation and confirmed that 10 μM losartan could fully inhibit the activation induced by 1 μM Ang II. In freshly isolated rat renal arterial smooth muscle cells (SMCs), extracellular application of 1 μM Ang II reduced whole-cell BK currents by 42.9±6.5% (n=5). Supporting the role of AT1R, BK currents were unaffected after Ang II stimulation in losartan-pretreated SMCs and remained at 99.5±2.8% (n=9) of their original amplitude. The full antagonistic action of losartan was also observed in Ang II pretreated HEK293T cells concurrently expressing AT1R and BK channels (n=6). On the other hand, intracellular application of 500 μM GDPβS via the pipette failed to prevent Ang II-induced inhibition of BK currents supporting a process that is independent of G-protein activation. In summary, the results demonstrate that AT1R mediates the Ang II-induced inhibition of BK currents in renal arterial SMCs, and support the hypothesis that the Ang II inhibitory effect on BK currents is mediated by an intimate interaction between AT1R and BK channels. Supported by NIH.

BK Gene Disruption Enhances Acute Renal Vascular Response to Angiotensin II

Renal vascular resistance is involved in the regulation of blood pressure through its contribution to the overall peripheral resistance and role in salt/fluid balance. Renal vascular tone is determined by the equilibrium between vasoconstrictive and vasodilative forces in renal arterial smooth muscle cells, where Angiotensin II type 1 receptor (AT1R) and BK channels are functionally coupled. In the present study, we investigated the role of BK in the maintenance of arterial blood flow and in its response to Angiotensin II (ANG II) stimulation. To this end, we used BK knockout (BK-/-) mice and ultrasound imaging to measure renal and aortic blood flow. In right renal arteries, peak systolic blood flow velocities (PSV) were similar in wild type, wt (502±122 mm/s, n=5) and BK-/- (482±64 mm/s, n=4) mice. End-diastolic velocities (EDV) also showed no differences (EDVwt=180±125 mm/s vs. EDVBK-/-=181±135 mm/s). Renal arterial resistive indexes (RI=[PSV-EDV]/PSV) appeared to be at the same level (RIwt=0.63±0.11, n=5 vs. RIBK-/-=0.63±0.05, n=4). Likewise, in abdominal aorta there were no significant differences in PSV, EDV, and RI. After administration of ANG II to wt mice (s.c., 500 ng/g body weight), right renal arterial PSV was reduced to 30.9±5.5% (n=6) of its initial value and recovered to 77.7±7.9% after 1 h. Remarkably, in BK-/- (n=3) PSV was reduced to 15.9±0.4%, and only recovered to 37.6±0.4% within the same time frame. Our findings show that loss of BK function does not cause alterations in basal arterial blood flow but critically potentiates the vasoconstrictor action of ANG II in renal arteries. Supported by NIH.

Effect of ranolazine on rat intrarenal arteries in vitro

Ranolazine is mainly used to treat patients with chronic stable angina in clinical practice. However, ranolazine does not lower significantly systemic blood pressure. The direct effect of ranolazine on vascular tone remains unknown. In the present study, we investigated the vascular effects and mechanisms of action of ranolazine in isolated rat intrarenal arteries. Rings of intrarenal arteries were mounted in a small vessel myography using two stainless steel wires for the measurement of isometric tension. L-type Ca2+ currents were recorded in isolated single renal arterial smooth muscle cells using patch clamp techniques in whole-cell mode. Ranolazine induced concentration-dependent relaxations in rings contracted with phenylephrine, but ranolazine failed to cause any relaxation in rings pre-contracted by U46619, 5-HT or endothelin-1. Ranolazine also induced relaxations in norepinephrine pre-contracted rings. Yohimbine failed to induce relaxation in rings pre-contracted by norepinephrine. Propranolol did not affect ranolazine-induced relaxation but the relaxant effect of ranolazine was much less than that of prazosin. Ranolazine-induced relaxations were slight but significantly attenuated by endothelial denudation. Partial inhibition was observed in endothelium-intact arteries exposed to a combination of iberiotoxin and apamin. Ranolazine at higher concentration (>30μM) inhibited Ca2+-induced contraction in a noncompetitive manner. Ranolazine reduced L-type Ca2+ currents at potentials between −30 and 50mV in isolated renal artery myocytes. Therefore it can be said that ranolazine has significant α1-adrenergic receptor and weak calcium channel antagonistic effects in rat intrarenal arteries.

Angiotensin II Type 1 Receptor Mediates Angiotensin II Inhibition on BKCa Channels

The renin-angiotensin system (RAS) is a critical regulator of sodium balance, extracellular fluid volume, vascular resistance, and, ultimately, arterial blood pressure. The key RAS molecule angiotensin II type 1 receptor (AT1R), and another major determinant of vascular tone, the large conductance calcium-activated potassium (BKCa) channel, are both highly expressed in renal arterial smooth muscle cells (SMCs). Our previous studies in expression systems revealed a physical association between AT1R and BKCa, and that AT1R association modified BKCa channel voltage sensitivity. However, the effect of Ang II on BKCa channels in renal arterial SMCs needs to be defined. Furthermore, whether AT1R association is critical for the alteration of channel activity in response to Ang II, and whether the coupling changes BKCa reactivity to specific inhibitors also remain unknown. Our present studies in rat renal arterial SMCs show that application of 100 nM Iberiotoxin (IbTx, a specific BKCa channel blocker) inhibits whole-cell BKCa currents by 63.0±12.5% (n=5). IbTx-sensitive BKCa currents were reduced by 44.4±8.7% (n=3) in response to 1 μM Ang II treatment. In AT1R-IRES-BKCa-transfected HEK293T cells, extracellular application of 1 μM Ang II also suppressed whole-cell BKCa currents by 19.7±0.3% (n=3), while this treatment made no significant changes in cells expressing only BKCa (n=6). IbTx reduced the whole-cell currents by 37.2±1.9% (n=4) in BKCa-transfected HEK293T cells and 37.7±12.5% (n=5) in AT1R-IRES-BKCa-transfected cells. Paxilline treatment (100 nM) produced a 66.8±8.0% (n=3) reduction of BKCa whole-cell currents in BKCa-transfected HEK293T cells and 65.8±9.2% (n=3) reduction in AT1R-IRES-BKCa-transfected cells. These results demonstrate that Ang II inhibits BKCa channel activity in renal arterial SMCs and suggest that AT1R serves as the mediator of the effect. However, the interaction between AT1R and BKCa does not change channel reactivity towards specific channel inhibitors. Supported by NIH.

Mitochondrial cytochrome redox states and respiration in acute pulmonary oxygen sensing

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism to optimise lung gas exchange. We aimed to decipher the proposed oxygen sensing mechanism of mitochondria in HPV. Cytochrome redox state was assessed by remission spectrophotometry in intact lungs and isolated pulmonary artery smooth muscle cells (PASMC). Mitochondrial respiration was quantified by high-resolution respirometry. Alterations were compared with HPV and hypoxia-induced functional and molecular readouts on the cellular level. Aortic and renal arterial smooth muscle cells (ASMC and RASMC, respectively) served as controls. The hypoxia-induced decrease of mitochondrial respiration paralleled HPV in isolated lungs. In PASMC, reduction of respiration and mitochondrial cytochrome c and aa3 (complex IV), but not of cytochrome b (complex III) matched an increase in matrix superoxide levels as well as mitochondrial membrane hyperpolarisation with subsequent cytosolic calcium increase. In contrast to PASMC, RASMC displayed a lower decrease in respiration and no rise in superoxide, membrane potential or intracellular calcium. Pharmacological inhibition of mitochondria revealed analogous kinetics of cytochrome redox state and strength of HPV. Our data suggest inhibition of complex IV as an essential step in mitochondrial oxygen sensing of HPV. Concomitantly, increased superoxide release from complex III and mitochondrial membrane hyperpolarisation may initiate the cytosolic calcium increase underlying HPV.

Novel Imaging Approaches Provide New Windows to View Subcellular Changes in the Redox State of Smooth Muscle Cells

See related articles, pages 526–535 In this issue of Circulation Research , Schumacker and colleagues1 describe the development of novel genetic targeting approaches that record unique information on subcellular changes in the thiol redox state. Ratiometric rhoGFP fluorescence detectors were used to examine hypoxia-induced redox changes in cultured rat pulmonary and systemic (renal) arterial smooth muscle cells. The role and the origins of peroxide contributing to the redox changes were dissected by genetically overexpressing catalase in subcellular compartments. Although the study was designed to provide further insight into the controversial hypothesis that hypoxia promotes contraction of pulmonary arterial smooth muscle by increasing the release of mitochondrial-derived hydrogen peroxide, the methodological approaches used may be viewed as a more important aspect of the work. In particular, the molecular transfection approaches used provide new windows of opportunity to examine the nature of subcellular redox signaling. Overall, the study contained impressive data showing that hypoxia increases the concentration of cytosolic hydrogen peroxide, based on the inhibition of thiol oxidation-dependent fluorescence responses by catalase overexpressed in the cytosol. Hypoxia also increased oxidation in the mitochondrial intermembrane space but, decreased it in the mitochondrial matrix in a manner that was not affected by the overexpression of catalase in the mitochondrial matrix. These results underscore the need …

Differential vasodilation response to olprinone in rabbit renal and common carotid arteries

Olprinone, one of the most frequently used phosphodiesterase-3 inhibitors, exerts its positive inotropic and vasodilation effects by inhibiting the degradation of intracellular cyclic adenosine monophosphate (cAMP). The vasodilation response to olprinone is not uniform among the different vascular beds. This study was designed to compare the vasorelaxation response to olprinone between renal and common carotid arteries, and investigate its underlying mechanisms. Isometric force measurement, enzyme immunoassay, and western blotting techniques were used to investigate the vasorelaxation action of olprinone in isolated rabbit renal and common carotid arteries. Olprinone inhibited the contractile response to phenylephrine (PE) both in the renal and carotid arteries in a concentration-dependent manner with IC50 values of 40 +/- 10 and 103 +/- 43 nM, respectively. The IC50 value was lower (P = 0.004) and the maximal inhibition was greater (P = 0.002) in the renal artery compared with the carotid artery. A cell-permeable cAMP analogue, 8-bromo-cAMP, also inhibited the contractile response to PE in the renal and carotid arteries with IC50 values of 581 +/- 150 and 740 +/- 179 microM, respectively; however no differences were observed both in the IC50 value and the maximal inhibition between two arteries. Olprinone (0.1 microM) increased the intracellular cAMP level in the renal arterial smooth muscle cells (ASMCs) but not in the carotid ASMCs. The expression of PDE3A was greater (P = 0.008) in the carotid ASMCs than the renal ASMCs. The enhanced vasodilator action of olprinone in the renal artery is presumably because of its ability to stimulate the cAMP production, which might be attributable to the heterogeneous expression of PDE3A.

Roles of Angiopoietins in Kidney Development and Disease

Angiopoietins are a family of growth factors, the best studied being angiopoietin 1 (Ang-1), which binds to and tyrosine-phosphorylates endothelial Tie-2, causing enhanced survival and cell-cell stabilization. Ang-2 and Tie-1 downregulate Ang-1-induced Tie-2 signaling, and angiopoietin actions are further modified by vascular endothelial growth factor A and integrins. Metanephric capillaries express Tie genes, whereas metanephric mesenchyme, maturing tubules, and mature podocytes express Ang-1. Ang-1 null embryos begin to form blood vessels, but subsequent vascular remodeling fails, and analyses of chimeric wild-type/Tie null mutant embryos show that Tie genes are needed for renal endothelial survival. Ang-2 is transiently expressed in renal arterial smooth muscle and mesangial cells, and tubules around adult vasa rectae express Ang-2. Ang-2 null mice have increased pericytes around kidney cortical peritubular capillaries, perhaps an indirect consequence of upregulated Tie-2 signaling. Ang-1 therapies attenuate peritubular capillary loss in adult models of tubulointerstitial disease, although, in one study, this was accompanied by enhanced inflammation and fibrosis. Podocyte-directed Ang-2 transgenic overexpression causes glomerular endothelial apoptosis, downregulated nephrin expression, and increased albuminuria, and glomerular Ang-2 is upregulated in hyperglycemic and immune-mediated glomerulopathies. Thus, angiopoietins affect podocyte as well as glomerular endothelial biology, and imbalanced angiopoietin signaling contributes to glomerular pathobiology.

Nebivolol Induces a Hyperpolarizing Effect on Smooth Muscle Cells in the Mouse Renal Artery by Activation of Beta-2-Adrenoceptors

Nebivolol is a highly selective β₁-adrenoceptor antagonist with vasodilator properties involving the vascular endothelium, but its effect on the smooth muscle cells (SMC) is still unclear. In this paper, we tested the effect of nebivolol on renal artery smooth muscle cells and investigated the cellular mechanism involved. To this purpose, the denuded renal arteries isolated from mice were studied in vitro using the myograph and the nitric oxide (NO) sensor techniques, while the SMC in culture were analyzed by the patch-clamp technique. The myograph technique was used to assay the vasodilator effect of nebivolol on the arterial muscular layer, and to establish the optimal dose of the drug to be tested on single SMC by the patch-clamp technique. Using both the myograph and the patch-clamp techniques, we examined the potential contribution of β₂-adrenoceptors and Ca²⁺-activated K⁺ channels to the nebivolol-induced effects, by exposing the denuded arteries and SMC cultures to specific inhibitors such as butoxamine (100 µmol/l), tetraethylammonium (TEA, 1 mmol/l), and iberiotoxin (100 nmol/l). The direct measurement of NO using the NO sensor enabled us to evaluate if nebivolol induces/or not the release of NO in denuded renal arteries. The results of this study show that nebivolol exerts vasodilator effects on the SMC in the denuded renal arteries and the maximal response is achieved at a concentration of 50 µmol/l. Nebivolol effects involve binding to the β₂-adrenoceptors and the subsequent activation of Ca²⁺-activated K⁺ channels in SMC, with no contribution of NO. Taken together, the study brings new insights into the mechanism underlying the nebivolol-induced arterial vasodilation.

Regional Brazilian diet-induced pre-natal malnutrition in rats is correlated with the proliferation of cultured vascular smooth muscle cells

Pre-natal malnutrition induces hypertension and insulin resistance, pathologies commonly linked to atherosclerotic disease. The proliferation of vascular smooth muscle cells (SMCs) is important during development of the atherosclerotic plaque. In this work, we investigated whether the serum of pre-natal malnourished Wistar rats could alter the proliferation of aortic and renal artery SMCs in culture. Malnutrition was induced by feeding a basic regional diet available in a rural area of Pernambuco State, Brazil. This diet was rich in carbohydrates and deficient in proteins, lipids, vitamins and minerals, including sodium chloride. Serum was obtained from the blood of 90-day-old control and pre-natal undernourished rats. SMCs from control Wistar rats at the 6th passage were allowed to adhere to plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal calf serum (10%). Subsequently, the SMCs were maintained in DMEM supplemented with rat serum (10%). The number of cells was counted on the 3rd, 6th and 8th days of culture into rat serum. [3H]-thymidine incorporation into SMCs was evaluated after 20 h or 6 days of incubation. The birth weight of male and female undernourished offspring was 25% (p<0.05) and 46% (p<0.05) lower, respectively, than their corresponding control groups. On the 8th day of culture, the number of aortic SMCs in the serum of undernourished male and female rats, as well as renal artery SMCs in the serum of undernourished female rats, was higher than in the serum of control rats. The [3H]-thymidine incorporation was higher in aortic SMCs incubated for 6 days in the serum of undernourished male and female rats. At confluence, the density of aortic SMCs was higher than that of renal artery SMCs. Pre-natal malnutrition produces serum with altered properties that can affect the proliferation of SMCs and may contribute to atherosclerotic disease.

Evaluation of the nitric oxide production in rat renal artery smooth muscle cells culture exposed to radiocontrast agents

Radiocontrast agents (RC), substances largely used in diagnostic procedures, present the nephrotoxicity as one of its major side effects, which could be due to an altered synthesis of vasodilators. The aim of the present study was to evaluate the nitric oxide (NO) production in rat renal artery smooth muscle cells primary culture (rVSMC) exposed to RC. The cells were treated for 72 hours with mannitol at 10% (MT10; 600 mOsm/kg H2O) or 35% (MT35; 2100 mOsm/kg H2O), with the nonionic iobitridol (IBT), the low-osmolality ioxaglate (IXG), the high-osmolality ioxitalamate (IXT), the nonionic, iso-osmolar iodixanol (IDX), and with lipopolysaccharide (LPS). We determined the NO and osmolality in the cell culture media and the cellular viability. By the Griess and chemiluminescence methods, the NO was not different in MT10 and IDX, but decreased in MT35, IBT, IXG, and IXT when compared with the control; it was increased in LPS and also decreased in all RC+LPS when compared with LPS. MT35, IXT, and IXT+LPS decreased the cellular viability, and the media osmolality was increased in MT35 and IXT compared with the control. The RC (except IDX) significantly reduced NO in rVSMC, which was more pronounced after IXT treatment (57.3%). This was not related to the reduced cell viability (15.8%) or to its high osmolality, because in MT35, with similar osmolality as IXT, NO decreased only 11.0% relatively to the control. Neither the media osmolality nor the cell viability was altered by IXG or IBT. The decreased NO could explain the vasoconstriction and, therefore, the acute renal failure by RC.

Nitric oxide in the kidney.

bastron and kaloyanides([2][1]) had already investigated the effects of sodium nitroprusside in the isolated kidney 8 years before the postulate of an endothelium-derived relaxing factor was made by Furchgott and Zawadzki ([14][2]). More recent studies aim at the importance of nitric oxide (NO) for