Rat Renal Interlobar Arteries Research Articles

Autoimmune activation and hypersensitization of the AT1 and ETA receptors contributes to vascular injury in scleroderma renal crisis.

Scleroderma renal crisis (SRC) is a rare vascular complication of systemic sclerosis with substantial risks for end-stage renal disease and premature death. Activating autoantibodies (Abs) targeting the angiotensin II type 1 (AT1R) and the endothelin-1 type A receptor (ETAR) have been identified as predictors for SRC. Here, we sought to determine their pathogenic significance for acute renal vascular injury potentially triggering kidney failure and malignant hypertension. IgG from patients with SRC was studied for AT1R and ETAR dependent biologic effects on isolated rat renal interlobar arteries and vascular cells including contraction, signalling and mechanisms of receptor activation. In myography experiments, patient IgG exerted vasoconstriction sensitive to inhibition of AT1R and ETAR. This relied on MEK-ERK signalling indicating functional relevance of anti-AT1R and anti-ETAR Abs. The contractile response to angiotensin II and endothelin-1 was amplified by patient IgG containing anti-AT1R and anti-ETAR Abs with substantial crosstalk between both receptors implicating autoimmune receptor hypersensitization. Co-immunoprecipitation experiments indicated heterodimerization between both receptor types which may enable the observed functional interrelation by direct structural interactions. We provide experimental evidence that agonistic Abs may contribute to SRC. This effect is presumably related to direct receptor stimulation and additional allosteric effects, at least in heterodimeric receptor constellations. Novel therapies targeted at autoimmune hyperactivation of AT1R and ETAR might improve outcomes in severe cases of SRC.

Hypoxia/Reoxygenation of Rat Renal Arteries Impairs Vasorelaxation via Modulation of Endothelium-Independent sGC/cGMP/PKG Signaling.

Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.

The impaired vasodilatation in rat renal interlobar arteries after hypoxia/re‐oxygenation is endothelium independent

Renal vasculature plays an important role in renal ischemia/reperfusion injury. We showed recently that hypoxia/re‐oxygenation combined with low dose norepinephrine treatment increases the angiotensin II responses in interlobar arteries. Since angiotensin II activates constrictor and dilator systems we hypothesized an impaired vasodilatation in this context.To test the hypothesis rat interlobar arteries were studied by using wire myography. The dilatory capacity was investigated by application of acetylcholine (10‐9 to 10‐5 mol/l). Hypoxia (95% N2, 5% CO2 for 60 min) alone followed by re‐oxygenation (95% O2, 5% CO2, 10 min) did not affect vasodilatation. However, norepinephrine in a non‐constrictor dose (10‐9 mol/l) applied during hypoxia resulted in a decreased response to acetylcholine. The contribution of the nitric oxide system, COX‐products, and endothelial derived hyperpolarizing factor to reduced dilatation was tested by pretreatment with L‐NAME (10‐4 mol/l), indomethacin (10‐5 mol/l), TRAM‐34 (10‐6 mol/), and UCL1684 (10‐7 mol/l), respectively. L‐NAME decreased the acetylcholine response after hypoxia/re‐oxygenation stronger compared to control conditions, while indomethacin, TRAM‐34, and UCL1684 did not affect it. Endothelium‐independent effects were analyzed by using the NO‐donor SNP and simultaneous blocking of endothelium‐dependent NO production. SNP significantly reduces vasodilation after hypoxia+norepinephrine/re‐oxygenation (H+NE/R). Passive stiffness of arteries was not altered in our experimental setup.H+NE/R provokes an impairment of the dilatory response to acetylcholine in renal arteries. Our data suggest that the endothelial nitric oxide system, COX products, or TRAM‐34 and UCL1684 inhibitable potassium channels do not contribute to reduced dilatation. The results indicate changed dilatory signaling pathways in vascular smooth muscle cells.Support or Funding InformationThis study was financially supported by a grant of the German Research Council to A.P and P.B.P (FG 1368, DFG PA 479/10‐1 and PA 479/10‐2).

CYP epoxygenase-derived H2O2 is involved in the endothelium-derived hyperpolarization (EDH) and relaxation of intrarenal arteries

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium–dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2•−) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.

Contribution of K(+) channels to endothelium-derived hypolarization-induced renal vasodilation in rats in vivo and in vitro.

We investigated the mechanisms behind the endothelial-derived hyperpolarization (EDH)-induced renal vasodilation in vivo and in vitro in rats. We assessed the role of Ca(2+)-activated K(+) channels and whether K(+) released from the endothelial cells activates inward rectifier K(+) (Kir) channels and/or the Na(+)/K(+)-ATPase. Also, involvement of renal myoendothelial gap junctions was evaluated in vitro. Isometric tension in rat renal interlobar arteries was measured using a wire myograph. Renal blood flow was measured in isoflurane anesthetized rats. The EDH response was defined as the ACh-induced vasodilation assessed after inhibition of nitric oxide synthase and cyclooxygenase using L-NAME and indomethacin, respectively. After inhibition of small conductance Ca(2+)-activated K(+) channels (SKCa) and intermediate conductance Ca(2+)-activated K(+) channels (IKCa) (by apamin and TRAM-34, respectively), the EDH response in vitro was strongly attenuated whereas the EDH response in vivo was not significantly reduced. Inhibition of Kir channels and Na(+)/K(+)-ATPases (by ouabain and Ba(2+), respectively) significantly attenuated renal vasorelaxation in vitro but did not affect the response in vivo. Inhibition of gap junctions in vitro using carbenoxolone or 18α-glycyrrhetinic acid significantly reduced the endothelial-derived hyperpolarization-induced vasorelaxation. We conclude that SKCa and IKCa channels are important for EDH-induced renal vasorelaxation in vitro. Activation of Kir channels and Na(+)/K(+)-ATPases plays a significant role in the renal vascular EDH response in vitro but not in vivo. The renal EDH response in vivo is complex and may consist of several overlapping mechanisms some of which remain obscure.

Induction of Angiotensin-Converting Enzyme and Activation of the Renin–Angiotensin System Contribute to 20-Hydroxyeicosatetraenoic Acid–Mediated Endothelial Dysfunction

20-hydroxyeicosatetraenoic acid (20-HETE) promotes endothelial dysfunction by uncoupling endothelial NO synthase, stimulating O(2)(-) production, and reducing NO bioavailability. Moreover, 20-HETE-dependent vascular dysfunction and hypertension are associated with upregulation of the renin-angiotensin system This study was undertaken to examine the contribution of renin-angiotensin system to 20-HETE actions in the vascular endothelium. In endothelial cells, 20-HETE induced angiotensin-converting enzyme (ACE) mRNA levels and increased ACE protein and activity by 2- to 3-fold; these effects were negated with addition of the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20 HEDE). 20-HETE induced ACE expression was protein kinase C independent and epidermal growth factor receptor tyrosine kinase and IκB kinase β dependent. ACE short interfering RNA abolished 20-HETE-mediated inhibition of NO production and stimulation of O(2)(-) generation, whereas angiotensin II type 1 receptor short interfering RNA attenuated these effects by 40%. 20-HETE-stimulated O(2)(-) production was negated by 20-HEDE and was attenuated by lisinopril and losartan. Importantly, 20-HETE-mediated impairment of acetylcholine-induced relaxation in rat renal interlobar arteries was also attenuated by lisinopril and losartan. These results indicate that ACE and angiotensin II type 1 receptor activation contribute to 20-HETE-mediated endothelial cell and vascular dysfunction and further enforce the notion that excessive production of 20-HETE within the vasculature leads to hypertension via mechanisms that include the induction of endothelial ACE, thus, perpetuating an increase in vascular angiotensin which, together with 20-HETE, promotes vascular dysfunction.

20-Hydroxy-5,8,11,14-eicosatetraenoic Acid Mediates Endothelial Dysfunction via IκB Kinase-Dependent Endothelial Nitric-Oxide Synthase Uncoupling

Endothelial dysfunction and activation occur in the vasculature and are believed to contribute to the pathogenesis of cardiovascular diseases. We have shown that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a cytochrome P450 4A-derived eicosanoid that promotes vasoconstriction in the microcirculation, uncouples endothelial nitric-oxide synthase (eNOS) and reduces nitric oxide (NO) levels via the dissociation of the 90-kDa heat shock protein (HSP90) from eNOS. It also causes endothelial activation by stimulating nuclear factor-kappaB (NF-kappaB) and increasing levels of pro-inflammatory cytokines. In this study, we examined signaling mechanisms that may link 20-HETE-induced endothelial dysfunction and activation. Under conditions in which 20-HETE inhibited NO production, it also stimulated inhibitor of NF-kappaB (IkappaB) phosphorylation. Both effects were prevented by inhibition of tyrosine kinases and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). It is noteworthy that inhibitor of IkappaB kinase (IKK) activity negated the 20-HETE-mediated inhibition of NO production. Immunoprecipitation experiments revealed that treatment of ionophore-stimulated cells with 20-HETE brings about a decrease in HSP90-eNOS association and an increase in HSP90-IKKbeta association, suggesting that the activation by 20-HETE of NF-kappaB is linked to its action on eNOS. Furthermore, addition of inhibitors of tyrosine kinase MAPK and IKK restored the 20-HETE-mediated impairment of acetylcholine-induced relaxation in rat renal interlobar arteries. The results indicate that 20-HETE mediates eNOS uncoupling and endothelial dysfunction via the activation of tyrosine kinase, MAPK, and IKK, and these effects are linked to 20-HETE-mediated endothelial activation.

A concise synthesis of 12( S),20-dihydroxyeicosa-5( Z),8( Z),10( E),14( Z)-tetraenoic acid, an endogenous vasoconstrictor

12( S),20-DiHETE, prepared by a combination of Evans–Crimmins asymmetric alkylation, Sonogashira alkynylation, and Suzuki–Miyaura cross-coupling, significantly sensitizes phenylephrine-induced vasoconstriction of rat renal interlobar arteries.

Carbon monoxide of vascular origin attenuates the sensitivity of renal arterial vessels to vasoconstrictors.

Rat renal interlobar arteries express heme oxygenase 2 (HO-2) and manufacture carbon monoxide (CO), which is released into the headspace gas. CO release falls to 30% and 54% of control, respectively, after inhibition of HO activity with chromium mesoporphyrin (CrMP) or of HO-2 expression with antisense oligodeoxynucleotides (HO-2 AS-ODN). Patch-clamp studies revealed that CrMP decreases the open probability of a tetraethylammonium-sensitive (TEA-sensitive) 105 pS K channel in interlobar artery smooth muscle cells, and that this effect of CrMP is reversed by CO. Assessment of phenylephrine-induced tension development revealed reduction of the EC(50) in vessels treated with HO-2 AS-ODN, CrMP, or TEA. Exogenous CO greatly minimized the sensitizing effect on agonist-induced contractions of agents that decrease vascular CO production, but not the sensitizing effect of K channel blockade with TEA. Collectively, these data suggest that vascular CO serves as an inhibitory modulator of vascular reactivity to vasoconstrictors via a mechanism that involves a TEA-sensitive K channel.

Vascular Reactivity to Phenylephrine Is Reciprocally Regulated by Carbon Monoxide and 20-Hydroxyeicosatetraenoic Acid of Vascular Origin

P107 Carbon monoxide (CO) inhibits CYP-450 enzymes that synthesize 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that enhances vascular reactivity to phenylephrine (PE). This study examines the hypothesis that an interaction between CO and 20-HETE of vascular origin regulates the reactivity of rat renal interlobar arteries (ILA) to PE. ILA incubated (1h) in Krebs buffer released CO into the headspace (95±16 pmol/mg) and manufactured 20-HETE (18±2 ng/mg). Chromium mesoporphyrin (CrMP, 30 μM), an inhibitor of heme oxygenase-dependent CO synthesis, reduced (p<0.05) CO release (29±5 pmol/mg) but not 20-HETE production (15±4 ng/mg). In studies conducted on ILA mounted on a wire-myograph, CrMP caused a leftward shift in the PE concentration-response curve, decreasing the EC 50 (0.27±0.03 to 0.12±0.05 μM, p<0.05) without affecting the maximal response. However, CrMP did not affect any aspect of the PE concentration-response relationship in ILA treated with dibromo-dodecenyl-methylsulfimide (DDMS, 30 μM), a 20-HETE synthesis inhibitor. Like CrMP, exogenous 20-HETE (10 μM) caused a leftward displacement of the PE concentration-response curve, decreasing (p<0.05) the EC 50 from 0.29±0.01 to 0.07±0.01 μM. Superimposed treatment with exogenous CO (1 μM) shifted back to the right the PE concentration-response curve, increasing the EC 50 to 0.31±0.03 μM (p<0.05). Exogenous CO had a similar effect in ILA treated with exogenous 20-HETE and DDMS in combination. These data suggest that CO of vascular origin inhibits vascular reactivity to PE via a mechanism independent of 20-HETE synthesis. However, vascular 20-HETE is a stimulatory regulator of vascular reactivity to PE and thus opposes at a functional level the inhibitory action of CO.

Vasoconstriction of rat renal interlobar arteries by noradrenaline and neuropeptide Y.

1. We have studied the contractile effects of noradrenaline and neuropeptide Y given alone and in combination on isolated rat renal interlobar arteries. 2. Noradrenaline contracted proximal and distal intrarenal microvessels in a concentration dependent manner, with similar potency (EC50 approximately equal to 550 nM), but maximal effects were greater in the proximal than in the distal vessel segments (approximately equal to 10 and 6 nM, respectively). 3. The noradrenaline-induced contraction was inhibited by low prazosin concentrations (3-10 nM) but not by 1 microM yohimbine indicating involvement of alpha(1)-but not alpha(2)-adrenoceptors. The alpha(1)A-adrenoceptor-selective antagonist, 5-methylurapidil and tamsulosin, had high potency (apparent affinities of approximately equal to 8 nM and 57 pM, respectively) while the alpha(1)D-adrenoceptor-selective antagonist, BMY 7378, had only low potency (apparent affinity approximately equal to 300 nM). The alpha(1)B-adrenoceptor-alkylating agent, chloroethylclonidine (10 microM for 30 min at 37 degrees C), had no inhibitory effects. The Ca2+ entry blocker, nitrendipine (300 nM), reduced the potency and maximal effects of noradrenaline. 4. Neuropeptide Y (1-100 nM) also contracted interlobar arteries in a concentration dependent manner, with greater effects in the proximal than in the distal segments, but maximal effects were only small in either segment (< 2 mN). In addition, neuropeptide Y also potentiated the response to noradrenaline, i.e. lowered its EC50 but this enhancement was also small. 5. We conclude that noradrenaline contracts rat interlobar arteries by an alpha(1)A-adrenoceptor; its co-transmitter, neuropeptide Y, affects the response only marginally in this vascular bed.

Alterations in rat interlobar artery membrane potential and K+ channels in genetic and nongenetic hypertension.

The renal vasculature plays an important role in the control of blood pressure. K+ channels have been demonstrated to regulate smooth muscle membrane potential and thereby control smooth muscle tone. However, few data are available on K+ channel function in the renal vasculature of hypertensive animals. This study details changes in K+ currents and membrane potential in genetic and nongenetic models of hypertension. The patch-clamp technique and Ca(2+)-imaging fluorescence were used to examine the differences in Wistar-Kyoto (WKY), Sprague-Dawley (SD), spontaneously hypertensive (SHR), and deoxycorticosterone acetate (DOCA) hypertensive single cells of rat kidney interlobar arteries. In current-clamp experiments, SHR and DOCA hypertensive cells were approximately 20 mV more depolarized than the control cells. In voltage-clamp experiments with 4-amino-pyridine and niflumic acid present to inhibit voltage-dependent K+ (K(v)) and Ca(2+)-activated CI- (CI(Ca)) currents, SHR and DOCA hypertensive Ca(2+)-activated K+ (K(Ca)) currents were significantly larger and activated at more negative potentials than the control. Conversely, with charybdotoxin and niflumic acid present to inhibit K(Ca) and CI(Ca) currents, SHR and DOCA hypertensive K(v) current was significantly smaller than the control. Finally, basal and angiotensin II-stimulated peak intracellular free [Ca2+] was greater in the SHR and DOCA hypertensive cells compared with control cells. These results suggest that membrane potential and the activity of K(Ca) and K(v) channels are altered in hypertensive rat renal interlobar arteries and may play a role in the regulation of renal blood flow under physiological and patho-physiological conditions.