Reduced KCNQ4-Encoded Voltage-Dependent Potassium Channel Activity Underlies Impaired β-Adrenoceptor–Mediated Relaxation of Renal Arteries in Hypertension
KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.
- Kv7 channels
- vascular smooth muscle
- renal artery
- spontaneously hypertensive rat
Hypertension is the leading cause of stroke, myocardial infarction, and kidney failure.1–3 The link between renal dysfunction and primary hypertension is well established, with increased renal artery resistance elevating blood pressure through the renin-angiotensin system4 and sodium retention.5 Moreover, it is widely recognized that altered sympathetic effects on the renal artery are strongly implicated in the initiation and perpetuation of the hypertensive state with dysfunction of the β-adrenoceptor pathway a dominant feature.6–11 However, little is known about the molecular mechanisms that contribute to renal artery vasospasm and decreased β-adrenoceptor–mediated dilation. Potassium (K+) channels regulate resting membrane potential in smooth muscle cells (SMCs) and are, thus, key determinants of smooth muscle contractility.12 Recently, our laboratory demonstrated that voltage-gated K+ channels encoded by KCNQ4 (Kv7.4) were drastically downregulated in the aorta and mesenteric artery of 2 different models of hypertension, spontaneously hypertensive rats (SHRs) and angiotensin II perfused mice.13 If a similar situation occurred in the renal artery, then the associated vasospasm and arterial stenosis would lead to reduced perfusion of the kidney and activation of the renin-angiotensin system. Although KCNQ gene expression and the functional role of Kv7 channels have been established in a number of different vascular beds,14–18 there is no information about these channels in the renal artery. Furthermore, although Kv7 channel activators are effective vasorelaxants,13,16–18 it is not known to what extent Kv7 channels contribute to endogenous dilator mechanisms, such as after β-adrenoceptor stimulation.
The aim of the present study was to ascertain whether Kv7 channels contribute to β-adrenoceptor–mediated vasodilation in renal arteries using a combination of pharmacological tools and small interference RNA (siRNA) technology. In addition, we determined whether the impaired β-adrenoceptor pathway in hypertension was linked to abrogation of Kv7.4 channel function. The findings of this study provide the first evidence that Kv7.4 channels have a central role in β-adrenoceptor–mediated vasodilation and provide a novel pathogenic link to the impaired activity of this endogenous pathway in hypertension.
Materials and Methods
Experiments were performed on male, 12- to 15-week–old Wistar (normotensive [NT]) rats and aged-matched SHRs euthanized by cervical dislocation.
KCNQ1 to 5 gene expression was measured using quantitative PCR. Kv7 protein was determined using immunohistochemistry and Western blotting (see the online-only Data Supplement).
Pharmacological tools and KCNQ4 siRNA transfection were used to assess functionality in either isometric tension recordings or an isolated perfused kidney preparation. Kv currents were recorded in a single renal artery myocyte by perforated whole-cell voltage clamp (see the online-only Data Supplement for details).
All of the responses are expressed as mean±SEM. Mann-Whitney tests were used to compare Kv7 channel modulator responses, as well as differences in maximum effect and pEC50 of methoxamine, isoproterenol, and forskolin. Isolation of Kv currents was assessed using a repeated-measure ANOVA followed by Bonferroni post hoc test. The effect of isoproterenol on Kv7 current was compared against the control using a paired Student t test. Difference in protein abundance between groups was determined as described previously.19 A value of P<0.05 was considered to indicate statistical significance.
KCNQ Expression in Rat Renal Artery
Quantitative relative abundance analysis of KCNQ1 to 5 mRNA was performed on NT rat renal artery samples. In accordance with all of the previous investigations,16–18 KCNQ2 mRNA was absent from the tissue preparation used in this study,16–18 whereas KCNQ1, 3, 4, and 5 were readily detected (Figure 1A). Immunodetection of Kv7 expression products in transverse sections of blood vessels confirms protein translation of KCNQ1, 4, and 5 to the smooth muscle layer of rat renal artery (Figure 1B). Overall, these studies show that rat renal artery expresses KCNQ1 and 3 to 5, and Kv7.1, 4, and 5 are abundant in the smooth muscle layer of these blood vessels.
Effect of Kv7 Channel Modulators on Renal Reactivity
Kv7 channel blockers XE991 and linopirdine have a depolarizing effect on vascular SMCs, which evokes contractions in a number of blood vessels.16,18,20 Conversely, Kv7 channel activators, such as retigabine and S-1, which enhance Kv7.2 to 7.5 but not Kv7.121 channel openings, hyperpolarize SMCs and induce relaxation in various blood vessels.16,18,20 Figure 2A shows that S-1 produced concentration-dependent relaxation of precontracted renal arteries that was completely abolished by previous application of linopirdine (Figure 2A and 2B). Figure 2C shows that 10 μmol/L of linopirdine produced robust contractions of the rat renal artery, which were fully reversed by application of the Ca2+ channel blocker nicardipine (1 μmol/L; data not shown). In addition to producing tonic contractions, application of a lower concentration of linopirdine (1 μmol/L) augmented methoxamine-induced contractions (pEC50: 6.20±0.10, n=7, versus vehicle 5.60±0.04, n=5; P=0.0339; Figure 2D). Linopirdine also enhanced responses to the thromboxane mimetic U46619 (Figure S1, available in the online-only Data Supplement). These data are consistent with Kv7 channels having a functional role in the renal artery. In line with the working hypothesis that Kv7 channels are key determinants of renal vascular reactivity, linopirdine increased the perfusion pressure in isolated kidney preparations (Figure 2E). Overall, these studies have established that renal arteries express KCNQ1, 4, and 5, and the translated proteins form channels that are key determinants of smooth muscle tone.
Kv7 Channels Contribute to β-Adrenoceptor–Mediated Relaxation
A common feature of the hypertensive vasculature is impairment of the β-adrenoceptor–mediated vasodilation, which is not attributed to a diminution of the adrenoceptor population.6–11 To date there is no information on the role of Kv7 channels in the physiological response to endogenous vasorelaxants, such as β-adrenoceptor stimulants. Figure 3A shows that the β-adrenoceptor agonist isoproterenol (0.001–3.000 μmol/L) caused concentration-dependent relaxation in renal arteries from NT animals, which was attenuated by 10 μmol/L of linopirdine (maximum effect: linopirdine 39.3±15.5, vehicle 103.6±4.2; n=5; P=0.0079). Figure 3B shows that relaxations produced by the adenylate cyclase activator forskolin were also attenuated by linopirdine (pEC50: vehicle 7.2±0.1, linopirdine 6.3±0.1; n=4; P=0.0285). In contrast, isoproterenol-induced relaxation was not attenuated by 4-AP at 1 mmol/L, a concentration known to block several Kv channels but not Kv7 channels (Figure S2A) or the ATP-sensitive K+ channel blocker glibenclamide (10 μmol/L; Figure S2B), which reduces hyperpolarization caused by β-adrenoceptor stimulation in small arteries.22,23
To support a role for Kv7 channel stimulation in the relaxant response to isoproterenol, perforated patch whole-cell recordings were made from isolated renal artery myocytes bathed in correolide (2 μmol/L) and ScTx1 (100 nmol/L) to inhibit Kv1 and Kv2 channels. Under these conditions a robust Kv current, which was abolished by linopirdine (1 μmol/L), was recorded (Figure S3). Isoproterenol (1 μmol/L) applied in the presence of correolide (2 μmols/L) and ScTx1 (100 nmol/L) significantly increased the linopirdine-sensitive current in all of the cells (Figure 4). Preincubation with linopirdine abrogated any increase in current produced by isoproterenol (Figure S4A and S4C; n=4), and isoproterenol was without an effect on residual current recorded in the presence of Kv1, Kv2, and Kv7 inhibitors (Figure S4B and S4D; n=4). These experiments show clearly that isoproterenol stimulates linopirdine-sensitive Kv7 channels but not Kv1 or Kv2 channels but was not intended to characterize the relative contribution of individual K+ channel subtypes.
KCNQ4 Knockdown Decreases Isoproterenol-Induced Responses
To consolidate our findings with linopirdine and to determine whether Kv7.4 channels contribute specifically to β-adrenoceptor-mediated vasodilation, we compared the effect of KCNQ4 targeted knockdown by targeted siRNAs on isoproterenol responses of renal arteries. The delivery of siRNAs by reverse permeabilization was validated by transfecting a nonselective siRNAs conjugated to the green 5′-carboxy fluorescein into an intact renal artery (Figure S5A through S5C). KCNQ4 targeted or scrambled siRNAs were transfected in segments of renal artery taken from the same animal. After 72 hours of incubation in culture medium, KCNQ4-encoded Kv7.4 protein expression (determined with a characterized antibody; Figure S6) was inhibited by ≈60% in vessels permeabilized with KCNQ4-targeted siRNAs compared with artery segments transfected with scrambled siRNAs (Figure 5A). Isometric tension studies demonstrated that relaxation evoked by S-1 (1 μmol/L) was reduced by 54.5±5.3% in arteries transfected with KCNQ4 siRNAs compared with scrambled siRNAs (P=0.0411; n=6; Figure 5B). This downregulation of Kv7.4 channels corresponded with hypersensitive responses to methoxamine (P=0.0306; Figure 5C) and a significant attenuation in isoproterenol-stimulated relaxation in KCNQ4 knockdown vessels (pEC50: scrambled siRNAs 8.2±0.1, KCNQ4 siRNAs 7.1±0.1; n=7; P=0.0084; Figure 5D). Overall, the molecular interference and pharmacological and electrophysiological studies provide considerable evidence for Kv7.4 channels contributing to β-adrenoceptor–mediated vasodilation of rat renal arteries.
Kv7 Channel Responses Are Lost in Renal Arteries From SHRs
Western blot analysis demonstrated that Kv7.4 abundance in rat renal arteries was markedly reduced in the SHR compared with the NT (Figure 6A). Strikingly, this was associated with a complete loss of a response to any Kv7 modulator. Hence, in SHRs, linopirdine had no effect on the basal tension of isolated arteries (Figure 6B) or the perfusion pressure of isolated kidneys (n=3–4; Figure 6C). Contractile responses to high K+ (60 mmol/L of KCl) were not different between NT rats and SHRs (Figure S7A). In addition, the ability of the Kv7 enhancer S-1 to relax preconstricted renal arteries from the SHR was completely abrogated (Figure 6D; P=0.0048). This was not because of the blood vessels being unable to relax as effectively or because of the contraction being less reliant on Ca2+ influx through voltage-dependent Ca2+ channels, because the relaxation produced by 1 μmol/L of nicardipine was not different between NT and SHRs (Figure S7B). As Figure 7 shows, renal arteries from SHRs exhibited altered responses to adrenoceptor agonists. Thus, the methoxamine-induced concentration-response curve was leftward-shifted compared with NT arteries (Figure 7A), and this was not affected by linopirdine pretreatment (Figure S8). In addition, consistent with a role for Kv7 channels in β-adrenoceptor dilations, renal arteries from SHRs did not respond to any concentration of isoproterenol tested (maximum effect: NT 81.6±4.4, SHR 2.3±3.8; n=4–7; P=0.0061; Figure 7B), and the relaxant responses of forskolin were markedly reduced in renal arteries from SHRs compared with vessels from NT animals (P=0.0477; Figure 7C).
In addition to their function in neurons and cardiomyocytes, Kv7 channels regulate resting vascular tone by acting as a “brake” on smooth muscle excitability.13,15,17,18,24 Further to their passive role as determinants of the membrane potential, a recent study suggested that Kv7 channels also contributed to the relaxant response of hydrogen sulfide and the anticontractile effects of adipose-derived molecules.25 The present study now shows that isoproterenol activates Kv7 channels in renal artery myocytes, and this contributes significantly to β-adrenoceptor- and forskolin-mediated relaxations in renal arteries. All of the Kv7 channels expressed in blood vessels contain a binding site in their C terminals that binds A-kinase anchor proteins26; however, very few data exist on the regulation of Kv7 channels by cAMP-dependent protein kinase A. In cardiomyocytes, channels formed by the association of Kv7.1 and KCNE1 expression products are enhanced through an interaction with the A-kinase anchor proteins Yotaio,27,28 and K+ channels produced by the overexpression of KCNQ4 in Chinese hamster ovary cells are enhanced by constitutively active protein kinase A.29 Consequently, the observations that isoproterenol increases linopirdine-sensitive currents and produces relaxations that are sensitive to Kv7 channel blockade or depletion of Kv7.4 protein are the first evidence that native Kv7 channels in vascular smooth muscle are regulated by cAMP-dependent processes. Because many potent vasodilators stimulate the production of cAMP, it is likely that recruitment of Kv7 channels may underpin a number of relaxant responses throughout the vasculature.
Importantly, with respect to the key physiological role of the renal artery in kidney perfusion and development of essential hypertension, the present study reveals that, in renal arteries from SHRs, the functional effect of Kv7 modulators was abolished. Responses to isoproterenol were also abrogated, further supporting a role for Kv7 channels in β-adrenoceptor–mediated vasorelaxation in this artery, although this does not confirm a causative link. Our previous work showed that responses to Kv7 activators were impaired in mesenteric arteries from hypertensive animals, and this was associated with a reduction in KCNQ4 expression and Kv7.4 protein.13 Interestingly, in SHR mesenteric, aortic, and renal arterial SMCs, Kv7.4 protein was reduced by similar amounts, but the functional effect was singularly pronounced in the renal artery. We hypothesize that this is because of a unique effect of hypertension on trafficking of mature Kv7.4 to functional microdomains in the renal artery. This is supported by our data showing that siRNA, which effects de novo synthesis, reduced protein levels by a similar extent in the same artery but had less functional impact. Presumably, longer periods of siRNA would be required to mimic chronic hypertension. Further studies are required to investigate vessel-specific effects of hypertension on Kv7 channels. Reduction in Kv7.4 abundance in renal arteries either by siRNA or as a consequence of hypertension produced a shift in the balance between α-adrenoceptor constriction and β-adrenoceptor dilation, which would increase renal artery resistance, as illustrated in Figure 8. Because renal artery stenosis is known to activate the renin-angiotensin system and sodium reabsorption that ultimately leads to hypertension, we propose that Kv7.4 channel downregulation may be a fundamental aspect in the development of long-term hypertension. It remains the focus of future studies to define the factors that regulate KCNQ gene expression in the vasculature.
Hypertension is the leading cause of stroke, myocardial infarction, and kidney failure. The link between renal dysfunction and primary hypertension is well established, with increased renal artery resistance elevating blood pressure through the renin-angiotensin4 and sodium excretion pathways. Moreover, an alteration in the balance of α-adrenoceptor–mediated vasoconstriction versus β-adrenoceptor–mediated vasodilation is a key feature of hypertension. However, little is known about the basic molecular mechanisms responsible for the aberrant control of renal artery contractility in this disorder. This study reveals that voltage-dependent potassium channels encoded by the KCNQ4 gene (Kv7.4) contribute significantly to β-adrenoceptor–mediated vasodilation in renal arteries and limit α-adrenoceptor–mediated vasoconstriction. Kv7 responses are abolished in the renal vasculature of SHRs, which results in impaired responsiveness to vasodilators and enhanced vasospasm in hypertension. These findings provide significant new insight concerning the mechanisms that regulate renal artery tone and provide a possible pathogenic link between arterial dysfunction and essential hypertension.
Sources of Funding
P.S.C. and A.J.D. were funded by British Heart Foundation grants awarded to I.A.G. (PG/09/104 and PG/07/127/24235). T.A.J. was funded by a Biotechnology and Biological Sciences Research Council-Collaborative Awards in Science and Engineering studentship (BB/G016321/1) in association with NeuroSearch A/S, and H.-L.Z. was supported by a fellowship from Alberta Innovates-Health Solutions. Patch clamp experiments were supported by funds from a Canadian Institutes of Health Research grant to W.C.C. (MOP-13505).
We thank all of the staff at the St George's Image Resource, Biomics Centre, and Biological Research Facilities for their assistance. We also thank Dr Ken Laing and Prof Alan Johnstone for their advice on quantitative PCR and immunohistochemistry, respectively.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.187427/-/DC1.
- Received November 8, 2011.
- Revision received December 5, 2011.
- Accepted February 2, 2012.
- © 2012 American Heart Association, Inc.
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