K_V7.1 channel blockade inhibits neonatal renal autoregulation triggered by a step decrease in arterial pressure

Abstract

K_V7 channels, the voltage-gated K⁺ channels encoded by KCNQ genes, mediate heterogeneous vascular responses in rodents. Postnatal changes in the functional expression of K_V7 channels have been reported in rodent saphenous arteries, but their physiological function in the neonatal renal vascular bed is unclear. Here, we report that, unlike adult pigs, only KCNQ1 (K_V7.1) out of the five members of KCNQ genes was detected in neonatal pig renal microvessels. KCNQ1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21. Activation of renal vascular smooth muscle cell (SMC) K_V7.1 stimulated whole cell currents, inhibited by HMR1556 (HMR), a selective K_V7.1 blocker. HMR did not change the steady-state diameter of isolated renal microvessels. Similarly, intrarenal artery infusion of HMR did not alter mean arterial pressure, renal blood flow, and renal vascular resistance in the pigs. An ∼20 mmHg reduction in mean arterial pressure evoked effective autoregulation of renal blood flow, which HMR inhibited. We conclude that 1) the expression of KCNQ isoforms in porcine renal microvessels is dependent on kidney maturation, 2) K_V7.1 is functionally expressed in neonatal pig renal vascular SMCs, 3) a decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs, and 4) SMC K_V7.1 does not control basal renal vascular tone but contributes to neonatal renal autoregulation triggered by a step decrease in arterial pressure.

NEW & NOTEWORTHY K_V7.1 is present in fetal pig kidneys as early as day 50 of gestation, and the level of expression remains the same up to postnatal day 21. K_V7.1 is functionally expressed in neonatal pig renal vascular smooth muscle cells (SMCs). A decrease in arterial pressure up to 20 mmHg induces renal autoregulation in neonatal pigs. Although SMC K_V7.1 does not control basal renal vascular resistance, its inhibition blunts neonatal renal autoregulation engendered by a step decrease in arterial pressure.

INTRODUCTION

The K_V7 family of voltage-gated K⁺ channels comprises five subtypes encoded by KCNQ genes (KCNQ1–KCNQ5) (1). K_V7 channels are expressed in a wide variety of blood vessels, including the aorta and portal veins and coronary, pulmonary, mesenteric, cerebral, saphenous, and renal arteries (2–6). K_V7 channels control vascular smooth muscle cell (SMC) intracellular Ca²⁺ oscillations, membrane potential, and vascular tone (3, 5, 7–10). Pan K_V7 blockers, including XE991 and linopirdine, induced vascular SMC depolarization and contraction of adult rodent portal veins and pulmonary, mesenteric, cerebral, and coronary arteries (11–15). The K_V7.2–K_V7.5 activator retigabine caused relaxation of the rodent aorta and femoral, mesenteric, and carotid arteries (6). Despite the near-ubiquitous expression of K_V7.1 in the vasculature, studies of the adult rat aorta and pulmonary and mesenteric arteries have suggested that the channels are not involved in the control of resting vascular tone (16, 17). Hence, K_V7-dependent regulation of vascular tone may be isoform specific. KCNQ1, KCNQ3, KCNQ4, and KCNQ5 are expressed in the main renal arteries isolated from adult rats (2). Immunostaining has also detected K_V7.4 in afferent arterioles of adult rats (18). The nonselective K_V7 blocker linopirdine and siRNA-mediated knockdown of KCNQ4 inhibited isoproterenol-induced relaxation of renal arteries (2). XE991 increased myogenic constriction in mesenteric arteries and isolated kidney perfusion pressure in adult mice (19).

Renal autoregulation, a reaction by which healthy kidneys maintain constant renal blood flow (RBF) and glomerular filtration rate (GFR), protects renal function despite physiological fluxes in arterial pressure and is mediated by two primary mechanisms: 1) the myogenic response and 2) tubuloglomerular feedback. The renal myogenic response is based on the intrinsic ability of afferent arterioles and distal interlobular arteries to swiftly constrict (myogenic vasoconstriction) or dilate (myogenic vasodilation) in response to an acute elevation or a reduction, respectively, in renal perfusion pressure (20–22). The slower tubuloglomerular feedback mechanism involves signal transduction events in which tubular flow rate-dependent changes in luminal NaCl concentration detected at the macula densa adjust renal vascular resistance (RVR) by stimulating the production of vasoactive mediators that alter preglomerular vascular tone (20–22).

Myogenic vasoconstriction is mediated by ion channels that respond to elevated intraluminal pressure by activating SMC depolarization and voltage-dependent Ca²⁺ channels (23, 24). Subsequent extracellular Ca²⁺ influx promotes vasoconstriction (23, 24). Our recent studies in newborn pigs have demonstrated myogenic autoregulation of RBF in response to increased arterial pressure (25). The autoregulation range in healthy adults is between ∼80 and 180 mmHg (21). The mean arterial pressure (MAP) of term newborns is less than the lower limit of the adult autoregulation range (26). Thus, renal autoregulation may occur at a lower perfusion pressure in neonates. However, it is unclear whether immature kidneys autoregulate RBF when renal perfusion pressure is decreased.

In adult rodent microvessels, K⁺ channels may limit the myogenic response by hyperpolarizing SMCs (5, 27, 28). At term, kidney maturation is functionally incomplete, and levels of expression and activity of vascular ion channels and vasoactive mediators may depend on postnatal kidney maturation (28–30). Although KCNQ4 expression was diminished at 10- to 15-day-old rat saphenous arteries compared with 2- to 3-mo-old rat saphenous arteries, KCNQ1, KCNQ2, and KCNQ5 expression levels were significantly higher in neonatal rats (5). Pharmacological inhibition of K_V7 also induced more pronounced arterial smooth muscle depolarization and contraction in neonatal rat saphenous arteries (5). The expression and function of K⁺ channels in resistance size renal microvessels of immature kidneys are unknown. Conceivably, the functional expression of K_V7 channels differs between vascular beds.

In the present study, we determined the expression of K_V7 channels in fetal and neonatal pig kidneys. We also studied the physiological function of K_V7 channels in neonatal renal vasculature. We used the translational porcine model because human and pig renal systems are similar, characterized by a minimal outer stripe of the outer medulla (OSOM) and multilobar/multipyramidal features in contrast to a well-defined OSOM and unilobar/unipyramidal kidneys of rodents (31–34).

METHODS

Animals

All experimental animal procedures were approved and performed following the Institutional Animal Care and Use Committee of the University of Memphis and University of Tennessee Health Science Center. Experiments also followed the National Institutes of Health and Animal Research: Reporting of In Vivo Experiments guidelines. Full-term neonatal domestic pigs (male, 3–5 days old) were obtained from Nichols Hog Farm (Olive Branch, MS). For experiments that examined the expression levels of K_V7.1 in the kidneys, fetal pigs were harvested from sows of the same genetic lineage at days 50 and 100 of gestation (43% and 87% of term, respectively). Kidneys were also collected from male pigs delivered naturally at term and raised until day 21 on commercial milk replacer. Adult male pig (∼6-mo old) kidneys were obtained from Midwest Research Swine (Glencoe, MN). Animals were randomly assigned to experimental groups in this study.

Tissue Preparation

The 50-day fetal pigs were euthanized by decapitation, and 100-day fetuses and postnatal 1- and 21-day-old pigs were euthanized by euthasol (1 mL/kg iv). Neonatal term pigs were euthanized by intramuscular injection of ketamine-xylazine (100/10 mg/kg) followed by exsanguination. The organs were immediately removed and processed. The kidneys were decapsulated, hemisected, and placed in ice-cold (4°C) modified Krebs solution with the following composition (in mM): 134 NaCl, 6 KCl, 2.0 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4). The kidneys were dissected to isolate interlobular arteries and afferent arterioles.

Isolation of Renal Vascular SMCs

For patch-clamp electrophysiology experiments, SMCs were freshly isolated from interlobular arteries and afferent arterioles, as we have previously described (25). For PCR, a high yield of SMCs without endothelial cell (EC) contamination was obtained using the Dynabeads CD31 EC isolation kit (Life Technologies, Grand Island, NY), as we have previously described (25). Briefly, the isolated cells were incubated in CD31-coated magnetic beads for ∼60 min with gentle rotation. A DynaMag magnet (Life Technologies) was used to separate EC-bound beads. The EC-bound beads were discarded, and the supernatant containing only SMCs was used for PCR.

PCR

Total RNA was isolated from SMCs using Direct-zol RNA Miniprep Plus Kits (Zymo Research). cDNAs were then synthesized from the RNA samples using a High-Capacity cDNA Reverse Transcription kit (Life Technologies). PCR amplification was performed using gene-specific oligonucleotide primer pairs (Table 1). Nested PCR was performed with the SMC samples to enhance sensitivity and specificity by increasing amplification cycles and binding of the outer primers (PCR 1; Table 1) and inner primers (PCR 2; Table 1) to the same target (35). The outer primer pairs amplify the target with expanded flanking regions. The first round’s products then serve as templates for the successive second round of PCR with inner (nested) primer pairs positioned within the first set of primer sequences. The following PCR reaction conditions were used: initial denaturation at 98°C for 2 min followed by 35 cycles {denaturation at 98°C for 10 s, annealing at 57°C [α-smooth muscle actin (ACTA2) or von Willebrand factor (vWF)] or 69°C (K_V7) for 30 s, and extension at 72°C for 30 s} with a final extension at 72°C for 10 min. Subsequently, PCR products were cleaned using a GenElute PCR Clean-up kit (Sigma-Aldrich), resolved on 1.5% agarose gels stained with SYBR Safe DNA gel stain (Life Technologies), and visualized on a Bio-Rad gel documentation system. Only SMCs positive for ACTA2 (SMC marker) and negative for vWF (EC marker) were used.

Table 1.

Oligonucleotide primer sequences

Gene	Sequence	Accession Number	Length, bp
PCR 1
ACTA2	Forward: 5′-CCCTGTGAAGCACCAGCCAGGA-3′ Reverse: 5′-GTAGAGGGACAGCACCGCCTGA-3′	NM_001164650.1	456
vWF	Forward: 5′-CGTGGAGAGTGCTGAGTGTT-3′ Reverse: 5′-GGCCATCCCAGTCTATCTGC-3′	NM_001246221.1	488
KCNQ1	Forward: 5′-CAGATCACGTGTGACCTCGC-3′ Reverse: 5′-CTCGGTCCTTGACCTTCTCTG-3′	XM_021082619.1	430
KCNQ2	Forward: 5′-TCGGCGTCTCTTTCTTTGCT-3′ Reverse: 5′-CAGCATCCACACAGGGACTT-3′	XM_021077758.1	388
KCNQ3	Forward: 5′-GGGAAGAGGGGGATTGTTGG-3′ Reverse: 5′-TTCTGGGCGGTCAAAGTCTC-3′	XM_021088897	417
KCNQ4	Forward: 5′-ACAAGCGCTACCGCCG-3′ Reverse: 5′-AGGAGGCAAAGATGAGCACC-3′	XM_021096970.1	531
KCNQ5	Forward: 5′-CTACAGCAGCGGCCAGAG-3′ Reverse: 5′-CGGAGCCCAGTAACTTCCAG-3′	XM_021073129.1	488
PCR 2
KCNQ1	Forward: 5′-GAGAACCAACAGCTTTGCCG-3′ Reverse: 5′-TTGAGGTGGCCTTGGGAGTA-3′	XM_021082619.1	213
KCNQ2	Forward: 5′-GGGTCTGGCTTTGCTCTGAA-3′ Reverse: 5′-GGCCCCGTAGGTTTGAGTTT-3′	XM_021077758.1	207
KCNQ3	Forward: 5′-AGATCCCAGTGGTTGAGCTG-3′ Reverse: 5′-TGGCTAGCATTGGTCCAACA-3′	XM_021088897	186
KCNQ4	Forward5′-ACTGGGTCTACAACGTGCTG-3′ Reverse5′-TTTCTGGCAAAACGGAAGCG-3′	XM_021096970.1	262
KCNQ5	Forward: 5′-ACAACGTGTTGGAGAGACCC-3′ Reverse: 5′-GTGCTCAGGGATGGTGGAAA-3′	XM_021073129.1	110
Quantitative PCR
KCNQ1	Forward: 5′-AGGTCATTCGACGTATGCAGT-3′ Reverse: 5′-TTGAGGTGGCCTTGGGAGTA-3′	XM_021082619.1	109
18S rRNA	Forward: 5′-CGAAAGCATTTGCCAAGAAT-3′ Reverse: 5′-AGTCGGCATCGTTTATGGTC-3′	NR_046261.1	102

ACTA2, α-smooth muscle actin; vWF, von Willebrand factor.

Quantitative RT-PCR was performed using an Applied Biosystems SYBR Green Master Mix kit (Life Technologies). The QuantStudio 3 Real-Time PCR System (Life Technologies) was used for PCR amplification. Relative gene expression levels were determined using 2^−ΔΔCt, where ΔC_t is the difference in threshold cycle (C_t) values between the gene of interest and 18S rRNA and ΔΔCt is the difference in ΔC_t values compared with gestational day 50.

Immunofluorescence

Formaldehyde-fixed (4%, ∼20 min) and Triton X-100-permeabilized (0.2%, ∼15 min) renal microvascular SMCs were incubated in MAXblock blocking medium (Active Motif, Carlsbad, CA) for 1 h to block nonspecific binding sites. Cells were treated overnight at 4°C with the respective primary antibodies and blocking peptide. The blocking peptide was preincubated with the primary antibody at a 5:1 ratio overnight. Kv7.1 antibody was used at a 1:100 dilution and caveolin-1 at a 1:75 dilution. The next day, SMCs were washed with PBS and incubated with secondary antibodies (1:400 dilution) for 1 h at room temperature. Following a wash and mount, images were acquired using a Zeiss LSM 710 laser scanning confocal microscope.

Patch-Clamp Electrophysiology

K⁺ currents were recorded at room temperature in freshly isolated SMCs attached to a glass-bottom chamber using the conventional whole cell configuration of the patch-clamp technique. Current recordings and data acquisition were made with Axopatch 200B, Digidata 1440A, and pCAMP 10 software (Molecular Devices, Sunnyvale, CA). A current-voltage relationship was generated by applying successive 300-ms voltage steps (−80 to +40 or +70 mV) in 10-mV increments every 5 s from a holding potential of −60 mV. Whole cell currents were filtered at 1 kHz and digitized at 5 kHz. The bath solution contained (in mM) 140 NaCl, 4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, and 5 glucose (pH 7.4). The pipette solution contained (in mM) 140 KCl, 2 MgCl₂, 5 EGTA, and 10 HEPES adjusted to pH 7.2 with KOH.

Pressurized Artery Myography

As previously described (25, 36, 37), interlobular artery luminal diameter changes were examined using the pressure myograph system (Living Systems Instrumentation, St. Albans, VT). Distal interlobular arteries were cannulated with a fabricated glass at each end in temperature-controlled chambers. The chambers were slowly and continuously perfused with modified Krebs solution (25, 36, 37) equilibrated with a 21% O₂-5% CO₂-74% N₂ gas mixture and maintained at 37°C. The arteries were pressurized to 60 mmHg, and their lumen diameter changes were determined using vessel dimension analysis software (IonOptix, Westwood, MA).

RBF Autoregulation

As we have previously described, neonatal term pigs were anesthetized, intubated, and mechanically ventilated (25, 37–39). We have also previously described the methods we used to administer pharmacological agents directly into the kidneys and measure MAP, RBF, and RVR in neonatal pigs (25, 37–39).

Step decreases in renal perfusion pressure were achieved by placing an inflatable 4-mm vascular occluder cuff (In Vivo Metrics, Healdsburg, CA) around the aorta immediately upstream of the main renal arteries. The cuff was inflated/deflated with a BasixCOMPAK inflation device (Merit Medical Systems, South Jordan, UT). The renal autoregulatory index (AI) in the pigs was calculated using the Semple and DeWardener equation as follows (40):

$$\left[\frac{\frac{{\text{RBF}}_2-{\text{RBF}}_1}{{\text{RBF}}_1}}{\frac{{\text{MAP}}_2-{\text{MAP}}_1}{{\text{MAP}}_1}}\right]$$

RBF₁ and MAP₁ are the baseline values taken before the step decrease in pressure, and RBF₂ and MAP₂ are the values taken after the step decrease in pressure. An AI of ≤0.2 signifies effective autoregulation, whereas an AI near or above 1.0 indicates ineffective autoregulation (21, 25, 40).

Antibodies and Reagents

Rabbit polyclonal anti-K_V7.1 antibody (Cat. No. APC-022) and Kv7.1 blocking peptide (Cat. No. BLP-PC022) were purchased from Alomone Labs (Jerusalem, Israel). Goat polyclonal anti-caveolin-1 antibody (Cat. No. ab36152) was purchased from Abcam (Waltham, MA). Highly cross-adsorbed CF488 donkey anti-rabbit IgG (Cat. No. 20015) and CF555 donkey anti-goat IgG (Cat. No. 20039) were purchased from Biotium (Fremont, CA). Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO). Mefenamic acid was purchased from Selleckchem (Houston, TX). Linopirdine was purchased from Alomone Labs (Jerusalem, Israel). ML277 and HMR1556 (HMR) were purchased from Tocris (Bristol, UK).

Dose-Response Curve Fit

Dose-response curves were fitted with Origin v9.8 (OriginLab, Northampton, MA) using the following formula:

$$y=A1+\frac{A2-A1}{1+{10}^{(\log \hspace{0.17em}x0-x)p}}$$

where A1 is the top asymptote, A2 is the bottom asymptote, logx0 is the center, and p is the Hill slope.

Statistical Analysis

Data are presented as means ± SE. Prism software (Graph Pad, Sacramento, CA) was used for data analysis. Student’s t test (paired or unpaired) and one- and two-way ANOVA with Bonferroni’s multiple comparisons test were used. P values of <0.05 were considered significant.

RESULTS

KCNQ1 Is the Predominant Isoform in the Kidney and Renal Vascular SMCs of Neonatal Pigs

Of the five members of KCNQ genes, only KCNQ1 was detected in neonatal term pig kidneys (Fig. 1A). In contrast, the neonatal pig brain expressed all five isoforms (Fig. 1A). The heart and pulmonary arteries of the pigs expressed KCNQ1, KCNQ4, and KNCQ5 (Fig. 1A). Quantitative RT-PCR showed that KCNQ1 expression levels were similar in the kidneys of fetal pigs at 50 and 100 days of gestation and remained comparable in the kidneys of naturally farrowed term neonatal pigs at postnatal day 1 (<24 h after birth) and postnatal day 21 (Fig. 1B). As shown in Fig. 1C, intact renal microvessels (interlobular arteries) contain ECs and SMCs, as confirmed by PCR amplification of vWF and ACTA2, EC, and SMC markers, respectively. Unlike KCNQ2–KCNQ5, clear bands corresponding to KCNQ1 were detected in intact interlobular arteries and SMCs (Fig. 1C). PCR was performed on adult male pig renal microvessels to verify whether KCNQ isoform expression is age dependent. As shown in Fig. 1D, adult pig renal microvessels expressed KCNQ1, KCNQ3, KCNQ4, and KNCQ5, indicating that KCNQ isoforms in porcine renal microvessels are related to kidney maturation. Confocal microscopy indicated that K_V7.1 was colocalized with caveolin-1 in the plasmalemma of the cells (Fig. 1, E and F). Of note, caveolin-1 also showed some degree of staining in parts of the cytoplasm (Fig. 1, E and F). This is not surprising as apart from the plasma membrane caveolae invaginations, caveolin-1 is also expressed in cytoplasmic caveolar vesicles (41–43). Our findings suggest that KCNQ1 is the predominant KCNQ gene in the developing pig kidney, resistance size vessels, and renal vascular SMCs.

Figure 1.

KCNQ1 is the predominant isoform in the neonatal pig kidney and renal vascular smooth muscle cells (SMCs). A: agarose gel images demonstrating the amplification of KCNQ isoforms in the neonatal pig (3–5 days old) kidneys, brain, heart, and pulmonary arteries. B: bar graphs summarizing mean data for quantitative RT-PCR experiments that compared KNCQ1 mRNA expression levels in fetal pig kidneys at gestational days 50 and 100 (GD50 and GD100, respectively) and postnatal days 1 and 21 (P1 and P21, respectively). C: agarose gels images demonstrating amplification of α-smooth muscle actin (ACTA2), von Willebrand factor (vWF), and KCNQ1 in interlobular arteries (ILA) and ACTA2 and KCNQ1 in ILA SMCs. D: agarose gel image demonstrating the amplification of KCNQ isoforms in adult pigs. E: confocal microscopy images of ILA SMCs immunostained with a selective K_V7.1 blocking peptide (BP) + K_V7.1 and caveolin-1 antibodies. F: images showing colocalization of caveolin-1 and K_V7.1 channels in the plasma membrane of SMCs. Product sizes for KNCQ1, KNCQ2, KNCQ3, KNCQ4, and KNCQ5 are 213, 207, 186, 262, and 110, respectively (one-way ANOVA with a Bonferroni’s post hoc test). ns, not significant; NTC, no template control.

K_V7.1 Is Functionally Expressed in Renal Vascular SMCs of Neonatal Pigs

To study Kv7.1 channel activity, we recorded whole cell currents in neonatal pig renal vascular SMCs in the presence of multiple activators and inhibitors. Responses to nonselective (linopirdine) (44, 45) and selective (HMR) (46, 47) K_V7.1 channel blockers were examined. Linopirdine inhibited the cells’ basal outward currents (Fig. 2, A–C), albeit at a high concentration (200 µM). The linopirdine-sensitive current was activated at around −60 mV and exhibited outward rectification (Fig. 2C). The magnitude of this current was ∼48% of the basal current at +40 mV. HMR reduced the basal outward currents concentration dependently (Fig. 2, D and E). HMR, at concentrations of 1, 3, and 10 µM, inhibited the currents at +40 mV by ∼11%, 42%, and 74%, respectively. A concentration-response curve fit of the uninhibited outward current normalized to the control at membrane voltages of +20, +30, and +40 mV exhibited a sigmoidal pattern with an IC₅₀ value of 2.66, 2.5, and 2.49 µM, respectively (Fig. 2E). ML277, a selective Kv7.1 activator (48), significantly increased outward membrane currents in SMCs (Fig. 2, F and G). Mefenamic acid, another activator of Kv7.1 channels (49, 50), elicited whole cell currents that were inhibited by 3 µM HMR (Fig. 2, H and I). HMR at a concentration of 3 µM inhibited 48% of the total current at +40 mV after mefenamic acid treatment, which is comparable with the HMR effect on basal currents. Taken together, these data confirm that K_V7.1 channels are functionally expressed in neonatal pig renal vascular SMCs.

Figure 2.

K_V7.1 is functionally expressed in neonatal pig renal vascular smooth muscle cells (SMCs). A: linopirdine (Lino; 200 µM) inhibited outward currents in renal vascular SMCs. B: current-voltage curves of whole cell currents in control and Lino-treated cells. C: current-voltage curve of Lino-sensitive current. D: HMR1556 (HMR) inhibited outward currents in a concentration-dependent manner. E: concentration-dependent curve fit of HMR shown on a semilog plot. The y axis represents the uninhibited current normalized to the mean of control (I/I_control) at membrane voltages of +20, +30, and +40 mV. F and G: whole cell currents in control and ML277 (1 µM)-treated renal vascular SMCs. H and I: mefenamic acid (MA; 300 nM)-induced whole cell currents in renal vascular SMCs, which were inhibited by HMR (3 µM). *P < 0.05 (two-way ANOVA with a Bonferroni’s post hoc test).

K_V7.1 Does Not Regulate Basal Renal Vascular Tone in Neonatal Pigs

To determine whether K_V7.1 controls basal renal vascular tone in neonates, we examined the effect of HMR on the diameter of pressurized newborn pig distal interlobular arteries. Unlike phenylephrine, HMR did not significantly change the diameter of neonatal interlobular arteries (Fig. 3, A and B). Similarly, intrarenal artery infusion of HMR did not change basal MAP, RBF, and RVR (Fig. 3, C–F). These data suggest that K_V7.1 does not control the basal RVR in neonatal pigs.

Figure 3.

K_V7.1 does not control the basal renal vascular tone of neonatal pigs. A trace (A) and bar chart (B) showing the effect of phenylephrine (PE; 10 µM) and HMR1556 (HMR; 10 µM) on pressurized interlobular arteries. C–F: traces and bar charts demonstrating that intrarenal artery infusion of HMR (20 ng/kg/min for 20 min) did not alter basal mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR). *P < 0.05 vs. baseline (two-tailed paired t test). ns, not significant.

Inhibition of K_V7.1 Channels Attenuated Renal Autoregulation in Neonatal Pigs by Opposing Vasodilation

To investigate whether K_V7.1 contributes to neonatal renal autoregulation in response to a reduction in arterial pressure, we first established whether a step decrease in arterial pressure induces RBF autoregulation in neonatal pigs. Steady-state autoregulation at 2 min following step decreases in MAP was determined (Fig. 4A). A reduction in MAP from ∼80 to 60 mmHg (Fig. 4, A and B) induced effective autoregulation of RBF in neonatal pigs (AI: ∼0.21; Fig. 4D). However, autoregulation of RBF was less effective when MAP was reduced by ∼23 mmHg (AI: ∼0.46; Fig. 4, B–D). These data suggest that RBF autoregulation efficiency in the pigs is impaired when physiological arterial pressure falls >20 mmHg.

Figure 4.

A step decrease in arterial pressure triggered renal autoregulation in neonatal pigs. A and B: traces and bar charts showing that an ∼20-mmHg decrease in mean arterial pressure (MAP) triggered renal autoregulation. C: traces demonstrating that renal autoregulation was ineffective by decreasing MAP by ∼23 mmHg. D: renal autoregulation indexes at ∼20 and 23 mmHg. *P < 0.05 (two-tailed unpaired t test). RBF, renal blood flow.

Next, renal autoregulation was determined in neonatal pigs infused (via the intrarenal artery) with HMR before a 20-mmHg step decrease in arterial pressure. HMR caused a dose-dependent impairment of RBF autoregulation (Fig. 5, A–D). The HMR dose-response fit for the AI within 5–20 ng/kg/min gave an EC₅₀ value of 13.73 ng/kg/min (Fig. 5D). These data reveal that K_V7.1 contributes to neonatal RBF autoregulation triggered by a step decrease in arterial pressure.

Figure 5.

Inhibition of K_V7.1 channels attenuated renal autoregulation in neonatal pigs by opposing vasodilation. A and B: traces illustrating the response of renal blood flow (RBF) to an ∼20 mmHg reduction in mean arterial pressure in the absence and presence of HMR1556 (HMR; 20 ng/kg/min for 20 min). C: bar charts summarizing renal autoregulation indexes in the absence and presence of HMR [5 ng/kg/min (a), 10 ng/kg/min (b), and 20 ng/kg/min (c) for 20 min]. D: HMR dose-response fit for the autoregulation index. *P < 0.05 (one-way ANOVA with Bonferroni’s post hoc test). MAP, mean arterial pressure.

DISCUSSION

Birth requires changes in homeostatic mechanisms for an infant to adapt to the transition from fetal to ex utero life. During the perinatal period, the kidney undergoes postnatal maturation that involves morphological and functional changes (29, 30). Perinatal adaptive changes in the kidney may involve alterations in the activity of signal transduction molecules and vasoactive mediators that control renal hemodynamics (29, 30). Developmental and maturational changes in KCNQ expression levels and activity have been reported in rodents’ brains, neurons, and saphenous arteries (5, 51–54). Whether KCNQ expression is altered during kidney development and maturation was unknown. This study shows differential expression of KCNQ isoforms in the neonatal pig kidneys, brain, and heart, with only KCNQ1 mRNA detected in renal microvessels and kidneys. We also demonstrate that KCNQ isoforms in porcine renal microvessels are dependent on kidney maturation. The average gestation period of a domestic pig is 115 days, and nephrogenesis continues after birth until postnatal day 21 (55–57). Our data indicate that KCNQ1 is expressed in fetal pig kidneys as early as gestational day 50 and that its expression level remains the same throughout the postnatal nephrogenic period. Whether KCNQ1 is required for kidney development remains to be determined.

Immunofluorescence staining detected K_V7.1, K_V7.4, and K_V7.5 in the smooth muscle layer of renal arteries (2) and K_V7.4 in afferent arterioles of adult rats (18). However, we detected only KNCQ1 mRNA in SMCs isolated from neonatal pig preglomerular microvessels. Perhaps renal vascular KCNQ isoform expression may vary due to interspecies differences, age, or detection methods. In addition to the data demonstrating plasma membrane localization of K_V7.1, we show here that activation of the channels stimulates outward currents in neonatal pig renal vascular SMCs. HMR, a selective K_V7.1 channel blocker, inhibited the outward currents at a concentration of ≤10 µM. However, a higher concentration of the nonselective K_V7 blocker linopirdine (200 µM) is required to inhibit the outward currents in the cells. These findings are consistent with published data demonstrating that 100 µM linopirdine is needed to completely block KCNQ2/KCNQ3 heteromers (45). Linopirdine (200 µM) also inhibited KCNQ4 and KCNQ3/KCNQ4 by ∼30% and 75%, respectively (44). For the first time, these data indicate functional expression of K_V7.1 channels in neonatal renal vascular SMCs and suggest that these channels may contribute to SMC-dependent renal vasoregulation.

Renal vasoregulation by K_V7 has been extensively studied in adult rats and controlled by signaling molecules, including sodium-myo-inositol transporter 1, G protein βγ-subunits, and exchange protein directly activated by cAMP (27, 58–61). XE991, a K_V7 inhibitor, attenuated isoprenaline-mediated renal artery relaxation induced by microtubule inhibition in adult rats (62). A previous study has also shown in adult rats that atrial natriuretic peptide-induced relaxations of the renal arteries and aorta were diminished by linopirdine but not HMR (63). Activation of K_V7 channels by flupirtine caused renal vasorelaxation and a slight increase in RBF in adult rats (18). On the other hand, XE991 contracted adult rat interlobar arteries and caused a slight reduction in afferent arteriolar diameter and RBF (18), suggesting that K_V7 channels control the resting renal vascular tone in adult rats (18). Since flupirtine and XE991 are nonselective K_V7 modulators and their effects on kidney perfusion were mild, the specific contributions of K_V isoforms to renal vascular tone were unclear. To investigate whether K_V7.1 maintains the resting tone of resistance size renal vessels in neonates, we examined the effect of K_V7.1 blockade on neonatal pig interlobular arteries. HMR did not significantly modulate the resting tone of the arteries. Correspondingly, direct infusion of HMR into the kidney to minimize its systemic effects did not alter basal MAP, RBF, and RVR in the pigs. These results suggest that K_V7.1 is unlikely to contribute to basal RVR in neonates. Our findings are consistent with previous reports showing that K_V7.1 channels do not regulate adult rodent aorta and mesenteric, intrapulmonary, and renal basal arterial tone (6, 16, 64).

The basal MAP of anesthetized and mechanically ventilated 3- to 5-day-old neonatal pigs is ∼80 mmHg. We show here that a reduction in MAP from ∼80 to 60 mmHg triggered an immediate passive fall in RBF, followed by vasodilation that caused blood flow to return toward the basal level. However, the effective RBF autoregulation was impaired when the arterial pressure was reduced by ∼23 mmHg, suggesting that the lower limit for renal autoregulation in neonatal pigs is ∼60 mmHg.

Increased intraluminal pressure may activate large-conductance Ca²⁺-activated K⁺ and 4-aminopyridine-sensitive K_V1 channels as a negative feedback mechanism to limit myogenic constriction (65–67). Whether this reactivity occurs in the renal microvasculature is unknown. Also, the cellular mechanisms by which a drop in arterial pressure within the autoregulation range promotes renal autoregulation remain unresolved but involve vasodilation. Since activation of SMC K_V channels and the resultant hyperpolarization induces vasodilation, we examined whether inhibition of the channels will alter renal autoregulation. Although blockade of K_V7.1 did not modulate basal RVR in the pigs, it impaired renal autoregulation evoked by a step decrease in arterial pressure, indicating that K_V7.1 contributes to renal autoregulation. Our study did not determine the links between arterial pressure reduction and K_V7.1 activation, but possibilities include local production of mediators that activate the channels. Also, a step decrease in arterial pressure and the accompanying transient reduction in RBF may result in acute moderate hypoxia, which causes vasodilation by activating K⁺ channels (67–71). These conjectures remain hypothetical and require further investigation.

In conclusion, our study demonstrates that the predominant K_V7 isoform in neonatal pig preglomerular vascular SMC is K_V7.1. K_V7.1 does not control the resting tone of neonatal renal microvessels but contributes to vasodilation that restores RBF to basal levels in the setting of a transient decrease in arterial pressure within the autoregulation range.

GRANTS

A.A. was supported by National Institutes of Health Grants R01DK101668, R56DK120595, R01DK120595, and R01DK127625 and by American Heart Association Grant-in-Aid 16GRNT30990069. D.P.-N. was supported by American Heart Association Postdoctoral Fellowship 18POST34030240.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.A. conceived and designed research; D.P.-N., P.K., J.M.A., H.S., and R.K.B. performed experiments; D.P.-N., P.K., J.M.A., H.S., and A.A. analyzed data; D.P.-N., P.K., J.M.A., H.S., and A.A. interpreted results of experiments; D.P.-N., P.K., and A.A. prepared figures; D.P.-N., P.K., R.K.B., and A.A. drafted manuscript; D.P.-N., P.K., J.M.A., H.S., R.K.B., and A.A. edited and revised manuscript; D.P.-N., P.K., J.M.A., H.S., R.K.B., and A.A. approved final version of manuscript.