Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure
- Department of Physiology University of Tennessee Health Science Center Memphis, United States
Figures
Figure 1 with 1 supplement
Generation and validation of Pkd2 ecKO mice.
(A) Representative Western blots illustrating the effect of tamoxifen-treatment of Pkd2fl/fl and Pkd2fl/fl: Cdh5(PAC)-creERT2 mice on PKD2, PKD1, Piezo1, GPR68, eNOS, SK3, IK and TPRV4, proteins in mesenteric arteries. (B) Mean data for proteins in mesenteric arteries of tamoxifen-treated Pkd2fl/fl: Cdh5(PAC)-creERT2 mice when compared to those in tamoxifen-treated Pkd2fl/fl mice. n = 3–8. * indicates p<0.05 versus Pkd2fl/fl. (C) En-face immunofluorescence imaging illustrating that PKD2 protein (Alexa Fluor 555) is abolished in endothelial cells of mesenteric arteries in tamoxifen-treated Pkd2fl/fl: Cdh5(PAC)-creERT2 mice (representative of 6 mesenteric arteries). CD31 (Alexa Fluor 488) and DAPI are also shown. Scale bars = 50 µm.
Figure 1—figure supplement 1
Genotyping of mouse lines.
Genomic PCR indicating that tamoxifen (1 mg/ml, i.p., 3 days) stimulated Cre-recombination in mesenteric arteries of Pkd2fl/fl: Cdh5(PAC)-creERT2 mice. Representative of n = 3.
Figure 2 with 4 supplements
PKD2 channels contribute to intravascular flow-, but not ACh-, mediated vasodilation.
(A) Original traces illustrating responses to ACh (10 µM) and Ca2+-free solution (passive diameter) in pressurized (80 mmHg) mesenteric arteries from Pkd2fl/fl and Pkd2 ecKO mice. (B) Original trace of flow-mediated dilation in pressurized (80 mmHg) mesenteric arteries from Pkd2fl/fl and Pkd2 ecKO mice. (C) Mean diameter changes in response to flow (15 dyn/cm2) or ACh (10 µM). *p<0.05 vs. Pkd2fl/fl. n = 8 for each. # p<0.05 vs. flow in the same genotype. (D) Original traces illustrating diameter responses to stepwise increases in intravascular flow in pressurized (80 mmHg) mesenteric arteries from Pkd2fl/fl and Pkd2 ecKO mice. (E) Mean data. The Pkd2-sensitive component of flow-mediated vasodilation is illustrated in blue. *p<0.05 vs. Pkd2fl/fl. n = 5 for Pkd2fl/fl, n = 4 for Pkd2 ecKO.
Figure 2—figure supplement 1
Endothelial denudation abolishes ACh-mediated vasodilation.
(A) Original traces demonstrating responses to ACh (10 µM) and SNP (10 µM) in EC-intact (black) and EC-denuded (blue) pressurized (80 mmHg) mesenteric arteries of Pkd2fl/fl. (B) Mean data. *p<0.05 vs. EC-intact. n = 8 for each.
Figure 2—figure supplement 2
Endothelial denudation abolishes flow-mediated dilation.
(A) Original traces demonstrating reproducible flow responses (15 dyn/cm2) in pressurized Pkd2fl/fl mesenteric arteries. (B) Mean diameter changes in response to two consecutive flow (15 dyn/cm2) stimuli. n = 3. (C) Original traces demonstrating flow responses (15 dyn/cm2) in pressurized (80 mmHg) EC-intact and EC-denuded mesenteric arteries from Pkd2fl/fl mice. (D) Mean data for Pkd2fl/fl arteries. n = 7 for EC-intact. n = 6 for Pkd2fl/fl EC-intact. *p<0.05 vs.EC-intact. n = 8 for each.
Figure 2—figure supplement 3
Endothelial cell PKD2 knockout attenuates flow-mediated vasodilation over a broad shear stress range.
Mean data illustrating relative dilation to shear stress in pressurized (80 mmHg) Pkd2 ecKO arteries compared with Pkd2fl/fl arteries. n = 4–5.
Figure 2—figure supplement 4
Smooth muscle-specific vasoconstriction and passive diameter are unaltered in Pkd2 ecKO arteries.
(A) Representative traces illustrating the development of myogenic tone in pressurized (80 mmHg) Pkd2fl/fl and Pkd2 ecKO arteries. (B) Mean myogenic tone in pressurized (80 mmHg) mesenteric arteries from Pkd2fl/fl and Pkd2 ecKO. n = 8 for each. (C) Representative traces illustrating 60 mm K+ constriction in pressurized (10 mmHg) arteries. (D) Mean data for 60 mM K+-induced constriction. n = 8. (E) Mean data for passive diameter (Ca2+- free PSS) in pressurized (80 mmHg) arteries. n = 8.
Figure 3
EC PKD2 channels contribute to flow-mediated arterial hyperpolarization.
(A) Original membrane potential recordings obtained from microelectrode impalements in pressurized mesenteric arteries of Pkd2fl/fl and Pkd2 ecKO mice at static (10 and 80 mmHg) and with 80 mmHg and flow (15 dyn/cm2). All three impalements in Pkd2fl/fl and Pkd2 ecKO were from the same two arteries. (B) Mean data (Pkd2fl/fl: 10 mmHg, n = 8; 80 mmHg, n = 10; 80 mmHg + flow, n = 18; Pkd2 ecKO: 10 mmHg, n = 7; 80 mmHg, n = 12; 80 mmHg + flow, n = 16). *p<0.05 for 80 mmHg static versus 10 mmHg static in same genotype. # p<0.05 for 80 mmHg + flow versus 80 mmHg static in the same genotype. and indicates p<0.05 versus Pkd2fl/fl under the same condition.
Figure 4
Flow reduces steady-state inward current through a PKD2-mediated, Ca2+ influx-dependent mechanism in voltage-clamped mesenteric artery endothelial cells.
(A) Original recordings of steady-state current modulation by flow (10 ml/min) and effect of removing bath Ca2+ at −60 mV in endothelial cells from Pkd2fl/fl and Pkd2 ecKO mice. (B) Mean data for flow-induced transient inward current. n = 9 for Pkd2fl/fl and n = 10 for Pkd2 ecKO. * indicates p<0.05 versus Pkd2fl/fl.(C) Mean data for steady-state currents in the presence and absence of flow and in the presence and absence of extracellular Ca2+ (Pkd2fl/fl: static + Ca2+, n = 9; static with zero Ca2+, n = 6; flow + Ca2+, n = 9; flow with zero Ca2+, n = 9 and Pkd2 ecKO: static + Ca2+, n = 9; static with zero Ca2+, n = 15; flow + Ca2+, n = 8; flow with zero Ca2+, n = 8). *p<0.05 versus static + Ca2+ conditions in the same genotype, and indicates p<0.05 vs Pkd2fl/fl under the same condition, # p<0.05 versus flow + Ca2+ in the same genotype.
Figure 5
Flow-mediated PKD2 channel activation stimulates SK/IK channels in mesenteric artery endothelial cells, leading to vasodilation.
(A) Original recordings of steady-state current modulation by flow (10 ml/min) and flow plus apamin/Tram-34 (300 nM of each) at −60 mV in mesenteric artery ECs from Pkd2fl/fl and Pkd2 ecKO mice. (B) Mean data (Pkd2fl/fl: static, n = 8; flow, n = 8; flow + apamin/Tram-34, n = 7. Pkd2 ecKO: static, n = 9, flow, n = 10; flow + apamin/Tram-34, n = 9). * indicates p<0.05 versus static in the same genotype and p<0.05 vs Pkd2fl/fl in the same conditions. # p<0.05 versus flow in the same genotype. (C) Original recordings of steady-state current modulation by apamin/Tram-34 (300 nM of each) in the absence of flow at −60 mV in ECs from Pkd2fl/fl and Pkd2 ecKO mice. (D) Mean data comparing responses to apamin/Tram-34 in static and flow conditions at −60 mV (Pkd2fl/fl: static, n = 6; flow, n = 7. Pkd2 ecKO: static, n = 6; flow, n = 9). *p<0.05 versus static control. # p<0.05 versus static + apamin/Tram-34 in the same genotype. and indicates p<0.05 for Pkd2 ecKO vs Pkd2fl/fl in the same condition.
Figure 6
PKD2 channels contribute to intravascular flow-mediated SK/IK channel activation and vasodilation.
(A) Representative traces illustrating responses to flow (15 dyn/cm2) and flow (15 dyn/cm2) + ACh (10 µM) in the presence and absence of apamin/Tram-34 (300 nM of each) in pressurized (80 mmHg) mesenteric arteries from Pkd2fl/fl and Pkd2 ecKO mice. (B) Representative traces illustrating responses to apamin/Tram-34 (300 nM of each) in the absence of intravascular flow in pressurized (80 mmHg) mesenteric arteries. (C) Mean data (Pkd2fl/fl: flow, n = 5; flow + apamin/Tram-34, n = 5; flow + ACh (10 µM), n = 5; flow + ACh (10 µM) + apamin/Tram-34, n = 5; static + apamin/Tram-34, n = 5. Pkd2 ecKO: flow, n = 5; flow + apamin/Tram-34, n = 5; flow + ACh (10 µM), n = 5; flow + ACh (10 µM) + apamin/Tram-34, n = 4; static + apamin/Tram-34, n = 5). * indicates p<0.05 versus Pkd2fl/fl in the same condition. # indicates p<0.05 for flow + apamin/Tram-34 versus flow in the same genotype. and indicates p<0.05 for flow + ACh versus flow + ACh + apamin/Tram-34 in the same genotype.
Figure 7
Flow-mediated PKD2 channel activation in ECs stimulates eNOS serine 1176 phosphorylation, leading to vasodilation.
(A) Original Western blots illustrating effects of flow (15 dyn/cm2) and Pkd2 ecKO on p-eNOS (S1176) and total eNOS proteins in Pkd2fl/fl and Pkd2 ecKO mesenteric arteries. (B) Mean data for flow-induced change (Δ) in proteins. n = 5 for Pkd2fl/fl. n = 4 for Pkd2 ecKO. * indicates p<0.05 versus static. # indicates p<0.05 versus same protein in Pkd2fl/fl. (C) Representative traces demonstrating flow (15 dyn/cm2)-mediated vasodilation in pressurized (80 mmHg) mesenteric arteries of Pkd2fl/fl and Pkd2 ecKO mice in the presence and absence of L-NNA (10 µM). (D) Mean data. n = 10 for Pkd2fl/fl. n = 5 for Pkd2 ecKO. * indicates p<0.05 versus Pkd2fl/fl in the same condition. # indicates p<0.05 versus flow in the absence of L-NNA (10 µM) in the same genotype.
Figure 8 with 1 supplement
Pkd2 ecKO elevates systemic blood pressure, but does not alter cardiac function or kidney histology.
(A) Mean diastolic and systolic blood pressures in Pkd2fl/fl and Pkd2 ecKO mice (n = 5 for each). * indicates p<0.05 versus Pkd2fl/fl. (B) Mean arterial blood pressure (MAP) (n = 5 for each). * indicates p<0.05 versus Pkd2fl/fl. (C–F) Mean echocardiography data. Heart rate (HR), Cardiac output (CO), fractional shortening (FS) and ejection fraction (EF) (n = 5 Pkd2fl/fl and n = 10 for Pkd2 ecKO). (G) Representative images of H and E stained kidney cortex used for histological assessment. Scale bars = 100 µm. (H) Mean proximal tubule length (n = 15 proximal tubules measured for each group from three individual mice). (I) Mean glomeruli surface area (n = 75 glomeruli measured per group from three individual mice).
Figure 8—figure supplement 1
Locomotion is similar in Pkd2fl/fl and Pkd2 ecKO mice (n = 5 for each).
Figure 9
Schematic illustration of the mechanisms by which endothelial cell PKD2 channels elicit flow-mediated vasodilation.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | Pkd2fl/fl | Baltimore PKD Core Center | PMID:20862291 | Mice with Pkd2 gene flanked by loxP regions. |
| Strain, strain background (M. musculus) | Cdh5(PAC)-creERT2 | Cancer Research UK | RRID:MGI:3848984 | Mice with tamoxifen-inducible Cre recombinase that is expressed specifically in endothelial cells. |
| Strain, strain background (M. musculus) | Pkd2fl/fl: Cdh5(PAC)-creERT2 | This paper | Mouse line created in-house by mating Pkd2fl/fl with Cdh5(PAC)-creERT2. Mice with inducible endothelial cell-specific deletion of PKD2. | |
| Antibody | Anti-PKD2 (rabbit polyclonal) | Baltimore PKD Core | Rabbit mAB 3374 CT-14/4 | IF 1:200 dilution |
| Antibody | Anti-PKD2 (mouse monoclonal) | Santa Cruz | Cat. # sc-100415 RRID:AB_1127284 | WB 1:100 dilution |
| Antibody | Anti-PKD1 (mouse monoclonal) | Santa Cruz | Cat. # sc-130554 RRID:AB_2163355 | WB 1:100 dilution |
| Antibody | Anti-Piezo1 (rabbit polyclonal) | Proteintech | Cat.# 15939–1-AP. | WB 1:100 dilution |
| Antibody | Anti-SK3 antibody | Abcam | Cat. # ab28631 RRID:AB_775888 | WB 1:100 dilution |
| Antibody | Anti-IK1 Antibody (D-5) (mouse monoclonal) | Santa Cruz | Cat. # sc-365265 RRID:AB_10841432 | WB 1:100 dilution |
| Antibody | Anti-eNOS (mouse monoclonal) | Abcam | Cat. # ab76198 RRID:AB_1310183 | WB 1:100 dilution |
| Antibody | Anti-p-eNOS (rabbit monoclonal) | Cell signaling Technology | Cat. # 9571 RRID:AB_329837 | WB 1:100 |
| Antibody | Anti-GPR68 | NOVUS Biologicals | Cat. # NBP2-32747 | WB 1:100 |
| Antibody | Anti-TRPV4 (clone 1B2.6) (mouse monoclonal) | Millipore Sigma | Cat. # MABS466 | WB 1:100 |
| Antibody | Anti-Actin (mouse monoclonal) | Millipore Sigma | Cat. # MAB1501 RRID:AB_2223041 | WB 1:5000 dilution |
| Antibody | Alexa 555 secondary antibodies (anti rabbit and anti mouse) | Thermo Fisher | Cat. # A-21429 (RRID:AB_141761) and # A-31570 (RRID:AB_2536180) | IF 1:400 dilution |
| Antibody | Alexa 488 secondary antibodies (anti rat) | Thermo Fisher | Cat. # A-21470 RRID:AB_2535873 | IF 1in 400 dilution |
| Other | Nuclear staining (DAPI) | Thermo Fisher | Cat. # 3571 RRID:AB_2307445 | IF 1:1000 dilution |
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Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure
eLife 9:e56655.
https://doi.org/10.7554/eLife.56655
