1. Structural Biology and Molecular Biophysics
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Intravascular flow stimulates PKD2 (polycystin-2) channels in endothelial cells to reduce blood pressure

  1. Charles E MacKay
  2. M Dennis Leo
  3. Carlos Fernández-Peña
  4. Raquibul Hasan
  5. Wen Yin
  6. Alejandro Mata-Daboin
  7. Simon Bulley
  8. Jesse Gammons
  9. Salvatore Mancarella
  10. Jonathan H Jaggar  Is a corresponding author
  1. Department of Physiology University of Tennessee Health Science Center Memphis, United States
Research Article
Cite this article as: eLife 2020;9:e56655 doi: 10.7554/eLife.56655
9 figures, 1 table and 1 additional file

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.

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.

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.

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.

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.

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).
Schematic illustration of the mechanisms by which endothelial cell PKD2 channels elicit flow-mediated vasodilation.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (M. musculus)Pkd2fl/flBaltimore PKD Core CenterPMID:20862291Mice with Pkd2 gene flanked by loxP regions.
Strain, strain background (M. musculus)Cdh5(PAC)-creERT2Cancer Research UKRRID:MGI:3848984Mice with tamoxifen-inducible Cre recombinase that is expressed
specifically in endothelial cells.
Strain, strain background (M. musculus)Pkd2fl/fl: Cdh5(PAC)-creERT2This paperMouse line created in-house by mating Pkd2fl/fl with Cdh5(PAC)-creERT2. Mice with inducible endothelial cell-specific deletion of PKD2.
AntibodyAnti-PKD2
(rabbit polyclonal)
Baltimore PKD CoreRabbit mAB 3374 CT-14/4IF 1:200 dilution
AntibodyAnti-PKD2 (mouse monoclonal)Santa CruzCat# sc-47734
RRID:AB_672380
WB 1:100 dilution
AntibodyAnti-PKD1 (mouse monoclonal)Santa CruzCat# sc-28331
RRID:AB_672377
WB 1:100 dilution
AntibodyAnti-Piezo1 (rabbit polyclonal)ProteintechCat 15939–1-AP.WB 1:100 dilution
AntibodyAnti-SK3 antibodyAbcamCat# ab28631
RRID:AB_775888
WB 1:100 dilution
AntibodyAnti-IK1 Antibody (D-5) (mouse monoclonal)Santa CruzCat# sc-365265
RRID:AB_10841432
WB 1:100 dilution
AntibodyAnti-eNOS (mouse monoclonal)AbcamCat# ab76198
RRID:AB_1310183
WB 1:100 dilution
AntibodyAnti-p-eNOS (rabbit polyclonal)Cell signaling TechnologyCat# 9571
RRID:AB_329837
WB 1:100 dilution
AntibodyAnti-GPR68NOVUS BiologicalsCat# NBP2-32747WB 1:100 dilution
AntibodyAnti-TRPV4
(clone 1B2.6) (mouse monoclonal)
Millipore SigmaCat# MABS466WB 1:100 dilution
AntibodyAnti-Actin (mouse monoclonal)Millipore SigmaCat# MAB1501
RRID:AB_2223041
WB 1:5000 dilution
AntibodyAlexa 555 secondary antibodies (anti rabbit and anti mouse)Thermo FisherCat# A-21429
(RRID:AB_141761) and # A-31570 (RRID:AB_2536180)
IF 1:400 dilution
AntibodyAlexa 488 secondary antibodies (anti rat)Thermo FisherCat# A-21470
RRID:AB_2535873
IF 1:400
dilution
OtherNuclear staining (DAPI)Thermo FisherCat# 3571
RRID:AB_2307445
IF 1:1000 dilution

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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