8 figures, 2 tables and 2 additional files

Figures

Figure 1 with 3 supplements
Activation of Cre recombinase abolishes PKD2 in arterial myocytes of Pkd2fl/fl:myh11cre/ERT2 mice.

(A) RT-PCR showing the absence of PKD2 transcript in isolated myocytes from tamoxifen-treated Pkd2fl/fl:myh11-cre/ERT2 mice. (B) Western blots illustrating the effect of tamoxifen-treatment in Pkd2fl…

https://doi.org/10.7554/eLife.42628.002
Figure 1—figure supplement 1
Genotyping of mouse lines.

Ethidium bromide gel illustrating PCR products in vasculature of C57BL/6J (WT) mice and tamoxifen-treated Pkd2fl/fl and Pkd2fl/fl:myh11cre/ERT2 mice.

https://doi.org/10.7554/eLife.42628.003
Figure 1—figure supplement 2
PKD2 protein is lower in aorta and mesenteric and hindlimb arteries from tamoxifen-treated Pkd2fl/fl:myh11-cre/ERT2 mice.

(A) Western blots illustrating PKD2 protein was lower in mesenteric arteries of tamoxifen-treated Pkd2fl/fl:myh11-cre/ERT2 mice, whereas other proteins were similar. (B) Mean data for proteins in …

https://doi.org/10.7554/eLife.42628.004
Figure 1—figure supplement 3
Several proteins that regulate arterial contractility are unchanged in tamoxifen-treated Pkd2fl/fl:myh11-cre/ERT2 mice.

(A) Western blots illustrating Angiotensin II type one receptor (AT1R), Piezo1, α1-adrenergic receptor A (α1A), α1-adrenergic receptor B (α1B), α1-adrenergic receptor D (α1D) and G protein-coupled …

https://doi.org/10.7554/eLife.42628.005
Figure 2 with 1 supplement
Pkd2 smKO mice are hypotensive with normal cardiac function and renal histology.

(A) Original telemetric blood pressure recordings from Pkd2 smKO and Pkd2fl/fl mice. (B) Mean systolic and diastolic blood pressures in Pkd2fl/fl (n = 11) and Pkd2 smKO (n = 12) mice. * indicates p<0…

https://doi.org/10.7554/eLife.42628.006
Figure 2—figure supplement 1
Lower blood pressure is sustained in Pkd2 smKO mice.

(A) Mean arterial blood pressure (MAP) in Pkd2 smKO and Pkd2fl/fl mice (n = 6 per group). * indicates p<0.05 versus Pkd2fl/fl. (B) Mean data of locomotor activity, n = 6 per group.

https://doi.org/10.7554/eLife.42628.007
Figure 3 with 1 supplement
Pressure-induced vasoconstriction is attenuated in Pkd2 smKO mouse hindlimb arteries.

(A) Representative traces illustrating diameter responses to intravascular pressure in gastrocnemius arteries of Pkd2fl/fl and Pkd2 smKO mice. (B) Mean data for myogenic tone in gastrocnemius …

https://doi.org/10.7554/eLife.42628.009
Figure 3—figure supplement 1
Myocyte PKD2 knockout does not alter phenylephrine or angiotensin II-induced vasoconstriction in hindlimb arteries.

(A) Mean passive diameter at 80 mmHg of first-order gastrocnemius arteries (G) and third-, fourth-and fifth-order mesenteric arteries (M) (Pkd2fl/fl: G, n = 5; M3rdn = 4; M4thn = 5; M5thn = 5 and Pkd

https://doi.org/10.7554/eLife.42628.010
Figure 4 with 1 supplement
PKD2 channels contribute to pressure-induced hindlimb artery depolarization and swelling-activated Na + currents in hindlimb artery myocytes.

(A) Representative traces of microelectrode impalements under indicated conditions illustrating that pressure-induced depolarization is attenuated in gastrocnemius arteries of Pkd2 smKO mice. …

https://doi.org/10.7554/eLife.42628.011
Figure 4—figure supplement 1
PKD2 knockout does not alter phenylephrine (PE)-activated ICat in isolated hindlimb artery myocytes.

(A) Representative I-V relationships recorded between −100 and +100 mV in the same hindlimb artery myocytes of Pkd2fl/fl or Pkd2 smKO mice in control and PE (10 µM). (B) Mean data for current …

https://doi.org/10.7554/eLife.42628.012
Figure 5 with 1 supplement
Pressure-induced vasoconstriction is unaltered, whereas phenylephrine-induced vasoconstriction is attenuated, in mesenteric arteries of Pkd2 smKO mice.

(A) Mean vasoconstriction over a range of pressures in resistance-size mesenteric arteries from Pkd2fl/fl (n = 7) and Pkd2 smKO (n = 9) mice. (B) Original recordings of concentration-dependent, …

https://doi.org/10.7554/eLife.42628.013
Figure 5—figure supplement 1
Myocyte PKD2 knockout attenuates phenylephrine-induced vasoconstriction, but does not alter pressure or angiotensin II-induced vasoconstriction in mesenteric arteries.

(A) Mean myogenic tone at 80 mmHg illustrating that myogenic tone is similar in third-, fourth-and fifth-order mesenteric arteries and unaltered by PKD2 knockout (Pkd2fl/fl: 3rd n = 4; 4th n = 5; 5th

https://doi.org/10.7554/eLife.42628.014
Figure 6 with 1 supplement
PKD2 channels contribute to phenylephrine-induced mesenteric artery depolarization and INa in mesenteric artery myocytes.

(A) Representative traces of microelectrode impalements illustrating that phenylephrine (PE, 1 µM)-induced depolarization is attenuated in mesenteric arteries of Pkd2 smKO mice. Scale bars: Y = 10 …

https://doi.org/10.7554/eLife.42628.015
Figure 6—figure supplement 1
PKD2 knockout does not alter swelling-activated Icat in isolated mesenteric artery myocytes.

(A) Representative I-V relationships from the same isolated mesenteric artery myocytes of Pkd2fl/fl or Pkd2 smKO mice in isosmosmotic (300 mOsm), hyposmotic (250 mOsm) and hyposmotic (250 mOsm) + Gd3…

https://doi.org/10.7554/eLife.42628.016
Angiotensin II-induced hypertension is attenuated in Pkd2 smKO mice.

(A) Telemetric blood pressure time course showing the development of angiotensin II-induced hypertension in Pkd2fl/fl (n = 6) and Pkd2 smKO mice (n = 9). Osmotic minipumps containing either saline …

https://doi.org/10.7554/eLife.42628.017
Arterial myocyte PKD2 knockout attenuates vasoconstriction and arterial wall remodeling during hypertension.

(A) Mean phenylephrine-induced vasoconstriction in pressurized (80 mmHg) mesenteric arteries from angiotensin II-treated mice (Pkd2fl/fl, n = 7–8; Pkd2 smKO, n = 8).

https://doi.org/10.7554/eLife.42628.018

Tables

Table 1
Plasma hormones and plasma and urine electrolytes.
https://doi.org/10.7554/eLife.42628.008
Pkd2fl/flPkd2 smKO
Plasma hormones (pg/ml)
Angiotensin II202.5 ± 17.2 (n = 16)204.7 ± 12.1 (n = 15)
Aldosterone341.0 ± 18.2 (n = 16)365.6 ± 14.0 (n = 10)
ANP107.2 ± 9.4 (n = 18)118.3 ± 12.3 (n = 18)
Plasma electrolytes (mM)
Na+142.3 ± 1.0 (n = 7)152.0 ± 5.5 (n = 6)
K+6.4 ± 0.4 (n = 7)6.8 ± 0.3 (n = 6)
Cl-78.8 ± 0.7 (n = 7)84.1 ± 4.3 (n = 6)
Urine electrolytes (mM)
Na+136.9 ± 9.3 (n = 15)147.0 ± 16.2 (n = 15)
K+563.2 ± 30.2 (n = 15)548.6 ± 37.6 (n = 15)
Cl-455.4 ± 24.7 (n = 15)479.1 ± 42.0 (n = 15)
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(Mus musculus)
Pkd2fl/flJohn Hopkins
PKD Core
PMID 20862291Mice with Pkd2 gene flanked
by loxP regions
Strain, strain
background
(M. musculus)
Myh11-cre/ERT2Jackson Laboratories.
PMID 18084302
Stock # 019079
RRID:IMSR_JAX:019079
Mice with tamoxifen-inducible
Cre recombinase that is
expressed under the
smooth muscle myosin
heavy polipeptide 11
(Myh11) promoter.
Strain, strain
background
(M. musculus)
Pkd2fl/fl:Myh11-cre/ERT2This paperMouse line created in-house
by mating Pkd2fl/fl with
Myh11-cre/ERT2. Mice with
inducible smooth
muscle-specific deletion
of PKD2.
Antibodyanti PKD2
(rabbit polyclonal)
Baltimore PKD CoreRabbit mAB
3374 CT-14/4,
IF 1:200 dilution
Antibodyanti PKD2
(mouse monoclonal)
Santa CruzCat. # sc-100415
RRID:AB_1127284
WB 1:100 dilution,
IF 1:100 dilution
Antibodyanti PKD1
(mouse monoclonal)
Santa CruzCat. # sc-130554
RRID:AB_2163355
WB 1:100 dilution
Antibodyanti CaV1.2
(mouse monoclonal)
UC Davis/NIH
NeuroMab
Cat# 73–053.
RRID:AB_10672290
WB 1:100 dilution
Antibodyanti ANO1
(rabbit monoclonal)
AbcamCat. # ab64085
RRID:AB_1143505
WB 1:100 dilution
Antibodyanti TRPC6
(rabbit polyclonal)
AbcamCat. # ab62461
RRID:AB_956401
WB 1:1000 dilution
Antibodyanti TRPM4
(rabbit polyclonal)
AbcamCat. # ab106200
RRID:AB_10865760
WB 1:500 dilution
Antibodyanti Actin
(mouse monoclonal)
Millipore SigmaCat. # MAB1501
RRID:AB_2223041
WB 1:5000 dilution
AntibodyAlexa 555 secondary
antibodies (anti
rabbit and anti mouse)
ThermofisherCat. # A-21429
(RRID:AB_141761)
and # A-31570
(RRID:AB_2536180)
IF 1:400 dilution
OtherNuclear staining (DAPI)ThermofisherCat. # 3571
RRID:AB_2307445
IF 1:1000 dilution
Commercial
assay or kit
EZ-Link Sulfo-NHS
-LC-LC-Biotin
ThermofisherCat. # 21338
Commercial
assay or kit
EZ-Link
Maleimide-PEG2-Biotin
ThermofisherCat. # 21901BID
Commercial
assay or kit
Mouse Angiotensin
II ELISA kit
ElabscienceCat. # E-EL-M2612
Commercial
assay or kit
Mouse Atrial Natriuretic
Peptide ELISA kit
ElabscienceCat. # E-EL-M0166
Commercial
assay or kit
Mouse Aldosterone
ELISA kit
MybiosourceCat. # MBS775626
Chemical
compound, drug
Angiotensin IISigma-AldrichCat. # A9525

Additional files

Supplementary file 1

Primers used for RT PCR.

The PKD2 forward primer recognizes nucleotides in exon 9 and 10 and the reverse primer was aligned with a sequence in exon 13.

https://doi.org/10.7554/eLife.42628.019
Transparent reporting form
https://doi.org/10.7554/eLife.42628.020

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