pkd2  (Alomone Labs)

Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Representative RT-PCR showing that mRNA for PKD1 is present in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl but absent in isolated endothelial cells from tamoxifen-treated Pkd1 fl/fl :Cdh5(PAC)-CreERT2 mice. Representative of 5 independent experiments for each genotype. (B) Representative Western blots illustrating PKD1, PKD2, AT1, eNOS, Fzd-7 and actin proteins in mesenteric arteries of Pkd1 fl/fl and Pkd1 ecKO mice. (C) Mean data from experiments shown in panel B. Significance was assessed using Student t-tests, n=5 independent mesenteric arterial lysates for each genotype and each protein. (D) Immunofluorescence images (representative of three mesenteric arteries from three mice for each genotype) illustrating that PKD1 (Alexa Fluor 546) is abolished in endothelial cells of en face mesenteric arteries from Pkd1 ecKO mice. CD31 (Alexa Fluor 488) and DAPI are also shown. Scale bars, 50 μm. (E) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt9b (3 μg/ml) applied in continuous flow in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (F) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt9b (3 µg/ml) applied under continuous flow (10 dyn/cm 2 ) in pressurized (80 mmHg) mesenteric arteries from Pkd1 fl/fl and Pkd1 ecKO mice. (G) Mean data from experiments shown in panels E and F, and the modulation of flow and Wnt9b-mediated vasodilation by SRI37892 (Fzd-7 Inh, 5 µM). Significance was assessed using a two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=10 arteries from 7 mice of each genotype for flow, flow+ Wnt9b and Wnt9b. n=7 arteries from 5 mice for Fzd-7 Inh+flow and Fzd-7 Inh+flow+Wnt9b. n=5 arteries from 5 mice for each genotype for flow + boiled Wnt9b. (H) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular Wnt5a (3 µg/ml). (I) Diameter responses to intravascular flow (10 dyn/cm 2 ) and intravascular boiled Wnt5a (3 µg/ml). (J) Mean data from experiments shown in panels H and I and the modulation of flow and Wnt5a-induced vasodilation by SRI37892 (Fzd-7 Inh, 5 μM). Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=8 arteries from 6 mice from Pkd1 fl/fl for flow, flow+ Wnt5a. n=5 arteries from 5 mice from Pkd1 ecKO for flow, flow+ Wnt5a. n=5 arteries from 5 mice of each genotype for flow + boiled Wnt5a. n=8 arteries from 6 mice from Pkd1 fl/fl for flow+ Fzd-7 Inh and flow+ Wnt5a+ Fzd-7 Inh.

Article Snippet: Membranes were blocked with 5% milk or 5% BSA and incubated with one of the following primary antibodies: PKD1 (Polycystic Kidney Disease Research Resource Consortium, Baltimore), PKD2 (Alomone), eNOS (Abcam), p-eNOS (Cell Signaling), Wnt9b (R&D Systems), Wnt5a (R&D Systems), AT1 receptor (Alomone) or actin (Cell Signaling) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Representative whole-cell current recordings obtained from freshly isolated mesenteric endothelial cells of a Pkd1 fl/fl and Pkd1 ecKO mouse in control (black, Ctrl), Wnt9b (blue, 1.5 μg/ml) or Wnt9b + Gd 3+ (green, 100 μM). (B) Mean data from experiments shown in panel A. Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=7 cells from 5 mice from each genotype. (C) Representative gel showing that mRNA for PKD2 is present in isolated endothelial cells from tamoxifen-treated Pkd2 fl/fl mice but absent in isolated endothelial cells from tamoxifen-treated Pkd2 fl/fl :Cdh5(PAC)-CreERT2 mice, representative of 5 independent experiments for each genotype. (D) Representative Western blots illustrating protein levels in mesenteric arteries of Pkd2 fl/fl and Pkd2 ecKO mice. (E) Mean data from experiment shown in panel C. Significance was assessed using Student t-tests, n=5 independent mesenteric arterial lysates from each genotype and for each protein. (F) En-face immunofluorescence imaging (representative of three mesenteric arteries from three mice for each genotype) illustrating that PC-2 (Alexa Fluor 546) is present in mesenteric artery endothelial cells of Pkd2 fl/fl mice, but absent in endothelial cells of Pkd2 ecKO mice. CD31 (Alexa Fluor 488) and DAPI are also shown. Scale bars, 50 μm. (G) Diameter responses to low flow (10 dyn/cm 2 ) and Wnt9b (3 µg/ml) in pressurized (80 mmHg) mesenteric arteries from Pkd2 fl/fl and Pkd2 ecKO mice. (H) Mean data for experiments shown in panel G. Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=9 arteries from 5 mice from Pkd2 fl/fl for flow, flow+ Wnt9b. n=7 arteries from 5 mice from Pkd2 ecKO for flow, flow+ Wnt9b.

Article Snippet: Membranes were blocked with 5% milk or 5% BSA and incubated with one of the following primary antibodies: PKD1 (Polycystic Kidney Disease Research Resource Consortium, Baltimore), PKD2 (Alomone), eNOS (Abcam), p-eNOS (Cell Signaling), Wnt9b (R&D Systems), Wnt5a (R&D Systems), AT1 receptor (Alomone) or actin (Cell Signaling) overnight at 4°C.

Techniques: Isolation, Control, Western Blot, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

doi: 10.64898/2026.03.17.712518

Figure Lengend Snippet: (A) Wnt9b stimulates dilation in pressurized (80 mmHg) mesenteric arteries of Pkd1 fl/fl mice through eNOS and SK channel activation. Low flow (10 dyn/cm 2 ) was applied and used to introduce Wnt9b (3 µg/ml) into the lumen, after which flow was stopped and L-NNA (100 µM) or apamin (300 nM) were applied abluminally. (B) Mean data for experiments shown in panel A. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=16 arteries from 10 Pkd1 fl/fl mice for flow, flow+ Wnt9b and Wnt9b. n=8 arteries from 5 Pkd1 fl/fl mice for Wnt9b+L-NNA, and Wnt9b+Apamin.(C) Western blot illustrating p-eNOS (serine1176), total eNOS, and actin in segments of first- to fifth-order mesenteric arteries of Pkd1 fl/fl and Pkd1 ecKO mice. Representative of 5 independent experiments. (D) Mean data for p-eNOS/total eNOS from experiments in panel C. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. n=5 independent mesenteric arterial lysates from each genotype (E) Mean data illustrating the effect of Wnt9b (3 µg/ml) on total eNOS from experiments in panel C. Significance was assessed using Student t-tests. n=5 independent mesenteric arterial lysates from each genotype. (F) Western blot illustrating p-eNOS (serine 1176), total eNOS, and actin in endothelial cells and modulation by Wnt9b (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM) and NSC668036 (Dvl Inh, 10 µM). Representative of 8 independent experiments. (G) Mean data from experiments shown in panel F. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. (H) Mean data illustrating nitric oxide generation from endothelial cells and modulation by Wnt9b (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM) and NSC668036 (Dvl Inh, 10 µM). Representative of 7 independent experiments. (I) Western blot illustrating p-eNOS (serine 1176), total eNOS, and actin in endothelial cells and their modulation by Wnt5a (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM), NSC668036 (Dvl Inh, 10 µM), and SP600125 (JNK Inh, 100 nM). Representative of 8 independent experiments. (J) Mean data from experiments shown in panel I. Significance was assessed using one-way ANOVA with Holm-Sidak post hoc multiple comparisons test. (K) Mean data illustrating nitric oxide generation by endothelial cells and modulation by Wnt5a (900 ng/ml), BAPTA-AM (BAPTA, 5 µM), SRI37892 (Fzd-7 Inh, 2.5 µM), NSC668036 (Dvl Inh, 10 µM), and SP600125 (JNK Inh, 100 nM). Representative of 8 independent experiments. (L) Mean data illustrating plasma Wnt9b at baseline and 5 min post Wnt9b intravascular infusion (30 μg/kg) in Pkd2 fl/fl and Pkd2 ecKO mice. n=6 mice for each genotype. Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test. (M) Mean data illustrating plasma nitric oxide at baseline and 5 min post Wnt9b infusion (30 μg/kg) in Pkd1 fl/fl , Pkd1 ecKO, Pkd2 fl/fl and Pkd2 ecKO mice. n=8 for Pkd1 fl/fl and Pkd1 ecKO mice. n=7 for Pkd2 fl/fl and n=7 Pkd2 ecKO mice. Significance was assessed using two-way ANOVA with Holm-Sidak post hoc multiple comparisons test.

Article Snippet: Membranes were blocked with 5% milk or 5% BSA and incubated with one of the following primary antibodies: PKD1 (Polycystic Kidney Disease Research Resource Consortium, Baltimore), PKD2 (Alomone), eNOS (Abcam), p-eNOS (Cell Signaling), Wnt9b (R&D Systems), Wnt5a (R&D Systems), AT1 receptor (Alomone) or actin (Cell Signaling) overnight at 4°C.

Techniques: Activation Assay, Introduce, Western Blot, Clinical Proteomics