RESULTS:
To determine the effects of RAAS activation on the renal vasculature, we used a two-kidney-one-clip (2K1C) model (Figure 1A), which involves surgical stenosis of one of the renal arteries. To assess the vasoreactivity responses of the unclipped contralateral (right) renal arteries, wire myography was performed on the right (unclipped) renal arteries of clipped and sham control animals. We observed that contralateral clipped arteries contracted significantly less to 10 μM phenylephrine than their sham counterparts (Figure 1B). We also observed a significant improvement in endothelium-dependent vasodilation in response to ACh (Figure 1C). The calculated values with respect to sham control and renal clip animals is 1.6x10-7M ± 4.0x10-8M and 1.1x10-7M ± 3.2x10-8M, respectively for EC50, and we observed 55.9 ± 5.2% and 76.8 ± 5.1%, respectively for Emax values. Similarly, a significant improvement in NO-dependent vasodilation in response to the NO-donor, sodium nitroprusside (SNP), was observed (Figure 1D) with calculated EC50 values of 3.99x10-5 M ± 3.95x10-5 M and 1.22x10-5 M ± 7.83x10-6 M for sham and renal clip groups, respectively. These data also show a calculated Emax of 44.5 ± 4.5% versus 66.3 ± 4.2% for sham control and clipped animals, respectively. These findings indicate a significant improvement in the vasorelaxation responses of the contralateral clipped arteries over sham controls to both an endothelium-dependent vasodilator and a NO-donor.
To determine if the increased NO-dependent vasodilatory response in contralateral clipped arteries were due to changes in sGC expression, we quantified protein expression within renal arteries. We found that the expression of the β subunit of sGC (sGCβ) was significantly elevated in contralateral renal arteries of clipped animals (Figure 2B, F) compared to sham controls. No significant differences were seen in nuclei staining (Figure 2A, E), smooth muscle α-actin (ACTA2) expression (Figure 2C, G), or the endothelial cell marker, von Willebrand Factor (vWF, Figure 2D, H), between groups.
Next, we sought to determine what drives sGC expression changes in renal artery smooth muscle. It is well established that reduced renal blood flow increases angiotensin II (Ang II) in models of 2K1C (Murphy, et al., 1984), (Sadjadi, et al., 2002). Therefore, we treated rat renal preglomerular smooth muscle cells (RPGSMCs) with vehicle or 10-6 M Ang II to test if Ang II increased sGC expression. Ang II treatment led to increased RPGSMC cell area and augmented filamentous (F)-actin expression (Figure 3B, E), indicating RPGSMC hypertrophy (Stephenson, et al., 1998). Additionally, Ang-II resulted in increased sGCβ protein expression by 1.7-fold via immunofluorescence and 4-fold via western blot analysis (Figure 3C, F, G). Consistent with increased sGC protein expression, we found that Ang II treatment also caused a 3.6-fold increase in sGCα mRNA (Figure 4A) and a 4.4-fold increase in sGCβ mRNA (Figure 4B). To test if increased sGC expression impacted cGMP production and PKG activity, RPGSMCs treated with Ang II or vehicle, and subjected to treatment with the NO donor, DEA-NONOate, for 15 minutes prior to harvest to induce sGC-mediated cGMP production. Quantification of vasodilator stimulated protein (VASP) phosphorylated at the serine 239 position, a surrogate indicator of cGMP-dependent protein kinase activity (Smolenski, et al., 1998), showed an 8-fold increase in pVASP in Ang II-treated cells stimulated with DEA-NONOate compared to vehicle controls (Figure 3H). Taken together, these data show that Ang II in vitro augments sGC expression and cGMP signaling, indicating that elevated RAAS activity increases sGC expression and downstream signaling in vivo .
We next tested which Ang II receptor subtype- either the AT1R or the Angiotensin Type 2 Receptor (AT2R) -was responsible for increasing sGC mRNA and protein expression. RPGSMCs co-treated Ang II and Losartan, an AT1R antagonist (Timmermans, et al., 1995), caused inhibition of Ang II – induced increases in sGCα mRNA (Figure 4A), sGCβ mRNA (Figure 4B), and sGCβ protein expression (Figure 4C & D). Conversely, RPGSMCs co-treated with Ang II and PD123319, an AT2R antagonist (Blankley, et al., 1991), showed no significant impact on the Ang II – induced increases in sGCα mRNA (Figure 4A), sGCβ mRNA (Figure 4B), or sGCβ protein expression (Figure 4C & D).
Recently, we published evidence that the FoxO family of transcription factors regulate the mRNA expression of sGC in aortic smooth muscle (Galley, et al., 2019). To determine whether the FoxO family of transcription factors also influence the function of renal smooth muscle, we treated RPGSMCs with 10-6 M AS1842856, a small molecule Fox O transcription factor inhibitor (Nagashima, et al., 2010), alone and in conjunction with Ang II. When AS1842856 was administered alone to RPGSMCs, a significant reduction in sGCα mRNA (Figure 5A), sGCβ mRNA (Figure 5B), and sGCβ protein expression (Figure 5C) was observed. When administered with Ang II, AS1842856 produced no effect on either sGCα mRNA (Figure 5A), sGCβ mRNA (Figure 5B), or sGCβ protein expression (Figure 5C) when compared to vehicle controls. These data indicate the FoxO transcription factors are necessary for the Ang II-mediated sGC expression increases in renal smooth muscle.
Next, we tested Ang II receptor antagonists and FoxO inhibitors on cGMP production, determined by pVASP expression following NO-stimulation with DEA-NONOate. At baseline, where no DEA-NONOate-stimulation occurred, no significant differences were observed between the treatment groups. Similar to the observed effect in sGC expression, co-treatment with Ang II and PD123319 produced significant increases in downstream sGC function via VASP phosphorylation following DEA-NONOate stimulation compared to controls treated with DEA-NONOate. AS1842856 or Losartan showed no significant differences from control-treated cells stimulated with DEA-NONOate (Figure 6A & B). These data show that PD123319 has no significant effect and that the blunting effect of Losartan or AS1842856 on the Ang II-mediated responses also inhibited downstream cGMP signaling following NO-dependent stimulation.