Hypoxia/Reoxygenation of Rat Renal Arteries Impairs Vasorelaxation via Modulation of Endothelium-Independent sGC/cGMP/PKG Signaling

Diana Braun; Christa Zollbrecht; Stefanie Dietze; Rudolf Schubert; Stefan Golz; Holger Summer; Pontus B Persson; Mattias Carlström; Marion Ludwig; Andreas Patzak

doi:10.3389/fphys.2018.00480

Hypoxia/Reoxygenation of Rat Renal Arteries Impairs Vasorelaxation via Modulation of Endothelium-Independent sGC/cGMP/PKG Signaling

Front Physiol. 2018 May 3:9:480. doi: 10.3389/fphys.2018.00480. eCollection 2018.

Authors

Diana Braun^{1

2}, Christa Zollbrecht³, Stefanie Dietze^{1

2}, Rudolf Schubert⁴, Stefan Golz⁵, Holger Summer⁵, Pontus B Persson^{1

2}, Mattias Carlström³, Marion Ludwig^{1

2}, Andreas Patzak^{1

2}

Affiliations

¹ Renal Vessel Physiology Group, Institute of Vegetative Physiology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
² Institute of Vegetative Physiology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
³ Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
⁴ Centre for Biomedicine and Medical Technology Mannheim, Research Division Cardiovascular Physiology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
⁵ Bayer Pharma AG, Wuppertal, Germany.

Abstract

Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N₂, 5% CO₂ for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O₂, 5% CO₂). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels K_Ca3.1 and K_Ca2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.

Keywords: cGMP; hypoxia; ischemia/reperfusion injury; nitric oxide; relaxation; sGC; vascular smooth muscle.