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

Figures

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  FIGURE 1. Illustration of applied chemicals (bold) to affect the NO/sGC/cGMP signaling pathway; SNP, sodium nitroprusside; NO, nitric oxide; NOS, nitric oxide synthase; L-NAME, Nω-nitro-L-arginine methylester hydrochloride; L-Arg, L-arginine; O2-, superoxide; GTP, guanosine triphosphate; sGC, soluble guanylyl cyclase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; GMP, guanosine monophosphate; cGMP, cyclic guanosine monophosphate; 8-Br-cGMP, 8-bromoguanosine 3’,5’-cyclic monophosphate sodium salt; PKG, cGMP-dependent protein kinase; DT-2, DT-2 trifluoroacetate salt; PDE, phosphodiesterase; tempol, 4-hydroxy-TEMPO. (∗) – activation, (–) – inhibition.

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  FIGURE 2. Effects of H/R and inhibitors of dilatory systems on endothelium-dependent relaxation in isolated rat renal interlobar arteries (ILAs) precontracted with PE. (A) Carbogen (control) showed no adverse effects on relaxation compared to 21% O2. EC50 of H/R compared to control was significantly different pointing to diminished ACh-induced relaxation following H/R [EC50: 2.37∗10-7 mol/l (control) vs. 4.75∗10-7mol/l (H/R); P < 0.05]. Inhibiting endothelial dilatory systems (B–D) did not reverse the effect of H/R on ILA relaxation. (B) NOS inhibition [with L-NAME, EC50: 3.33∗10-7 mol/l (control) vs. 9.54∗10-7 mol/l (H/R); P < 0.05]. The maximum ACh-induced relaxation was further significantly different comparing H/R and control (P < 0.05; Brunner test). (C) COX inhibition [with indomethacin (Indo), EC50: 1.63∗10-7 mol/l (control) vs. 4.00∗10-7 mol/l (H/R); P < 0.05]. (D) KCa3.1 inhibition [with TRAM34, EC50: 2.02∗10-7 mol/l (control) vs. 4.05∗10-7 mol/l (H/R): P < 0.05] and KCa2.1 inhibition [with UCL 1684, EC50: 3.52∗10-7 mol/l (control) vs. 2.07∗10-6 mol/l (H/R): P < 0.05]. Mean ± SEM. (∗) indicates significant differences. (E) Relative x-fold mRNA expression of nNOS, iNOS and eNOS…

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  FIGURE 3. Effect of H/R on endothelium independent relaxation in isolated rat renal ILA. (A) H/R and simultaneous inhibition of the endothelial dilatory systems did not reverse the effect of H/R on ILA relaxation as indicated by significantly different EC50 [3.32∗10-8 mol/l (control) vs. 9.37∗10-8 mol/l (H/R); P < 0.05]. (B) Vascular wall stiffness parameter b (beta) did not significantly differ between both groups. Mean ± SEM. (∗) indicates significant differences.

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  FIGURE 4. Analysis of superoxide in renal rat ILA. (A) Representative images of vascular rings loaded with DHE under control conditions and after H/R treatment (DHE concentration: 2∗10-5 mol/l, magnification: 200×; scale bar = 400 μm) (B) Analysis of DHE fluorescence showed no significant differences between both groups. (C) H/R and simultaneous inhibition of superoxide, using the superoxide dismutase mimetic tempol, did not reverse the H/R effect on endothelium-independent relaxation in isolated rat renal ILA precontracted with PE. [EC50: 3.34∗10-8 mol/l (control) vs. 6.75∗10-8 mol/l (H/R); P < 0.05]. (D) Relative x-fold mRNA expression of NADPH oxidase enzymes (NOX) normalized to 18SrRNA after H/R relative to control conditions. The analyzed NOX were not differentially regulated following H/R. The average mRNA expression level under control conditions was arbitrarily given a value of 1 (20) (∗P < 0.05; compared to control; Mann–Whitney Test; n = 8). Mean ± SEM. (∗) indicates significant differences.

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  FIGURE 5. Effect of H/R on the cGMP pathway in rat renal ILA. (A) The H/R effect on ILA relaxation was not abolished by PDE5 inhibition using sildenafil as indicated by significant differences in the EC50 [1.18∗10-6 mol/l (control) vs. 7.14∗10-6 mol/l (H/R); P < 0.05]. (B) The cGMP analog 8-Br-cGMP reversed the H/R effect and abolished significant differences between the two groups. (C) H/R significantly decreased cGMP concentration in ILA (P < 0.05). Mean ± SEM. (∗) indicates significant differences.

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  FIGURE 6. Effect of H/R on sGC-dependent relaxation in isolated rat renal interlobar arteries (ILAs) precontracted with PE. (A) Inhibition of PKG kinase by DT-2 could not reverse H/R effect on ILA relaxation [EC50: 2.14∗10-8 mol/l (control) vs. 4.88∗10-8 mol/l (H/R); P < 0.05]. Mean ± SEM. (∗) indicates significant differences. (B) The sGC inhibitor ODQ abolished the H/R effect on ILA relaxation and aligned control and H/R dose–response relationships [8.90∗10-7 mol/l (control) vs. 1.70∗10-6 mol/l (H/R); EC50 not significant, ANOVA not significant].