Sildenafil effects on blood pressures.

<p>Sildenafil (100 mg/kg d) effects on systolic blood pressures measured in conscious sham- and 2K1C-operated WT (WT-sham 118 ± 1 mmHg without, and 112 ± 1 mmHg with sildenafil; WT-2K1C 130 ± 4 mmHg without, and 118 ± 2 mmHg with sildenafil) and NO-GC1 KO mice (KO-sham 118 ± 1 mmHg without, and 113 ± 2 mmHg with sildenafil; KO-2K1C 125 ± 2 mmHg without, and 121 ± 2 mmHg with sildenafil) using tail-cuff manometer (n=4 WT-sham, 5 WT-2K1C, 6 KO-sham, and 10 KO-2K1C mice). Sildenafil administration and SBP measurements were performed during the 4^th week after operation. *P< 0.05 2K1C WT compared to sham WT, unpaired Student's t test. SBP indicates systolic blood pressure. </p

2K1C operation did not reduce vascular relaxation in NO-GC1 KOs but in WT mice.

<p><b>A</b>, endothelium-dependent relaxation induced by carbachol (30 µM) in NA-contracted aortic rings and isolated perfused kidneys of sham- and 2K1C-operated WT mice (36 aortic rings of n=9 mice per group; kidneys of n=21 and 8 mice, respectively). * P< 0.05 versus aorta of WT-sham, paired 2-tailed Student's t test; ** P<0.001 versus kidney of WT-sham, unpaired 2-tailed Student's t test. <b>B</b>, concentration-response curves for GSNO-induced relaxation determined in NA-contracted isolated perfused kidneys of sham- and 2K1C-operated WT mice in the presence of L-NAME (n=5 and 7 mice, respectively). P=0.037, ANOVA for repeated measurements. <b>C</b>, endothelium-dependent relaxation induced by carbachol (30 µM) in NA-contracted aortic rings and isolated perfused kidneys of sham- and 2K1C-operated NO-GC1 KO mice (28 aortic rings of n=7 mice per group; kidneys n=12 and 7 mice, respectively). <b>D</b>, concentration-response curves for GSNO-induced relaxation determined in NA-contracted isolated perfused kidneys of sham- and 2K1C-operated NO-GC1 KO mice in the presence of L-NAME (n=8 and 6 mice, respectively). Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

2K1C operation does not alter cGMP synthesis or cGMP sensitivity in WT kidneys.

<p><b>A</b>, NO-stimulated cGMP-forming activity (DEA-NO 100 µM) determined in kidney homogenates of sham- and 2K1C-operated WT mice (n=6 mice per group). <b>B</b>, quantification of the β₁ subunit content with respect to the subunit amount in sham-operated WT mice using subunit specific antibodies (n=12 mice per group). <b>C</b>, concentration-response curves for 8-pCPT-cGMP of NA-contracted isolated perfused kidneys of sham- and 2K1C-operated WT mice in the presence of L-NAME (n=13 and 7 mice, respectively). Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

Reduced cGMP response in kidneys of NO-GC1 KO mice.

<p><b>A</b>, NO-stimulated cGMP-forming activity (100 µM DEA-NO) determined in kidney homogenates of WT and NO-GC1 KO mice (n=19 and 6 mice, respectively). PDE activity in WT and KO kidneys measured with 1 µM cGMP as substrate (n=11 and 7 mice, respectively). ** P< 0.01 versus WT, unpaired Student's t test. B, absent of the α1 subunit in NO-GC1 KO kidney homogenates and quantification of the α2 and β1 subunit content with respect to the subunit amount in WT using subunit specific antibodies (n=8 mice per genotype). Representative strips with α1, α2, β1 and the respective β tubulin bands of the same lanes are shown above. *** P< 0.001 versus WT, unpaired Student's t test. C, cyclic GMP content determined in renal cortical slices of WT and KO mice without any addition (n=20 and 10 mice, respectively), or in the presence of ODQ (20 µM, 15 min; n=5 and 4 mice, respectively) or carbachol (30 µM, 3 min; n=10 and 6 mice, respectively) or DEA-NO (100 µM 3 min; n=10 and 7 mice, respectively). # P< 0.01 versus untreated slices in the same group; *P< 0.01 versus WT, unpaired Student's t test. D, carbachol-induced relaxation of NA-contracted isolated perfused kidneys of NO-GC1 KO (n=12) and WT mice (n=21). ** P< 0.01 versus WT, unpaired Student's t test. E, concentration-response curves for GSNO-induced relaxation of NA-contracted isolated perfused kidneys in the presence of L-NAME (n=5 WT and 7 KO mice). The white dots indicate the Cch-induced relaxation plotted on the curves to determine the corresponding GSNO concentration. F, Western blot detection of eNOS in 50 µg kidney homogenates of NO-GC1 KO (n=8) and WT mice (n=5) and quantification with respect to the eNOS amount in WT kidneys. A representative strip with eNOS and the respective β tubulin bands of the same lanes is shown above. * P< 0.05 versus WT, unpaired Student's t test.</p

2K1C operation did not up-regulate PDE5.

<p><b>A</b>, PDE5 mRNA in preglomerular vessels of sham- and 2K1C-operated WT mice detected by quantitative real-time PCR and quantified relative to GAPDH with the ∆C_T method (n=8 and 9 mice, respectively). <b>B</b>, PDE5 protein detected by Western blot in 50 µg kidney homogenates of sham- and 2K1C-operated WT mice and expressed as % of PDE5 content in kidneys of sham-WT mice (n=7 mice per group). A representative strip with PDE5 and the respective β tubulin bands of the same lanes is shown below. PDE activities in homogenates of kidneys and preglomerular vessels of sham- and 2K1C-operated WT mice in the absence and presence of sildenafil (100 nM) measured by 1 µM (<b>C</b>) or 30 nM cGMP (<b>D</b>). PDE activities were obtained of 3 mice per group in kidneys by 1 µM cGMP; n=13 and 10 mice per group, preglomerular vessels; n=6 per group kidneys by 30 nM. * P< 0.001 versus corresponding PDE activity without sildenafil, paired 2-tailed Student's t test. Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

Evidence of enhanced eNOS-catalyzed NO formation induced by the 2K1C operation.

<p><b>A</b>, Western blot detection of p-eNOS (serine 1177) and eNOS in 50 µg kidney homogenates of sham- and 2K1C-operated WT mice and quantification with respect to the ratio of p-eNOS to eNOS in kidneys of sham-operated WT (n=8 and 7 mice, respectively). * P< 0.05 versus WT-sham, unpaired Student's t test. <b>B</b>, concentration-response curves for angiotensin II-induced vasoconstriction of isolated perfused kidneys of sham- and 2K1C-operated WT mice in the presence of L-NAME (n=9 and 6 mice, respectively). * P< 0.05, and ** P< 0.01 versus WT-sham, unpaired Student's t test. <b>C</b>, cyclic GMP content determined in renal cortical slices of sham- and 2K1C-operated WT mice without any addition (15 slices of 5 mice per group) or in the presence of sildenafil (100 µM, 10 min; 15 slices of 5 mice per group). ***P< 0.001 versus WT-sham, unpaired Student's t test. Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p