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 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

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 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

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

Albuminuria and nephrin surface abundance in early nephrotoxic nephritis.

<p><b>(A)</b> Coomassie gel shows albuminuria in mice injected with NTN serum (NTN 1d). No albuminuria was detected in mice injected with normal saline (control). <b>(B)</b> Quantitative evaluation of albumin-creatinine excretion in healthy mice (control) and NTN mice (NTN 1d). Statistical analysis: Mann-Whitney U test, ** p < 0.01 (control: n = 4; NTN: n = 6). <b>(C, D)</b> Immunofluorescence staining of murine kidney sections of healthy mice (control) and mice injected with NTN serum (NTN 1d). Staining was performed with an anti-nephrin antibody (red), an anti-EEA1-antibody (green) and nuclear DNA with Draq5 (blue). White arrows indicate colocalization of nephrin with EEA1 positive vesicles. <b>(E)</b> Representative western blot of total and biotinylated nephrin detected with streptavidin (streptavidin). In comparison to untreated mice (control) less surface nephrin (WB: streptavidin) was detected in NTN mice (NTN 1 d). Total nephrin immunoprecipitates and lysates (WB: nephrin) showed equal expression of total nephrin in both groups. β-actin (WB: actin) was used as loading control. <b>(F, G)</b> Densitometric analysis of western blots: <b>(F)</b> Amount of nephrin at the cell surface graphed as biotinylated nephrin to total nephrin ratio; <b>(G)</b> Amount of total nephrin graphed as total nephrin to β-actin ratio. <b>(H)</b> Immunohistochemistry: staining of p57 indicates podocytes in healthy mice (control) and NTN mice (NTN d1). <b>(I)</b> Number of p57 positive cells per glomerular tuft <b>(J)</b> Glomerular tuft area (μm²) <b>(K)</b> Percentage of p57 positive cells in relation to tuft area. (40 gloms per mouse quantified). Western Blot data show means ± SD. Podocyte counts show means ± SEM. Statistical analysis: unpaired <i>t</i>-test with Welch’s correction. Significance level was set to 5%, *p < 0.01, ** p<0.001, (non-significant differences (ns)).</p

Nephrin surface abundance in adriamycin nephropathy at day 7.

<p><b>(A)</b> Coomassie gel shows albuminuria in ADR mice at day 7 (ADR 7d), which was not detected in healthy mice (control). <b>(B)</b> Quantitative analysis of the albumin to creatinine ratio in healthy mice (control) versus ADR mice on days 1 and 7 (ADR1 and ADR 7). Statistical analysis: <i>t</i>-test with Welch’s correction was performed on day 1 and 7. **** p < 0.0001 (non-significant differences (ns); control: n = 9; ADR: n = 12). <b>(C, D)</b> Immunofluorescence staining of glomeruli from healthy mice (control) or ADR mice (ADR 7 d). Staining was performed with an anti-nephrin antibody (red), and nuclei were stained with DAPI (blue). White arrows indicate colocalization of nephrin with EEA1 positive vesicles. <b>(E)</b> Western blot analysis of surface nephrin (streptavidin) and total nephrin immunoprecipitates and lysates (WB nephrin). In comparison to healthy mice (control), less surface nephrin (WB: streptavidin) was detected in ADR mice at day 7 (ADR 7d). Total nephrin immunoprecipitates and lysates (WB: nephrin) showed less expression of total nephrin at day 7. β-actin (WB: actin) was used as loading control. <b>(F, G)</b> Densitometric analysis of western blots: <b>(F)</b> Amount of cell surface nephrin graphed as biotinylated nephrin to total nephrin ratio; <b>(G)</b> Amount of total nephrin graphed as total nephrin to β-actin ratio. <b>(H)</b> Immunohistochemistry: staining of p57 indicates podocytes in healthy mice (control) and NTN mice (NTN d1). <b>(I)</b> Number of p57 positive cells per glomerular tuft <b>(J)</b> Glomerular tuft area (μm²) <b>(K)</b> Ratio of p57 positive cells in relation to tuft area (control n = 2, ADR n = 3, 40 gloms per mouse quantified). Western blot data show means ± SD. Podocyte counts show means ± SEM. Statistical analysis: unpaired <i>t</i>-test with Welch’s correction. Significance level was set to 5%, *** p < 0.0001, **** p<0.00001, (non-significant differences (ns)).</p

Detection of biotin in murine glomeruli.

<p><b>(A)</b> Representative immunofluorescence staining of kidney sections of C57Bl/6 mice. Podocyte nuclei were labeled by WT1 staining (red). Biotin was detected with streptavidin (green). In biotin-perfused mice (biotin), biotin was detected along the glomerular capillaries. PBS-perfused mice (control) did not show any biotin deposition. <b>(B)</b> Western blot analysis after immunoprecipitation of nephrin (IP α-nephrin) from biotin-perfused (biotin) and PBS-perfused (control) murine kidneys. A biotinylated fraction of nephrin (WB streptavidin) was only detectable in biotin-perfused mice. Total nephrin immunoprecipitates and lysates (WB nephrin) showed equal expression of total nephrin. Beta-actin (actin) was used as a loading control. <b>(C)</b> Western blot analysis after immunoprecipitation of biotinylated surface proteins (IP streptavidin agarose beads) from biotin-perfused (biotin) and PBS-perfused (control) murine kidneys. Biotinylated nephrin (WB nephrin) and podocalyxin (WB podocalyxin) were only detectable in biotin-perfused mice. Extracellular-signal regulated kinases p42 and p44 (ERK) could not be detected at all. Total nephrin, podocalyxin and ERK lysates (WB nephrin, WB podocalyxin and WB ERK) showed equal expression of nephrin, podocalyxin and ERK. Beta-actin (actin) was used as a loading control.</p