Bradykinin/B₂receptor activation regulates renin in M-1 cells via protein kinase C and nitric oxide

Figures

• 

  Figure 1. M‐1 cells express B2R. (A) Representative image of a PCR product detection of a specific single band of 572 bp, using primers for the Bdkrb2, in two different cell passages (P12 and P34) (n = 3). (B) Representative image of the immunodetection of a 42 KDa band comparable with the B2R molecular weight in different cell passages (P4, P9, P12, and P34) (n = 3). (C) Colocalization of B2R (green) and renin (red) in M‐1 cells. Image was captured using a 100× oil immersion objective and a DS‐U2/L2 USB digital camera attached to a Nikon Eclipse 50i fluorescence microscope.

• 

  Figure 2. Bradykinin stimulates renin expression in M‐1 cells. (A) qRT‐PCR amplification of M‐1 cells renin (Ren1C) gene. M‐1 cells were incubated in the absence (control) or in the presence of different bradykinin concentrations (from 10−14 to 10−6 mol/L). Results were expressed as mean ± SE in arbitrary unities. (B) Representative image of renin protein detection by Western blot. Three bands were detected corresponding to pre‐prorenin (~48 kDa), prorenin (~45 kDa) and renin (~40 kDa). Densitometric analysis of either prorenin (C) or renin (D) bands was normalized against β‐actin densitometry. Results were expressed as mean ± SE in arbitrary units. (E) Immunofluorescence of renin (red) expression in M‐1 cells incubated without (top panels) or with bradykinin 10−10 mol/L (bottom panels) for 6 h. Representative images were obtained using a 100× oil immersion objective. (F) Renin content in the cell culture media after incubation with varying concentrations of bradykinin (from 10−14 to 10−6 mol/L). Results were expressed as mean ± SE in ng of Ang I formed/h/mL. In all graphs, significance (*) was defined as P < 0.05 compared to control (n = 5–6; one‐way ANOVA followed by Dunnet's post‐test).

• 

  Figure 3. Bradykinin stimulates CD renin expression via B2R activation. (A) qRT‐PCR amplification of M‐1 cells renin (Ren1C) gene. Where indicated 10−10 mol/L bradykinin and 10−6 mol/L Icatibant (ICA), the B2R antagonist were added in the M‐1 cell culture media for 6 h. Results were expressed as mean ± SE in arbitrary unities. (B) Representative image of prorenin and renin detection by Western blot. Densitometric analysis of either prorenin (C) or renin (D) bands was normalized against β‐actin densitometry. Results were expressed as mean ± SE in arbitrary unities. In all graphs, significance (*) was defined as P < 0.05 compared to control (n = 5–6; one‐way ANOVA followed by Dunnet's post‐test).

• 

  Figure 4. Bradykinin stimulates CD renin expression through a PKA‐independent and PKC‐dependent pathway. (A) and (D) qRT‐PCR amplification of M‐1 cells renin gene (Ren1C). Where indicated 10−10 mol/L bradykinin, 10−7 mol/L H89, a PKA inhibitor and 10−7 mol/L calphostin C, a PKC inhibitor were added in the M‐1 cell culture media for 6 h. Densitometric analysis of the specific bands (B) and (E) of either prorenin or (C) and (F) renin were normalized to β‐actin expression. Results were expressed as mean ± SE in arbitrary unities. In all graphs, significance (*) was defined as P < 0.05 compared to control (n = 5–6; one‐way ANOVA followed by Dunnet's post‐test).

• 

  Figure 5. Bradykinin stimulates CD renin expression via NO release. (A) Measurement of cGMP levels (in pmol/mg of tissue) in the presence of 10−10 mol/L bradykinin and 10−6 mol/L Icatibant (ICA), the B2R antagonist were added in the M‐1 cell culture media for 6 h. Results were expressed as mean ± SE. (B) qRT‐PCR amplification of M‐1 cells renin gene (Ren1C). Where indicated 10−10 mol/L bradykinin, 10−7 mol/L L‐NAME, a NOS inhibitor were added in the M‐1 cell culture media for 6 h. Densitometric analysis of either prorenin (C) or renin (D) bands were normalized to β‐actin expression. Results were expressed as mean ± SE in arbitrary unities. In all graphs, significance (*) was defined as P < 0.05 compared to control (n = 5–6; one‐way ANOVA followed by Dunnet's post‐test).

• 

  Figure 6. CD renin is decreased in mice lacking the Bdkrb2 gene. Immunofluorescence was performed as described in Material and Methods section. Representative digital images from kidneys of wild‐type (A)–(B) and Bdkrb2−/− (D)–(E) mice taken respectively at low (4× objective) and high (40× objective) magnifications show specific renin staining (green). (C) Co‐localization of renin (green) and B2R (red) in CD cells of kidneys from wild‐type mice. (F) Principal cells of the CD in Bdkrb2−/− mice labeled with aquaporin‐2 (APQ‐2, red) showing the fainted renin (green) immunoexpression. Representative images were obtained using a 40× objective. The inset to Figure 1D denotes specific renin immunoexpression in JGA cells (*) in the Bdkrb2−/− mice. Glomerulus (Glom) is highlighted by a dashed circle.

• 

  Figure 7. Specific renin immunofluorescence is decreased in the conditional UBBdkrb2−/− mice. (A) Co‐localization of renin (green) and B2R (red) in CD cells of a kidney section (3–4 μm) from a control UB flox/flox mouse. (B) Localization of aquaporin 2 (AQP‐2, green) and B2R (red) in kidney CD cells in the conditional UBB dkrb2−/−. B2R immunoexpression was detected only in interstitial cells but not in the CD cells. (A)–(B) are representative images obtained using a 40× objective. The inset displays renin expression in JGA cells (asterisk) in the kidney of a Bdkrb2−/− mouse. (C)–(F) Renin (green), B2R (red) and aquaporin‐2 (APQ‐2, violet) colocalization in the kidney CD cells in Bdkrb2−/− mice. To obtain a 4‐color stain, we used two consecutive sections: in one section the antibodies against renin (green, C) and B2R (red, D) were incubated according to the Material and Methods section and in the second section renin (green, not showing) and AQP‐2 (red, E) antibodies were used. The red color of AQP‐2 detection was digitally changed by violet for better illustration (E). Renin immunoexpression was markedly reduced. The images obtained were merged (F). Representative images were obtained…