J Korean Med Sci.  2016 Apr;31(4):535-541. 10.3346/jkms.2016.31.4.535.

Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase

• Ha TS
• Park HY
• Seong SB
• Ahn HY

• KMID: 2363691
• DOI: http://doi.org/10.3346/jkms.2016.31.4.535

Abstract

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.

Keyword

AMP-activated Protein Kinase; Angiotensin II; p130Cas; Podocyte; Angiotensin II Type 1 Receptor

MeSH Terms

AMP-Activated Protein Kinases/antagonists & inhibitors/chemistry/*metabolism
Aminoimidazole Carboxamide/analogs & derivatives/pharmacology
Angiotensin II/*pharmacology
Angiotensin II Type 1 Receptor Blockers/pharmacology
Animals
Blotting, Western
Cell Line
Cell Nucleus/metabolism
Crk-Associated Substrate Protein/*metabolism
Cytoplasm/metabolism
Focal Adhesion Kinase 1/metabolism
Losartan/pharmacology
Metformin/pharmacology
Mice
Microscopy, Confocal
Podocytes/cytology/drug effects/metabolism
Protein Kinase Inhibitors/*pharmacology
Ribonucleotides/pharmacology
Signal Transduction/*drug effects
AMP-Activated Protein Kinases
Aminoimidazole Carboxamide
Angiotensin II
Angiotensin II Type 1 Receptor Blockers
Crk-Associated Substrate Protein
Focal Adhesion Kinase 1
Losartan
Metformin
Protein Kinase Inhibitors
Ribonucleotides

Figure

• 

  Fig. 1 Effects of Ang II on the localization of p130Cas in podocytes. p130Cas stainings are located in the cytoplasm of cultured podocytes diffusely, whereas, F-actin fibers are distributed in peripheral cytoplasm and run over nucleus, apart from p130Cas. (A) Ang II reduces and re-localizes the immunofluorescent intensities of p130Cas in internal cytoplasm and peri- and intra-nuclear areas of podocytes with concentrations. Such changes by Ang II are associated with disrupted F-actin fibers. (B) Ang II (10-6 M) also decreases and concentrates the immunofluorescent intensities of FAK (arrow) in internal cytoplasm and peri-nuclear areas of podocytes, which become to be separated partially from p130Cas (arrow head). Magnification, × 1,000.

• 

  Fig. 2 Effects of Ang II on the p130Cas protein assayed by Western blotting. (A) The bands for p130Cas protein at 130 kDa are reduced by 10-7 M Ang II in a time-dependent manner. Such changes are further aggravated by compound C, a potent AMPK inhibitor. (B) At 24 hours, the density values for p130Cas decreased significantly at doses of 10-7 M and 10-6 M Ang II, respectively, compared to control (without Ang II) after correcting for β-tubulin levels. Data on the densitometric analysis of p130Cas/β-tubulin ratio are expressed as mean ± SD (n = 3). Control (100%); the value of no Ang II conditions. *P < 0.05 and † P < 0.01 vs. control.

• 

  Fig. 3 Effects of AMPK activators on the distributional changes of p130Cas induced by Ang II. (A) AMPK activators, metformin and AICAR, restore the mal-localized p130Cas, whereas, AMPK inhibitor, compound C further aggravates the distributional changes of p130Cas (arrowheads). (B) AICAR also restores the mal-localized p130Cas (arrowheads) and FAK (arrows) induced by Ang II. Magnification, × 1,000.

• 

  Fig. 4 Effects of AMPK activators on the changes of p130Cas protein induced by Ang II. Ang II suppresses p130Cas protein significantly in a time-dependent manner. (A) Compound C further exaggerates the reduced p130Cas protein significantly (all P < 0.01, not denoted) at all experiment times. (B) Concerning the concentration of 10-7 M Ang II at 24 hours, Ang II suppresses p130Cas protein significantly, which is also reversed by AICAR and metformin. Compound C further aggravates the changes of p130Cas. Data on the densitometric analysis of p130Cas/β-tubulin ratio are expressed as mean ± SD (n = 3). Control (100%); the value of no Ang II conditions. *P < 0.05 and † P < 0.01 vs. control. ‡ P < 0.05 and § P < 0.01 vs. Ang II-treated groups.

• 

  Fig. 5 Suppression of p130Cas by Ang II via AT1R. AT1R antagonist, losartan, ameliorates the mal-localized p130Cas (arrowheads, A) and reverses the suppression of p130Cas induced by high doses (10-7 M and 10-6 M) of Ang II (B). Data on the densitometric analysis of p130Cas/β-tubulin ratio are expressed as mean ± SD (n = 3). Control (100%); the value of no Ang II conditions. *P < 0.05 and † P < 0.01 vs. control. ‡ P < 0.05 vs. Ang II-treated groups.

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