Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: AngII increases the Etd + uptake in MES-13 cells. ( A ) Phase contrast and fluorescence images show MES-13 cells exposed to ethidium (5 μM Etd + ) for 13 min under AngII stimulation (10 −7 M) at different periods of time (0, 24, 48 and 72 h). Scale bar = 50 μm; ( B ) Representative time lapse of Etd + uptake in mesangial cells recorded every 30 s, each plotted point corresponds to the mean of 20 cells, mean value ± SE. Dye uptake curve after application of AngII at different times (0, 24, 48 and 72 h); ( C ) Etd + uptake rate of mesangial cells under basal conditions (0 h) or after treatment with AngII for different times (24, 48, and 72 h), with or without FBS (Fetal bovine serum) and Losartan (20 μM). Inset shows propidium iodide uptake rate of mesangial cells under basal conditions or after treatment with AngII for 72 h. Each bar represents the mean value ± SE of ≥3 independent experiments. Statistical significance *** p < 0.001 vs. 0 h; ### p < 0.001 vs. AngII 72 h. Treatments with AngII for 72 h are denoted below each bar with a plus sign (+). Losartan was co-treated with AngII for 72 h.

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: ROCK, Cx43 HC, Panx1 Ch and P2X 7 R blockers reduce the AngII-induced cell membrane permeabilization in MES-13 cells. Quantification of the Etd + uptake rate in MES-13 cells under control conditions, exposed to AngII (10 −7 M) for 72 h with or without different blockers: Fasudil (15 μM) and Y-27632 (15 μM), ROCK Inhibitors; apyrase (2 units/mL), ATP hydrolase; probenecid (PBC, 500 µM), Panx1 Ch blocker. Lanthanum ion (La 3+ , 200 μM), Cx43 HC and P2X 7 R blocker; the mimetic peptide Gap27 (100 µM), a selective Cx43 HCblocker; carbenoxolone (CBX 10 μM), a Panx1 Ch blocker; A740003 (100 μM), a selective P2X 7 R blocker. Each bar represents the mean value ± SE of ≥3 independent experiments. Statistical significance *** p < 0.001 vs. control; ### p < 0.001 vs. AngII. Gap27, apyrase, PBC, Fasudil and Y-27632, were added during the last 24 h of treatment with AngII. Acutely were added Gap27 (added 15 min before each Etd + uptake measurement), La 3+ , CBX, A740003, fasudil and Y27632 (each was added 10 min after Etd + uptake). Each treatment with AngII for 72 h is denoted below each bar with a plus sign (+).

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: AngII decreases gap junctional coupling between mesangial cells. ( A ) Etd + was microinjected into the brightest cell (arrow) and diffused to neighboring cells. The right panels show fluorescence of Etd + at different times (0 and 72 h) and the left panels are the phase contrast images. Coupling incidence ( B ) and coupling index ( C ) were evaluated in confluent mesangial cell cultures with AngII for different time periods (0 and 72 h) using dye (Etd + , 25 μM) coupling technique (black bars). Each bar represents the mean value ± SE of 4 independent experiments. In each experiment the dye was microinjected into at least 10 cells. Statistical significance *** p < 0.001 vs. Control. Scale bar = 20 μm.

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: Fasudil increases gap junctional coupling between mesangial cells treated with AngII for 72 h. Coupling incidence ( A ) and coupling index ( B ) were evaluated in confluent mesangial cells (black bars) using a dye (Etd + , 25 μM) coupling technique, under control conditions or exposed at AngII (10 −7 M) for 72 h. Fasudil (15 µM) was added during the last 24 h of incubation with AngII. Each treatment is denoted below each bar with a plus sign (+). Each bar represents the mean value ± SE of 4 independent experiments. In each experiment the dye was microinjected into at least 10 cells. Statistical significance ** p < 0.01 and *** p < 0.001 vs. Ctrl; ## p < 0.001 and ### p < 0.001 vs. AngII. ( C ) Etd + was microinjected into the brightest cell (arrow) and diffused to neighboring cells. Top panels are the phase contrast images and bottom panels show Etd + fluorescence in cells under different treatments. Scale bar = 20 μm.

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques: Incubation, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: Fasudil or Y-27632 decreases the amount of phosphorylated MYPT and Cx43 without affecting the amount of Panx1 and P2X 7 R in MES-13 cells treated with AngII. Graphs show phosphorylation of MYPT-1 ( A ), the relative amount of Cx43 ( B ), Panx1 ( C ) and P2X 7 R ( D ) determined by western blot analysis in MES-13 cells under control conditions or exposed to AngII (10 −7 M) for 72 h. Fasudil (15 µM) or Y-27632 (15 µM) were added 24 h before harvesting the cells. Each treatment is denoted below each bar with a plus sign (+). Each bar represents the mean value ± SE of 4 independent experiments. Statistical significance *** p < 0.001 vs. Ctrl; ### p < 0.001 vs. AngII. Under the graph representative pictures of Cx43, phosphorylated MYPT (p-MYPT) and unphosphorylated MYPT positive bands and its loading control (α-tubulin) are shown.

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: Blockade of ROCK, Cx HCs or P2X 7 Rs decreases the amount of TBARS, TNF-α and IL-1β in AngII-treated MES-13 cells. Graphs showing amount of ( A ) TBARS, ( B ) IL-1β and ( C ) TNF-α in the culture medium of MES-13 cells under control conditions or after 72 h exposure to AngII (10 −7 M) in the presence of different blockers: Gap27 (100 µM), a selective Cx43 HC blocker; probenecid (PBC, 500 µM), Panx1 Ch blocker; A740003 (100 μM), a selective P2X 7 R blocker or Fasudil (15 μM), ROCK inhibitor applied 24 h before harvesting the medium samples. Each bar represents the mean value ± SE of 4 independent experiments. Statistical significance, *** p < 0.001 vs. Ctrl; ### p < 0.001 vs. AngII; &&& p < 0.001 vs. AngII + PBC.

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Angiotensin II-Induced Mesangial Cell Damage Is Preceded by Cell Membrane Permeabilization Due to Upregulation of Non-Selective Channels

doi: 10.3390/ijms19040957

Figure Lengend Snippet: Scheme of possible signaling pathways involved in regulating the functional state of Cx43 HCs, Panx1 Chs, P2X 7 Rs and Cx GJs in mesangial cells stimulated with AngII. High AngII concentrations (continuous black arrows) could promote Ca 2+ release from the endoplasmic reticulum (ER) and activation of a RhoA/ROCK-dependent pathway through angiotensin type 1 receptors (AT1R). The latter is inhibited by losartan. Once the RhoA/ROCK-dependent pathway is activated, the expression and release of pro-inflammatory cytokines such as TNF-α and IL-1β as well as formation of reactive oxygen species (ROS) that generate thiobarbituric acid reactive substances (TBARS) upon reaction with lipid of cell membranes can occur. The activated RhoA/ROCK-dependent pathway could affect the function of Cx43 HCs by opening them, since Fasudil or Y-27632 (ROCK blockers) inhibit this response. The resulting increase in intracellular Ca 2+ can also activate Panx1 Chs and together with activated Cx HCs enable release of ATP to the extracellular medium. Then, the extracellular ATP activates P2X 7 receptors that together with Cx43 HCs permit a drastic increase in Ca 2+ influx. The resulting intracellular Ca 2+ concentration, as already seen in other systems, promote the expression and release of pro-inflammatory cytokines such as TNF-α, IL-1β, and the generation of ROS. The release of ATP and influx of Ca 2+ establish a positive feedback loop. This loop is inhibited by different compounds: Apyrase, ATP hydrolase; the mimetic peptide Gap27, a selective Cx43 HCs blocker; A740003, a selective P2X 7 R blocker or probenecid (PBC), an inhibitor of Panx1 Chs. This increase in the cellular activity caused by AngII, where the RhoA/ROCK pathway could be involved, also reduces cell–cell communication through GJs, further affecting the cellular integrity. Discontinuous red and black arrows indicate cell responses identified in other systems, whereas continuous black arrows denote responses identified in the present work.

Article Snippet: Angiotensin II (AngII) was obtained from Alomone (Jerusalem, Israel); Fasudil, Y-27632, ethidium (Etd + ) bromide, lanthanum (La 3+ ) chloride, Losartan and malondialdehyde (MDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); Gap27—a peptide that inhibits Cx43 HCs—was obtained from NeoMPS, SA (Strasbourg, France); A740003—a specific P2X 7 Rblocker—was obtained from Tocris Biosciences (Bristol, UK).

Techniques: Functional Assay, Activation Assay, Expressing, Concentration Assay, Activity Assay