AT1 and AT2 receptors modulate renal tubular cell necroptosis in angiotensin II-infused renal injury mice

Figures

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  Figure 1. Inhibition of AT1R and AT2R suppresses renal tubular epithelial cell necroptosis in Ang II-treated renal injury mice. (A) Representative TEM images of necrotic tubular epithelial cells (as shown by the arrow) in the kidney tissues of Ang II-treated mice. (B) TUNEL-stained (green fluorescence) cells in the mouse renal tubules were costained to detect RIP3 (red fluorescence) and the nuclei (DAPI, blue fluorescence). The scale bars represent 10 μm. (C) The % ratio of necrotic tubular epithelial cells (as shown by the arrow) was analyzed. (D) The data are presented as the % ratio of TUNEL-positive and RIP3-positive tubular cells (necroptotic tubular cells). For the data in (C, D), the values are reported as the means ± S.E.M. of n = 6; **p < 0.01, versus control; #p < 0.05, ##p < 0.01, versus the Ang II-treated group. Ang II: angiotensin II.

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  Figure 2. Inhibition of AT1R and AT2R diminishes necroptosis-related proteins in Ang II-induced renal injury mice. Tissue lysates of the retrieved mouse kidneys were subjected to SDS-PAGE and Western blotting with RIP3, p-RIP3, MLKL and p-MLKL (A), AT1R and AT2R (F) antibodies and a β-actin antibody. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 (B–E,G,H). For the data in (B–E,G,H), the values are reported as the means ± S.E.M. of n = 6; *p < 0.05 and **p < 0.01, versus control; #p < 0.05, ##p < 0.01, versus the Ang II-treated group. Ang II: angiotensin II.

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  Figure 3. Necroptosis inhibition improves renal function and renal injury in a mouse model of Ang II-induced renal injury. (A) H&E staining of the kidney tissues retrieved from Ang II-infused mice treated with or without PD123319 or losartan or Nec-1 (n = 6 per group). Representative images are shown. (B) The tubular damage scores were assessed using a light microscope. Ang II-induced increases in blood urea nitrogen (C) and serum creatinine (D) levels were significantly reversed by PD123319 or losartan or Nec-1. (E) The systolic blood pressure was assessed. **p < 0.01 versus control; ##p < 0.01 versus the Ang II-treated group. Ang II: angiotensin II.

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  Figure 4. Detection of necrotic tubular epithelial cells using a flow cytometer. (A,B) HK-2 cells were treated with varying concentrations of Ang II (10−10–10−5 M) for 24 h. (C,D) HK-2 cells were exposed to 10−9 M Ang II for different times. After treatment, HK-2 (upper panel) cells were stained with annexin V-FITC and PI to determine cell necrosis (annexin V+/PI+ cells) using a flow cytometry assay. The bar chart shows that the ratio of annexin V+/PI+ cell numbers was highest in the HK-2 cells treated with 10−9 M Ang II for 24 h.

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  Figure 5. Ang II induces RIPK3-MLKL-mediated necroptosis in HK-2 cells. HK-2 cells were pretreated with or without 10 µM losartan and 10 µM PD123319 for 30 min or 50 µM Nec-1 for 30 min, followed by exposure to 10−9 M Ang II for 24 h. Representative Representative blots (A) and Western blot analysis (B–E) for necroptosis marker proteins: RIP3, MLKL, p-RIP3, and p-MLKL antibodies were used; β-actin was used as a loading control. The results are representative of three independent experiments. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 **p < 0.01 versus control; #p < 0.05, ##p < 0.01 versus the Ang II-induced group. Ang II: angiotensin II.

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  Figure 6. Inhibition of AT1R and AT2R diminishes Ang II-induced necroptotic HK-2 cells. (A) TUNEL-stained (green fluorescence) HK-2 cells treated with 10−9 M Ang II for 24 h were costained to detect RIP3 (red fluorescence) and the nuclei (DAPI, blue fluorescence). The scale bars represent 50 μm. (B) Representative TEM images of necroptotic HK-2 cells (as shown by the arrow) after treatment with 10−9 M Ang II for 24 h. The scale bars represent 5 μm. (C) The data are presented as the % ratio of necroptotic HK-2 cells(as shown by the arrow). (D) The data are presented as the % ratio of necrotic cells among all HK-2 cells treated with 10−9 M Ang II for 24 h. For the data in (C, D), the values are reported as the means ± S.E.M. of n = 6; **p < 0.01, versus control; ##p < 0.01, versus the Ang II-treated group. Ang II: angiotensin II.

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  Figure 7. The AT2 agonist CGP42112A facilitates necroptosis of HK-2 cells. Effects of CGP42112A on HK-2 cells necroptosis were determined. HK-2 cells stimulated with CGP42112A at a concentration of 0, 0.1, 1, and 10µmol/L for 24 h. Then, qPCR and Western Blot analysis was performed to detect the level of RIP3 and MLKL mRNA and protein (A–E). Next, HK-2 cells were pretreated with an AT2R antagonist (10 µM PD123319) and a RIP1 inhibitor (50 µM Nec-1) for 30 min, then the cells were treated with 0.1 µM CGP42112A for 24 h. F shows representative images of immunofluorescence staining for RIP3 (red fluorescence) and in situ fluorescence TUNEL staining (green fluorescence). Scale bars represent 50 μm. (G) The data are presented as the % ratio of necroptotic HK-2 cells (TUNEL-positive and RIP3-positive cells). Representative blots (H) and Western blot analysis (I) for RIP3, MLKL, p-RIP3, and p-MLKL proteins; β-actin was used as a loading control. The results shown are representative of three independent experiments. *p < 0.05 versus control; **p < 0.01 versus control; ##p < 0.01 versus the CGP42112A group.

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  Figure 8. Inhibition of AT1R and AT2R mitigates the expression of Fas/FasL signaling molecules in Ang II-induced HK-2 cells. Effects of FasL blockade via neutralizing human Fas ligand/TNFSF6 antibody on Ang II-induced necroptosis in HK-2 cells were determined. HK-2 cells were pretreated with 3 µg/ml neutralizing human Fas ligand/TNFSF6 antibody for 2 h and exposed to 10−9 M Ang II for 24 h. Then, Western blotting was performed to detect Fas and FasL (A,D,E), RIP3, MLKL, p-RIP3, and p-MLKL (B,F–I) levels and β-actin was used as a loading control. C shows representative images of immunofluorescence staining for RIP3 (red fluorescence) and in situ fluorescence TUNEL staining (green fluorescence). Scale bars represent 50 μm. (J) The data are presented as the % ratio of necroptotic HK-2 cells (TUNEL-positive and RIP3-positive cells). The results shown are representative of three independent experiments. The intensities of the bands were determined quantitatively using Image-Pro Plus 6.0 **p < 0.01 versus control; ##p < 0.01 versus the Ang II-induced group. Ang II: angiotensin II.