Angiotensin II type-2-receptor stimulation ameliorates focal and segmental glomerulosclerosis in mice

Abstract

Podocyte damage and loss are the early event in the development of focal segmental glomerulosclerosis (FSGS). Podocytes express angiotensin II type-2-receptor (AT2R), which may play a key role in maintaining kidney integrity and function. Here, we examined the effects of AT2R deletion and AT2R agonist compound 21 (C21) on the evolution of FSGS. FSGS was induced by adriamycin (ADR) injection in both male wild-type (WT) and AT2R knockout (KO) mice. C21 was administered to WT-FSGS mice either one day before or 7 days after ADR (Pre-C21 or Post-C21), using two doses of C21 at either 0.3 (low dose, LD) or 1.0 (high dose, HD) mg/kg/day. ADR-induced FSGS was more severe in AT2RKO mice compared with WT-FSGS mice, and included profound podocyte loss, glomerular fibrosis, and albuminuria. Glomerular cathepsin L expression increased more in AT2RKO-FSGS than in WT-FSGS mice. C21 treatment ameliorated podocyte injury, most significantly in the Pre C21-HD group, and inhibited glomerular cathepsin L expression. In vitro, Agtr2 knock-down in mouse podocyte cell line given ADR confirmed the in vivo data. Mechanistically, C21 inhibited cathepsin L expression, which protected synaptopodin from destruction and stabilized actin cytoskeleton. C21 also prevented podocyte apoptosis. In conclusion, AT2R activation by C21 ameliorated ADR-induced podocyte injury in mice by the inhibition of glomerular cathepsin L leading to the maintenance of podocyte integrity and prevention of podocyte apoptosis.

Keywords: AT2R; C21; FSGS; cathepsin L; podocytes.

Figure 1. Physiological parameters of FSGS mice with AT₂R deletion and C21 treatment.

(A) Schematic design of the study protocol. Both wild-type (WT) and AT₂RKO mice received adriamycin (ADR) injection to induce FSGS. Separate groups of WT-FSGS mice were treated with either 0.3 (low dose, LD) or 1.0 (high dose, HD) mg/kg/day of Compound 21 (C21), 1-day before (Pre-C21) or 7-day after (Post- C21) the ADR injection. D, day. (B-C) Body weight (g) change at day 28 (N=12–20 in each group). (D-E) Systolic blood pressure (mmHg) at day 28 (N=8–12 in each group). (F-G) Kidney weight (KW)/tibia length (TL) ratio in WT and AT₂RKO mice with and without FSGS (F) and WT-FSGS mice treated with C21 (G) (N=9–12 in each group). Data are mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. Deletion of AT₂RKO exacerbated and C21 ameliorated ADR-induced FSGS.

(A; C; E; G) WT and AT₂RKO with and without FSGS; (b; d; f; h) WT-FSGS mice treated with C21; (A-B) Longitudinal measurements of urinary ACR (N= 8–13 in each group). * p<0.05 in WT-FSGS vs AT₂RKO-FSGS. ^# p<0.05 in WT-FSGS vs Pre C21-LD, ^† p<0.05 in WT-FSGS vs Pre C21-HD; (C-F) Coomassie Blue gel staining of urine at week 2 (C, D) and week 4 (E, F) of FSGS. MSA, mouse serum albumin (μg); (G, H) Urinary Ang II/creatinine value at week 4 of FSGS. Data are mean ± SEM. * p<0.05, ** p<0.01. *** p<0.001.

Figure 3. The effect of AT₂R deletion and C21 treatment on renal histology in ADR-induced FSGS.

(A-D) PAS staining of glomeruli in outer cortex in WT and AT₂RKO mice with and without FSGS (A-B) and WT-FSGS mice treated with C21 (C-D). Glomerular injury score based on the PAS images in WT and AT₂RKO mice with and without FSGS (B) and WT-FSGS mice treated with C21 (D). Each mouse is represented by a dot, which is the average of scores of the 30–35 glomeruli (n = 6–7 in each group). (E-H) Masson’s trichrome staining of glomeruli in outer cortex in WT and AT₂RKO mice with and without FSGS (E-F) and WT-FSGS mice treated with C21 (G-H) and the quantification of glomerular fibrosis (F, H). Scale bar, 50 μm; Data are mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. The effect of AT₂R deletion and C21 treatment on glomerular molecular changes in ADR-induced FSGS.

(A-D) RT-qPCR of Bax and Bcl2 mRNA (A, B) and Tgf-β1 and Col4α1 mRNA (C, D) in freshly isolated glomeruli. Rpl13α was used as a reference gene. Data are mean ± SEM, N=6 mice per group. (E; F) TUNEL staining of glomeruli in WT and AT₂RKO mice with and without FSGS (E) and WT-FSGS mice treated with C21 (F) and their semi-quantifications. Scale bar, 50 μm; (−) TUNEL negative staining; (+) TUNEL positive staining. Scale bar, 10 μm; (G; H) Collagen IV-IHC staining of glomeruli in WT and AT₂RKO mice with and without FSGS (G) and WT-FSGS mice treated with C21 (H) and their quantifications. Scale bar, 50 μm; Data are mean ± SEM. N=4–5 mice in each group; * p<0.05, *** p<0.001.

Figure 5. The effect of AT₂R deletion and C21 treatment on podocyte injury in ADR-induced FSGS.

(A; C; E; G) WT and AT₂RKO with and without FSGS; (B; D; F; H) WT-FSGS mice treated with C21; (A-B) Electron microscopy. Original magnification x5000. Scale bar, 2 μm; (C-D) RT-qPCR of synaptopodin (Synpo) and Wilms tumor 1 (Wt-1) mRNA in freshly isolated glomeruli. Rpl13α was used as a reference gene; (E; F) Synpo-IF staining and their quantifications (N=5 in each group). (G-H) Podocyte number per glomerular section based on p57-positive cells (red) in glomeruli of the kidneys with DAPI (blue). Each mouse is represented by a dot, which is the average of the p-57 positive cells in 30–35 glomeruli (N = 4–6 mice in each group). Scale bar, 50 μm; Data are mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 6. The effect of AT₂R deletion and C21 treatment on glomerular cathepsin L expression in ADR-induced FSGS.

(A; B) RT-qPCR of inflammatory cytokines (C–C motif chemokine ligand 2 (Ccl2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10) in freshly isolated glomeruli. Rpl13α was used as a reference gene; (C; D) Co-IF staining of glomerular cathepsin L (red) and the merge with Synpo (green) or DAPI (blue) in WT and AT₂RKO mice with and without FSGS (C) and WT-FSGS mice treated with C21 (D). Scale bar, 50 μm. (E; F) Semi-quantification of cathepsin L positive staining area/glomerular area (N=6 in each group). Data are mean ± SEM. * p<0.05, *** p<0.001.

Figure 7. Podocyte integrity and apoptosis induced by ADR in vitro.

(A) Synpo-IF (upper panel, white) and F-actin-IF (lower panel, white) with DAPI (blue) in mPODs, in the presence or absence of C21 and/or ADR. (B) Effect of Agtr2 siRNA (siAgtr2) or scrambled siRNA (siCon) with and without ADR on Synpo and F-actin expressions. (C) Western blot analysis in mPODs with and without ADR, treated with different doses of C21 for 24 hours. (D) Western blot analysis in mPODs with siAgtr2 vs siCon with and without ADR. Scale bar, 50 μm. Data are mean ± SEM. * p<0.05, *** p<0.001.

Figure 8. ADR increases and C21 decreases Cathepsin L expression

(A) Co-IF staining of Cathepsin L (red) and the merge with F-actin (green) and DAPI (blue) in mPODs in vitro. Control, Agtr2 siRNA (siAgtr2), C21-treated, and CAA0225 (a cathepsin L inhibitor)-treated cells were assessed with and without ADR. Scale bar, 50 μm. (B) A schematic illustration of how Cathepsin L potentially mediates the podocyte actin instability and proteinuria in AT₂R deletion on FSGS development in mice; and how the administration of AT₂R agonist (C21) would ameliorate FSGS-induced podocyte injury. In brief, increased inflammatory cytokines (infl. cyto) by ADR likely stimulate Cathepsin L expression, which causes podocyte actin instability, leading to podocyte loss (left orange podocyte). In contrast, C21 prevents the ADR-induced inflammatory cytokines and Cathepsin L expression, thereby maintaining the podocyte integrity (right green podocyte).