Reversal of albuminuria by combined AM6545 and perindopril therapy in experimental diabetic nephropathy

Abstract

Background and purpose: The endocannabinoid (EC) system has been implicated in the pathogenesis of diabetic nephropathy (DN). We investigated the effects of peripheral blockade of the cannabinoid CB₁ receptor as an add-on treatment to ACE-inhibition in type 1 diabetic mice (DM) with established albuminuria.

Experimental approach: Renal functional parameters (albumin excretion rate, creatinine clearance), tubular injury, renal structure, both EC and CB receptor levels and markers of podocyte dysfunction, fibrosis and inflammation were studied in streptozotocin-induced DM treated for 14 weeks with vehicle, the ACE-inhibitor perindopril (2 mg·kg^-1 ·day^-1 ), peripherally-restricted CB₁ receptor antagonist AM6545 (10 mg·kg^-1 ·day^-1 ) or both. Treatments began at 8 weeks after diabetes onset, when early DN is established.

Key results: CB₁ receptors were overexpressed in DM and neither perindopril nor AM6545 altered this effect, while both drugs abolished diabetes-induced overexpression of angiotensin AT₁ receptors. Single treatment with either AM6545 or perindopril significantly reduced progression of albuminuria, down-regulation of nephrin and podocin, inflammation and expression of markers of fibrosis. However, reversal of albuminuria was only observed in mice administered both treatments. The ability of the combination therapy to completely abolish slit diaphragm protein loss, monocyte infiltration, overexpression of inflammatory markers and favour macrophage polarization towards an M2 phenotype may explain this greater efficacy. In vitro experiments confirmed that CB₁ receptor activation directly inhibits retinoic acid-induced nephrin expression in podocytes and IL-4-induced M2 polarization in macrophages.

Conclusion and implications: Peripheral CB₁ receptor blockade used as add-on treatment to ACE-inhibition reverses albuminuria, nephrin loss and inflammation in DM.

Figure 1

Effect of treatment with AM6545 and/or perindopril on CB₁, CB₂ and AT₁ receptors in DM. Control ND and DM were treated with vehicle, AM6545 (AM), perindopril (ACE‐I) or AM6545 plus perindopril (Combo) (n = 6 mice per group). Representative immunohistochemistry analysis of CB₁ receptors (A) and CB₂ receptors (D) are shown (original magnification ×400) and quantification of glomerular positive staining reported in the graphs (B–E). CB₁, CB₂ and AT₁ receptor mRNA levels were measured by real‐time PCR on total RNA extracted from the renal cortex. Results were corrected for the expression of the housekeeping gene HPRT and shown in the graphs (C, F, I). Representative immunoblot (G) and densitometry analysis (H) of AT₁ receptor protein expression in total protein extracts from the total renal cortex are shown. Tubulin was used as internal control (*P < 0.05, vs. ND).

Figure 2

Effect of treatment with AM6545 and/or perindopril on nephrin and podocin expression and podocyte number in DM. Control ND and DM were treated with vehicle, AM6545 (AM), perindopril (ACE‐I) or AM6545 plus perindopril (Combo) (n = 6 mice per group). Both nephrin (A–C) and podocin (D–F) mRNA and protein expression was assessed. Representative immunofluorescence images of nephrin (A) and podocin (D) are shown (original magnification ×400). Quantification of both glomerular staining (B, E) and mRNA levels (C, F) is reported in the graphs. Representative immunoblots (G) and results of densitometry analysis (H, I) of nephrin and podocin protein expression in total protein extracts from the total renal cortex. Tubulin was used as an internal control. Representative immunohistochemistry images (J original magnification ×400) and quantification of glomerular positive staining of p57 (K) are shown (*P < 0.05 vs. ND; ^# P < 0.05 vs. DM; °P < 0.05 dual treatment vs. single treatment). (L) Nephrin mRNA levels were measured by real‐time PCR on total RNA extracts from podocytes exposed to RA (1 μmol·L⁻¹)/vehicle for 24 h in the presence and in the absence of ACEA (1 μmol·L⁻¹) (*P < 0.05 RA vs. vehicle; ^§ P < 0.05 RA vs. ACEA and RA + ACEA; ^# P < 0.05 RA + ACEA vs. vehicle).

Figure 3

Effect of AM6545 and/or perindopril on markers of fibrosis in DM. ND and DM were treated with vehicle, AM6545 (AM), perindopril (ACE‐I) or AM6545 plus perindopril (Combo) (n = 6 mice per group) for 14 weeks. PAS staining was assessed in renal cortex sections. Representative images (scale bar: 50 μm) and percentage PAS glomerular‐positive areas are shown (A, B). Both fibronectin (C) and collagen type I (D) mRNA expression was measured by real‐time PCR on total RNA extracted from the renal cortex and results are presented in the graphs. Representative images of glomerular fibronectin protein expression (E), picrosirius red staining (G) and quantitation of glomerular staining are shown (F, H) (*P < 0.05 vs. ND).

Figure 4

Effect of AM6545 and/or perindopril on diabetes‐induced inflammation. ND and DM were treated with vehicle, AM6545 (AM), perindopril (ACE‐I) or AM6545 plus perindopril (Combo) (n = 6 mice per group) for 14 weeks. Representative images of glomerular F4/80 (A) and MAC‐2 (C) positive immunostaining are shown and quantitation reported in the graph (B, D). Total RNA was extracted from the renal cortex and levels of mRNA encoding for CD68 (E), ICAM‐1 (F), MCP‐1 (H) and CCR2 (J), measured by real‐time PCR. Protein levels of ICAM‐1 (G) and MCP‐1 (I) in renal tissue were determined by elisa and normalized to total protein (*P < 0.05 vs. ND; ^# P < 0.05 vs. DM group; °P < 0.05 dual vs. single treatment; ^§ P < 0.05 combo vs. ND).

Figure 5

Effect of AM6545 and/or perindopril on macrophage phenotype. ND and DM were treated with vehicle (DM), AM6545 (AM), perindopril (ACE‐I) or AM6545 plus perindopril (Combo) (n = 6 mice per group) for 14 weeks. Total RNA was extracted from the renal cortex and levels of mRNA encoding for TNF‐α (A), NOS2 (C), CCL3 (D), CD163 (E), CD206 (F), arginase‐1 (G), measured by real time‐PCR. (B) Levels of TNF‐α in kidney tissue were determined by elisa and normalized to total protein (*P < 0.05 vs. ND; ^# P < 0.05 vs. DM; °P < 0.05 dual vs. single treatment; ^§ P < 0.05 combo vs. ND). (H) Arginase‐1 mRNA levels were measured by real‐time PCR on total RNA extracts from RAW 264.7 cells exposed to WIN55,212‐2 (1 μmol·L⁻¹)/vehicle for 18 h in the presence and in the absence of AM6545 (1 μmol·L⁻¹) and AM630 (1 μmol·L⁻¹), followed by incubation with IL‐4 (5 ng·mL⁻¹) for 6 h (*P < 0.05 WIN and AM6545 vs. vehicle).