Cannabinoid receptor 1 blockade ameliorates albuminuria in experimental diabetic nephropathy

Abstract

Objective: Cannabinoid receptor 1 (CB1) is localized in the central nervous system and in peripheral tissues involved in energy metabolism control. However, CB1 receptors are also expressed at low level within the glomeruli, and the aim of this study was to investigate their potential relevance in the pathogenesis of proteinuria in experimental type 1 diabetes.

Research design and methods: Streptozotocin-induced diabetic mice were treated with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,3-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), a selective CB1-receptor antagonist, at the dosage of 1 mg x kg(-1) x day(-1) via intraperitoneal injection for 14 weeks. Urinary albumin excretion was measured by enzyme-linked immunosorbent assay. CB1 receptor expression was studied by immunohistochemistry, immunoblotting, and real-time PCR. Expression of nephrin, podocin, synaptopodin, and zonula occludens-1 (ZO-1) was assessed by immunofluorescence and real-time PCR. Fibronectin, transforming growth factor-beta1 (TGF-beta1), and connective tissue growth factor (CTGF) mRNA levels were quantitated by real-time PCR.

Results: In diabetic mice, the CB1 receptor was overexpressed within the glomeruli, predominantly by glomerular podocytes. Blockade of the CB1 receptor did not affect body weight, blood glucose, and blood pressure levels in either diabetic or control mice. Albuminuria was increased in diabetic mice compared with control animals and was significantly ameliorated by treatment with AM251. Furthermore, CB1 blockade completely prevented diabetes-induced downregulation of nephrin, podocin, and ZO-1. By contrast overexpression of fibronectin, TGF-beta1, and CTGF in renal cortex of diabetic mice was unaltered by AM251 administration.

Conclusions: In experimental type 1 diabetes, the CB1 receptor is overexpressed by glomerular podocytes, and blockade of the CB1 receptor ameliorates albuminuria possibly via prevention of nephrin, podocin, and ZO-1 loss.

FIG. 1.

The CB1 receptor is overexpressed in experimental diabetes. CB1 protein expression was assessed in renal sections from nondiabetic (A and C) and diabetic (B and D) mice by immunohistochemistry as described in the “Research Design and Methods” section (magnification ×400: in A and B; ×100 in C and D). Percentage area of staining for CB1, quantified by a computer-aided image analysis system, is shown for both diabetic and nondiabetic mice (*P < 0.05 diabetic vs. nondiabetic) (E). CB1 mRNA levels in renal cortex from both diabetic and nondiabetic mice were measured by real-time PCR and corrected for the expression of the housekeeping gene HPRT as described in the “Research Design and Methods” section (*P < 0.05 diabetic vs. nondiabetic) (F). CB1 protein expression was assessed by immunoblotting in total protein extracts of renal cortex from diabetic and nondiabetic mice. Tubulin was used as internal control. A representative immunoblot and results of densitometry analyses are shown (*P < 0.05 diabetic vs. nondiabetic) (G and H). Double immunofluorescence for CB1 (I) and nephrin (L) performed on diabetic glomeruli showed colocalization of the positive staining, as demonstrated by merging (M). The dashed square in M delimits the area shown at higher magnification in N. Binding of AM251 fluorescent analog (O) and CB1 immunostaining (P) overlapped in the diabetic glomeruli as shown by merging (Q). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

CB1 blockade abolished downregulation of nephrin, podocin, and ZO-1 protein expression in diabetic mice. Renal cryostatic sections from both diabetic and nondiabetic mice, treated with either vehicle or the CB1 antagonist AM251 for 14 weeks, were stained for nephrin (A), podocin (C), ZO-1 (E), and synaptopodin (G) by immunofluorescence as described in the “Research Design and Methods” section (magnification ×400). Quantification of glomerular staining for nephrin (B), podocin (D), ZO-1 (F), and synaptopodin (H) is shown (*P < 0.01 diabetic vs. others; #P < 0.001 diabetic vs. others). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

CB1 blockade prevented diabetes-induced reduction of both nephrin and podocin gene expression. Total RNA was extracted from the renal cortex of nondiabetic and diabetic mice treated with either vehicle (diabetic mice) or the CB1 antagonist AM251 (diabetic + AM251) for 14 weeks. Nephrin (A), podocin (B), and ZO-1 (C) mRNA levels were measured by real-time PCR and corrected for the expression of WT-1 as described in the “Research Design and Methods” section (*P < 0.05 diabetic vs. nondiabetic and diabetic + AM251; #P < 0.05 diabetic vs. nondiabetic).

FIG. 4.

Podocytes number in diabetic and nondiabetic mice treated with either vehicle or AM251. WT-1 protein expression was assessed by immunohistochemistry in the glomeruli from nondiabetic and diabetic mice treated for 14 weeks with either vehicle or the CB1 receptor antagonist AM251. Quantification of WT-1–positive cells per glomerular area is shown in the graph.

FIG. 5.

Transmission electron microscopy analysis. Representative electron microscopy images of glomeruli (upper panels) and tubuli (lower panels) from nondiabetic (A, B, G, and H: magnification ×2600) and diabetic mice (C, D, I, and L: magnification ×2600; E and F: magnification ×6000) treated for 14 weeks with either vehicle (A, C, E, G, and I) or the CB1 receptor antagonist AM251 (B, D, F, H, and L). In A and B the glomerular structure is normal. Glomeruli from diabetic mice (C and D) display some degree of mesangial expansion, independent of treatment. At higher magnification (E and F) the podocyte foot processes are regularly shaped and only a minimal irregularity in the thickness of the GBM can be observed in E. Tubular structure appears normal in both control and diabetic mice (G and I) and unaffected by treatment with AM251 (H and L).

FIG. 6.

CB1 blockade did not affect overexpression of fibronectin, TGF-β1, and CTGF in diabetic mice. Total RNA was extracted from the renal cortex of nondiabetic and diabetic mice treated with either vehicle (diabetic mice) or the CB1 antagonist AM251 (diabetic + AM251) for 14 weeks. TGF-β1 (A), CTGF (B), and fibronectin (C) mRNA levels were measured by real-time PCR and corrected for the expression of the gene housekeeping HPRT as described in the “Research Design and Methods” section (*P < 0.05 diabetic and diabetic + AM251 vs. nondiabetic).