The Endocannabinoid System

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The two endogenous cannabinoids (ECs), anandamide and 2-arachidonoylglycerol (2-AG), bind to the endocannabinoid receptor of type 1 (CB1) and type 2 (CB2) that are coupled to G proteins. The CB1 receptor is expressed predominantly in the central nervous system [118], but is also exposed by several other cell types in peripheral tissues where it displays potent oxidative, inflammatory and profibrotic activity [119-121]. By contrast, the CB2 receptor is mainly expressed by immune cells and has strong anti-inflammatory properties [119-122]. We have recently reported that a full EC system is present within the kidney, comprising ECs, EC receptors, and enzymes involved in EC synthesis and degradation [123, 124]. In the normal glomeruli, constitutive EC receptor expression is low for CB1 and high for CB2 and localises predominantly to podocytes [123, 124]. In addition, both receptors are also expressed by monocytes/macrophages, implying relevance to inflammatory processes, and a recent study in monocytes has shown that CB1 activates an intracellular cascade leading to inflammatory cytokine production through induction of oxidative stress that is inhibited by CB2 activation [125].

Recently, we have reported that in STZ-induced diabetes the CB1 receptor is overexpressed within the glomeruli predominantly by podocytes [124]. On the contrary, podocyte CB2 expression was strongly downregulated in human biopsies from patients with advanced DN and the only CB2 positive cells within the glomeruli were infiltrating monocytes [123]. In early experimental diabetes, CB2 expression was still unaltered, but there was a relative deficiency of 2-AG, the major CB2 ligand, in the renal cortex [123]. Collectively, these data suggest that in DN signalling through the deleterious CB1 receptor is enhanced, while the protective CB2 signalling is reduced. The underlying cellular mechanisms are unknown; however, in vitro high glucose increased CB1 expression in podocytes [126] and mesangial cells [127], whereas mechanical stretch downregulates CB2 in cultured podocytes [128], suggesting that the major insults involved in the pathogenesis of DN can modulate the response of glomerular cells to EC. Furthermore, exposure of cultured proximal tubular epithelial cells to albumin reduces CB2 expression, suggesting that proteinuria may diminish the constitutive anti-inflammatory activity of the tubulo-interstitium in advanced DN [129].

Recently, we have provided evidence of the important role of the EC system in the pathogenesis of DN. We have shown that blocking of CB1 receptors with AM251, a selective CB1 receptor antagonist, ameliorates albuminuria by preventing the downregulation of both nephrin and podocin in STZ-induced diabetic mice [124]. Other studies have shown that treatment with selective CB1 antagonists has also renoprotective and proteinuric effects in obesity-induced nephropathy [130] and db/db mice [126], though beneficial effects may be partially ascribed to amelioration of metabolic control in these models. We have also demonstrated that activation of the CB2 receptor, using the selective CB2 agonist AM1241, reduces albuminuria, glomerular monocyte infiltration, and nephrin loss in STZ-induced diabetic mice [123], indicating that a strategy compensating for the relative deficiency of CB2 signalling can be beneficial. Recently, we have further confirmed the protective role of CB2 by showing that in diabetic mice deletion of the CB2 gene worsens proteinuria, mesangial matrix expansion, renal function loss, monocytes infiltration, downregulation of slit diaphragm proteins, and overexpression of extracellular matrix components [128]. Taken together these results demonstrated that both CB1 overexpression and CB2 downregulation play an important role in the pathogenesis of experimental DN and may thus represent novel targets for treatment.

The anti-proteinuric and renoprotective effect of CB2 is likely due to inhibition of inflammatory processes and we have shown the existence of an interaction between the CB2 receptor and the MCP-1/CCR2 system. Although pharmacological/genetic modulation of CB2 does not alter MCP-1 expression, CB2 activation reduces [123], whereas CB2 deletion strongly enhances CCR2 expression [128] in the renal cortex of both diabetic and non-diabetic mice. Consistently, in both cultured podocyte [123] and monocytes [1301, CCR2 expression is downregulated by CB2 agonists and upregulated by CB2 antagonists. Therefore, CB2 appears an endogenous modulator of the MCP-1/CCR2 system and may represent a physiological target of therapies aiming to lower MCP-1 signalling (Figure 2).

Of interest, we have recently shown using adaptive transfer techniques that worsening of DN in CB2 knockout mice is due to CB2 deficiency on podocytes rather than on monocytes [128], suggesting that the predominant mechanism of kidney damage in response to MCP-1 is not monocyte recruitment, but the direct MCP-1 effect on podocytes. Lowering of inflammatory processes may also contribute to the beneficial effects of CB1 blockade observed in animal model of DN. Consistently, CB1 antagonists reduce monocyte infiltration in STZ-diabetic mice (unpublished data) as well as MCP-1 expression in db/db mice [126]. The underlying mechanism remains elusive; however, CB1 is a potent inducer of oxidative stress pathways that are strictly interconnected with inflammatory cascades. Furthermore, CB1 promotes pro-inflammatory responses of macrophages through ROS production [125] and favors macrophage polarization towards a M1 phenotype in various tissues.