The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-beta, increases podocyte motility and albumin permeability

Abstract

The role of monocyte chemoattractant protein-1 (MCP-1) in diabetic nephropathy is typically viewed through the lens of inflammation, but MCP-1 might exert noninflammatory effects on the kidney cells directly. Glomerular podocytes in culture, verified to express the marker nephrin, were exposed to diabetic mediators such as high glucose or angiotensin II and assayed for MCP-1. Only transforming growth factor-β (TGF-β) significantly increased MCP-1 production, which was prevented by SB431542 and LY294002, indicating that signaling proceeded through the TGF-β type I receptor kinase and the phosphatidylinositol 3-kinase pathway. The TGF-β-induced MCP-1 was found to activate the podocyte's cysteine-cysteine chemokine receptor 2 (CCR2) and, as a result, enhance the cellular motility, cause rearrangement of the actin cytoskeleton, and increase podocyte permeability to albumin in a Transwell assay. The preceding effects of TGF-β were replicated by treatment with recombinant MCP-1 and blocked by a neutralizing anti-MCP-1 antibody or a specific CCR2 inhibitor, RS102895. In conclusion, this is the first description that TGF-β signaling through PI3K induces the podocyte expression of MCP-1 that can then operate via CCR2 to increase cellular migration and alter albumin permeability characteristics. The pleiotropic effects of MCP-1 on the resident kidney cells such as the podocyte may exacerbate the disease process of diabetic albuminuria.

mononocyte chemoattractant protein-1 (MCP-1), a member of the cysteine-cysteine ligand chemokine family that regulates the recruitment and activation of monocytes and macrophages, is believed to play a major role in many kidney diseases, including diabetic nephropathy. MCP-1 (also known as CCL2) is upregulated in the diabetic kidney and is excreted in increased amounts in the urine of diabetic subjects (20, 26). The elevation in urinary MCP-1 has been correlated with progression and renal outcomes in humans (1). In culture, various kidney cell lines such as renal tubular cells and mesangial cells have been shown to increase their MCP-1 expression in response to diabetic conditions, as mimicked by high ambient glucose or advanced glycation end products (13). When the MCP-1 increase in diabetes is prevented by a genetic knockout, the mouse model displays amelioration of diabetic albuminuria, reduction in renal fibrosis, preservation of kidney clearance function, and decreased accumulation of macrophages (8). The latter finding of macrophage infiltration in the kidney suggests that diabetic nephropathy can be considered, in part, an inflammatory-mediated disease, and certain immunosuppressive therapies have successfully delayed the development of diabetic renal disease (28).

However, the deleterious actions of MCP-1 in the diabetic kidney may accrue beyond its well-characterized ability to attract macrophages and promote inflammation. The secreted MCP-1 could have substantive effects on the resident kidney cells themselves. The podocyte seems an apt choice for further investigation, as MCP-1 gene expression appears to be predominantly localized to podocytes in the glomeruli of diabetic mice (7), and advanced glycation end products (AGE) stimulate MCP-1 mRNA and protein expression in mouse podocytes (12), raising the possibility that MCP-1 might in turn have a pathogenetic effect on the podocytes. Thus far, it has been shown that the binding of MCP-1 to a specific receptor stimulates the podocyte to enhance its migratory capacity, with possible implications for the progression of crescentic glomerulonephritis (5). Given that the podocyte is subjected to the heightened MCP-1 levels of the diabetic state, further investigation is warranted into the noninflammatory mechanisms whereby MCP-1 participates in the pathogenesis of diabetic nephropathy, especially considering that podocytes play a role in the albuminuria of diabetes.

MCP-1 acts on its target cells by binding to the cognate cysteine-cysteine chemokine receptor 2 (CCR2), a member of the seven-transmembrane, G protein-coupled receptor family that is the main signaling partner of MCP-1 (29). CCR2 probably exists in the podocyte, with mRNA expression demonstrated in glomerular podocytes and CCR2 protein observed in cultured mouse and human podocytes (5, 27). The CCR2 receptor can be inhibited by a nonpeptide molecule dubbed RS102895, which belongs to the structural class of spiropiperidine. The RS102895 compound prevents MCP-1 binding by occupying the interhelical bundle region on the extracellular side of the CCR2 receptor; RS102895 does not antagonize other chemokine receptors such as CXCR1, CCR1, or CCR3 (21). Therefore, RS102895 is a useful tool for interrogating the role of MCP-1/CCR2 signaling in various models of disease.

To isolate the contribution of the podocytic MCP-1/CCR2 system, a cultured line of conditionally immortalized mouse podocytes was utilized. In this study, we determine the diabetic metabolic mediators that can induce MCP-1 expression [e.g., transforming growth factor-β (TGF-β)] and the signaling pathways involved, confirm the expression of CCR2 in podocytes, and establish the paradigm of an inducible podocyte MCP-1/CCR2 loop. The functionality of this loop, demonstrated by the use of a neutralizing anti-MCP-1 antibody and a CCR2 inhibitor, is evidenced in the significant effects on podocyte motility, the actin cytoskeleton, and the permeability to albumin, phenomena that might underlie the pathophysiology of diabetic albuminuria.