Fibrosis is a hallmark of progressive organ diseases. Monocyte chemoattractant protein (MCP)-1, also termed as macrophage chemotactic and activating factor (MCAF/CCL2) and its receptor, CCR2 are presumed to contribute to progressive fibrosis. However, the therapeutic efficacy of MCP-1/CCR2 blockade in progressive fibrosis remains to be investigated. We hypothesized that blockade of CCR2 may lead to the improvement of fibrosis. To achieve this goal, we investigated renal interstitial fibrosis induced by a unilateral ureteral obstruction in CCR2 gene-targeted mice and mice treated with propagermanium or RS-504393, CCR2 inhibitors. Cell infiltrations, most of which were F4/80-positive, were reduced in CCR2 knockout mice. In addition, dual staining revealed that CCR2-positive cells were mainly F4/80-positive macrophages. Importantly, CCR2 blockade reduced renal interstitial fibrosis relative to wild-type mice. Concomitantly, renal transcripts and protein of MCP-1, transforming growth factor-␤, and type I collagen were decreased in CCR2-null mice. Further, this CCR2-dependent loop for renal fibrosis was confirmed by treatment with CCR2 antagonists in a unilateral ureteral obstruction model. These findings suggest that the therapeutic strategy of blocking CCR2 may prove beneficial for progressive fibrosis via the decrease in infiltration and activation of macrophages in the diseased kidneys. Fibrosis is characteristic in progressive diseases, resulting in organ failure. Monocyte chemoattractant protein (MCP)-1 (also termed as monocyte chemotactic and activating factor/CCL2) is presumed to be a key molecule in chemotaxis and activation of macrophages.

Abstract. Examined were CCR2-deficient mice to clarify the contribution of macrophages via monocyte chemoattractant protein 1 (MCP-1 or CCL2)/CCR2 signaling to the pathogenesis of renal ischemia-reperfusion injury. Also evaluated was the therapeutic effects via the inhibition of MCP-1/CCR2 signaling with propagermanium (3-oxygermylpropionic acid polymer) and RS-504393. Renal artery and vein of the left kidney were occluded with a vascular clamp for 60 min. A large number of infiltrated cells and marked acute tubular necrosis in outer medulla after renal ischemia-reperfusion injury was observed. Ischemia-reperfusion induced the expression of MCP-1 mRNA and protein in injured kidneys, followed by CCR2-positive macrophages in interstitium in wild-type mice. The expression of MCP-1 was decreased in CCR2-deficient mice compared with wild-type mice. The number of interstitial infiltrated macrophages was markedly smaller in the CCR2-deficient mice after ischemia-reperfusion. CCR2-deficient mice decreased the number of interstitial inducible nitric oxide synthase-positive cells after ischemia-reperfusion. The area of tubular necrosis in CCR2-deficient mice was significantly lower than that of wild-type mice after ischemia-reperfusion. In addition, CCR2-deficient mice diminished KC, macrophage inflammatory protein 2, epithelial cell-derived neutrophil-activating peptide 78, and neutrophil-activating peptide 2 expression compared with wild-type mice accompanied with the reduction of interstitial granulocyte infiltration. Similarly, propagermanium and RS-504393 reduced the number of interstitial infiltrated cells and tubular necrosis up to 96 h after ischemia-reperfusion injury. These results revealed that MCP-1 via CCR2 signaling plays a key role in the pathogenesis of renal ischemia-reperfusion injury through infiltration and activation of macrophages, and it offers a therapeutic target for ischemia-reperfusion.

Abstract. Monocyte chemoattractant protein (MCP)-1, also termed monocyte chemotactic and activating factor (MCAF)/ CCL2, plays an important role in progressive organ fibrosis. It was hypothesized that MCP-1, through its cognate receptor, CCR2, regulates the pathogenesis and is therapeutically of importance for renal fibrosis. To achieve this goal, the therapeutic efficacy and efficiency in renal fibrosis induced by a unilateral ureteral obstruction nephropathy model in mice by the blockade of MCP-1/CCR2 signaling was studied. The delivery of N-terminal deletion mutant of the human MCP-1 gene, 7ND, into a skeletal muscle ameliorated renal fibrosis by resulting in decrease in the deposit of type I collagen and in reduced expression of TGF-␤. Concomitantly, gene transfer of 7ND reduced the cell infiltration, most of which were CCR2-positive macrophages, followed by the decrease in MCP-1 expression in the diseased kidneys. These observations suggest that MCP-1 through CCR2 signaling is responsible for M recruitment, which augments downstream events, resulting in renal fibrosis. Moreover, these findings imply that gene therapy against MCP-1/CCR2 signaling via the mutant gene transferred strategy may serve a beneficial therapeutic application for renal fibrosis.

A novel lipophilic zinc(II) complex with 1-hexadecyl-1,4,7,10-tetrazacyclododecane (hexadecylcyclen, L) has been synthesized, which is almost insoluble in water, but becomes soluble in the presence of Triton X-100 surfactant. Analysis of the potentiometric pH titration of ZnII−hexadecylcyclen (1 mM) in the presence of Triton X-100 (10 mM) disclosed monodeprotonation of the ZnII-bound H2O, yielding an OH--bridged complex (ZnL)2OH- (pK d = −log([(ZnL)2OH-]a H + /[ZnL]2) = 3.92 ± 0.05) and a monomeric ZnII−OH- complex ZnL−OH- (pK a = −log([ZnL−OH-]a H + /[ZnL]) = 7.56 ± 0.05) at 25 °C with I = 0.10 (NaNO3). The zinc(II) complex (ZnL) possesses higher catalytic activity than the parent zinc(II) complex of 1,4,7,10-tetrazacyclododecane (cyclen, L‘) in hydrolysis of 4-nitrophenyl acetate (NA), bis(4-nitrophenyl) phosphate (BNP-), and tris(4-nitrophenyl) phosphate (TNP) in aqueous comicellar solution with 10 mM Triton X-100. The NA hydrolysis activity of ZnII−hexadecylcyclen increased with the pH and leveled off at pH > 10 and 25 °C, from which the active species was estimated to be a ZnL−OH- complex. The second-order rate (first-order in [NA] and [ZnL−OH-]) of NA hydrolysis (k NA = 5.0 ± 0.2 M-1 s-1) in the presence of 10 mM Triton X-100 is 50 times greater than that with a reference ZnL‘−OH- in 10% (v/v) CH3CN aqueous solution. Hydrolysis of TNP in 10% (v/v) MeOH aqueous solution with ZnL−OH- and 10 mM Triton X-100 (k TNP = (1.1 ± 0.1) × 103 M-1 s-1) is 290 times more efficient than with ZnL‘−OH- (3.8 ± 0.2 M-1 s-1) at 25 °C. The higher effective molarity of the lipophilic substrate coexisting with ZnL−OH- in the micelles accounts for the extraordinary catalytic activity. From comparison with previously reported metal catalysts, the present lipophilic zinc(II) complex with hexadecylcyclen is probably one of the best candidate catalyst for detoxification of poisonous phosphotriesters.

a b s t r a c tIn this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFa remarkably enhanced GPR84 expression. In the presence of TNFa, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFa from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity.