Kengo Furuichi

Blockade of CCR2 Ameliorates Progressive Fibrosis in Kidney

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-beta, 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.

Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1).

We investigated the pathophysiological role of a potent macrophage (Mϕ) chemotactic cytokine (chemokine), monocyte chemotactic and activating factor/monocyte chemoattractant protein‐1 (MCAF/MCP‐1), in an animal model of crescentic glomerulonephritis. Administration of a small dose of nephrotoxic sera induced severe proliferative and necrotizing glomerulonephritis, with crescentic formation in the early phase and glomerulosclerosis in the later phase, in Wistar‐Kyoto rats. MCAF/MCP‐1 protein was detected immunohistochemically in glomeruli, vascular endothelial cells, and tubular epithelial cells in the early phase of injured kidney tissues but not in normal ones. Anti‐MCAF/MCP‐1 antibodies decreased the number of Mϕ in glomeruli, and prevented crescentic formation and the fusion of epithelial cell foot process in nephritic rats, thereby decreasing the excreted amounts of protein to normal levels on days 3 and 6. Furthermore, anti‐MCAF/MCP‐1 antibodies remarkably reduced glomerulosclerosis and improved renal dysfunction as well as proteinuria in the later phase (56 days). These results indicate that MCAF/MCP‐1 essentially participates in the impairment of renal functions associated with crescentic glomerulonephritis by recruiting and activating Mϕ.—Wada, T., Yokoyama, H., Furuichi, K., Kobayashi, K.‐i., Harada, K., Naruto, M., Su, S.‐B., Akiyama, M., Mukaida, N., Matsushima, K. Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP‐1). FASEB J. 10, 1418‐1425 (1996)

CCR2 Signaling Contributes to Ischemia-Reperfusion Injury in Kidney

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.

Gene Therapy via Blockade of Monocyte Chemoattractant Protein-1 for Renal Fibrosis

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-beta. 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 Mphi 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.

Blockade of a Chemokine, CCL2, Reduces Chronic Colitis-Associated Carcinogenesis in Mice

Accumulating evidence indicates the crucial contribution of chronic inflammation to various types of carcinogenesis, including colon carcinoma associated with ulcerative colitis and asbestosis-induced malignant mesothelioma. Ulcerative colitis-associated colon carcinogenesis can be recapitulated in mice by azoxymethane administration followed by repetitive dextran sulfate sodium ingestion. In the course of this carcinogenesis process, the expression of a macrophage-tropic chemokine, CCL2, was enhanced together with intracolonic massive infiltration of macrophages, which were a major source of cyclooxygenase (COX)-2, a crucial mediator of colon carcinogenesis. Mice deficient in CCL2-specific receptor, CCR2, exhibited less macrophage infiltration and lower tumor numbers with attenuated COX-2 expression. Moreover, CCL2 antagonists decreased intracolonic macrophage infiltration and COX-2 expression, attenuated neovascularization, and eventually reduced the numbers and size of colon tumors, even when given after multiple colon tumors have developed. These observations identify CCL2 as a crucial mediator of the initiation and progression of chronic colitis-associated colon carcinogenesis and suggest that targeting CCL2 may be useful in treating colon cancers, particularly those associated with chronic inflammation.

Urinary levels of chemokines (MCAF/MCP-1, IL-8) reflect distinct disease activities and phases of human IgA nephropathy.

Leukocytes have been implicated to be involved in the pathogenesis of IgA nephropathy (IgAN). To clarify the precise molecular mechanism of recruitment and activation of leukocytes in the subgroups of IgAN, latent, acute, and chronic types, we studied monocyte chemotactic and activating factor (MCAF/MCP‐1) and interleukin (IL)‐8 in urines and renal expression of these cytokines. Urinary MCAF levels were significantly higher in chronic type, and were correlated with pathological progressive factors such as mesangial proliferation and interstitial cellular infiltration associated with CD68‐positive macrophage. On the other hand, urinary IL‐8 elevated only in acute type and were correlated with glomerular endocapillary proliferation and the degree of hematuria. In immunohistochemical study, IL‐8 was mainly observed in glomeruli, otherwise MCAF in vascular endothelial cells, tubular epithelial cells, and infiltrated mononuclear cells in the interstitial lesions. These observations demonstrated that MCAF and IL‐8 were differentially expressed in kidneys with IgAN, and their subtypes, and suggest that chemokines may be involved in the pathogenesis of IgAN at distinct phases or pathological lesions, possibly through the recruitment and activation of a distinct type of leukocyte. J. Leukoc. Biol. 63: 493–499; 1998.

Gene Therapy Expressing Amino-Terminal Truncated Monocyte Chemoattractant Protein-1 Prevents Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion is closely associated with tissue damage in various organs, including kidney. Despite clinical investigations, useful therapy for renal ischemia-reperfusion injury is not available so far. This study evaluated therapeutic effects of gene therapy expressing an amino-terminal deletion mutant of MCP-1 called 7ND to inhibit monocyte chemoattractant protein (MCP)-1/CCR2 signaling in vivo on renal ischemia-reperfusion injury. 7ND gene was transferred into the femoral muscle of Balb/c mice. Renal artery and vein of the left kidney were occluded with a vascular clamp for 60 min. A large number of infiltrated cells were observed, as was marked acute tubular necrosis in outer medulla after renal ischemia-reperfusion injury in control mice, while these lesions were significantly decreased in 7ND gene-transfected mice. Macrophages in the interstitial region, most of which were CCR2-positive, were markedly decreased in 7ND gene-transfected mice after reperfusion. Although macrophages infiltrated around MCP-1-positive cells in control mice, the smaller number of F4/80-positive cells could infiltrate into the neighbor of MCP-1-positive cells in 7ND-treated mice. These results provide evidence that gene therapy by 7ND is potentially a powerful therapeutic approach to inhibit MCP-1/CCR2 signaling, resulting in rescue from renal ischemia-reperfusion injury.

Distinct expression of CCR1 and CCR5 in glomerular and interstitial lesions of human glomerular diseases.

We investigated the presence of CCR1- and CCR5-positive cells immunohistochemically in the kidneys of 38 patients with several renal diseases, including 13 crescentic glomerulonephritis patients. In addition, we determined cell phenotypes of CCR1- and CCR5-positive cells using a dual immunostaining technique. Urinary levels of their ligands, for CCR1 and CCR5; macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated upon activation in normal T cells expressed and secreted (RANTES) were evaluated by enzyme-linked immunosorbent assay. CCR1- and CCR5-positive cells were detected in both glomeruli and interstitium of the diseased kidneys. Using a dual immunostaining technique, these positive cells were CD68-positive macrophages (MΦ) and CD3-positive T cells. The number of CCR1-positive cells in glomeruli was correlated with urinary levels of MIP-1α. The number of CCR1-positive cells in the interstitium was correlated with both urinary MIP-1α and RANTES levels. CCR1-positive cells in the interstitium remained after glucocorticoid therapy, most of which were MΦ, and were correlated with the intensity of interstitial fibrosis and tubular atrophy. Glomerular CCR5-positive cells were well correlated with extracapillary lesions and urinary MIP-1α levels, while interstitial CCR5-positive cells, mainly CD3-positive T cells, were correlated with interstitial lesions and urinary RANTES levels. Renal CCR5-positive cells were dramatically decreased during convalescence induced by glucocorticoids. These results suggest that chemokine receptor signaling may be pivotal for human renal diseases through the recruitment and activation of MΦ and T cells; CCR5-positive cells may participate in glomerular lesions including extracapillary lesions via MIP-1α and in interstitial lesions via RANTES. CCR1 may be involved in interstitial lesions in resolving phase after glucocorticoid therapy.

Interleukin-1-dependent sequential chemokine expression and inflammatory cell infiltration in ischemia-reperfusion injury.

Objective:Ischemia-reperfusion injury is known to cause organ failure, but the mechanisms of pathogenesis remain unclear. Inflammation is a factor in tissue destruction in ischemia reperfusion injury, and interleukin (IL)-1 is a key promoter of inflammation. Design:Prospective, randomized, and controlled study. Setting:University laboratory. Subjects:Male mice 6–8 wks of age, in which genes for IL-1&agr; and IL-1&bgr; (IL-1&agr;/&bgr; deficient) and IL-1 receptor antagonist (IL-1RA deficient) are deleted by homologous recombination, and wild-type controls on a Balb/c background. Interventions:In this study, the role of IL-1 on inflammatory cascades, including chemokine expression, inflammatory cell infiltration, and tissue destruction, was investigated in 45 mins of unilateral renal ischemic injury using IL-1&agr;/&bgr;-deficient mice and IL-1RA-deficient mice. In addition, the effects of IL-1 on chemokine expression in cultured tubular epithelial cells were investigated. Measurements and Main Results:In vivo study revealed that the number of interstitial infiltrated neutrophils and macrophages, which accompanied the increase of the serum levels of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-1&agr;, respectively, significantly increased in IL-1RA-deficient mice. The number of interstitial infiltrated neutrophils correlated well with serum levels of KC at 24 hrs after reperfusion, whereas the number of interstitial infiltrated macrophages correlated well with the serum levels of MIP-1&agr; and monocyte chemoattractant protein (MCP)-1 at 24 and 48 hrs after reperfusion, respectively. Likewise, in vitro study revealed that stimulation of tubular epithelial cells by IL-1&bgr; and/or H2O2 sequentially induced KC, MIP-1&agr;, and MCP-1 in both protein and messenger RNA levels, which is consistent with in vivo results. Conclusion:IL-1-dependent inflammatory cascades, followed by inflammatory cell infiltration and subsequent tissue destruction, may affect pathogenesis of renal ischemia-reperfusion injury.

p38 Mitogen-Activated Protein Kinase Contributes to Autoimmune Renal Injury in MRL-Faslpr Mice

ABSTRACT. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) is responsible for the production and signal transduction of cytokines and chemokines. This study hypothesized that p38 MAPK activation is required for spontaneous autoimmune renal injury in MRL- Fas lpr mice, resembling human lupus erythematosus. FR167653, a specific inhibitor of p38 MAPK, is orally administrated from 3 or 4 mo of age in MRL- Fas lpr mice (at doses of 10 or 32mg/kg per day) until 6 mo of age. The phosphorylated p38 MAPK in kidneys of MRL- Fas lpr mice was evaluated. The number of phosphorylated p38 MAPK-positive cells was increased in diseased kidneys. The daily oral administration of FR167653 decreased p38 MAPK phosphorylation in kidneys, especially in a group of mice administered FR167653 (32 mg/kg per day) daily from 3 to 6 mo of age. FR167653 reduced the accumulation of macrophages and T cell and prevented kidney pathology, resulting in prolonged survival. In addition, FR167653 reduced expression of MCP-1 and TNF-α in the diseased kidneys and cultured tubular epithelial cells. Furthermore, FR167653 decreased IgG levels in the diseased kidneys and circulation. These results suggest that the phosphorylation of p38 MAPK is required for the pathogenesis of renal injury in MRL- Fas lpr mice followed by subsequent expression of renal cytokine/chemokine and IgG production. This study provides evidence that the regulation of p38 MAPK is a novel target for the therapy of renal injury in systemic lupus erythematosus. twada@medf.m.kanazawa-u.ac.jp