Norihiko Sakai

Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis

Fibrocytes are a distinct population of bloodborne cells that share markers of leukocytes as well as mesenchymal cells. We hypothesized that CCR7-positive fibrocytes migrate into the kidney in response to secondary lymphoid tissue chemokine (SLC/CCL21) and contribute to renal fibrosis. To investigate this hypothesis, renal fibrosis was induced by unilateral ureteral obstruction in mice. A considerable number of fibrocytes dual-positive for CD45 and type I collagen (ColI) or CD34 and ColI infiltrated the interstitium, reaching a peak on day 7. Most fibrocytes were positive for CCR7, and CCL21/CCR7 blockade reduced the number of infiltrating fibrocytes. CCL21 and MECA79 dual-positive vessels were also detected in the interstitium. The blockade of CCL21/CCR7 signaling by anti-CCL21 antibodies reduced renal fibrosis, which was confirmed by a decrease in fibrosis in CCR7-null mice with concomitant reduction in renal transcripts of pro α1 chain of ColI and TGF-β1. The number of F4/80-positive macrophages decreased along with renal transcripts of monocyte chemoattractant protein 1 (MCP-1/CCL2) after the blockade of CCL21/CCR7 signaling. These findings suggest that CCR7-positive fibrocytes infiltrate the kidney via CCL21-positive vessels, thereby contributing to the pathogenesis of renal fibrosis. Thus, the CCL21/CCR7 signaling of fibrocytes may provide therapeutic targets for combating renal fibrosis.

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.

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.

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

LPA1-induced cytoskeleton reorganization drives fibrosis through CTGF-dependent fibroblast proliferation

There has been much recent interest in lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, in fibrotic diseases, but the mechanisms by which LPA‐LPA1 signaling promotes pathological fibrosis remain to be fully elucidated. Using a mouse peritoneal fibrosis model, we demonstrate central roles for LPA and LPA1 in fibroblast proliferation. Genetic deletion or pharmacological antagonism of LPA1 protected mice from peritoneal fibrosis, blunting the increases in peritoneal collagen by 65.4 and 52.9%, respectively, compared to control animals and demonstrated that peritoneal fibroblast proliferation was highly LPA1 dependent. Activation of LPA1 on mesothelial cells induced these cells to express connective tissue growth factor (CTGF), driving fibroblast proliferation in a paracrine fashion. Activation of mesothelial cell LPA1 induced CTGF expression by inducing cytoskeleton reorganization in these cells, causing nuclear translocation of myocardin‐related transcription factor (MRTF)‐A and MRTF‐B. Pharmacological inhibition of MRTF‐induced transcription also diminished CTGF expression and fibrosis in the peritoneal fibrosis model, mitigating the increase in peritoneal collagen content by 57.9% compared to controls. LPA1‐induced cytoskeleton reorganization therefore makes a previously unrecognized but critically important contribution to the profibrotic activities of LPA by driving MRTF‐dependent CTGF expression, which, in turn, drives fibroblast proliferation.—Sakai, N., Chun, J., Duffield, J. S., Wada, T., Luster, A. D., Tager, A. M. LPA1‐induced cytoskeleton reorganization drives fibrosis through CTGF‐dependent fibroblast proliferation. FASEB J. 27, 1830–1846 (2013). www.fasebj.org

Exacerbation of glomerulonephritis in subjects with chronic hepatitis C virus infection after interferon therapy

We previously reported the glomerular deposition of hepatitis C virus (HCV) core antigen (Ag) in HCV-related nephropathy. In this study, we analyzed 23 HCV-positive subjects with exacerbation of proteinuria and/or hematuria during interferon (IFN) therapy and measured urinary protein selectivity. We also examined the involvement of HCV-related Ag using anti-HCV core (capside) Ag murine monoclonal antibody (Ab) and anti-core2 rabbit polyclonal Abs in nine subjects. Of 17 subjects, 13 (78%) showed low selective proteinuria. We found mesangial proliferative glomerulonephritis in 9 subjects, membranoproliferative glomerulonephritis in 1 subject, and nephrosclerosis in 1 subject. Immunofluorescence study showed the glomerular deposition of immunoglobulin G (IgG) or IgA and complements in all 9 subjects examined. Trace amounts only of HCV core Ag were detected along the glomerular capillary wall in 3 of 9 subjects (33%). Electron microscopy showed subendothelial or mesangial electron-dense deposits and also foot process effacement (20% to 72.5% of glomerular capillary walls) in all subjects and endothelial swelling in 4 subjects. In conclusion, IFN therapy for HCV may exacerbate the underlying glomerulopathies, unrelated to HCV Ags, through direct or indirect effects on glomerular endothelial and epithelial cells. Physicians should carefully distinguish HCV-related nephropathy from other glomerular diseases when they administer IFN therapy to HCV-positive subjects.

MCP-1/CCR2-dependent loop for fibrogenesis in human peripheral CD14-positive monocytes.

Monocyte/macrophage (Mο) migration to sites of inflammation is a prerequisite cause of organ fibrosis. The recruitment and activation of Mo are regulated by C‐C chemokines, especially monocyte chemoattractant protein‐1 [(MCP‐1)/CC chemokine ligand 2], which interacts with CC chemokine receptor 2 (CCR2). However, the mechanisms leading to fibrosis via MCP‐1/CCR2 signaling in Mo remain to be investigated. The effect of MCP‐1 on the expression of MCP‐1, CCR2, transforming growth factor‐β1 (TGF‐β1), and type I collagen in circulating human CD14‐positive Mo was investigated. In addition, the impact of MCP‐1‐specific or TGF‐β1‐specific antisense (AS) phosphorothioate oligodeoxynucleotides (ODN) was examined to explore the involvement of autocrine/paracrine production of MCP‐1 and TGF‐β1 by human CD14‐positive Mo. Furthermore, specific CCR2 inhibitors were applied to examine the involvement of CCR2 signaling for the promotion of a fibrogenic response. The stimulation of Mo with MCP‐1 increased mRNA levels of TGF‐β1 and a pro‐α1 chain of type I collagen (COL1A1) as well as protein synthesis. Similarly, the expression of MCP‐1 and CCR2 was enhanced by the stimulation with MCP‐1 in dose‐ and time‐dependent manners. This positive loop via MCP‐1 was reduced by pretreatment with MCP‐1‐specific AS‐ODN. It was also noted that pretreatment with TGF‐β1‐specific AS‐ODN partially reduced COL1A1 mRNA levels. Finally, transcripts of these molecules were suppressed by pretreatment with specific CCR2 inhibitors. The present study demonstrated that human peripheral CD14‐positive Mo contribute directly to fibrogenesis by a MCP‐1/CCR2‐dependent amplification loop. These data suggest that fibrogenic processes in Mo regulated by MCP‐1/CCR2 may be novel, therapeutic targets for combating organ fibrosis.

The renin-angiotensin system contributes to renal fibrosis through regulation of fibrocytes.

Background The renin–angiotensin system is a major pathway in the pathogenesis of cardiovascular and renal diseases. Bone marrow-derived fibrocytes, which are dual positive for CD45 and type I collagen, are now considered to contribute to the pathogenesis of various fibrotic diseases. We hypothesized that fibrocytes might contribute to renal fibrosis by an angiotensin II dependent pathway. Results In murine models of renal fibrosis, angiotensin II type 2 receptor (AT2R)-deficient mice, when compared with wild-type mice, showed increased renal fibrosis and fibrocyte infiltration with a concomitant upregulation of renal transcripts of procollagen type I (α) (COL1A1). Fibrocyte numbers in the bone marrow also were increased in AT2R-deficient mice. By contrast, pharmacological inhibition of angiotensin II type 1 receptor (AT1R) with valsartan reduced the degree of renal fibrosis and the number of fibrocytes in both the kidney and the bone marrow. In isolated human fibrocytes, inhibition of AT2R signaling increased the angiotensin II-stimulated expression of type I collagen, whereas inhibition of AT1R decreased collagen synthesis. These results suggest that AT1R/AT2R signaling may contribute to the pathogenesis of renal fibrosis by at least two mechanisms: by regulating the number of fibrocytes in the bone marrow, and by activation of fibrocytes.

Upregulation of Fractalkine in Human Crescentic Glomerulonephritis

Background/Aim: To evaluate the importance of fractalkine, a novel member of the CX3C chemokine, and natural killer (NK) cells in human crescentic glomerulonephritis, we determined the presence of fractalkine in the diseased kidneys immunohistochemically, and the correlation among fractalkine, NK cells and the degree of renal damage. Methods: Twenty-three patients (13 males and 10 females) with primary or secondary crescentic glomerular disease were evaluated in this study. Fractalkine and CD16-positive cells including NK cells were detected immunohistochemically. Results: Fractalkine-positive cells were detected in the interstitium of 23 patients with crescentic glomerulonephritis, while they were not detected in the glomeruli. In addition, CD16-positive cells were detected in both the glomeruli (1.3 ± 0.2/glomerulus) and interstitium (1.3 ± 0.2/visual field). The number of fractalkine-positive cells in the interstitium correlated with the number of CD16-positive cells before glucocorticoid therapy (r = 0.43, p = 0.047, n = 23). The number of fractalkine-positive cells in the interstitium before glucocorticoid therapy (0.2 ± 0.1/visual field) decreased after therapy (0.1 ± 0.1/visual field, p = 0.050) in 11 cases tested. The number of CD16-positive cells in the diseased kidneys did not change after glucocorticoid therapy. Conclusion: These results suggest that the local production of fractalkine may explain the presence of CD16-positive cells including NK cells, which may participate in the interstitial lesions of human crescentic glomerulonephritis before corticoid therapy.

Long-term Outcomes of Japanese Type 2 Diabetic Patients With Biopsy-Proven Diabetic Nephropathy

OBJECTIVE We evaluated the structural-functional relationships and the prognostic factors for renal events, cardiovascular events, and all-cause mortality in type 2 diabetic patients with biopsy-proven diabetic nephropathy. RESEARCH DESIGN AND METHODS Japanese type 2 diabetic patients with biopsy-proven diabetic nephropathy (n = 260) were enrolled. Patients were stratified by albuminuria (proteinuria) and estimated glomerular filtration rate (eGFR) at the time of renal biopsy. The outcomes were the first occurrence of renal events (requirement of dialysis or a 50% decline in eGFR from baseline), cardiovascular events (cardiovascular death, nonfatal myocardial infarction, coronary interventions, or nonfatal stroke), and all-cause mortality. RESULTS The factors associated with albuminuria (proteinuria) regardless of eGFR were hematuria, diabetic retinopathy, low hemoglobin, and glomerular lesions. The factors associated with low eGFR regardless of albuminuria (proteinuria) were age and diffuse, nodular, tubulointerstitial, and vascular lesions. The glomerular, tubulointerstitial, and vascular lesions in patients with normoalbuminuria (normal proteinuria) and low eGFR were more advanced compared to those in patients with normoalbuminuria (normal proteinuria) and maintained eGFR. In addition, compared to patients with micro-/macroalbuminuria (mild/severe proteinuria) and low eGFR, their tubulointerstitial and vascular lesions were similar or more advanced in contrast to glomerular lesions. The mean follow-up period was 8.1 years. There were 118 renal events, 62 cardiovascular events, and 45 deaths. The pathological determinants were glomerular lesions, interstitial fibrosis and tubular atrophy (IFTA), and arteriosclerosis for renal events, arteriosclerosis for cardiovascular events, and IFTA for all-cause mortality. The major clinical determinant for renal events and all-cause mortality was macroalbuminuria (severe proteinuria). CONCLUSIONS Our study suggests that the characteristic pathological lesions as well as macroalbuminuria (severe proteinuria) were closely related to the long-term outcomes of biopsy-proven diabetic nephropathy in type 2 diabetes.