Cardiovascular disease is a major cause of death in chronic kidney disease (CKD) 2 (1). Atherosclerosis is highly prevalent in patients with severe renal failure and advances more rapidly in individuals with renal dysfunction compared with the general population (2). Reduced kidney function is associated with the risk of cardiovascular events, even when the dysfunction is mild (3).Leukocyte-endothelial interactions play an important role in the development of atherosclerosis (4). Cell adhesion molecules belonging to the immunoglobulin superfamily, such as ICAM-1 (intercellular cell adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1), together with members of the selectin family, including E-selectin, are upregulated to mediate monocyte/macrophage infiltration into atherosclerotic lesions (4, 5).Indoxyl sulfate is a uremic toxin synthesized in the liver from indole, a metabolite of tryptophan produced by the intestinal flora (6). In CKD patients, the serum levels of indoxyl sulfate are increased significantly compared with those in healthy individuals (7), and a number of studies have indicated that indoxyl sulfate accelerates glomerular sclerosis, whereas its accumulation promotes renal failure (8 -10). Other studies also showed that indoxyl sulfate induces endothelial dysfunction by releasing endothelial microparticles (11) and producing reactive oxygen species (ROS) (12). However, its effect on endothelial inflammatory processes such as leukocyte recruitment to vascular endothelium has not been reported.We report for the first time that indoxyl sulfate enhances monocyte adhesion to vascular endothelium through up-regulation of E-selectin and augmentation of oxidative stress in both in vitro and in vivo models. The underlying mechanisms seem to involve activation of JNK and NF-B. Our findings reveal a previously unrecognized molecular link between uremic toxins and cardiovascular diseases. EXPERIMENTAL PROCEDURESReagents-Indoxyl sulfate, N-acetylcysteine, probenecid, RPMI 1640 medium, and Dulbecco's PBS were obtained from Sigma. The JNK phosphorylation inhibitor SP600125, the p38 MAPK phosphorylation inhibitor SB203580, the ERK1/2 inhibitor U0126, and the IB phosphorylation inhibitor BAY11-7082 were purchased from Calbiochem. Recombinant human TNF-␣ was obtained from R&D Systems (Minneapolis, MN). A monoclonal antibody against E-selectin (clone 7A9) was obtained from American Type Culture Collection (Manassas, VA) (13). Antibodies against ICAM-1, VCAM-1, the NF-B p65 subunit, and the phospho-NF-B p65 subunit and a monoclonal blocking antibody against mouse E-selectin (clone UZ4) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-ERK, anti-phospho-ERK, anti-p38 MAPK, anti-phospho-p38 MAPK, anti-JNK, and anti-phos-* This work was supported in part by Ministry of Education, Science, and Technology Grant-in-aid for Scientific Research 10178102 and special coordination funds, a grant-in-aid from the Ministry of Culture of Japan, a grant from the Ministry of Health, Labor, and We...
Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed ␣, , and ␥. The putative binding site for integrins has been mapped to the G domain of the ␣ chain, although trimerization with  and ␥ chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of  and ␥ chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the ␥ chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the ␥1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the ␥2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either ␥1 or ␥2 chains. However, the peptide segment modeled after the C-terminal region of ␥1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.Laminins are a family of glycoproteins present in the basement membrane (1-3). All laminins are large heterotrimeric glycoproteins composed of ␣, , and ␥ chains that assemble into a cross-shaped structure. To date, five ␣ chains (␣1-␣5), three  chains (1-3), and three ␥ chains (␥1-␥3) have been identified, combinations of which yield at least 15 isoforms with distinct subunit compositions (4). Laminins contribute to basement membrane architecture and influence cell adhesion, spreading, and migration through binding to their cell surface receptors, particularly the integrin family of cell adhesion receptors (5-9).Integrins play important roles in cell-matrix adhesion and signaling events regulating proliferation and differentiation of cells. Among the various integrin family members, ␣61, ␣64, ␣31, and ␣71 have been shown to be the major laminin receptors expressed in many cell types (10). Binding sites for these integrins have been mapped to the C-terminal globular (G) 3 domain of the laminin ␣ chains (6, 11-15). The G domain consists of five tandemly repeated LG modules of ϳ200 amino acid residues, designated LG1 through LG5. By analogy with the identification of the Arg-Gly-Asp (RGD) cell-adhesive motif in fibronectin, many attempts have been made to identify specific sequences mimicking the integrin binding activity of laminins. However, neither recombinant fragments of the G domain nor synthetic peptides modeled after the sequences in the G domain ...
Chronic kidney disease (CKD) has been considered a major risk factor for cardiovascular diseases. Although great advances have recently been made in the pathophysiology and treatment of cardiovascular diseases, CKD remains a major global health problem. Moreover, the occurrence rates of cardiovascular events among CKD patients increase even in cases in which patients undergo hemodialysis, and the mechanisms underlying the so-called “cardiorenal syndrome” are not clearly understood. Recently, small-molecule uremic toxins have been associated with cardiovascular mortality in CKD and/or dialysis patients. These toxins range from small uncharged solutes to large protein-bound structures. In this review, we focused on protein-bound uremic toxins, such as indoxyl sulfate and p-cresyl sulfate, which are poorly removed by current dialysis techniques. Several studies have demonstrated that protein-bound uremic toxins, especially indoxyl sulfate, induce vascular inflammation, endothelial dysfunction, and vascular calcification, which may explain the relatively poor prognosis of CKD and dialysis patients. The aim of this review is to provide novel insights into the effects of indoxyl sulfate and p-cresyl sulfate on the pathogenesis of atherosclerosis.
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