Renal Collagen Synthesis and Its Control

The Maillard reaction has been implicated in cross-linking and fluorescence formation of collagen exposed to high glucose in vitro. However, several pharmacologic agents, whose action seems unrelated to pathways of nonenzymatic glycation, have been demonstrated to prevent cross-linking in diabetes. To clarify this discrepancy, kinetic changes in glycation, glycoxidation (carboxymethyllysine, CML), and cross-linking (measured as tendon breaking time, TBT) were evaluated in rat tail tendons incubated in 5 and 30 mM glucose in vitro and in tendons implanted in vivo into diabetic rat peritoneal cavity. In vitro, rates were found to be both O2- and glucose-dependent. Tendon preglycation and presence of added 2 mM glycosylamine and Amadori compounds (Amadori product of glucose and propylamine) catalyzed these changes in a primarily O2-dependent manner. In the presence of Amadori compounds, kinetic changes were dramatically increased and were preventable by addition of catalase to the medium. Tendons implanted into diabetic rat peritoneum became more rapidly glycoxidized and cross-linked when implanted at day 30 from diabetes onset (high tissue glycation) compared to day 3 (low tissue glycation) in spite of similar glycation kinetics, suggesting a mechanistic dissociation between glycation, glycoxidation, and cross-linking in diabetes. Indeed, intraperitoneal injection of catalase and other antioxidants dramatically suppressed cross-linking, fluorescence formation, and, to some extent, glycoxidation, without affecting glycation. This study confirms the role of oxidative stress in protein cross-linking by the Maillard reaction in vitro and provides the first evidence for a role of H2O2 in cross-linking in diabetes. Whereas Amadori products are a potent source of H2O2 formation in vitro, their precise contribution to H2O2 generation and the actual role of Maillard reaction products in collagen cross-linking in diabetes requires further investigation.

Regardless of its involvement in either wound healing or excessive fibrosis, the interstitial fibroblast can now be considered an important early participant in inflammatory responses. Although it is recognized that certain immune cells and proinflammatory mediators are intricately linked to fibrotic disease, little is presently known about the manner in which these mediators and cells are orchestrated to a fibrotic finale. Experimental studies have shown that interstitial fibroblasts are capable of participating in an inflammatory response by promoting direct fibroblast-to-immune cell communication and/or modulating the release of soluble mediators that are mutually recognized by both types of cells. Primary cultures of murine fibroblasts were recovered from either normal tissue or tissue undergoing a cell-mediated inflammatory response. These stromal cells were assessed for the expression of various cytokines and chemokines indicative of a type 1 or type 2 response. In addition, the fibroblasts were co-cultured with mononuclear cells to assess the cell-to-cell communication. Fibroblasts recovered from different cell-mediated inflammatory responses demonstrated a dramatic alteration in their cytokine profile. Fibroblasts recovered from the type 2 immune response produced high levels of monocyte chemotactic protein-1 (MCP-1), as compared to the normal fibroblasts and fibroblasts recovered from the type 1 lesion. Mononuclear cells co-cultured with fibroblasts induced a contact-dependent expression of elevated levels of chemokines, especially the macrophage-derived MIP-1 alpha. Thus, both fibroblasts themselves and fibroblasts co-cultured with immune-inflammatory cells have the ability to participate in the maintenance of an inflammatory response via the expression of chemokines. Our laboratory and others have addressed the role of chemotactic cytokines or chemokines in the fibrotic process, and have demonstrated that fibroblasts are capable of modulating the activation of various immune cells that have been implicated in fibrotic disease. In addition, the interstitial fibroblast is capable of regulating its own behavior within the interstitial environment via the expression of chemokines and chemokine receptors. Thus, novel strategies aimed at preventing fibrotic disease will likely need to address the early engagement of inflammatory cells by fibroblasts, and possibly modulate the ability of fibroblasts to generate and/or recognize profibrotic signals supplied by chemokines.