The lectin-like domain of thrombomodulin interferes with complement activation and protects against diabetic nephropathy

Introduction: Thrombomodulin (TM) is predominantly a vascular endothelial cell plasma membrane glycoprotein that, via distinct structural domains, interacts with multiple ligands, thereby modulating coagulation, fibrinolysis, complement activation, inflammation and cell proliferation. We have previously shown that TM protects against diabetic nephropathy (DN) through activation of protein C(PC), which inhibits glomerular apoptosis. Recent studies showed that TM mediates cytoprotective effects independent of PC activation through it's lectin-like domain (LLD). The LLD directly interferes with complement activation and protect against arthritis. DN is associated with an infiltration of inflammatory cells, enhanced cytokine production, and complement activation raising the question whether endothelial TM may protect against DN through a dual mechanism depenending on (a) PC activation and (b) complement inhibition through the LLD.

Methods: DN was evaluated in mice with targeted deletion of the LLD of TM (TMLed/Led mice). Persistent hyperglycemia was induced using streptozotocin in TMLed/Led mice and wild type mice (wt). Subgroups of mice were either anticoagulated (low molecular heparin (LMWH), fondaparinux) or treated with minocycline to inhibit glomerular apoptosis.. After 28 weeks albuminuria was determined and mice were sacrificed to isolated tissues for analyses.

Results: Albuminuria, histological indices of DN and glomerular C3 deposition increased in diabetic wt mice and further increased in diabetic TMLed/Led mice. Only treatment with LMWH, but not with fondaparinux corrected the aggravated DN in TMLed/Led mice, despite equal anticoagulation in both groups. Unlike fondparinux LMWH is also a potent inhibitor of complement activation. Indeed, we observed less complement deposits in LMWH treated diabetic TMLed/Led mice than in fondaparinux treated mice. In vitro, endothelial cells expressing a TM mutant lacking the LLD had diminished capacity to bind and inactivate C3b and were thus sensitive to complement mediated cell lysis. Of note, apoptosis (TUNEL) and expression levels of apoptosis regulators (p53, Bax/Blc-2 ratio, Western) did no differ between untreated or LMWH/fondaparinux treated diabetic TMLed/Led mice, indicating that the protective effect of LLD is independent of apoptosis inhibition. Consistently, inhibition of glomerular apoptosis with minocycline failed to normalize albuminuria in diabetic TMLed/Led mice.

Discussion and conclusion: We identify a novel pathway through which endothelial TM protects against DN. TM protects against DN by inhibiting complement activation through its LLD. This function is independent of the recently identified activated PC dependent inhibition of glomerular apoptosis. Thus, endothelial TM prevents DN through two independent pathways. These results provide further experimental support for a causative role of endothelial dysfunction for DN.