The lectin-like domain of thrombomodulin ameliorates diabetic glomerulopathy via complement inhibition

Summary

Coagulation and complement regulators belong to two interactive systems constituting emerging mechanisms of diabetic nephropathy. Thrombomodulin (TM) regulates both coagulation and complement activation, in part through discrete domains. TM’s lectin like domain dampens complement activation, while its EGF-like domains independently enhance activation of the anticoagulant and cytoprotective serine protease protein C (PC). A protective effect of activated PC in diabetic nephropathy is established. We hypothesised that TM controls diabetic nephropathy independent of PC through its lectin-like domain by regulating complement. Diabetic nephropathy was analysed in mice lacking TM’s lectin-like domain (TM^LeD/LeD) and controls (TM^wt/wt). Albuminuria (290 μg/mg vs. 166 μg/mg, p=0.03) and other indices of experimental diabetic nephropathy were aggravated in diabetic TM^LeD/LeDmice. Complement deposition (C3 and C5b-9) was markedly increased in glomeruli of diabetic TM^LeD/LeDmice. Complement inhibition with enoxaparin ameliorated diabetic nephropathy in TM^LeD/LeDmice (e.g. albuminuria 85 μg/mg vs. 290 μg/mg, p <0.001). In vitroTM’s lectin-like domain cell-autonomously prevented glucose-induced complement activation on endothelial cells and –notably –on podocytes. Podocyte injury, which was enhanced in diabetic TM^LeD/LeDmice, was reduced following complement inhibition with enoxaparin. The current study identifies a novel mechanism regulating complement activation in diabetic nephropathy. TM’s lectin-like domain constrains glucose-induced complement activation on endothelial cells and podocytes and ameliorates albuminuria and glomerular damage in mice.

Keywords

Thrombomodulin - complement - endothelial dysfunction - podocyte - diabetic nephropathy

• References

• 1 Stewart JH, McCredie MR, Williams SM. Geographic, ethnic, age-related and temporal variation in the incidence of end-stage renal disease in Europe, Canada and the Asia-Pacific region, 1998-2002. Nephrol Dial Transplant 2006; 21: 2178-2183.
• 2 Isermann B, Vinnikov IA, Madhusudhan T. et al. Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. Nat Med 2007; 13: 1349-1358.
• 3 Amara U, Flierl MA, Rittirsch D. et al. Molecular intercommunication between the complement and coagulation systems. J Immunol 2010; 185: 5628-5636.
• 4 Onat A, Can G, Rezvani R. et al. Complement C3 and cleavage products in cardiometabolic risk. Clin Chim Acta 2011; 412: 1171-1179.
• 5 Badeanlou L, Furlan-Freguia C, Yang G. et al. Tissue factor-protease-activated receptor 2 signaling promotes diet-induced obesity and adipose inflammation. Nat Med 2011; 17: 1490-1497.
• 6 Walport MJ. Complement. First of two parts. N Engl J Med 2001; 344: 1058-1066.
• 7 Walport MJ. Complement. Second of two parts. N Engl J Med 2001; 344: 1140-1144.
• 8 Saraheimo M, Forsblom C, Hansen TK. et al. Increased levels of mannan-binding lectin in type 1 diabetic patients with incipient and overt nephropathy. Diabetologia 2005; 48: 198-202.
• 9 Hovind P, Hansen TK, Tarnow L. et al. Mannose-binding lectin as a predictor of microalbuminuria in type 1 diabetes: an inception cohort study. Diabetes 2005; 54: 1523-1527.
• 10 Hansen TK, Gall MA, Tarnow L. et al. Mannose-binding lectin and mortality in type 2 diabetes. Arch Intern Med 2006; 166: 2007-2013.
• 11 Hansen TK, Forsblom C, Saraheimo M. et al. Association between mannose-binding lectin, high-sensitivity C-reactive protein and the progression of diabetic nephropathy in type 1 diabetes. Diabetologia 2010; 53: 1517-1524.
• 12 Ostergaard J, Hansen TK, Thiel S. et al. Complement activation and diabetic vascular complications. Clin Chim Acta 2005; 361: 10-19.
• 13 Acosta J, Hettinga J, Fluckiger R. et al. Molecular basis for a link between complement and the vascular complications of diabetes. Proc Natl Acad Sci USA 2000; 97: 5450-5455.
• 14 Qin X, Goldfine A, Krumrei N. et al. Glycation inactivation of the complement regulatory protein CD59 a possible role in the pathogenesis of the vascular complications of human diabetes. Diabetes 2004; 53: 2653-2661.
• 15 Davies CS, Harris CL, Morgan BP. Glycation of CD59 impairs complement regulation on erythrocytes from diabetic subjects. Immunology 2005; 114: 280-286.
• 16 Weiler H, Isermann BH. Thrombomodulin. J Thromb Haemost 2003; 1: 1515-1524.
• 17 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 18 Gil-Bernabe P, D’Alessandro-Gabazza CN, Toda M. et al. Exogenous activated protein C inhibits the progression of diabetic nephropathy. J Thromb Haemost 2012; 10: 337-346.
• 19 Conway EM, Van de Wouwer M, Pollefeyt S. et al. The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. J Exp Med 2002; 196: 565-577.
• 20 Abeyama K, Stern DM, Ito Y. et al. The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. J Clin Invest 2005; 115: 1267-1274.
• 21 Van de Wouwer M, Plaisance S, De Vriese A. et al. The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis. J Thromb Haemost 2006; 4: 1813-1824.
• 22 Wang H, Madhusudhan T, He T. et al. Low but sustained coagulation activation ameliorates glucose-induced podocyte apoptosis: protective effect of factor V Leiden in diabetic nephropathy. Blood 2011; 117: 5231-5242.
• 23 Lin M, Yiu WH, Wu HJ. et al. Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol 2012; 23: 86-102.
• 24 Harper PA, Robinson JM, Hoover RL. et al. Improved methods for culturing rat glomerular cells. Kidney Int 1984; 26: 875-880.
• 25 Conway EM, Pollefeyt S, Collen D. et al. The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 1997; 89: 652-661.
• 26 Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nat Med 2004; 10: 1222-1226.
• 27 Seehaus S, Shahzad K, Kashif M. et al. Hypercoagulability inhibits monocyte transendothelial migration through protease-activated receptor-1-, phospholipase-Cbeta-, phosphoinositide 3-kinase-, and nitric oxide-dependent signaling in monocytes and promotes plaque stability. Circulation 2009; 120: 774-784.
• 28 Hansen TK, Tarnow L, Thiel S. et al. Association between mannose-binding lectin and vascular complications in type 1 diabetes. Diabetes 2004; 53: 1570-1576.
• 29 Falk RJ, Sisson SP, Dalmasso AP. et al. Ultrastructural localization of the membrane attack complex of complement in human renal tissues. Am J Kidney Dis 1987; 9: 121-128.
• 30 Wehner H, Hohn D, Faix-Schade U. et al. Glomerular changes in mice with spontaneous hereditary diabetes. Lab Invest 1972; 27: 331-340.
• 31 Mauer SM, Michael AF, Fish AJ. et al. Spontaneous immunoglobulin and complement deposition in glomeruli of diabetic rats. Lab Invest 1972; 27: 488-494.
• 32 Michels LD, O’Donnell MP, Keane WF. Glomerular hemodynamic and structural correlations in long-term experimental diabetic rats. J Lab Clin Med 1984; 103: 840-847.
• 33 Ostergaard J, Thiel S, Gadjeva M. et al. Mannose-binding lectin deficiency attenuates renal changes in a streptozotocin-induced model of type 1 diabetes in mice. Diabetologia 2007; 50: 1541-1549.
• 34 Weiler-Guettler H, Christie PD, Beeler DL. et al. A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state. J Clin Invest 1998; 101: 1983-1991.
• 35 Wang W, Nagashima M, Schneider M. et al. Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation. J Biol Chem 2000; 275: 22942-22947.
• 36 Campbell WD, Lazoura E, Okada N. et al. Inactivation of C3a and C5a octapeptides by carboxypeptidase R and carboxypeptidase N. Microbiol Immunol 2002; 46: 131-134.
• 37 Sumi A, Yamanaka-Hanada N, Bai F. et al. Roles of coagulation pathway and factor Xa in the progression of diabetic nephropathy in db/db mice. Biol Pharm Bull 2011; 34: 824-830.
• 38 Zhou W, Marsh JE, Sacks SH. Intrarenal synthesis of complement. Kidney Int 2001; 59: 1227-1235.
• 39 Accardo-Palumbo A, Triolo G, Colonna-Romano G. et al. Glucose-induced loss of glycosyl-phosphatidylinositol-anchored membrane regulators of complement activation (CD59, CD55) by in vitro cultured human umbilical vein endothelial cells. Diabetologia 2000; 43: 1039-1047.
• 40 Fujiwara Y, Tagami S, Kawakami Y. Circulating thrombomodulin and hematological alterations in type 2 diabetic patients with retinopathy. J Atheroscler Thromb 1998; 5: 21-28.
• 41 Borcea V, Morcos M, Isermann B. et al. Influence of ramipril on the course of plasma thrombomodulin in patients with diabetes mellitus. Vasa 1999; 28: 172-180.
• 42 Rosoklija GB, Dwork AJ, Younger DS. et al. Local activation of the complement system in endoneurial microvessels of diabetic neuropathy. Acta Neuropathol 2000; 99: 55-62.
• 43 Zhang J, Gerhardinger C, Lorenzi M. Early complement activation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimental diabetic retinopathy. Diabetes 2002; 51: 3499-3504.