The Role of the N-Terminal Domain of Thrombomodulin and the Potential of Recombinant Human Thrombomodulin as a Therapeutic Intervention for Shiga Toxin-Induced Hemolytic-Uremic Syndrome

Abstract

Hemolytic-uremic syndrome (HUS) is a rare complication of an infection with Shiga toxin (Stx)-producing Escherichia coli (STEC-HUS), characterized by severe acute kidney injury, thrombocytopenia and microangiopathic hemolytic anemia, and specific therapy is still lacking. Thrombomodulin (TM) is a multi-domain transmembrane endothelial cell protein and its N-terminal domain has been implicated in the pathophysiology of some cases of HUS. Indeed, the administration of recombinant human TM (rhTM) may have efficacy in HUS. We used a Stx-based murine model of HUS to characterize the role of the N-terminal domain of TM. We show that mice lacking that domain (TMLed (-/-)) are more sensitive to Stx, with enhanced HUS progression seen at 4 days and increased mortality at 7 days post-HUS induction. In spite of these changes, renal function was less affected in surviving Stx-challenged TMLed (-/-) mice compared to their wild-type counterparts TMLed (+/+) at 7 days. Contrary to few clinical case reports from Japan, the administration of rhTM (0.06 mg/kg) to wild-type mice (C57BL/6J) with HUS did not protect against disease progression. This overall promising, but also contradictory body of evidence, requires further systematic preclinical and clinical investigations to clarify the role of TM in HUS as a potential therapeutic strategy.

Keywords: Shiga toxin; experimental HUS model; hemolytic-uremic syndrome; kidney injury; recombinant human thrombomodulin; thrombomodulin.

Figure 1

Disease progression of TMLed (+/+) and TMLed (−/−) mice with HUS at day 4 and day 7. Survival was followed up for (A) 4 days or (B) 7 days in sham and Stx-challenged mice at days 0, 3, and 6. Displayed are (A,B) survival; (C,D) analysis of HUS progression indicated by HUS score over the duration of experiment (ranging from 1 = no signs of illness to 5 = dead); (E,F) progression of weight loss over the duration of experiment; and (G,H) weight loss at the end of the experiment. (A,C): n = 7 for TMLed (−/−) sham; n = 8 for other groups; (B,D): n = 6 for TMLed (+/+) Stx and n = 7 for TMLed (−/−); (E–H): n = 3–8 mice per group (only surviving mice on day 4 or day 7). Data are presented with mean + SD. (A,B) Survival by Kaplan-Meier survival analysis + post hoc test. (C,D) Two-way-ANOVA with Tukey’s multiple comparisons test. (G) One-way ANOVA + Sidak’s multiple comparisons test and (H) Mann-Whitney test. * p < 0.05 vs. corresponding sham group; # p < 0.05 TMLed (+/+) Stx vs. TMLed (−/−) Stx. HUS, hemolytic-uremic syndrome; Stx, Shiga toxin; ns, not significant.

Figure 2

Parameters of renal injury, hemolysis, and liver injury of TMLed (+/+) and TMLed (−/−) mice with HUS at day 4 and day 7. Determination of plasma (A) creatinine, (B) urea, (C) cholesterol, (D) LDH activity, (E) ALAT, (F) ASAT, and (G) albumin. (A–C,E) One-way ANOVA + Sidak’s multiple comparisons test. (D,F,G) Kruskal–Wallis test + Dunn’s multiple comparisons test. n = 3–8 per group (only surviving mice with blood withdrawal on day 4 or day 7). Data are presented with mean + SD. * p < 0.05 vs. corresponding sham group. $ p < 0.05 Stx 4 days vs. Stx 7 days (same genotype). # p < 0.05 TMLed (+/+) Stx vs. TMLed (−/−) Stx. Stx, Shiga toxin; LDH, lactate dehydrogenase; ALAT, alanine transaminase; ASAT, aspartate transaminase.

Figure 3

Hematological parameters of TMLed (+/+) and TMLed (−/−) mice with HUS at day 4 and day 7. Determination of whole blood (A) RBC, (B) HGB, (C) HCT, (D) MCV, (E) MCH, (F) MCHC, (G) RDW-SD, (H) RDW-CV, (I) PLT, (J) WBC, (K) W-SCR, and (L) W-LCR. (A–C,F,J–L) One-way ANOVA + Sidak’s multiple comparisons test. (D,E,G–I) Kruskal–Wallis test + Dunn’s multiple comparisons test. n = 3−8 per group (only surviving mice with blood withdrawal on day 4 or day 7). Data are presented with mean + SD. * p < 0.05 vs. corresponding sham group. $ p < 0.05 Stx 4 days vs. Stx 7 days (same genotype). # p < 0.05 TMLed (+/+) Stx vs. TMLed (−/−) Stx. Stx, Shiga toxin; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume, MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood cell distribution width; PLT, platelet; WBC, white blood cell; W-SCR, white cell–-small cell ratio (including lymphocytes); W-LCR, white cell- large cell ratio (including neutrophils).

Figure 4

Clinical presentation and parameters of kidney injury of mice with HUS treated with rhTM. HUS was followed up for 7 days in sham and Stx-challenged mice with i.v. injection of rhTM (0.06 mg/kg bodyweight) on day 0, 3, and 6. (A) Survival is displayed by Kaplan–Meier survival analysis + post hoc test. (B) HUS progression is indicated by HUS score (ranging from 1 = no signs of illness to 5 = dead) and (C) progression of weight loss over the duration of experiment. (D) Plasma NGAL was determined on humane endpoint or day 7. Quantification of (E) PAS reaction, (F) fibrin deposits and (G) relative CD31 expression in renal sections at the end of the experiment (day 7 or humane endpoint). (B) Two-way-ANOVA with Tukey’s multiple comparisons test. (D,G) One-way ANOVA + Holm–Sidak’s multiple comparison test. (E,F) Kruskal–Wallis test + Dunn’s multiple comparison test. n = 8 for sham + vehicle; n = 10 for Stx + vehicle and Stx + rhTM; n = 7 for sham + rhTM. Data are presented with mean + SD. * p < 0.05 vs. corresponding sham group; # p < 0.05 Stx + vehicle vs. Stx + rhTM. HUS, hemolytic–uremic syndrome; Stx, Shiga toxin; rhTM, recombinant human thrombomodulin; NGAL, neutrophil gelatinase-associated lipocalin; PAS, periodic acid Schiff; CD31, cluster of differentiation 31.

Figure 5

Hematological parameters of mice with HUS treated with rhTM. Determination of whole blood (A) RBC, (B) HGB, (C) HCT, (D) MCV, (E) MCH, (F) MCHC, (G) WBC, (H) lymphocytes, and (I) granulocytes on humane endpoint or day 7. (A–D,F,H,I) Kruskal–Wallis test + Dunn’s multiple comparison test and (E,G) one-way ANOVA + Holm–Sidak’s multiple comparison test. n = 8 for sham + vehicle; n = 10 for Stx + vehicle and Stx + rhTM; n = 7 for sham + rhTM. Data are presented as mean + SD. * p < 0.05. HUS, hemolytic–uremic syndrome; rhTM, recombinant human thrombomodulin; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBC, white blood cells.