Reduced cleavage of von willebrand factor by ADAMTS13 is associated with microangiopathic acute kidney injury following trauma

Abstract

Acute kidney injury (AKI) is common after trauma, but contributory factors are incompletely understood. Increases in plasma von Willebrand Factor (vWF) with concurrent decreases in ADAMTS13 are associated with renal microvascular thrombosis in other disease states, but similar findings have not been shown in trauma. We hypothesized that molecular changes in circulating vWF and ADAMTS13 promote AKI following traumatic injury. VWF antigen, vWF multimer composition and ADAMTS13 levels were compared in plasma samples from 16 trauma patients with and without trauma-induced AKI, obtained from the Prehospital Air Medical Plasma (PAMPer) biorepository. Renal histopathology and function, vWF and ADAMTS13 levels were assessed in parallel in a murine model of polytrauma and haemorrhage. VWF antigen was higher in trauma patients when compared with healthy controls [314% (253-349) vs. 100% (87-117)] [median (IQR)], while ADAMTS13 activity was lower [36.0% (30.1-44.7) vs. 100.0% (83.1-121.0)]. Patients who developed AKI showed significantly higher levels of high molecular weight multimeric vWF at 72-h when compared with non-AKI counterparts [32.9% (30.4-35.3) vs. 27.8% (24.6-30.8)]. Murine plasma cystatin C and vWF were elevated postpolytrauma model in mice, with associated decreases in ADAMTS13, and immunohistologic analysis demonstrated renal injury with small vessel plugs positive for fibrinogen and vWF. Following traumatic injury, the vWF-ADAMTS13 axis shifted towards a prothrombotic state in both trauma patients and a murine model. We further demonstrated that vWF-containing, microangiopathic deposits were concurrently produced as the prothrombotic changes were sustained during the days following trauma, potentially contributing to AKI development.

Fig. 1

High molecular weight multimeric vWF is elevated in patients who develop post-traumatic acute kidney injury. (a) vWF multimeric form was evaluated by means of 1.75% vertical agarose gel electrophoresis. Each lane evaluates a single traumatic injury patient who did or did not develop AKI; the red dotted lines denote various molecular weight (MW) cutoffs. (b) Patients who developed AKI demonstrated significantly elevated HMWM vWF forms, when compared to non-AKI counterparts, 72 h after injury [32.9% (30.4–35.3) vs. 27.8% (24.6–30.8) P.0.02]. (c) A significant inverse relationship is generated when plotting HMWM-vWF composition against ADAMTS13 activity of all trauma samples (Spearman r= −0.35, P=0.018).

Fig. 2

vWF and ADAMTS13 levels in patients suffering traumatic injury. (a,b) vWF levels of both AKI and non-AKI traumatic injury patients at 0-, 24-, and 72-h time points compared to healthy controls ([Healthy Control: 4.57mg/ml (3.99–5.35)], [ControlTrauma (Non-AKI) 0 h: 13.37mg/ml (9.56–15.11), 24 h: 13.48mg/ml (10.98–18.07), 72 h: 14.54mg/ml (8.43–17.11)], [AKI Patient 0 h: 11.18mg/ml (6.28–11.18), 24 h: 11.93mg/ml (6.05–15.72), 72 h: 14.36mg/ml (12.58–15.35)]. ADAMTS13 activity levels of both AKI and non-AKI traumatic injury patients at 0-, 24-, and 72-h time points compared to healthy controls ([Healthy Control: 1.3 IU/ml (1.06–1.53)], [Control Trauma (Non-AKI) 0 h: 0.50 IU/ml (0.32–0.65) 24 h: 0.58 IU/mL (0.43–0.68) 72 h: 0.48 IU/ml (0.43–0.58), [AKI Patient 0.44 IU/ml (0.37–0.54) 24 hr: 0.57 IU/ml (0.43–0.68) 72 h: 0.37 IU/ml (0.34–0.43)]. (c) vWF Antigen:ADAMTS13 Activity Ratio of control trauma patients was significantly different than those that developed AKI at the 72-h time point [29.3 (18.6–33.9) vs. 37.8 (34.6–41.8) P.0.02]. (d) vWF Antigen levels strongly correlated with type III collagen binding assay (CBA) activity of samples from 0 to 72 h (r=0.83, P<0.0001). (e) The ADAMTS13 activity level was inversely related to CBA:vWF antigen ratio for samples from 24 and 72 h (r= −0.46, P.0.01). The red dotted line represents the median CBA:vWF Antigen ratio of healthy control samples [0.166IU/ml:μg/ml (0.149-0.202)].

Fig. 3

Haematological and renal effects of a murine polytrauma model. Mice underwent a polytrauma model. (a,b) Plasma samples from mice demonstrated a significant increase in cystatin C levels over 6 and 24-h post polytrauma model [496 ng/ml (439–575) vs. 553 ng/ml (479–640) vs. 550 ng/ml (466–701)]. (c) vWF levels showed significant increases over this 24-h time period as well [0 h = 18.0 μg/ml (11.6–22.8) vs. 6 h = 26.5 μg/ml (23.4–36.9) P < 0.0001 vs. 24 h = 63.7 μg/ml (59.9–81.0)]. (d) ADAMTS13 antigen decreased following the murine polytrauma model [0 h = 0.200 μg/ml (0.167–0.255) vs. 6 h = 0.146 (0.118–0.175) P < 0.001 vs. 24 h = 0.102 (0.066–0.163) P < 0.0001]. (e) There was a significant inverse relationship between murine ADAMTS13 antigen levels and vWF antigen levels pooled from both 6 and 24 h post polytrauma model [spearman r = −0.49 (−0.75 to −0.08) P = 0.02]. (f,g) A matched analysis of murine HMWM-vWF forms demonstrated significant increases 6hrs following the polytrauma model [30.0% (25.9–32.61 vs. 34.8% (29.0–36.1)]. Two lanes were utilized for each mouse, denoted above the gels as ‘pre’ or ‘post’ traumatic injury, which refer to plasma samples obtained immediately prior to injury (’pre’) or 6hrs after injury (’post’). Horizontal red dotted lines designate various MW cutoffs. Vertical white dotted lines indicate cropped standard lanes.

Fig. 4

Murine histologic and immunohistochemical vWF deposition analysis. (a) Left: H&E slide showing a 20um thick PFA fixed healthy kidney section with both 20x and 40x optics. Right: H&E slide demonstrating a 20um thick PFA fixed kidney section from a mouse with a >200 ng/ml increase in cystatin C following traumatic injury. Note dilated lumens with denudation of tubule cells (Black Arrows). (b) A 20x optic, healthy kidney section with a 100x optic blow out of generalized small vessels utilizing RGB-colour channels is shown. vWF is shown utilizing red, fibrinogen with blue, and generalized structure using green. Weak fibrinogen staining can be seen within the vessel lumen. (c) A 20x optic, 24-h post polytrauma model kidney section is shown. Under the 100x optic, small vessel plugs are visible, staining strongly for fibrinogen with a central region containing vWF; this overlap appears purple within the images.