Novel Ex-Vivo Thrombolytic Reconditioning of Kidneys Retrieved 4 to 5 Hours After Circulatory Death

Abstract

Background: Due to organ shortage, many patients do not receive donor organs. The present novel thrombolytic technique utilizes organs from donors with uncontrolled donation after circulatory deaths (uDCD), with up to 4-5 h warm ischemia, without advanced cardiopulmonary resuscitation (aCPR) or extracorporeal circulation (EC) after death.

Methods: The study group of pigs (n = 21) underwent simulated circulatory death. After 2 h, an ice slush was inserted into the abdomen. Kidneys were retrieved 4.5 h after death. Lys-plasminogen, antithrombin-III (ATIII), and alteplase (tPA) were injected through the renal arteries on the back table. Subsequent ex vivo perfusion at 15 °C was continued for 3 h, followed by 3 h with red blood cells (RBCs) at 32 °C. Perfusion outcome and histology were compared between uDCD kidneys, receiving no thrombolytic treatment (n = 8), and live donor kidneys (n = 7). The study kidneys were then transplanted into pigs as autologous grafts with a single functioning autologous kidney as the only renal support. uDCD control pigs (n = 8), receiving no ex vivo perfusion, served as controls.

Results: Vascular resistance decreased to <200 mmHg/mL/min ( P < 0.0023) and arterial flow increased to >100 mL/100 g/min ( P < 0.00019) compared to controls. In total 13/21 study pigs survived for >10 days, while all uDCD control pigs died. Histology was preserved after reconditioning, and the creatinine level after 10 days was next to normal.

Conclusions: Kidneys from extended uDCD, not receiving aCPR/EC, can be salvaged using thrombolytic treatment to remove fibrin thrombi while preserving histology and enabling transplantation with a clinically acceptable early function.

FIGURE 1.

(A) Retrieved uDCD kidneys én-bloc after 4–5 h ischemia. The kidneys will be reconditioned and one of the kidneys will be transplanted to a recipient. (B) Kidneys after completed reconditioning, ready for transplantation. uDCD, uncontrolled donation after circulatory death.

FIGURE 2.

(A) shows the changes in vascular resistance and (B) the arterial flow in kidneys perfused using the study group I protocol (n = 21), in black squares with solid lines. The flow and resistance were compared to a group of healthy normal kidneys, retrieved as live donor kidneys, labeled as group II (n = 8) in blue solid line and triangles perfused with only perfusion solution without thrombolytic drugs, and to a group of kidneys from uDCD without thrombolytic drugs (n = 7), but reconditioned with perfusion solution, labeled group III (n = 7) in red solid line and circles. The dotted horizontal black line marks the 200 mm Hg * min/L limit of vascular resistance in (A) and the 100 mL/100 g/min limit of arterial flow in (B). The dotted vertical black line marks the shift in temperature from 15 °C to 32 °C, and the increase in pressure from 20 to 30 mm Hg. Group I had a significantly lower vascular resistance (P < 0.0023) and higher arterial flow (P < 0.000019) than group III at the end of the evaluation, using the Mann-Whitney U test. uDCD, uncontrolled donation after circulatory death.

Figure 3.

Box chart of vascular resistance in mm Hg * min/L after 360 min of ex-vivo perfusion. The box shows 25%–75% percentile range of datapoints. Diamonds, in black or red, show individual datapoints, round symbols show outliers. Whiskers show 1 SD, mean value are marked as a dotted line, median value as a solid line. A normal distribution line is plotted beside the data points. In (A) the study group (group I), in black, is compared to the nonthrombolysis group (group III), in red. Group I had a significant lower vascular resistance (P < 0.0023) and higher arterial flow (P < 0.000019) than group III at the end of evaluation, using Mann-Whitney U. In (B), a post hoc analyzes was performed to study any difference between pigs that had a functioning kidney >10 d (n = 13) vs <7 d (n = 8). No significant difference in vascular resistance could be seen.

FIGURE 4.

Box chart of arterial flow in anesthetized pigs. The box shows 25%–75% percentile range of datapoints. Diamonds, in black or red, show individual datapoints, round symbols show outliers. Whiskers show 1 SD, mean value are marked as a dotted line, median value as a solid line. A normal distribution line is plotted beside the data points. In (A) arterial flow was measured in anesthetized pigs, before any intervention of the kidney in 14 pigs, in (B) arterial flow was measured at reperfusion of study group (n = 21) kidneys in black and uDCD control (n = 8) group in red. In (C) arterial flow 90 min after reperfusion of study group (n = 21) kidneys in black and uDCD control (n = 8) group kidneys in red. Flow was significantly better in the study group vs the uDCD control group at reperfusion (P < 0.00003), but not significantly different 90 min after reperfusion, as measured with Mann-Whitney U-test. uDCD, uncontrolled donation after circulatory deaths.

FIGURE 5.

Box chart of creatinine in transplanted study group (I) pigs. The box shows 25%–75% percentile range of datapoints. Diamonds, in black or red, show individual datapoints, round symbols show outliers. Whiskers show 1 SD, mean value are marked as a dotted line, median value as a solid line. A normal distribution line is plotted beside the data points. In (A) creatinine was measured in anesthetized pigs, before any intervention of the kidney, in (B) creatinine was measured at 90 min after reperfusion of the study group (n = 21) kidneys. In (C) creatinine was measured at 10 d in the study group (n = 8) kidneys creatinine was measured in 8/13 pigs surviving days or longer.

FIGURE 6.

Box chart of creatinine in anesthetized pigs receiving uDCD control kidneys, without any perfusion or thrombolytic treatment (group IV). The box shows 25%–75% percentile range of datapoints. Diamonds, in black, show individual datapoints, round symbols show outliers. Whiskers show 1 SD, mean value are marked as a dotted line, median value as a solid line. A normal distribution line is plotted beside the data points. In (A) creatinine was measured in anesthetized pigs, before any intervention of the kidney; in (B) creatinine was measured 90 min after reperfusion of the uDCD control group (n = 8) kidneys; and in (C) creatinine was measured in the two uDCD control group (n = 2) kidneys surviving 5 d after transplantation. None of the eight animals survived beyond 5 d. uDCD, uncontrolled donation after circulatory deaths.

FIGURE 7.

Evaluation of the kidney histology of the study groups. In (A) histology of kidneys from the study group following ischemia but before reconditioning. Signs of tubular injury can be seen as cellular cytoplasmic vacuolization, epithelial simplification and sloughing of apical membranes into the lumen. In (B) and (C) the postperfusion histology is shown. Again, signs of tubular injury are seen as indicated above, but with less observable apical membrane remnants in the lumen of the tubules. In (D) the histology is shown in kidneys from healthy pigs, retrieved as live donor kidneys, after perfusion with base solution without thrombolytic factors added. The histological changes are similar to those described above. In (E) histology of uDCD kidneys, perfused with base solution and no thrombolytic treatment. Acute tubular injury is seen with simplification, vacuolization, and sloughing apical membranes. In (F) histology from uDCD kidneys subjected to ischemia followed by immediate transplantation without perfusion. Signs of tubular injury was seen with vacuolization and apical sloughing, in two cases this was severe and with distinct interstitial edema. No changes were seen in glomeruli or vessels. Scale bars = 200 µm. Inserts shows higher magnification. Stained by Hematoxylin/Eosin. uDCD, uncontrolled donation after circulatory deaths.

FIGURE 8.

Fibrin(ogen) presence in kidneys treated or not treated with fibrinolytic drugs (immunohistochemistry) (A.1) and (A.2) represent uDCD kidney biopsies taken immediately after 4.5 h of death. In (A.3 and A.4) and (A.5 and B.6) biopsies collected after cold perfusion and the end of machine perfusion respectively, from uDCD kidneys that were treated with Lys-plasminogen, alteplase and anti-thrombin III (group 1). In (B.1) and (B.2) biopsies collected before machine perfusion of live donor kidneys (group 2). In (B.3) and (B.4) biopsies from live donor kidneys after cold perfusion (group 2), whereas (B.5) and (B.6) show biopsies at the end of machine perfusion of the same group. In (C.1) and (C.2) biopsies collected at end of machine perfusion of uDCD kidneys after 4.5 h of death, which were not treated with thrombolytic drugs but were machine perfused (group 3). In (D.1) and (D.2) uDCD kidneys after 4.5 h of death that were transplanted without thrombolytic treatment or machine perfusion (group 4). Blue arrows show fibrin(ogen). All images on the right are 20× images of images represented on the left at 10×. uDCD, uncontrolled donation after circulatory deaths.

FIGURE 9.

Representative images from biopsies before (A.1–A.3) and during machine perfusion (B.1–D.3) of uDCD kidneys (group I) with 4.5 h of WIT and of a normal kidney (E.1–E.3) using a high-resolution transmission electron microscopy. Fig A.1–A.3 shows fibrinogen and RBC rich microvascular plugs (red arrows) before any treatment in uDCD kidney biopsies 4.5 h after death. Platelets (marked in red asterisk) could be seen in many of the capillaries in biopsies taken before any treatment (A.1). No RBC plugs were found in biopsies taken after the flushing phase, although there were platelets observed in some of the capillaries (B.1–B.3). Capillaries appear clear and without any clots or platelets in biopsies taken at the end of the cold perfusion (C.1–C.3) and the end of the RBC phase (D.1–D.3). Capillaries in biopsies taken from normal pig kidneys (group II), can be seen as comparison in the last panel (E.1–E.3). RBCs are depicted as red arrows and platelets as red asterisks. RBC, red blood cell; uDCD, uncontrolled donation after circulatory deaths; WIT, warm ischemia time.