Long-term Transplant Function After Thrombolytic Treatment Ex Vivo of Donated Kidneys Retrieved 4 to 5 H After Circulatory Death

Abstract

Background: Using a novel thrombolytic technique, we present long-term transplant function, measured by creatinine and iohexol clearance, after utilizing kidneys from porcine donors with uncontrolled donation after circulatory deaths, with 4.5-5 h of warm ischemia.

Methods: Pigs in the study group were subjected to simulated circulatory death. After 2 h, ice slush was inserted into the abdomen and 4.5 h after death, the kidneys were retrieved. Lys-plasminogen, antithrombin-III, and alteplase were injected through the renal arteries on the back table. Subsequent ex vivo perfusion was continued for 3 h at 15°C, followed by 3 h with red blood cells at 32°C, and then transplanted into pigs as an autologous graft as only renal support. Living-donor recipient pigs that did not receive ex vivo perfusion, and unilateral nephrectomized pigs served as the controls.

Results: Pigs in the study group (n = 13), surviving 10 d or more were included, of which 7 survived for 3 mo. Four animals in the living-donor group (n = 6) and all 5 nephrectomized animals survived for 3 mo. Creatinine levels in the plasma and urine, neutrophil gelatinase-associated lipocalin levels, Kidney Injury Marker-1 expression, and iohexol clearance at 3 mo did not differ significantly between the study and living-donor groups. Histology and transmission electron microscopy after 3 mo showed negligible fibrosis and no other damage.

Conclusions: The present method salvages kidneys from extended unontrolled donation after circulatory death using thrombolytic treatment while preserving histology and enabling transplantation after ex vivo reconditioning, with clinically acceptable late function after 3 mo, as measured by creatinine and iohexol clearance.

Conflict of interest statement

M.O. is the inventor of several patents. The other authors declare no conflicts of interest.

Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.

Figures

Graphical abstract

FIGURE 1.

The study group I (A–E) is seen to the left: (A) The left kidney, in green, was transplanted from the recipient (red pig) to the donor pig (blue pig) after (B) the right kidney of the donor had been removed and discarded. C, The donor pig was then converted to a uDCD pig. D, After 4.5 h, both the remaining donor kidney and the transplanted recipient kidney (in green) were removed en bloc with vena cava and aorta, followed by treatment according to the protocol. E, After completed reconditioning, the original recipient kidney, in green, was transplanted back to the recipient after the remaining right kidney of the recipient had been removed. The transplanted kidney is now an autotransplant, in a uDCD model. The recipient is followed for 3 mo.The live-donor group II (F–H) is seen in the middle (F) The right kidney is removed from a healthy pig. The pig is kept under anesthesia during the same time period as the study group. G, After 15–16 h, the remaining left kidney is removed and flushed with StoreProtect. H, After the flush, the kidney is transplanted back to the right side of the pig, without any reconditioning protocol, and then observed for 3 mo.The nephrectomy group III (I,J) is seen to the right: (I) The right kidney is removed and the pig is observed for 3 mo with no surgery (J) until the end of the experiment. uDCD, uncontrolled donation after circulatory death.

FIGURE 2.

Box chart of creatinine in anesthetized pigs receiving study (black) and live-donor (red) group kidneys. Unilaterally nephrectomized controls, in blue, served as a second control group. The box shows 25%–75% percentile range of datapoints. Diamonds, in black, red, or blue, show individual datapoints, round symbols show outliers. Whiskers show 1 SD, mean values 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, after nephrectomy, of the study group (n = 13) kidneys in black, in the live-donor group (n = 4) kidneys in red and in the nephrectomy group (n = 4) kidneys in blue. In (B) creatinine was measured at 10 d after transplantation of the study group (n = 13) kidneys in black, in the live-donor group (n = 4) kidneys in red and in the nephrectomy group (n = 5) kidneys in blue. In (C) creatinine was measured at the 1-mo timepoint in the study group (n = 6) kidneys, in black, and nephrectomy group (n = 5) kidneys in blue, no samples were drawn from the sham group at the 1-mo timepoint. In (D) creatinine was measured at the 3-mo timepoint after transplantation of the study group (n=7) kidneys in black, in the live-donor group (n = 4) kidneys in red and in the nephrectomy group (n = 5) kidneys in blue. No statistical difference in creatinine levels was observed between the groups at the 3-mo timepoint.

FIGURE 3.

Normal looking kidney 3 mo after transplantation, in the study group (A), cut surface of the same kidney to the right (B). Normal looking kidney 3 mo after transplantation, in the live-donor group (C), cut surface of the same kidney to the right (D). Nephrectomy group kidney at exploration (E), and cut surface of the same kidney at the 3-mo time point to the right (F).

FIGURE 4.

Box chart of iohexol clearance in at 3-mo after transplantation. The box shows 25%–75% percentile range of datapoints. Diamonds, in black, red or blue, show individual data points, round symbols show outliers. Whiskers show 1 SD, mean values are marked as a dotted line, median value as a solid line. A normal distribution line is plotted beside the data points. The black box shows data from study group animals surviving 3 mo (n = 4). The red box shows data from live-donor group pigs surviving 3 mo (n = 4). The blue box shows data from pigs with 2 normal kidneys (n = 7). There was no significant difference in clearance between the study and the sham groups.

FIGURE 5.

NGAL levels at start, 90 min after reperfusion of the transplanted kidney and 3 mo after transplantation. There was no difference between the study group I (n = 4) and the live-donor group (n = 4). At 3 mo comparison included the Nephrectomized Group (n = 5) as well, showing no statistical difference in NGAL expression. NGAL, neutrophil gelatinase-associated lipocalin.

FIGURE 6.

IHC staining of KIM-1 in biopsies taken after 90 min of reperfusion in the study group I (A and B) and in the live-donor operated group II (C and D). (A) and (C) represent positively stained biopsies for KIM-1 in groups I and II, respectively. Whereas (B) and (D) represent negatively stained biopsies for KIM-1 in the same groups. IHC, immunohistochemistry; KIM-1, Kidney Injury Marker-1.

FIGURE 7.

IHC staining on tissues taken from all 3 groups at the end of the 3 mo evaluation. (A) represents positive staining for KIM-1 and (B) shows no presence of KIM-1 in tissues taken from 2 different animals from the 3 mo study group. (C) shows positive staining for KIM-1 and (D) shows no presence of KIM-1 in tissues taken from 2 different animals of the live-donor operated group. Similarly, (E) represents positive staining for KIM-1 in the nephrectomized control group, whereas (F) shows no presence of KIM-1 in the same group. In (G) and (H) normal kidneys are seen. KIM-1 stains as red color and is pointed out with black arrows. HE stains the nucleus blue. All images are 10×. IHC, immunohistochemistry; KIM-1, Kidney Injury Marker-1.

FIGURE 8.

Histological evaluation of the kidney tissue (hematoxylin). (A) and (B) shows the renal morphology of the study group. No major histological changes apart from a slight focal simplification of the proximal tubular epithelium and slight swelling of the tubular epithelium. No fibrosis or inflammatory infiltrates. (C) and (D) show the morphology of the living-donor operated group. No distinct histological changes can be seen. (E) and (F) show the results from histological analysis of the normal untreated control kidney group. Again, no major histological aberrations can be seen apart from apical sloughing of the plasma membranes. No chronic changes. (A, C, and E) show ×10 magnification, whereas (B, D, and F), represent areas of higher magnification (×30). All images were stained by hematoxylin/eosin. CD, collecting duct. Scale bars are 100 µm; PT, proximal tubule.

FIGURE 9.

Ultrastructural evaluation of kidney tissue at the 3-mo evaluation by TEM. In the study group (A), the tubular epithelium is generally well preserved with most of the apical brush border membranes intact (arrowheads). Signs of intracytoplasmic vacuolization can be seen focally (*). Focal signs of minor interstitial fibrosis could be seen (arrow). In some tubular profiles of the study group (B), edematous swelling of the epithelium occluded the lumen of the tubules and the apical brush border was reduced (arrowhead). The peritubular capillaries were patent without thrombi (*). In the live-donor operated control group (C), a patent peritubular capillary is seen (*). The proximal tubular epithelium displays a relatively normal configuration, but also signs of apical blebbing (arrowhead) and focal reduction of brush border height. Morphology from a control case with focus on the tubulointerstitium is seen in (D). In the center a peritubular capillary can be seen (*), surrounded by 3 proximal tubular profiles. These have a high mitochondrial content and preserved apical brush border membrane. Scale bars 10 µm. TEM, transmission electron microscopy.