Rapid or Slow Time to Brain Death? Impact on Kidney Graft Injuries in an Allotransplantation Porcine Model

Abstract

The use of donors deceased after brain death (DBD) with extended criteria in response to the shortage of grafts leads to the removal of more fragile kidneys. These grafts are at greater risk of not being grafted or delayed function. A better knowledge of the pathophysiology of DBDs would improve this situation. There is a difference between the results from animal models of DBD and the clinical data potentially explained by the kinetics of brain death induction. We compared the effect of the induction rate of brain death on the recovery of post-transplant renal function in a pig model of DBD followed by allografts in nephrectomized pigs. Resumption of early function post-transplant was better in the rapidly generated brain death group (RgBD) and graft fibrosis at three months less important. Two groups had identical oxidative stress intensity but a greater response to this oxidative stress by SIRT1, PGC1-α and NRF2 in the RgBD group. Modulation of mechanistic target of rapamycin (mTOR) stimulation by NRF2 would also regulate the survival/apoptosis balance of renal cells. For the first time we have shown that an allostatic response to oxidative stress can explain the impact of the rapidity of brain death induction on the quality of kidney transplants.

Keywords: allostasis; brain death; kidney; mechanistic target of rapamycin; nuclear factor erythroid-2-related factor 2; oxidative stress; transplantation.

Figure 1

Renal function evaluation from day 0 to day 14 after transplantation. Renal function evaluation of the first 14 days after allotransplantation of kidneys subjected to rapid brain death induction (RgBD) or slow brain death induction (SgBD). Blood creatinine (A,B), glomerular filtration rate in mL/min (C,D), fractional sodium excretion (E,F), blood LDH (G,H), blood high mobility group box 1 (HMGB1; I,J), blood NGAL (K,L) and blood IL-18 (M,N) were analyzed from the day before transplantation (D–L) to day 14 after transplantation. (n = 5 to 7 per group). Kinetic results (A,C,E,G,I,K,M) are expressed as mean ± SD, statistical analysis was performed with Kruskal–Wallis multiple-comparison Dunn’s test. Areas under the curve (AUC; B,D,F,H,J,L,N) are expressed in a scatter plot with the median, statistical analysis was performed with a Mann–Whitney test. * RgBD vs SgBD; ** vs D-1 p < 0.05.

Figure 2

Renal function evaluation at day 90 after transplantation. Renal function evaluation at day 90 after allotransplantation of kidneys submitted to rapid brain death induction (RgBD) or slow brain death induction (SgBD). Blood creatinine (A), glomerular filtration rate in mL/min (B), ratio proteinuria/creatininuria (C), blood NGAL (D), blood HMGB1 (E), blood IL-18 (F) and kidney tissue fibrosis (G) evaluated by histological red Sirius staining magnification × 6.3 (H) analyzed at day 90 after transplantation. (n = 6 per group). Results are expressed in a scatter plot with the median, statistical analysis was performed with the Mann–Whitney test. * p < 0.05.

Figure 3

Donor management: Biological stability of the two groups during reanimation. Evaluation of kidney injury marker evolution during BD donor management after rapid brain death induction (RgBD) or slow brain death induction (SgBD). Blood creatinine (A,B), blood NGAL (C,D), blood aspartate aminotransferase activity (ASAT) (E,F) and blood LDH (G,H) concentration blood evolution, investigated at control time (baseline), brain death time (B,D), and per donor management at 60, 120, 180 and 240 min after brain death (abd; n = 5–6 per group). Kinetic results (A,C,E,G) are expressed as mean ± SD, statistical analysis was performed with a Kruskal–Wallis multiple-comparison Dunn’s test. Areas under the curve (B,D,F,H) are expressed in a scatter plot with the median, statistical analysis was performed with a Mann–Whitney test. * p < 0.05.

Figure 4

Donor management: Oxidative stress and response to oxidative stress by SIRTs-PGC-1a-NRF2-mTOR path and mitochondrial integrity at the end of reanimation. Protein expression level of nitrotyrosine (A), aconitase (B), 4HNE (C), ratio phosphorylated SIRT-1 to total SIRT-1 (D), SIRT-3 (E), PGC-1a (F), NRF2 (G), ratio phosphorylated mTOR to total mTOR (H), ratio phosphorylated mTOR to NRF2 (I) and ratio Bcl2 to Bax (J), evaluated by western blot at the end of 4 h donor management in renal tissue of rapid brain death induction (RgBD) and slow brain death induction (SgBD) groups versus control kidney (n = 5–6 per group). Results are expressed in a scatter plot with the median, statistical analysis was performed with a Kruskal–Wallis multiple-comparison Dunn’s test. * p < 0.05.

Figure 5

Donor management: Endothelial activation; genomic expression of adherence molecules and protein expression of endothelial nitric oxide synthase (eNOS) and P-Serine-1177-eNOS at end of reanimation. RNA expression level of E-Selectin (A), CCL2/MCP-1 (B) and ICAM-1 (C), and protein expression level, by western blot, of eNOS (D) and ratio Serine 1177 phosphorylated eNOS to eNOS total (E) evaluated in renal tissue of rapid brain death induction (RgBD) and slow brain death induction (SgBD) groups at the end of cold storage following 4 h donor management (called RgBD + CS and SgBD + CS) versus control kidney (n = 5–6 per group). Results are expressed in a scatter plot with the median, statistical analysis was performed with a Kruskal–Wallis multiple-comparison Dunn’s test. * p < 0.05.

Figure 6

Experimental design.