Role of warm ischemia on innate and adaptive responses in a preclinical renal auto-transplanted porcine model

Abstract

Background: Deceased after cardiac arrest donor are an additional source of kidney graft to overcome graft shortage. Deciphering the respective role of renal warm and cold ischemia is of pivotal interest in the transplantation process.

Methods: Using a preclinical pig model of renal auto-transplantation, we investigated the consequences of warm and cold ischemia on early innate and adaptive responses as well as graft outcome. Kidneys were subjected to either 60 min-warm ischemia (WI) or auto-transplanted after cold storage for 24 h at 4°C (CS), or both conditions combined (WI+CS). Renal function, immune response and cytokine expression, oxidative stress and cell death were investigated at 3 h, 3 and 7 days (H3, D3 and D7) after reperfusion. At 3 months, we focused on cell infiltration and tissue remodelling.

Results: WI + CS induced a delayed graft function linked to higher tubular damage. Innate response occurred at D3 associated to a pro-oxidative milieu with a level dependent on the severity of ischemic injury whereas adaptive immune response occurred only at D7 mainly due to CS injuries and aggravated by WI. Graft cellular death was an early event detected at H3 and seems to be one of the first ischemia reperfusion injuries. These early injuries affect graft outcome on renal function, cells infiltration and fibrosis development.

Conclusions: The results indicate that the severe ischemic insult found in kidneys from deceased after cardiac arrest donor affects kidney outcome and promotes an uncontrolled deleterious innate and adaptive response not inhibited 3 months after reperfusion.

Figure 1

Intensive ischemic injury induces early pro oxidative milieu. The mRNA expressions of p47phox and gp91phox (a), and protein expression of cytoplasmic Cu/Zn SOD (SOD1, b) were investigated in control, WI, CS and WI + CS groups. Ischemia induces early necrosis and apoptosis. The numbers of necrotic tubular cells (c) and positively cleaved caspase 3 tubular cells (d) per field were counted on ten different tissue sections for each experimental condition in the first week of reperfusion. Shown are mean ± SEM. *p < 0.05 versus control. $ p < 0.05 between groups.

Figure 2

Chronological analysis of early immune process in reperfused kidneys. The P-selectin mRNA was studied as marker of endothelial activation (a). Innate immunity markers like Toll-like receptors 2 (TLR2) and TLR4 mRNA (b), MCP-1 protein (c), IL-1β and IL-6 cytokines mRNA (d) were investigated in kidney tissue. IL-1Rn and IL-10 cytokine mRNA expressions were also explored (e). The numbers of positively ED1 and CD3 stained cells per surface area (10⁴/μm²) was counted on five different tissue sections for each experimental conditions (f). Shown are mean ± SEM. *p < 0.05 versus control. $ p < 0.05 between groups.

Figure 3

WI + CS maintained inflammatory cells recruitment 3 months after reperfusion and induced tubular injury and fibrosis. VCAM-1 and P-selectin mRNA and MCP-1 protein expressions were investigated in each studied conditions (a-b).The numbers of positively ED1 and CD3 (c) stained cells per surface area (10⁴/μm²) was counted on ten different tissue sections for each experimental conditions. Tubular atrophy, Red Sirius staining (e), and protein expression (d) of mediators of TGF beta and tPA pathways were studied in control, WI, CS and WI + CS groups at 3 months post-reperfusion. * p < 0.05 vs. control, $ p < 0.05 between ischemic groups.

Figure 4

Adaptive response affects early and chronic graft function. Invading CD3+ and ED1+ cells in kidney graft correlate with the severity of ischemic injuries (a, c). The creatininemia after 7 days of reperfusion and fibrosis percentage evaluated at 3 months correlate with the number of lymphocytes invading the tissue at the end of the first week of reperfusion (b, d). r² and Spearman rank are indicated.