Donor treatment with a PHD-inhibitor activating HIFs prevents graft injury and prolongs survival in an allogenic kidney transplant model

Abstract

Long-term survival of renal allografts depends on the chronic immune response and is probably influenced by the initial injury caused by ischemia and reperfusion. Hypoxia-inducible transcription factors (HIFs) are essential for adaptation to low oxygen. Normoxic inactivation of HIFs is regulated by oxygen-dependent hydroxylation of specific prolyl-residues by prolyl-hydroxylases (PHDs). Pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of tissue-protective genes. We examined the effect of donor treatment with a specific PHD inhibitor (FG-4497) on graft function in the Fisher-Lewis rat model of allogenic kidney transplantation (KTx). Orthotopic transplantation of the left donor kidney was performed after 24 h of cold storage. The right kidney was removed at the time of KTx (acute model) or at day 10 (chronic model). Donor animals received a single dose of FG-4497 (40 mg/kg i.v.) or vehicle 6 h before donor nephrectomy. Recipients were followed up for 10 days (acute model) or 24 weeks (chronic model). Donor preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond cold storage. It reduced acute renal injury (serum creatinine at day 10: 0.66 +/- 0.20 vs. 1.49 +/- 1.36 mg/dL; P < 0.05) and early mortality in the acute model and improved long-term survival of recipient animals in the chronic model (mortality at 24 weeks: 3 of 16 vs. 7 of 13 vehicle-treated animals; P < 0.05). In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcomes after allogenic KTx. Inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves clinical evaluation.

Fig. 1.

Induction of HIF-α and HIF target genes by FG-4497 in human proximal tubular cells. (A) HKC-8 cells were exposed to dipyridyl (DP, positive control) or FG-4497 (0.5, 1, 5, 10, 25, 50, and 100 μM) for 6 h. Immunoblotting for HIF-α protein shows dose-dependent accumulation of HIF-1α and HIF-2α by FG-4497, with a maximal level of HIF-α stabilization at concentrations above 10 μM. (B) Fifteen minutes after FG-4497 treatment (150 μM), HIF-1α and HIF-2α were stabilized and remained stable over the observation period of 6 h. (C) With a delay of approximately 2 h, the mRNA (real-time PCR) of the HIF target genes AngPTL4, Glut-1, and VEGF was significantly up-regulated compared with baseline (set as “1”) and further increased up to 6 h after treatment with FG-4497. Normoxic untreated cells (N) served as a negative control.

Fig. 2.

Kinetics of HIF accumulation after FG-4497 treatment in the kidneys of Fisher (F-344) rats before and after cold ischemia. b.w., body weight. (A) At 1, 4, and 6 h after i.v. injection of FG-4497, HIF-1α was immunohistochemically detectable in tubular epithelial cells of rat kidneys and HIF-2α was detectable in interstitial, endothelial, and glomerular cells. (B) After FG-4497 and cold ischemia of 24 h, HIF-α was still detectable to a comparable degree as before cold ischemia. Veh treatment with or without cold ischemia did not lead to detectable HIF-α accumulation.

Fig. 3.

Effect of FG-4497 on the expression of HIF target genes. Single potentially protective HIF target genes were quantified by real-time PCR 6 h after FG-4497 treatment without cold ischemia (c.i.) or after an additional 24-h period of cold ischemia. The nephroprotective genes HO-1 and EPO were strongly up-regulated after FG-4497 treatment irrespective of cold ischemia (*, P < 0.05).

Fig. 4.

Effect of FG-4497 on early graft function and renal morphology. In the acute setting, FG-4497 significantly improved survival within the initial days after KTx (A), indicating a lower incidence of delayed graft function. In addition, in surviving animals, FG-4497 donor pretreatment resulted in an earlier and more pronounced decrease in serum creatinine from days 3–10 after allogenic transplantation (Allo) when compared with donor pretreatment with Veh only (B). In isogenic KTx, there was no measurable difference in serum creatinine and survival between FG-4497 and Veh. (C) Blinded scoring for acute tubular necrosis revealed better preservation of renal morphology after FG-4497 compared with Veh independent of the immunological constellation (*, P < 0.05).

Fig. 5.

Effect of FG-4497 on long-term graft survival. Kaplan-Meier curves after allograft KTx in animals with and without pretreatment of the donor with FG-4497. Animals that received a renal transplant from an FG-4497-treated donor had significantly better survival rates (black line) than animals transplanted with a kidney from a Veh-treated donor (dotted gray line). None of the isogenic control animals died (dashed gray line). (*, P < 0.05).