Defining the molecular response to ischemia-reperfusion injury and remote ischemic preconditioning in human kidney transplantation

Abstract

Background: Ischemia-reperfusion injury (IRI) inevitably occurs during kidney transplantation and extended ischemia is associated with delayed graft function and poor outcomes. Remote ischemic preconditioning (RIPC) is a simple, noninvasive procedure aimed at reducing IRI and improving graft function. Experimental studies have implicated the kynurenine pathway as a protective mechanism behind RIPC.

Methods: First, paired biopsies from 11 living kidney donors were analyzed to characterize the acute transcriptomic response to IRI. Second, 16 living kidney donors were subjected to either RIPC (n = 9) or no pretreatment (n = 7) to evaluate the impact of RIPC on the transcriptomic response to IRI. Finally, the effect of RIPC on plasma metabolites was analyzed in 49 healthy subjects.

Results: There was a robust immediate response to IRI in the renal transcriptomes of living-donor kidney transplantation, including activation of the mitogen-activated protein kinase (MAPK) and epidermal growth factor receptor (EGFR) pathways. Preconditioning with RIPC did not significantly alter the transcriptomic response to IRI or the concentration of plasma metabolites.

Conclusions: The present data validate living-donor kidney transplantation as a suitable model for mechanistic studies of IRI in human kidneys. The failure of RIPC to alter transcriptomic responses or metabolites in the kynurenine pathway raises the question of the robustness of the standard procedure used to induce RIPC, and might explain the mixed results in clinical trials evaluating RIPC as a method to attenuate IRI.

Fig 1. Study outlines.

A) In the IRI biopsy group (n = 11 donor-recipient pairs) the kidney’s immediate cellular response to IRI was characterised by RNA sequencing in paired biopsies. During surgery, a first kidney biopsy was taken at back-table with 3–15 minutes of warm ischemia (Baseline) and a second biopsy was taken after 60–120 minutes of cold ischemia and approximately 60 minutes after reperfusion in the recipient (Ischemia-reperfusion). B) In the RIPC biopsy group (n = 16 donor-recipient pairs), nine donors received RIPC immediately prior to surgery and seven donors received no preconditioning, and the potential impact of RIPC on the response to IRI was evaluated by RNA sequencing. C) In the RIPC blood sample group, samples were collected from living donors (n = 23) at the morning of surgery and approximately 2 h after the first blood sample, and in healthy controls (n = 26) immediately before RIPC and 2 h later. Thereafter, the potential impact of RIPC on KYNA and other metabolites of the kynurenine pathway was analyzed using mass spectrometry.

Fig 2. Immediate response to ischemia-reperfusion.

(A) Histological features were similar between baseline and ischemia-reperfusion samples (HE staining, black bar = 50 μm). (B) Gene expression across samples for the top 50 significant differentially expressed genes (adjusted p-value < 0.05; |logFC| > 1.5). (C) Dimensionality reduction of the transcriptomic profiles across samples using the two first principal components revealed no clear separation between timepoints. (D) Transcription factor activities for the top 20 transcription factors that change the most in response to IRI. (E) Pathway activities in response to IRI.

Fig 3. Modulation of the immediate response to IRI by RIPC.

(A) Histological features were not affected by preconditioning with RIPC (HE staining, black bar = 50 μm). (B) Gene expression across samples for the top 10 significant differentially expressed genes (p-value < 0.05; |logFC| > 0.3). (C) Transcription factor activities for the top 20 transcription factors that changed the most in response to RIPC. (D) Pathway activities in response to RIPC. (E and F) Volcano plots of the target genes of the JAK-STAT pathway for IRI in Control and RIPC groups.

Fig 4. Plasma metabolites in response to RIPC.

(A) Plasma metabolites in samples obtained from living donors before RIPC/no preconditioning and during surgery. (B) Plasma metabolites in samples obtained from healthy controls before RIPC/no preconditioning and 2 h after. All values are presented as μM.