MOLECULAR MECHANISMS OF DELAYED KIDNEY GRAFT FUNCTION

A. Lau¹, Z. Zhang², S. Wang³, A.M. Jevnikar^4
1Pathology, University of Western Ontario, London/ON/CANADA, ²Department Of Medicine, University of Western Ontario, London/CANADA, ³Department Of Medicine, Unuiversity of Western Ontario, London/CANADA, ⁴Department Of Medicine, Multi-Organ Transplant Program-London Health Sciences Centre, London/CANADA

Body: INTRODUCTION: Ischemia reperfusion injury (IRI) occurs in all transplanted kidneys as a consequence of organ procurement. With IRI, the death of kidney epithelial cells (TEC) and other parenchymal cells releases High Mobility Group Box-1 (HMGB1) and other damage associated molecular pattern (DAMP) proteins that promote inflammation by known effects on dendritic and infiltrating immune cells. However the role of DAMPs in propagating renal injury by effects on viable TEC remains unknown. METHODS/RESULTS: Uni-nephrectomized C57BL/6J mice were subjected to clamping of the remaining renal pedicle for 45 min to induce IRI. In western blot analyses, HMGB1 increased progressively in kidney samples in 24 hours post ischemia (naïve density ratio: 0.953±0.7107 vs. 24h post IRI density ratio: 5.3768±0.2439, p<0.001, n=3) without a change in HMGB1 mRNA expression. Correspondingly there was increased mRNA expression of TLR4, a key receptor for HMGB1 up to 24 hours post ischemia (relative fold increase: 4.654±2.554). Released extracellular HMGB1 was evident diffusely in sections by immuno-histochemistry, including tubular lumen. In vitro, TEC lines (NG1.1) subjected to hypoxia (2-5% oxygen for 20 min) underwent death (co-Annexin V/PI positivity) and progressively released HMGB1 into the supernatant up to 24 hours post ischemia (density ratio: 0.574 in no treatment vs. 8.88 in 24h post hypoxia). Conditioned media from hypoxia exposed TEC induced death in untreated TEC when added to viable monolayers and Annexin V/PI positivity was increased (media control: 36.1±4.4% vs. conditioned media: 51.25±9.75%). In addition to reducing survival of viable TEC, hypoxia induced TEC to a 6.86±2.067 fold increase of monocyte chemotactic protein-1 (MCP-1) mRNA expression at 24 hours compared to no treatment. Similarly, MCP-1 mRNA increased in vivo up to 24h after IRI (sham: 1±0 vs. 24h post IRI: 8.6328±3.56). Glycyrrhizic acid (GZA), a functional inhibitor of HMGB1 prevented this increase in hypoxic TEC in vitro in a dose dependent manner and reduced MCP-1 mRNA expression to levels observed in naive TEC (800ng/mL GZA: 1.587±0.345 vs. 24h post hypoxia: 6.86±2.067 ), indicating that the effect of HMGB1 on MCP-1 was mediated by preventing interaction with DAMP receptors on viable TEC. We found that MCP-1 is a chemoattractant for Natural Killer (NK) cells in transwell assay (migration index=4.19). In previous studies we have shown that NK cells are important effector cells in kidney IRI. To test the effect of DAMP on NK cells activation, purified NK cells were exposed to highly purified endotoxin free HMGB1, and showed subsequently mRNA increase in IFN-γ (2.51±0.52 vs. naïve NK cells, p<0.05, n=3) and Granzyme B (2.43±0.58 vs. naïve NK cells, p<0.05, n=3). CONCLUSION: These data demonstrate for the first time that HMGB1 released following hypoxic kidney injury has a previously unrecognized capacity to propagate renal injury by altering the survival of TEC as well as attracting and activating NK cells. Inhibition of HMGB1 release by TEC or blocking HMGB1 interaction with TEC may provide a therapeutic strategy that could limit renal IRI following transplantation.

Disclosure: All authors have declared no conflicts of interest.