Systemic overexpression of endogenous secretory RAGE attenuates renal ischaemia-reperfusion injury

Miriam Habib¹, Rebecca Morton¹, Gerard Adrianus¹, Moumita Paul¹, Eithne Cunningham¹, Yuan Min Wang², Stephen I Alexander², Huiling Wu¹, Ian Alexander³, Michael Crawford¹, Steven Chadban¹, Alexander Bishop¹, Alexandra Sharland¹

¹Collaborative Transplantation Research Group, Bosch Institute, The University of Sydney; ²Centre for Kidney Research, Children's Hospital Westmead; ³Gene Therapy Research Unit, Children's Hospital Westmead; Australia

Aims: Ischaemia-reperfusion (IR) injury in transplanted organs has both short and long-term sequelae. Signalling through toll-like receptors (TLRs) and the receptor for advanced glycation end products (RAGE) intensifies tissue injury after IR. Endogenous secretory (es)RAGE is a naturally-occurring soluble decoy receptor that can competitively bind ligands for TLRs and RAGE. Liver-specific recombinant adeno-associated viral vectors of serotype 2/8 (rAAV2/8) yield robust longterm transgene expression and often immunological tolerance to transgene products. In this study, we aim to determine whether overexpression of esRAGE using this system can protect mice against development of renal IR injury.

Methods: We created a liver-specific rAAV encoding human esRAGE (rAAV-esRAGE) and a control vector encoding human serum albumin (rAAV-HSA). Expression was quantitated using ELISA specific for human esRAGE or HSA. C57BL/6 mice underwent bilateral renal warm ischaemia with or without IP injection of 5x10¹¹ vector genomes rAAV-esRAGE or rAAV-HSA 7 days prior to ischaemia. Mice were sacrificed at intervals between 1 and 7 days post-reperfusion. Serum and tissue were collected for analysis.

Results: High-level esRAGE expression ranging from 4000-40000ng/ml was detectable over a period between D2 and D100 after injection. Physiological levels are 0.1-1ng/ml in humans and negligible in mouse. HSA concentrations in injected mice were 50-100mg/L. ALT and liver histology were comparable between injected mice and controls. Following renal warm ischaemia, serum creatinine peaked at 24 hrs. Median creatinine in the uninjected group was 173µmol/l compared with 17 µmol/l in sham-operated mice and 21.5 µmol/l in rAAV-esRAGE injected mice (p<0.001). An inverse trend was seen between serum esRAGE and creatinine level. Tubular injury scores in the rAAV-esRAGE-injected mice were 2.0±0.7 compared with 3.1±0.7 in uninjected animals, 3.4±0.5 in rAAV-HSA-injected mice and 0.4±0.5 in sham-operated controls. Evaluation of the effects of systemic esRAGE overexpression on cellular infiltration and production of inflammatory mediators is ongoing.

Conclusion: High-level expression of serum esRAGE using a liver-specific rAAV gene transfer system is well-tolerated and appears to confer protection against renal IR injury in an animal model.