Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB264

Poster Communications

Activation of the receptor for advanced glycation end products mediates sensitization of primary afferents in diabetic neuropathy

S. M. Bestall1,2, R. Hulse2, N. Ved2, K. Paton1, N. Beazley-Long1, D. Bates2, L. F. Donaldson1

1. School of Life Sciences, University of Nottingham, Nottingham, Nottinghamshire, United Kingdom. 2. School of Medicine, University of Nottingham, Nottingham, United Kingdom.

  • AUC values for capsaicin-evoked calcium response in DRG neurons for different drug treatment groups (Ca2+ fluorescence F/F0)<\#13>

    Data are mean

Painful neuropathy is a serious diabetic complication that affects up to 20% of diabetic patients. Transient receptor potential vanilloid 1 (TRPV1) sensitization has been implicated in the development of diabetic pain and is ameliorated by vascular endothelial growth factor-A(VEGF-A)165b treatment. We hypothesized that the activation of the receptor for advanced glycation end products (RAGE) by high mobility group box-1 (HMGB1) sensitizes neuronal TRPV1, and this would be prevented with VEGF-A165b. Diabetes was induced in adult female Sprague Dawley rats (250-350g) with streptozotocin (STZ, 50mg/kg, i.p) and maintained for 3 weeks. Diabetic rats were insulin treated (LinShin, 1/3 slow release pellet, implanted under 2% isoflurane in O2 anesthesia), and treated bi-weekly with recombinant human VEGF-A165b (20ng/g, i.p, n=4) or vehicle (PBS, i.p, n=4). At 3 weeks, animals were killed with an overdose of sodium pentobarbital (60mg/kg, i.p), plantar skin was dissected, protein extracted, and Western blots performed for HMGB1 and actin. Dorsal root ganglion (DRG) neurons were isolated from naïve adult male Wistar rats, plated onto 96 well plates for measurement of intracellular calcium changes in response to TRPV1 (1µM in 0.1% DMSO) in a high throughput Fluo-4 based assay. DRG neurons were treated for 24h before assay with HMGB1 (RAGE agonist, 10nM), HMGB1+FPSZM1 (RAGE antagonist, 10nM), HMGB1+rhVEGF-A165b (2.5nM), and HMGB1+BIM1 (PKC inhibitor, 1µM). Calcium responses were calculated as AUC for each assay. HMGB1 expression in the skin was significantly increased in STZ injected rats compared to naïve (STZ + vehicle 5.4±1.8 fold change vs. naïve 1±0.3 fold change, n=4, 1 way ANOVA + post hoc Bonferroni p<0.05). There was no effect of VEGF-A165b treatment on HMGB1 expression. HMGB1 alone increased capsaicin-evoked TRPV1 activity in DRG neurons and treatment with all inhibitors reduced the response (Table 1). HMGB1 induces TRPV1 sensitization in DRG neurons, through activation of RAGE and PKC. VEGF-A165b had no effect in vivo on expression of HMGB1, but prevented the HMGB1-RAGE mediated TRPV1 sensitization in vitro. HMGB1 expression is increased in the peripheral skin in diabetic rats. These data suggest peripheral sensory neuronal RAGE activation may contribute to peripheral neuronal sensitization in diabetes through modulation of TRPV1.

Where applicable, experiments conform with Society ethical requirements