The role of urea in neuronal degeneration and sensitization: An in vitro model of uremic neuropathy

BACKGROUND: Uremic neuropathy commonly affects patients with chronic kidney disease, with painful sensations in the feet, followed by numbness and weakness in the legs and hands. The symptoms usually resolve following kidney transplantation, but the mechanisms of uremic neuropathy and associated pain symptoms remain unknown. As blood urea levels are elevated in patients with chronic kidney disease, we examined the morphological and functional effects of clinically observed levels of urea on sensory neurons. METHODS: Rat dorsal root ganglion neurons were treated with 10 or 50 mmol/L urea for 48 h, fixed and immunostained for PGP9.5 and βIII tubulin immunofluorescence. Neurons were also immunostained for TRPV1, TRPM8 and Gap43 expression, and the capsaicin sensitivity of urea- or vehicle-treated neurons was determined. RESULTS: Urea-treated neurons had degenerating neurites with diminished PGP9.5 immunofluorescence, and swollen, retracted growth cones. βIII tubulin appeared clumped after urea treatment. After 48 hours urea treatment, neurite lengths were significantly reduced to 60 ± 2.6% (10 mmol/L, **P < 0.01), and to 56.2 ± 3.3% (50 mmol/L, **P < 0.01), compared with control neurons. Fewer neurons survived urea treatment, with 70.08 ± 13.3% remaining after 10 mmol/L (*P < 0.05) and 61.49 ± 7.4% after 50 mmol/L urea treatment (**P < 0.01), compared with controls. The proportion of neurons expressing TRPV1 was reduced after urea treatment, but not TRPM8 expressing neurons. In functional studies, treatment with urea resulted in dose-dependent neuronal sensitization. Capsaicin responses were significantly increased to 115.29 ± 3.4% (10 mmol/L, **P < 0.01) and 125.3 ± 4.2% (50 mmol/L, **P < 0.01), compared with controls. Sensitization due to urea was eliminated in the presence of the TRPV1 inhibitor SB705498, the mitogen-activated protein kinase kinase inhibitor PD98059, the PI3 kinase inhibitor LY294002 and the TRPM8 inhibitor N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide (AMTB hydrochloride). CONCLUSION: Neurite degeneration and sensitization are consistent with uremic neuropathy and provide a disease-relevant model to test new treatments.