The role of AQP9 in mannitol-resistant oedema

Abstract

The aquaglyceroporin, AQP9, facilitates the transport of water and other small neutral solutes through the cell membrane. It is expressed widely in the liver, while mRNA and/or protein expression has also been reported in lung, testis and brain tissue. The extent to which AQP9 is expressed in the human brain remains unclear. In rodents, it was shown that the expression of AQP9 is upregulated under hypoxia and/or hyperosmolality induced by mannitol. Mannitol is a sugar alcohol that is used medically as a hyperosmotic medication in the treatment of patients with cerebral oedema post-stroke or traumatic-brain injury (TBI). Mannitol exerts its effect by increasing blood osmolarity, causing accumulated fluid in swollen brain tissue to move into the blood stream for excretion by the kidneys. However, resistance to mannitol therapy has been documented after multiple doses of mannitol. The hypothesis tested in this thesis is that hypoxia and/or hyperosmolarity induced by mannitol increase AQP9 mRNA and protein expression. This enables mannitol to enter swollen astrocyte brain cells through the AQP9 channel, thereby ablating the osmotic gradient. AQP9 mRNA and protein expression were investigated in human astrocytes through qPCR and western blot, respectively. The mannitol permeability of these cells was probed using a calcein quenching permeability assay following hypoxia and/or mannitol treatment with/without known AQP9 inhibitors. AQP9 mRNA and protein expression were both upregulated in a time dependent manner following 1% hypoxic incubation for 24-72 h, although the magnitude of the response was subject to inter-donor variation between the astrocytes being assayed. Astrocytes incubated under hypoxia for 24-72 h with/without 5% mannitol showed a time-dependent elevation in their mannitol permeability which was reversed by two established AQP9 inhibitors, phloretin and RG100204. A novel AQP9 inhibitor, EATA-2A, produced by Dr Zaid Alobaidi at Aston University was also characterised. Protein kinase A and/or calmodulin may play a role in AQP9 expression and/or surface localisation. Overall, the work in this thesis shows that AQP9 is expressed in the human brain following hypoxia and/or mannitol treatment and that AQP9 is a mannitol channel. These findings would explain the resistance to mannitol therapy observed in patients suffering cerebral oedema; in vivo experiments are now required to validate this hypothesis.