Introduction: Hydrocephalus is a disease caused by an overproduction of cerebrospinal fluid (CSF), blockage of flow, or decreased reabsorption. When CSF accumulates in the brain it causes ventriculomegaly, increased intracranial pressure, inflammation, and cell damage. The choroid plexus within the brain's ventricles consists of a fenestrated capillary network surrounded by an epithelial monolayer. This tissue produces CSF. Choroid plexus epithelial cells (CPe) are polarized, meaning that the ion channels are located apically (CSF-facing) or basolaterally (blood-facing) and regulation of these channels is responsible for the composition and production of CSF. Some notable channels include transient receptor potential vanilloid 4 (TRPV4), calcium-activated chloride channel (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR), and Na+/Cl- co-transporter (NCC). Activation of TRPV4, a nonselective cation channel that is osmo-, shear-, temperature-, and pressure-sensitive, allows Ca2+ and Na+ influx into the CPe which, secondarily, causes transepithelial electrolyte flux that involves multiple electrolyte transporters. In Tmem67-/- rats, a genetic form of hydrocephalus was reversed with TRPV4 antagonist treatment [1]. TMEM16A and CFTR are located basolaterally on the CPe while NCC is apical. Interestingly, patients with posthemorrhagic hydrocephalus (PHH) have elevated Cl- levels in their CSF [2]. This could be caused by an interaction of blood components with chloride channels. We investigated hemoglobin, a major protein found in red blood cells.  

Aims: The goal of this study was to determine if there was intercellular signaling between activated TRPV4, TMEM16A, CFTR, and NCC. By investigating the role of these channels in the CPe, we aim to understand the regulation of CSF production in normal and pathophysiological states. Additionally, we investigated the role of hemoglobulin as a potential pathological factor in hydrocephalus development.  

Methods: Human choroid plexus papilloma (HIBCPP) cells were grown in Dulbecco’s modified eagle media (DMEM) with sodium bicarbonate, 10% fetal bovine serum, 1% penicillin/streptomycin, and 5 ng/L insulin. Ussing-style electrophysiology was used to measure net transepithelial electrolyte flux and transepithelial permeability in the cultures. To determine the relationship between the ion channels, cells were pre-treated with the following compounds and concentrations: Ani9 10 uM (TMEM16A inhibitor), FeCl3 10 nM (TMEM16A activator), CFTR Inhibitor 172 5 µM, metolazone 0.2 mM (NCC inhibitor), and bovine hemoglobulin 140 mg/mL . Compounds were added apically, basolaterally, or bilaterally to determine polarity. 10 minutes after effector treatments, TRPV4 agonist GSK1016790A (GSK) was used to stimulate the influx of Ca2+ and Na+ into the cells. Each drug treatment group (n=6) was analyzed for statistical significance with a multiple T-test.  

Results: TMEM16A activation or inhibition substantially altered the complex, multiphase transepithelial ion flux that is activated in response to TRPV4 agonist stimulation. When CFTR was inhibited basolaterally, the membrane permeability increased. NCC inhibition had no significant effect. Hemoglobulin treatment potentiated the agonist-stimulated transepithelial ion flux and increased conductance.  

Conclusion: There is a relationship between TRPV4 and chloride channels. By investigating these interactions, we hope to understand PHH pathophysiology and identify drug targets for non-invasive hydrocephalus treatments.