Investigating hydrocephalus pathophysiology: TRPV4-mediated transepithelial electrolyte flux in relation to the WNK/SPAK pathway in choroid plexus epithelial cells

Hydrocephalus is caused by dysregulation of cerebrospinal fluid (CSF). CSF is produced by the choroid plexus which is a fenestrated capillary network surrounded by a specialized epithelium. The choroid plexus epithelium (CPe) is polarized with ion channels, transporters and pumps located apically (CSF-facing) or basolaterally (blood-facing). These proteins are important for CSF production and composition. Transient receptor potential vanilloid 4 channel (TRPV4) is a nonselective cation channel in the CPe. Inhibition of TRPV4 in a genetic rat model of hydrocephalus ameliorated fluid accumulation and ventriculomegaly (1). In CPe cells, we identified TRPV4 as a regulator of ion influx that secondarily activates K+ and Cl- channels. The with no lysine/STE20/SPS1-related proline/alanine-rich kinase (WNK/SPAK) pathway is well-characterized in the kidney (2) where it regulates transporters that are also present in the CPe including the Na+/Cl- co-transporter (NCC), K+ Cl- cotransporter 3 (KCC3) and the Na+-K+-2Cl- cotransporter (NKCC1). KCC3 mutations are implicated in congenital hydrocephalus (3) and other groups have shown that NKCC1 appears to be involved in CSF production. It is our hypothesis that electrolyte channels that are regulated by the WNK/SPAK pathway play a role in TRPV4-mediated electrolyte and fluid movement during the production of CSF. We grew the human choroid plexus papilloma (HIBCPP) cell line to confluence and used Ussing-style electrophysiology to measure two values: short-circuit current (net transepithelial electrolyte flux) and transepithelial resistance (membrane permeability). To determine the role of each of the transporters in TRPV4-stimulated ion flux, the cells were pre-incubated for 10 minutes with the following effectors: STOCK2S 20 µM (SPAK inhibitor), bumetanide (NKCC1 inhibitor), metolazone 0.2 mM (NCC inhibitor), DIOA 50 µM (KCC inhibitor). Subsequently, the TRPV4 agonist GSK1016790A (15 nM) was added to stimulate net ion and fluid flux. Data were analyzed with a multiple paired t-test. Inhibition of SPAK completed blocked TRPV4 agonist stimulated ion flux and permeability changes. Interestingly, inhibition of either NKCC1 or NCC had no effect on TRPV4-mediated ion flux while inhibition of KCC caused a decrease in the stimulated ion flux. Our results agree with previous studies showing the importance of the SPAK pathway in CSF production. However, our data indicate the involvement of ion transporters which are different than those reported previously. By investigating these interactions, we aim to understand hydrocephalus pathophysiology and identify targets for non-invasive treatments. References 1. Hochstetler, A.E. et al. (2020). JCI Insight 5, 1-14. 2. Richardson, C. & Alessi D.R. (2008). J Cell Sci 121, 3293-3304. 3. Jin, S.C. et al. (2019). Mol Genet Genom Med 7, e892. These studies were supported by DoD Congressionally Directed Medical Research Program Grants #HT94252310296 and #HT94252310401 (BBY) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.