Role of Glial TRPV4 in Hydrocephalus Pathology and Treatment

Transient receptor potential vanilloid member 4 (TRPV4) is a mechanosensitive cation channel implicated in osmotic regulation. Our laboratory found that a TRPV4 antagonist ameliorates hydrocephalus, a disease caused by cerebrospinal fluid (CSF) accumulation in the brain’s ventricles, resulting from an imbalance in CSF secretion, flow, and/or absorption. A genetic model of hydrocephalus in rodents is caused by a single missense point mutation in the transmembrane 67 (TMEM67) gene, leading to ciliopathy and hydrocephalic pathology.TRPV4 antagonist treatment of Tmem67-/- rats attenuated the development of hydrocephalus presumably by slowing production of CSF by the choroid plexus, a tissue that is responsible for CSF production. However, hydrocephalus pathology affects the entire brain and TRPV4 is expressed throughout the brain in several different cell types. This study focuses on changes occurring in glia, which have roles in brain-barrier maintenance and brain fluid/electrolyte regulation. TRPV4 regulates both CSF and brain interstitial fluid in combination with other transport proteins. Aquaporins (AQP), specifically AQP4, have been implicated in diseases associated with fluid regulation and may interact with TRPV4. We hypothesize that TRPV4 and AQPs are important in hydrocephalus pathology, and that changes may be attenuated with RN1734 treatment. Postnatal day 15 (P15) untreated (untr) and TRPV4 antagonist treated (RN1734; 4mg/kg intraperitoneal injection daily P7-P14) wildtype and Tmem67-/- rats were utilized in this study. Changes in glial cell morphology, protein expression, and localization of TRPV4 and AQP4 were observed. Periventricular glia were visualized using fluorescent immunohistochemistry (IHC) of GFAP. Using IHC, astrocyte cell volume and process morphology of glia were found to be altered in Tmem67-/- rats. The TRPV4 antagonist returned process morphology to relatively normal, but not the amount of GFAP fluorescent intensity. GFAP protein and mRNA expression increased in hydrocephalic animals, but RN1734 treated animals were not significantly different from untreated. Next, we used IHC to examine localization of TRPV4 and AQP4. Immunoreactivity of TRPV4 increased in the subventricular zone and white matter of Tmem67-/- untreated rats and colocalized with GFAP and a marker of cell death, cleaved caspase-3 (CC3). AQP4 also increased at the blood-brain-barrier and brain-CSF interface in hydrocephalic animals, occurring in the same regions as GFAP and CC3 labeling. TRPV4 expression was normalized with RN1734, but not AQP4 expression. By utilizing western blot analysis, in the cortex, AQP4, but not TRPV4, protein was increased in hydrocephalic rats. With RN1734, AQP4 protein was not normalized. In summary, TRPV4 changes in the Tmem67-/- rats returned to normal with RN1734, glial morphology and AQP4 changes did not. This may indicate residual inflammation in the Tmem67-/- rats, which we aim to address in future studies which will elucidate TRPV4’s role in hydrocephalus pathology by exploring transcriptional, post-translational, and membrane localization and expression changes. Overall, investigating cell and molecular changes that occur in hydrocephalus will aid in the development of better pharmacological targets and treatments. Funding: This research was conducted with the support of the Congressionally Directed Medical Research Program Grants #HT94252310296 and #HT94252310401; Hydrocephalus Association This abstract was presented at the American Physiology Summit 2026 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.