PMP22 Regulates Fluid Movement Across the Intestinal Epithelium Through Effects on NHE3 Activity

Background: In addition to its roles in nutrient uptake and barrier function, the intestinal epithelium is essential for maintaining water and electrolyte homeostasis. We previously identified the claudin-like protein peripheral myelin protein 22 (PMP22) as a player in tight junction function in the gut. Here, we investigated PMP22’s role in maintaining electrolyte and fluid balance. We hypothesized that PMP22 regulates ion transport mechanisms and fluid movement across the intestinal epithelium. Methods: Caco-2BBe cells stably transfected with non-targeting (control) or PMP22-targeting shRNA constructs were grown on Transwells, either on top of the filter or on the underside. Medium volume in the apical and basolateral chambers was measured every 3-4 days. Barrier permeability was measured by transepithelial electrical resistance (TEER), and bulk RNA sequencing performed at 21 days. We inhibited SLC9A3 (NHE3) in monolayers at 21 days with tenapanor (1µM) and measured fluid movement, pH, and TEER for four days. Results: Compared to controls, shPMP22 Caco2-BBe monolayers had reduced TEER, indicating increased barrier permeability. shPMP22 monolayers also rapidly pumped medium from the apical to the basolateral chamber, regardless of whether the apical side was facing up or down. The pH in shPMP22 cultures was lower in the basolateral compartment compared to controls, while the apical medium was no different. RNA-seq analysis identified SLC9A3 (NHE3) as one of the top differentially expressed genes in shPMP22 monolayers. Treatment with the NHE3 inhibitor tenapanor normalized apical to basolateral fluid movement in shPMP22 monolayers, restoring medium levels to control amounts. While tenapanor moderately increased TEER in both shPMP22 and control monolayers, it was not sufficient to rescue shPMP22 TEER to that of control levels. pH was not altered by tenpanaor treatment in either shPMP22 or control cells. Conclusions: Not only does PMP22 contribute to maintaining epithelial barrier integrity, but also it appears to have a role in regulating fluid movement and pH across the intestinal epithelium. Inhibiting the sodium-hydrogen exchanger NHE3 was sufficient to rescue shPMP22 Caco2-BBe fluid volumes to control levels, but did not rescue barrier integrity. Thus, PMP22 regulates fluid balance via NHE3 but this effect is uncoupled from its effects on tight junction stability and barrier integrity. Future work is required to define the interaction between PMP22 and NHE3 and the impact this mechanistic axis has in health and disease. This research was funded by Senior Research Award from the Crohn’s and Colitis Foundation and NIH award R01DK095004 (MRF) and CIRM Scholar Fellowship (OSS). 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.