Stretch‐Regulated Tight Junction Dynamics, and Paracellular Permeability of the Urothelium
Authors: Eaton Amity F , Clayton Dennis R , Ruiz Wiley G , Apodaca Gerard L 
Published: November 2023

Tight junctions (TJ) are responsible for regulating the paracellular pathway across epithelia, which is the passive movement of ions, and water through the lateral space between adjacent epithelial cell membranes. Despite the fact that paracellular flux is passive, it is still controlled by differential claudin expression, and specific assembly into TJ. Previously, there has not been a comprehensive characterization of claudin expression in the bladder. Currently, our lab has positively identified claudin −2, −3, −4, −6, −7, −8, −10, and −11 in the urothelium via western blot (WB), and immunofluorescence (IF); and has confirmed the specificity of the antibodies. The urothelium is composed of a single outer layer of umbrella cells, multiple layers of intermediate cells, and a layer of basal cells. Under relaxed conditions, claudin −2, −6, −8, and −10 localize to the TJ of the umbrella cells; claudin −4, −7, and −11 localize from the TJ of the umbrella cells, down the lateral membrane, and into the intermediate cell layer; finally, claudin‐3 localizes, exclusively, to the basal cell layer.

The urothelium is an exceptionally “tight” epithelium, with a transepithelial resistance (TER) ranging from 75,000–135,000 Ω·cm2, which makes it an ideal model for examining the dynamics of paracellular permeability. It is, also, an interesting system to study, because it is subjected to stretch over time; which our lab has previously shown decreases junctional resistance (RJ) across the umbrella cell TJ, while the integrity of the epithelial membrane is maintained (Carattino, et al. AJP Renal 2013 Oct 15; 305(8): F1158–68). Since the paracellular permeability, and selectivity of an epithelial monolayer is dependent upon the unique assembly of claudins in TJ, our lab has begun characterizing claudin remodeling under stretched conditions to elucidate the underlying mechanism of the observed decrease in RJ. Claudins can be cation, or anion‐selective pore‐formers; or alternatively, can occlude the paracellular pathway. Claudin‐4 is a well‐characterized, anion‐selective, pore‐forming claudin. Although WB showed no change in total expression of claudin‐4 under stretched conditions in the bladder, by IF claudin‐4 was shown to localize to the TJ of the umbrella cells from the lateral membrane after bladder filling, as confirmed by its colocalization with the TJ protein ZO‐1. This would explain our previous observation that umbrella cell RJ was, significantly, reduced after bladder filling.

We have, also, shown that there is a physical expansion of the TJ ring upon bladder filling (Carattino, et al. AJP Renal 2013 Oct 15; 305(8): F1158–68). In addition to this observation, we have previously published that the apical membrane of the umbrella cell layer expands upon filling, via Rab‐dependent exocytosis; and contracts via compensatory endocytosis, upon voiding (Khandelwal et, al. Mol Biol Cell 24: 1007–1019, 2013; EMBO J 29: 1961–1975, 2010). Based on these prior observations, it seems likely that the TJ ring is expanded via exocytosis when the urothelium is subjected to stretch. This hypothesis is supported by our observation that whole‐mount bladders filled in the presence cytochalasin D are unable to recover TJ size upon voiding, like control bladders. Understanding the dynamics of this change in the TJ under stretched conditions is relevant as it could be applicable to any of the ubiquitous epithelia subjected to mechanical stretch under normal conditions; such as the lungs, and vasculature.

Support or Funding Information

1R01 DK099196 (Apodaca and Carattino – multi‐PI) NIDDK Biology and Function of the Bladder Umbrella Cell Paracellular Barrier The goals of this grant are to explore how claudin‐based pores in the tight junctions of adjacent umbrella cells are altered during stretch and relaxation as the bladder fills and empties. We also seek to understand the role of paracellular transport in signaling epithelial stretch to cells underyling the epithelium.