Dual Regulation of Ascending Thin Limb Structure and Function

Background: The ability to concentrate urine is a fundamental physiologic adaptation that generates a steep osmotic gradient across the renal medulla and enables the production of hyperosmotic urine. The contribution of the inner medullary loop of Henle (LOH) to urinary concentration is incompletely understood, with the role of the ascending thin limb (aTL) remaining unknown. The aTL passively transports chloride transcellularly through Clck1 and sodium paracellularly through unclear mechanisms. Methods: We identified a novel genetic reporter of the ascending LOH that recombines specifically in the aTL following neonatal tamoxifen administration. Furthermore, low dose tamoxifen yields mosaic GFP labelling, which allows for the visualization of single aTL cells. This offers a unique genetic tool for studies of aTL structure and function. Results: Visualization of single GFP labelled aTL cells revealed branched, finger-like lateral interdigitations that structurally resemble those of podocytes. Through a time-course a single nucleus RNA sequencing and RT-PCR, we identified that claudin-10b is highly enriched during aTL maturation. Claudin-10b is a tight junction protein that forms a cation-selective pore, making it a possible mediator of paracellular sodium transport in the aTL. aTL-specific deletion of claudin-10 resulted in a urinary concentrating defect, marked by decreased urine osmolality and increased urine output at baseline and following water deprivation. Conditional deletion of claudin-10 also profoundly disrupted aTL lateral protrusions, suggesting claudin-10 is necessary for both aTL structure and function. Mechanistically, in vitro studies found claudin-10b promotes membrane remodeling through ZO1- mediated trans-cellular adhesion, rather than its established role in ion transport. Conclusion: These studies offer the first in vivo evidence that the aTL is required for urine concentration. We characterized the previously unappreciated cell architecture of the aTL, whose disruption is associated with urinary concentrating defects. Lastly, we identified claudin-10b as a dual regulator of both aTL structure and function. Funding: NIDDK Support