A novel, tractable, and physiologically relevant primary human cortical collecting duct model

Mechanistic studies of regulated ion transport processes in the human collecting duct (CD) have largely relied on non-human models because a tractable human epithelial system has been lacking. This is particularly important because much of what we know about CD ion transport derives from rodent or heterologous systems, where species differences limit direct translation to human physiology. We have established a primary human cortical CD (CCD) culture from kidney cortex obtained from non-cancerous regions of nephrectomy specimens (Western General Hospital, Edinburgh). Branched tubules were microdissected and fixed for immunofluorescence labelling, confirming CCD identity by the predominant expression of principal cell markers (AQP2, γ-ENaC, 11βHSD2), interspersed with V-ATPaseβ1-positive intercalated cells. Separate tubules were enzymatically dissociated to generate a single-cell suspension from which a population of primary human CCD cells was expanded in culture. When grown on permeable supports and fixed after electrophysiological experiments, the monolayers exhibited an epithelial (E-cadherin-positive) but not fibroblast (vimentin-negative) phenotype and expressed principal cell markers (AQP2, γ-ENaC, 11βHSD2). Interestingly, these monolayers retained occasional V-ATPaseβ1-positive cells interspersed among principal cell-like epithelial cells, a pattern not typically observed in established murine CCD cell models. Monolayers developed transepithelial resistance (Rt) of 3.5 ± 0.4 kΩ·cm 2 and voltage (Vt) of -37.7 ± 4.1 mV after 7d culture in fully supplemented media (n = 10). These properties remained measurable across five consecutive passages, with Vt decreasing and Rt showing a modest increase. Following standard protocols used to assess regulated ion transport in murine CCD cells, media supplements were withdrawn for 48h before electrophysiological measurements. The removal of supplements markedly reduced Vt to near zero coupled with a small increase in Rt (n = 3). To identify the component responsible, supplements were individually withdrawn for 24h. Only withdrawal of dexamethasone (dex, 50nM) reproduced the loss of Vt, indicating the importance of the glucocorticoid in maintaining measurable, amiloride-sensitive electrogenic Na + transport. Consistent with this, cells maintained for 24h in fully supplemented media (50nM dex) showed no aldosterone (ALDO, 3nM) response, whereas cells transferred to supplement-free media or to media containing 5nM dex exhibited clear ALDO-induced increases in equivalent short-circuit current (~2–3 fold), despite differences in starting Vt. In all responding conditions, the ALDO-induced current was amiloride-sensitive, confirming ENaC-mediated Na + transport. These findings demonstrate the generation of a primary human CCD model with epithelial identity, principal- and intercalated-cell markers, and steroid-responsive ENaC activity, providing a foundation for future studies of regulated ion transport in the human CD. Supported by the School of Medicine, University of St Andrews. 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.