Development and characterization of cellular models for the study of the distal convoluted tubule and the thick ascending limb

The distal convoluted tubule (DCT) and the thick ascending limb (TAL) of the loop of Henle’s play essential roles in the maintenance of blood pressure, electrolyte balance, and water homeostasis. The study of the molecular physiology of these nephron segments has been hindered by the absence of adequate cellular models. The use of primary cultures is complicated by the difficulty of obtaining cell-type-homogeneous preparations and by the dedifferentiation that occurs upon culturing. On the other hand, available cell lines show little resemblance to native cells. Thus, our objective was to test whether the Terminator mouse-based system is useful for generating primary cultures enriched in these cell types and to develop culture conditions that maintain their differentiated phenotype. Briefly, the Terminator mouse was engineered to express the Diphtheria toxin (DT) receptor (normally absent in mice) in all cell types except those in which the encoding sequence is deleted by Cre-mediated recombination. These mice were crossed with PV-Cre mice (which, in the kidney, express Cre only in the DCT) and with NKCC2-Cre mice (which express Cre only in the TAL). Primary cell cultures were prepared and treated with DT to achieve DCT- or TAL-enriched cultures. This was confirmed by observation of enrichment of cell-type specific markers. A dedifferentiation process was observed shortly after seeding; however, following a period of cellular expansion, redifferentiation was achieved by culturing the cells on transwell inserts with a carefully defined differentiation medium. In DCT cultures, we observed NCC expression by immunoblot, apical NCC localization by immunofluorescence, and robust thiazide-sensitive Na + and Cl - transport. Unexpectedly, γENaC expression was also observed, along with amiloride-sensitive Na + and K + transport. In TAL cultures, we observed NKCC2 expression by immunoblot, apical NKCC2 localization by immunofluorescence, and bumetanide-sensitive Na + and Cl - transport. Future studies will include RNA sequencing, evaluation of responses to physiological stimuli, and the development of immortalized cell lines. In conclusion, we established conditions to enrich and maintain differentiated DCT and TAL primary cultures with functional NCC and NKCC2 expression, respectively. Although further characterization is required, these models provide a valuable tool for studying the molecular physiology these nephron segments. 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.