A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing
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The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm2) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.
KeywordsEpithelial transport NKCC2 ROMK Loop of Henle TAL
The authors would like to thank Gery Barmettler, Soline Bourgeois, Huguette Debaix, David Hoogewijs and Klaus Marquardt for assistance and helpful suggestions. Prof. Jan Loffing kindly provided the parvalbumin-EGFP mouse. The uromodulin knockout mouse was kindly provided by Prof. X-R. Wu. These studies were supported in part by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 246539 (Marie Curie) and grant no. 305608 (EURenOmics); an Action de Recherche Concertée (ARC, Communauté Française de Belgique); the FNRS and FRSM; the Inter-University Attraction Pole (IUAP, Belgium Federal Government); the NCCR Kidney. CH program (Swiss National Science Foundation); the Gebert Rüf Stiftung (Project GRS-038/12); and the Swiss National Science Foundation 31003A-125422/1 (to OS) and 310030–146490 (to OD).
Conflict of interest
The authors declare no competing interests.