*Article* **Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells**

**Carlos Ayala-Torres, Susanne M. Krug, Rita Rosenthal † and Michael Fromm \*,†**

> Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology and Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany; carlos-mario.ayala-torres@charite.de (C.A.-T.); susanne.m.krug@charite.de (S.M.K.); rita.rosenthal@charite.de (R.R.)

**\*** Correspondence: michael.fromm@charite.de

† These authors contributed equally to this work.

**Abstract:** Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (*alias* lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines.

**Keywords:** angulin-1; LSR; tricellulin; tricellular tight junction; paracellular water transport; tight epithelium; MDCK C7 cells; intermediate-tight epithelium; HT-29/B6 cells
