**1. Introduction**

Adequate transport of solutes and water across epithelial barriers is indispensable for maintaining normal physiological homeostasis in all animals [1,2]. Fluid is moved either across the plasma membranes of the cells that comprise the epithelial layer (transcellular transport) or between these cells (paracellular transport). The discovery of aquaporin water channels provided a first molecular basis for transcellular water movement [3].

Paracellular transport involves specialized structures called tight junctions (TJ) that regulate the flow of solutes through paracellular pathways and maintain cell polarity, thereby functioning as a barrier of epithelial and endothelial cellular sheets [4]. Tricellular tight junctions (tTJs) form at the convergence of bicellular tight junctions (bTJs) where three epithelial cells meet in polarized epithelia [5,6]. Claudin family proteins and occludin are main components of bicellular TJs, which are important for the barrier function and permselectivity [7–10]. To date, 27 members of the claudin family have been identified in humans. Many of these have sealing functions (claudins 1, 3, 5, 11, 14, 19) while some claudins form channels across TJs, which are permeable either for cations (claudins 2, 10b, 15) or for anions (claudins 10a, 17). For several claudins, their effects on epithelial barriers are inconsistent and the function is unclear (claudins 4, 7, 8, 16) [11]. The claudin composition of the TJ mainly determines the tightness of an epithelium or an epithelial cell line, respectively.

**Citation:** Ayala-Torres, C.; Krug, S.M.; Rosenthal, R.; Fromm, M. Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells. *Int. J. Mol. Sci.* **2021**, *22*, 7827. https://doi.org/ 10.3390/ijms22157827

Academic Editor: Masoud Jelokhani-Niaraki

Received: 31 May 2021 Accepted: 20 July 2021 Published: 22 July 2021

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In addition to their role as channels for small cations, Rosenthal et al. demonstrated that claudin-2 and claudin-15 also transport water [12–14].

The mechanisms that determine the use of paracellular versus transcellular routes for water transport are multifaceted. For example, in the kidney, transport of water is achieved in part through the paracellular pathway in the proximal nephron [15], whereas in the distal nephron and collecting duct, it takes place through transcellular routes [16]. Mutations in claudins have been identified to cause hereditary diseases, demonstrating that regulation of the paracellular permeability is crucial to the normal functions of various organs [11].

The tricellular TJ is more complex than bTJ. In the center of tTJ, bTJ elements from each of the three cells come together to form a central tube of about 1 μm in length and 10 nm in diameter [17]. To date, two types of integral membrane proteins, tricellulin and angulin family proteins, are identified as molecular components of tTJ [18]. Tricellulin, which was the first tTJ protein to be identified [5], plays a critical role in sealing the tTJ against the passage of solutes of up to 10 kDa [19,20], and it was recently found that it also plays a role in the paracellular water transport in the tight epithelial cell line, MDCK C7 [21].

As said, angulins contribute to forming the central element of the tTJ. Angulin family proteins, comprising lipolysis-stimulated lipoprotein receptor (LSR), immunoglobulin-like domain containing receptor-1 (ILDR1) and -2 (ILDR2), are type-I transmembrane proteins with an extracellular immunoglobulin-like domain. Due to their common structure and function as tTJs-associated membrane proteins, LSR, ILDR1, and ILDR2 were designated as angulin-1, angulin-2, and angulin-3, respectively [22].

The original role of LSR/angulin-1 was described in lipid metabolism studies, as LSR was first discovered to be expressed in hepatocytes, where it plays a role in the clearance of triglyceride-rich lipoproteins and low-density lipoproteins, and also acts as an apolipoprotein B/E–containing lipoprotein receptor [23]. At tricellular contacts, angulin-1 recruits tricellulin, and the interaction between the cytoplasmic domain of angulin-1 and the Cterminal cytoplasmic domain of tricellulin is required for this recruitment [24,25]. Angulin-1 together with tricellulin are required for full barrier function of epithelial cells with high transepithelial electrical resistance [25]. Loss or downregulation of angulin-1 disrupts the barriers with relocalization of tTJ molecule tricellulin in various cell types [24,26]. In addition, loss of angulin-1 enhances cancer cell motility [27,28] and the LSR knockout mice die before embryonic day 15.5 (E15.5), but the cause of death remains unclear [29].

The LSR-related proteins immunoglobulin-like domain containing receptors angulin-2 and angulin-3 are also expressed complementarily in many epithelial cell types at tricellular contacts of many epithelial cells and recruit tricellulin [22], for instance, it is known that angulin-1 and angulin-2 are co-expressed in the large intestine and the kidney [22].

As for the tTJ, Gong et al. provided evidence that water transport is increased in isolated kidney tubules in the absence of angulin-2, but is normally inhibited in the presence of angulin-2 [30]. In contrast to this, Hempstock et al. found in the colon and kidney of angulin-2 KO mice no detectable abnormalities in water transport and maintained barrier function of the epithelia. They concluded that angulin-1 changes its expression pattern and location, which compensates for the loss of angulin-2 [31].

These findings led us to hypothesize that angulin-1 is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium, in this respect similar to tricellulin. Therefore, we aimed to characterize the effect of angulin-1 expression on water transport in a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, respectively. In order to define the tightness of these cell lines, we previously performed a two-path impedance spectroscopy study where we analyzed paracellular, transcellular, and transepithelial resistance in MDCK C7, HT-29/B6, and MDCK C11 cells. As a result, MDCK C7 cells were defined as a tight and HT-29/B6 cells as a moderate tight epithelial cell line, as defined by the ratio Rpara/Rtrans and the absolute resistance values of the two pathways [32]. In our present study, we found that the water passage through the tTJ was changed in the absence of angulin-1 only in MDCK C7 cells, thus in a tight epithelium.
