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ILDR1 Is Important for Paracellular Water Transport and Urine Concentration Mechanism

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ILDR1 Is Important for Paracellular Water Transport and Urine Concentration Mechanism ILDR1 is important for paracellular water transport and urine concentration mechanism Yongfeng Gonga,1, Nina Himmerkusb,1, Abby Sunqa, Susanne Milatzb, Cosima Merkelb, Markus Bleichb,2, and Jianghui Houa,2 aDepartment of Internal Medicine, Washington University in St. Louis, St. Louis, MO 63110; and bDepartment of Physiology, University of Kiel, 24118 Kiel, Germany Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 6, 2017 (received for review January 18, 2017) Whether the tight junction is permeable to water remains highly chromosome 12, and the sites of mutations were found to be controversial. Here, we provide evidence that the tricellular tight functionally important in making the water permeation pore (12) junction is important for paracellular water permeation and that Ig- or facilitating proper intracellular trafficking (13). Whether there like domain containing receptor 1 (ILDR1) regulates its permeability. may exist any previously unknown gene important for renal water In the mouse kidney, ILDR1 is localized to tricellular tight junctions reabsorption is a tantalizing question. Here, we have revealed Ildr1 of the distal tubules. Genetic knockout of in the mouse kidney that homozygous deletion of the ILDR1 gene, which encodes a causes polyuria and polydipsia due to renal concentrating defects. TJ protein controlling the putative paracellular water pathway, Microperfusion of live renal distal tubules reveals that they are im- can cause NDI-like phenotypes in the mouse kidney. permeable to water in normal animals but become highly perme- able to water in Ildr1 knockout animals whereas paracellular ionic Results permeabilities in the Ildr1 knockout mouse renal tubules are not The Gene Expression of ILDR1 in the Kidney. Whether the kidney ex- affected. Vasopressin cannot correct paracellular water loss in Ildr1 presses ILDR1 has not been determined before. We first performed knockout animals despite normal effects on the transcellular aqua- porin-2–dependent pathway. In cultured renal epithelial cells nor- microdissection on mouse kidneys to isolate each nephron segment, mally lacking the expression of Ildr1, overexpression of Ildr1 including the glomerulus, the proximal tubule (PT), the thin limb PHYSIOLOGY significantly reduces the paracellular water permeability. Together, (TL) and the thick limb (TAL) of the loop of Henle, the distal our study provides a mechanism of how cells transport water and convoluted tubule (DCT), and the collecting duct (CD), obeying a shows how such a mechanism may be exploited as a therapeutic rigorous anatomic criterion (14) (Fig. S1A). Quantitative RT-PCR approach to maintain water homeostasis. revealed that the distal nephron, from the TAL to the CD, expressed the highest levels of ILDR1 mRNA, which were over 58- tight junction | water channel | nephrology | diuresis | diabetes insipidus fold higher than in the glomerulus and over 454-fold higher than in the PT (Fig. S1B). To reveal the cellular localization pattern of ight junctions (TJs) are known to be leaky to ions and, thus, to ILDR1 protein along the nephron, we performed colocalization Tconstitute a paracellular pathway with ionic permselectivity analyses using molecular markers against each nephron segment. In similar to that of transmembrane channels (1). Whether the TJ is WT mouse kidneys, ILDR1 proteins were detected in the tricellular permeable to water, on the other hand, has been highly contro- junctions of renal tubules lacking the Lotus tetragonolobus lectin (a versial. Jorge Fischbarg noticed that the corneal endothelium PT marker), but expressing the claudin-19 protein (a TAL marker) retained over 60% of water permeability even when AQP1, the only (15), the NCC protein (a DCT marker) (16), and the Aqp2 protein aquaporin expressed by these cells, was knocked out by genetic (aCDmarker)(17)(Fig.1).TheILDR1 antibody specificity was deletion (2). Rosenthal et al. have demonstrated that, in cultured demonstrated by staining the ILDR1 knockout mouse kidney (Fig. renal epithelium expressing the claudin-2 protein, transepithelial S2). Thus, ILDR1 is expressed in the distal tubules of the kidney. water permeability was significantly higher than in cells without its expression (3). Using an optical microscopic approach, Spring and Significance coworkers have directly measured the velocity of water flow across the tight junction and have ruled out the existence of any significant transjunctional water flow, at least for the bicellular TJ (bTJ) (4). The role of tight junction in water homeostasis is an important The tricellular tight junction (tTJ) is a specialized cell junction but understudied area. Here, we reveal a paracellular water different in ultrastructure from that of the bTJ (5). Unlike the bTJ, permeation pathway made by the tricellular tight junction and which is made of claudin and occludin (6), the proteins making the regulated by its integral protein, known as Ildr1. The impor- tTJ include tricellulin and angulins (LSR/angulin-1, ILDR1/angulin-2, tance of paracellular water permeation is demonstrated here and ILDR2/angulin-3) (7, 8). Transgenic knockout (KO) of either with emphasis of its potential role in causing urinary concen- tricellulin or Ildr1 in mice causes hearing loss due to degeneration of trating defects in the kidney. Ildr1 and its related proteins, mechanosensory cochlear hair cells in the organ of Corti (9, 10). Ildr2 and Ildr3, may play broader physiologic roles to establish Peculiarly, neither the endocochlear potential nor the paracellular the water homeostasis in other organs, such as the skin, the permeability in the stria vascularis changed in these mutant animals. lung, and the intestine. The permeation property of tTJ therefore remains a major mystery. Author contributions: Y.G., N.H., M.B., and J.H. designed research; Y.G., N.H., A.S., S.M., There are two forms of diabetes insipidus (DI): central (neu- and C.M. performed research; Y.G., N.H., M.B., and J.H. analyzed data; and Y.G., N.H., rogenic) and nephrogenic. The nephrogenic DI (NDI) is caused M.B., and J.H. wrote the paper. by the inability of the kidney to reabsorb water. NDI is hereditary The authors declare no conflict of interest. and has been linked to two genetic loci. The most common ge- This article is a PNAS Direct Submission. netic cause (in 95% of cases) is an X-linked disorder mapped to 1Y.G. and N.H. contributed equally to this work. the type-2 vasopressin receptor (AVPR2) gene in the X chro- 2To whom correspondence may be addressed. Email: [email protected] or m.bleich@ mosome, mutation of which makes the collecting duct cells in- physiologie.uni-kiel.de. sensitive to vasopressin (11). The second cause is an autosomal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. recessive disorder linked to the aquaporin-2 (Aqp2) gene in 1073/pnas.1701006114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1701006114 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 levels (PNa,PCl) were not altered in KO animals (Table 1). Compatible with well-preserved salt handling by the kidney, the mean blood pressure (BP) was not different between KO and control animals (Table 1), indicating that the urinary volume loss did not elicit a significant effect on the extracellular fluid volume (ECFV). Hypokalemia has been associated with polyuria due to + down-regulation of Aqp2 in the collecting duct (19). No K deregulation was present in KO mice (Table 1), clearly dis- tinguishing their phenotypes from Bartter syndrome or Gitelman syndrome. Hypercalcemia or hypercalciuria has also been asso- ciated with polyuria, presumably due to activation of the calcium-sensing receptor (CaSR) located in the basolateral membrane of the thick ascending limb or the luminal membrane of the collecting duct, respectively, and impairment of renal concentrating ability (20, 21). There was no difference in plasma + Ca2 levels between KO and control animals (Table 1). The urinary excretion rates (absolute excretion, ECa, and fractional excretion, FECa), however, were significantly increased by 3.0- and 3.3-fold, respectively. Because KO animals excreted more water than calcium, the urinary calcium concentration was in fact lower in KO than in control animals. Thus, loss of function in ILDR1 can cause primary renal concentrating defect in mice. Microperfusion of Thick Ascending Limb Tubules from ILDR1 KO Mice. Since the late1950s, the countercurrent multiplication hypothesis has been the widely accepted explanation for the generation of the osmolality gradient along the cortico-medullary axis, which in turn drives water reabsorption in the collecting duct through Aqp2 (22). This hypothesis holds that the osmotic pressure dif- ference between the lumen and the interstitium generated by the thick ascending limb (TAL) of the loop of Henle is multiplied because the fluid flows in the opposite direction from the thin descending limb to the TAL. The driving force of this process depends upon the effective separation of salt reabsorption from water impermeation in the TAL, which creates the osmotic pressure difference that can then be multiplied. The TAL was previously considered water impermeable. We performed ex vivo perfusion experiments on microdissected TAL tubules from control and ILDR1 KO animals (Fig. 2A). To record the water Fig. 1. ILDR1 protein localization in the kidney. Double staining of ILDR1 protein permeability across the microperfused tubule, a high molecular with a PT marker (LTL), with a TAL marker (CLDN19), with a DCT marker (NCC), and weight fluorescent dye (FITC-dextran, 150 kDa, 30 μM) was with a CD marker (Aqp2). (Scale bars: 10 μm.) included in the perfusate (Table S1), which was too large to permeate through the epithelium. Then, the open end of the microperfused tubule was sealed, allowing stepwise increases in The Renal Phenotypes of ILDR1 KO Mice. ILDR1 KO mice have basolateral osmolality to generate an outward osmotic gradient been generated, described, and validated before (18). The age- that drives water to leave the tubule.
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