Am J Physiol Renal Physiol 306: F51–F52, 2014; doi:10.1152/ajprenal.00545.2013.
Editorial Focus
Significant water absorption goes paracellular in kidney proximal tubules Rita Rosenthal and Michael Fromm Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Submitted 11 October 2013; accepted in final form 16 October 2013 IT HAD BEEN A FUNDAMENTAL dispute for several decades, whether water passes epithelia such as kidney proximal tubule or small intestine along the transcellular, paracellular, or both pathways. By discovery of the transmembranal aquaporin water channels (AQP), the molecular basis of the transcellular pathway was uncovered (1), but the molecular basis of the tight junctional pathway was not resolved at that time. Transcellular and paracellular water transport was, therefore, measured by methods that attempted to technically discriminate the two pathways. This turned out to be difficult, and as a result, the contribution of the paracellular pathway was estimated in the maximal range between 0 and 100%, depending on the method used for analysis (3). In recent years, several tight junction proteins were identified to form paracellular channels with selectivity for small cations (claudin-2, claudin-10b, and claudin-15) or anions (claudin-10a and claudin-17) (4). In consequence, new approaches were developed to measure transepithelial water transport before and after molecular perturbation of the tight junction by overexpression or downregulation of specific junctional proteins. In this way, the tight junctional contribution to transepithelial water transport could be analyzed. Using an overexpression study in the epithelial cell line MDCK-C7, Rosenthal et al. (8) identified claudin-2 to form a paracellular water channel. Claudin-2 is highly expressed in the proximal tubule of the kidney and is known as a paracellular cation channel (2). Thus, claudin-2 forms channels for both, small cations and water. Whereas this in vitro study employed claudin-2 overexpression, others were performed on claudin-2-deficient mice. In a very evident study published in the American Journal of Physiology—Renal Physiology, Schnermann et al. (9) measured proximal fluid reabsorption in wild-type mice and mice deficient for claudin-2, AQP-1, and both. In claudin-2-deficient mice, fluid reabsorption was reduced by 23% compared to wild-type mice. This result corroborates the concept of a claudin-2-based paracellular water permeability and convincingly demonstrates a paracellular contribution to proximal tubule fluid reabsorption of 25%. Schnermann et al. (9) performed a comprehensive analysis not only of proximal fluid reabsorption, but also on kidney and single-nephron filtration rate on a large number of mice. Furthermore, in a detailed expression analysis of wild-type mice and mice deficient for claudin-2, AQP1, and both, the authors demonstrated that the findings are due to the deletion of the specific protein and not to further alterations in gene expression.
Address for reprint requests and other correspondence: M. Fromm, Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany (e-mail: michael. [email protected]). http://www.ajprenal.org
They showed that in mice deficient of AQP-1, which is highly expressed in both apical and basolateral membranes of proximal tubule cells and, thus, represents the main pathway for transcellular water movement, fluid reabsorption is reduced by 20%. The absence of AQP-1 causes a transepithelial osmotic difference, which induces fluid reabsorption across AQP1-independent transcellular pathways. This was observed both in the absence and presence of claudin-2. In mice deficient of both AQP-1 and claudin-2, a reduction of fluid flow by 26% was found, which was not significantly different from values measured in singly deficient animals (9). This was an unexpected finding of the study, which has not be sufficiently explained yet. A possible interpretation could be the assumption that AQP-1- and claudin-2-mediated water fluxes are interdependent, as it has been shown for paracellular permeability and AQP-5 function in salivary glands (5). These findings are consistent with results obtained by Muto et al. (7), who found a decreased net transepithelial reabsorption of Na, Cl, and water in isolated, perfused S2 segments of proximal tubules of claudin-2-deficient mice. Aquaporin expression was not investigated in that study. Proximal tubule net water reabsorption in Cldn2⫺/⫺ mice was decreased by about 30% compared with that in Cldn2⫹/⫹ mice. A possible alternative explanation for the role of claudin-2 in kidney tubular water flux has to be discussed, namely, that claudin-2 mediates Na⫹ reabsorption through the paracellular pathway, but water follows only along the transcellular pathway, mediated, e.g., by AQP-1. However, this possibility was excluded in the study on MDCK C7 cells, as it demonstrated that in claudin-2-expressing cell layers, water flux was induced not only by an osmotic gradient, but also by a NaCl gradient in the absence of an osmotic gradient. As a second piece of evidence, net water flux induced by an osmotic gradient caused a net Na⫹ transport in claudin-2-expressing cells, indicating that water and Na⫹ were transported through the same channel (8). For claudin-2-based paracellular channels, the characteristics of the pore have been determined. A negatively charged site within the pore formed by aspartate-65 causes the cation selectivity by electrostatic interaction. The pore radius was estimated to be 3.25 Å, which appears large enough for the passage of both partially dehydrated cations and water molecules (diameter 2.8 Å) (10). Among other claudins exhibiting undisputed cation or anion permeability (4), neither overexpression of claudin-10b nor of claudin-17 altered water permeability of MDCK C7 cell layers (6, 8). Thus, claudin-2 so far is the only claudin for which water permeability has been demonstrated. The present study by Schnermann et al. (9) has convincingly proven and quantified that concept for kidney proximal tubules. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.
1931-857X/14 Copyright © 2014 the American Physiological Society
F51
Editorial Focus F52 AUTHOR CONTRIBUTIONS Author contributions: R.R. and M.F. drafted manuscript; R.R. and M.F. edited and revised manuscript; R.R. and M.F. approved final version of manuscript.
6.
7.
REFERENCES 1. Agre P, Preston GM, Smith BL, Jung JS, Raina S, Moon C, Guggino WB, Nielsen S. Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol Renal Fluid Electrolyte Physiol 265: F463–F476, 1993. 2. Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, Fromm M. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 115: 4969 –4976, 2002. 3. Fischbarg J. Fluid transport across leaky epithelia: central role of the tight junction and supporting role of aquaporins. Physiol Rev 90: 1271–1290, 2010. 4. Günzel D, Yu AS. Claudins and the modulation of tight junction permeability. Physiol Rev 93: 525–569, 2013. 5. Kawedia JD, Nieman ML, Boivin GP, Melvin JE, Kikuchi K, Hand AR, Lorenz JN, Menon AG. Interaction between transcellular and
8.
9.
10.
paracellular water transport pathways through Aquaporin 5 and the tight junction complex. Proc Natl Acad Sci USA 104: 3621–3626, 2007. Krug SM, Günzel D, Conrad MP, Rosenthal R, Fromm A, Amasheh S, Schulzke JD, Fromm M. Claudin-17 forms tight junction channels with distinct anion selectivity. Cell Mol Life Sci 69: 2765–2778, 2012. Muto S, Hata M, Taniguchi J, Tsuruoka S, Moriwaki K, Saitou M, Furuse K, Sasaki H, Fujimura A, Imai M, Kusano E, Tsukita S, Furuse M. Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules. Proc Natl Acad Sci USA 107: 8011–8016, 2010. Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M. Claudin-2, a component of the tight junction, forms a paracellular water channel. J Cell Sci 123: 1913–1921, 2010. Schnermann J, Huang Y, Mizel D. Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin. Am J Physiol Renal Physiol 305: F1352–F1364, 2013. Yu ASL, Cheng MH, Angelow S, Günzel D, Kanzawa SA, Schneeberger EE, Fromm M, Coalson RD. Molecular basis for cation selectivity in claudin-2-based paracellular pores: Identification of an electrostatic interaction site. J Gen Physiol 133: 111–127, 2009.
AJP-Renal Physiol • doi:10.1152/ajprenal.00545.2013 • www.ajprenal.org
Copyright of American Journal of Physiology: Renal Physiology is the property of American Physiological Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Editorial Focus
Significant water absorption goes paracellular in kidney proximal tubules Rita Rosenthal and Michael Fromm Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Submitted 11 October 2013; accepted in final form 16 October 2013 IT HAD BEEN A FUNDAMENTAL dispute for several decades, whether water passes epithelia such as kidney proximal tubule or small intestine along the transcellular, paracellular, or both pathways. By discovery of the transmembranal aquaporin water channels (AQP), the molecular basis of the transcellular pathway was uncovered (1), but the molecular basis of the tight junctional pathway was not resolved at that time. Transcellular and paracellular water transport was, therefore, measured by methods that attempted to technically discriminate the two pathways. This turned out to be difficult, and as a result, the contribution of the paracellular pathway was estimated in the maximal range between 0 and 100%, depending on the method used for analysis (3). In recent years, several tight junction proteins were identified to form paracellular channels with selectivity for small cations (claudin-2, claudin-10b, and claudin-15) or anions (claudin-10a and claudin-17) (4). In consequence, new approaches were developed to measure transepithelial water transport before and after molecular perturbation of the tight junction by overexpression or downregulation of specific junctional proteins. In this way, the tight junctional contribution to transepithelial water transport could be analyzed. Using an overexpression study in the epithelial cell line MDCK-C7, Rosenthal et al. (8) identified claudin-2 to form a paracellular water channel. Claudin-2 is highly expressed in the proximal tubule of the kidney and is known as a paracellular cation channel (2). Thus, claudin-2 forms channels for both, small cations and water. Whereas this in vitro study employed claudin-2 overexpression, others were performed on claudin-2-deficient mice. In a very evident study published in the American Journal of Physiology—Renal Physiology, Schnermann et al. (9) measured proximal fluid reabsorption in wild-type mice and mice deficient for claudin-2, AQP-1, and both. In claudin-2-deficient mice, fluid reabsorption was reduced by 23% compared to wild-type mice. This result corroborates the concept of a claudin-2-based paracellular water permeability and convincingly demonstrates a paracellular contribution to proximal tubule fluid reabsorption of 25%. Schnermann et al. (9) performed a comprehensive analysis not only of proximal fluid reabsorption, but also on kidney and single-nephron filtration rate on a large number of mice. Furthermore, in a detailed expression analysis of wild-type mice and mice deficient for claudin-2, AQP1, and both, the authors demonstrated that the findings are due to the deletion of the specific protein and not to further alterations in gene expression.
Address for reprint requests and other correspondence: M. Fromm, Institute of Clinical Physiology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany (e-mail: michael. [email protected]). http://www.ajprenal.org
They showed that in mice deficient of AQP-1, which is highly expressed in both apical and basolateral membranes of proximal tubule cells and, thus, represents the main pathway for transcellular water movement, fluid reabsorption is reduced by 20%. The absence of AQP-1 causes a transepithelial osmotic difference, which induces fluid reabsorption across AQP1-independent transcellular pathways. This was observed both in the absence and presence of claudin-2. In mice deficient of both AQP-1 and claudin-2, a reduction of fluid flow by 26% was found, which was not significantly different from values measured in singly deficient animals (9). This was an unexpected finding of the study, which has not be sufficiently explained yet. A possible interpretation could be the assumption that AQP-1- and claudin-2-mediated water fluxes are interdependent, as it has been shown for paracellular permeability and AQP-5 function in salivary glands (5). These findings are consistent with results obtained by Muto et al. (7), who found a decreased net transepithelial reabsorption of Na, Cl, and water in isolated, perfused S2 segments of proximal tubules of claudin-2-deficient mice. Aquaporin expression was not investigated in that study. Proximal tubule net water reabsorption in Cldn2⫺/⫺ mice was decreased by about 30% compared with that in Cldn2⫹/⫹ mice. A possible alternative explanation for the role of claudin-2 in kidney tubular water flux has to be discussed, namely, that claudin-2 mediates Na⫹ reabsorption through the paracellular pathway, but water follows only along the transcellular pathway, mediated, e.g., by AQP-1. However, this possibility was excluded in the study on MDCK C7 cells, as it demonstrated that in claudin-2-expressing cell layers, water flux was induced not only by an osmotic gradient, but also by a NaCl gradient in the absence of an osmotic gradient. As a second piece of evidence, net water flux induced by an osmotic gradient caused a net Na⫹ transport in claudin-2-expressing cells, indicating that water and Na⫹ were transported through the same channel (8). For claudin-2-based paracellular channels, the characteristics of the pore have been determined. A negatively charged site within the pore formed by aspartate-65 causes the cation selectivity by electrostatic interaction. The pore radius was estimated to be 3.25 Å, which appears large enough for the passage of both partially dehydrated cations and water molecules (diameter 2.8 Å) (10). Among other claudins exhibiting undisputed cation or anion permeability (4), neither overexpression of claudin-10b nor of claudin-17 altered water permeability of MDCK C7 cell layers (6, 8). Thus, claudin-2 so far is the only claudin for which water permeability has been demonstrated. The present study by Schnermann et al. (9) has convincingly proven and quantified that concept for kidney proximal tubules. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.
1931-857X/14 Copyright © 2014 the American Physiological Society
F51
Editorial Focus F52 AUTHOR CONTRIBUTIONS Author contributions: R.R. and M.F. drafted manuscript; R.R. and M.F. edited and revised manuscript; R.R. and M.F. approved final version of manuscript.
6.
7.
REFERENCES 1. Agre P, Preston GM, Smith BL, Jung JS, Raina S, Moon C, Guggino WB, Nielsen S. Aquaporin CHIP: the archetypal molecular water channel. Am J Physiol Renal Fluid Electrolyte Physiol 265: F463–F476, 1993. 2. Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, Fromm M. Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J Cell Sci 115: 4969 –4976, 2002. 3. Fischbarg J. Fluid transport across leaky epithelia: central role of the tight junction and supporting role of aquaporins. Physiol Rev 90: 1271–1290, 2010. 4. Günzel D, Yu AS. Claudins and the modulation of tight junction permeability. Physiol Rev 93: 525–569, 2013. 5. Kawedia JD, Nieman ML, Boivin GP, Melvin JE, Kikuchi K, Hand AR, Lorenz JN, Menon AG. Interaction between transcellular and
8.
9.
10.
paracellular water transport pathways through Aquaporin 5 and the tight junction complex. Proc Natl Acad Sci USA 104: 3621–3626, 2007. Krug SM, Günzel D, Conrad MP, Rosenthal R, Fromm A, Amasheh S, Schulzke JD, Fromm M. Claudin-17 forms tight junction channels with distinct anion selectivity. Cell Mol Life Sci 69: 2765–2778, 2012. Muto S, Hata M, Taniguchi J, Tsuruoka S, Moriwaki K, Saitou M, Furuse K, Sasaki H, Fujimura A, Imai M, Kusano E, Tsukita S, Furuse M. Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules. Proc Natl Acad Sci USA 107: 8011–8016, 2010. Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M. Claudin-2, a component of the tight junction, forms a paracellular water channel. J Cell Sci 123: 1913–1921, 2010. Schnermann J, Huang Y, Mizel D. Fluid reabsorption in proximal convoluted tubules of mice with gene deletions of claudin-2 and/or aquaporin. Am J Physiol Renal Physiol 305: F1352–F1364, 2013. Yu ASL, Cheng MH, Angelow S, Günzel D, Kanzawa SA, Schneeberger EE, Fromm M, Coalson RD. Molecular basis for cation selectivity in claudin-2-based paracellular pores: Identification of an electrostatic interaction site. J Gen Physiol 133: 111–127, 2009.
AJP-Renal Physiol • doi:10.1152/ajprenal.00545.2013 • www.ajprenal.org
Copyright of American Journal of Physiology: Renal Physiology is the property of American Physiological Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
