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Unraveling the relationship between macula densa cell volume and luminal solute concentration/osmolality

2006; Elsevier BV; Volume: 70; Issue: 5 Linguagem: Inglês

10.1038/sj.ki.5001633

1523-1755

Péter Komlósi, Attila Fintha, P. Darwin Bell,

Neuroscience and Neuropharmacology Research

At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium–chloride–potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process. At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium–chloride–potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process. With increased luminal flow in the loop of Henle, macula densa cell activation occurs in response to a concomitant elevation in luminal fluid sodium chloride concentration ([NaCl]L) and osmolality (osmL).1.Bell P.D. Navar L.G. Ploth D.W. et al.Tubuloglomerular feedback responses during perfusion with nonelectrolyte solutions in the rat.Kidney Int. 1980; 18: 460-471Abstract Full Text PDF PubMed Scopus (13) Google Scholar,2.Blantz R.C. Konnen K.S. Relation of distal tubular delivery and reabsorptive rate to nephron filtration.Am J Physiol. 1977; 233: F315-F324PubMed Google Scholar This results in paracrine signaling to the afferent arteriole to adjust preglomerular vascular tone (a phenomenon called tubuloglomerular feedback (TGF) response) and to contribute to the regulation of renin release from the granular cells. The alterations in [NaCl]L at the macula densa segment are thought to occur between ~15 and 60 mmol/l, with concomitant changes in osmL between ~100 and 150–200 mOsm/kg H2O.3.Bell P.D. Lapointe J.Y. Peti-Peterdi J. Macula densa cell signaling.Annu Rev Physiol. 2003; 65: 481-500Crossref PubMed Scopus (93) Google Scholar Early studies suggested that macula densa cells transport NaCl via a furosemide-sensitive Na+:2Cl−:K+ cotransporter, and that these cells exhibited at least some degree of apical water permeability.4.Laamarti M.A. Bell P.D. Lapointe J.Y. Transport and regulatory properties of the apical Na–K–2Cl cotransporter of macula densa cells.Am J Physiol. 1998; 275: 703-709PubMed Google Scholar, 5.Hoyer J.R. Sisson S.P. Vernier R.L. Tamm-Horsfall glycoprotein: ultrastructural immunoperoxidase localization in rat kidney.Lab Invest. 1979; 41: 168-173PubMed Google Scholar, 6.Sikri K.L. Foster C.L. Bloomfield F.J. et al.Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm-Horsfall glycoprotein in adult hamster kidney.Biochem J. 1979; 181: 525-532Crossref PubMed Scopus (65) Google Scholar, 7.Obermuller N. Kunchaparty S. Ellison D.H. et al.Expression of the Na–K–2Cl cotransporter by macula densa and thick ascending limb cells of rat and rabbit nephron.J Clin Invest. 1996; 98: 635-640Crossref PubMed Scopus (88) Google Scholar The finding that macula densa cells might be a water-permeable ‘window’ in the otherwise water-impermeant cortical thick ascending limb (cTAL) was the base of observations by Kirk et al.,8.Kirk K.L. Bell P.D. Barfuss D.W. et al.Direct visualization of the isolated and perfused macula densa.Am J Physiol. 1985; 248: 890-894PubMed Google Scholar using differential interference contrast microscopy. It was found that parallel increases in [NaCl]L and osmL from 26 to 146 mmol/l and from 70 to 290 mOsm/kg H2O, respectively, led to reversible decreases in macula densa cell height and narrowing of the lateral intercellular spaces. Also, transmission electron microscopic studies demonstrated closure of intercellular spaces between macula densa cells in cases where [NaCl]L and osmL at the macula densa were expected to rise.9.Kaissling B. Kriz W. Variability of intercellular spaces between macula densa cells: a transmission electron microscopic study in rabbits and rats.Kidney Int Suppl. 1982; 12: S9-S17PubMed Google Scholar According to both studies, changes in lateral intercellular spaces and cell height were specific to the macula densa cells and there were no changes in the surrounding cTAL. Studies by Gonzalez et al.10.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. I. Isosmotic and anisosmotic cell volume changes.Acta Physiol Scand. 1988; 133: 149-157Crossref PubMed Scopus (18) Google Scholar,11.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.Acta Physiol Scand. 1988; 133: 159-166Crossref PubMed Scopus (20) Google Scholar also suggested that parallel increases in [NaCl]L and osmL lead to decreases in macula densa cell height. Thus, these studies indicate that concordant elevations in [NaCl]L and osmL lead to cell shrinkage owing to a change in the osmotic gradient across the apical membrane. In contrast, others reported [NaCl]L elevation-induced increases in macula densa cell volume, using fluorescence microscopy.12.Liu R. Pittner J. Persson A.E. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration.J Am Soc Nephrol. 2002; 13: 2688-2696Crossref PubMed Scopus (59) Google Scholar,13.Liu R. Persson A.E. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration.J Physiol. 2005; 563: 895-901Crossref PubMed Scopus (28) Google Scholar Also, a study from our laboratory concluded that elevations in [NaCl]L result in macula densa cell swelling.14.Peti-Peterdi J. Morishima S. Bell P.D. et al.Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.Am J Physiol Renal Physiol. 2002; 283: F197-F201Crossref PubMed Scopus (72) Google Scholar However, these studies were performed by altering [NaCl]L while maintaining osmL constant or minimally altered.14.Peti-Peterdi J. Morishima S. Bell P.D. et al.Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.Am J Physiol Renal Physiol. 2002; 283: F197-F201Crossref PubMed Scopus (72) Google Scholar Thus, there appears to be a conundrum regarding the effect of increasing [NaCl]L on macula densa cell volume. The importance of a detailed knowledge of macula densa cell volume regulation is best demonstrated by speculations that cell swelling might play a role in adenosine 5′ triphosphate release from macula densa cells induced by changes in [NaCl]L,15.Komlosi P. Peti-Peterdi J. Fuson A.L. et al.Macula densa basolateral ATP release is regulated by luminal [NaCl] and dietary salt intake.Am J Physiol Renal Physiol. 2004; 286: F1054-F1058Crossref PubMed Scopus (67) Google Scholar,16.Bell P.D. Lapointe J.Y. Sabirov R. et al.Macula densa cell signaling involves ATP release through a maxi anion channel.Proc Natl Acad Sci USA. 2003; 100: 4322-4327Crossref PubMed Scopus (234) Google Scholar and that macula densa cell shrinkage would participate in the signaling of [NaCl]L-dependent prostaglandin E2 release from macula densa cells.17.Peti-Peterdi J. Komlosi P. Fuson A.L. et al.Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.J Clin Invest. 2003; 112: 76-82Crossref PubMed Scopus (121) Google Scholar,18.Harris R.C. Cyclooxygenase-2 and the kidney: functional and pathophysiological implications.J Hypertens Suppl. 2002; 20: S3-S9Crossref PubMed Scopus (70) Google Scholar The present studies were performed to re-evaluate changes in macula densa cell and cTAL epithelial cell volume upon alterations in [NaCl]L at constant or varying osmL and upon changes in luminal urea concentration. One approach to determine changes in cell volume is to use the fluorescent probe calcein, which can be loaded into cells as an ester form and is trapped inside the cells upon cleavage of the methyl ester. Unlike fura-2 or other ionic probes, this dye is not sensitive to changes in intracellular ionic composition. However, when cells swell or shrink, changes in calcein dye concentration can be used as an index of alterations in cell volume. For example, an increase in cell volume is expected to result in a dilution of the dye and a decline in fluorescence. Studies were performed to compare cell volume responses in macula densa versus adjacent cTAL cells (Figure 1). Increases in osmL caused increases in calcein fluorescence, whereas decreases in osmL led to decreases in intensity, indicating an inverse relationship between cell volume and calcein fluorescence intensity. As shown in Figure 2a, increases in [NaCl]L from 0 to 80 mmol/l at constant osmL of 210 mOsm/kg H2O produced reversible, dose-dependent decreases in calcein intensity in the macula densa cells, suggesting reversible cell swelling. Luminal application of 10−4 mol/l furosemide, inhibitor of Na+:2Cl−:K+ cotransporter, reduced the magnitude of changes in calcein intensity upon increases in [NaCl]L at constant osmL by 83±11% (n=6; P<0.05). In contrast, parallel increases n [NaCl]L and osmL from 0 to 80 mmol/l and from 60 to 210 mOsm/kg H2O (Figure 2b) caused increases in calcein fluorescence, indicating cell shrinkage. As shown in Figure 2c, concomitant increases in [NaCl]L and osmL produced dose-dependent decreases in macula densa cell volume. The comparison of volume responses to changes in luminal fluid composition between macula densa and cTAL produced strikingly different responses. As shown in Figure 3a, both cell types produced cell swelling in response to increases in [NaCl]L at constant osmL. In contrast, increases in both [NaCl]L and osmL produced cell shrinkage in macula densa cells and cell volume increase in cTAL cells. Interestingly, cell volume responses in macula densa cells were sustained, whereas the volume responses in cTAL were transient, suggesting active volume-regulatory mechanisms in cTAL cells (Figure 3b).Figure 2Effect of [NaCl]L and osmL on macula densa cell volume. Representative tracings of calcein fluorescence intensity recorded from macula densa plaques upon changes in [NaCl]L (shown in mmol/l above the tracings) (a) at constant osmL (in mOsm/kg H2O) and (b) with concomitant changes in osmL. (c) Dose–response relationship between [NaCl]L and calcein fluorescence intensity in macula densa cells upon concomitant alterations in osmL (n=4–9; all values are different from each other, P<0.05).View Large Image Figure ViewerDownload (PPT)Figure 3[NaCl]L-dependent changes in macula densa and cTAL cell volume. Representative tracings of calcein fluorescence intensity recorded from macula densa plaques and cTAL cells (cTAL) upon changes in [NaCl]L (shown in mmol/l above the tracings) (a) at constant osmL (in mOsm/kg H2O) and (b) with concomitant changes in osmL. Cell volume changes in macula densa cells were sustained, and cTAL cells exhibited cell volume regulatory responses.View Large Image Figure ViewerDownload (PPT) As determined by visualizing cell membranes with a membrane-staining dye 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) (Figure 4) and multiphoton excitation confocal microscopy, increases in [NaCl]L from 0 to 80 mmol/l at constant osmL of 210 mOsm/kg H2O produced reversible swelling of macula densa cells with an increase in cross-sectional area of 24±4%, whereas parallel increases in [NaCl]L and osmL from 0 to 80 mmol/l and from 60 to 210 mOsm/kg H2O, respectively, caused reversible shrinkage of macula densa cells (Figures 4c, d and 7 and Supplementary Material) with a decrease in cross-sectional area of 10±2% (Figure 5). These changes in macula densa cross-sectional area were caused by alterations in macula densa cell height.Figure 5[NaCl]L- and osmL-dependent changes in macula densa cross-sectional area. Effect of modulation of [NaCl]L (in mmol/l; see legend) and/or osmL (in mOsm/kg H2O; see legend in parentheses) on the macula densa cross-sectional area (n=6; *P<0.05 as compared to values obtained with mock changes).View Large Image Figure ViewerDownload (PPT) Under normal physiological conditions, changes in osmL occur predominantly either through alterations in [NaCl]L or [urea]L. Studies in Figure 6 compare the changes in macula densa cell volume with addition of either NaCl or urea. As shown in Figure 6b, increases in [urea]L produced cell shrinkage, although at comparable osmotic concentration it was less effective than NaCl. Increases in [urea]L from 0 to 160 mmol/l (60–217 mOsm/kg H2O) caused a reversible, dose-dependent increase in calcein fluorescence of 2.7±0.6%. The present studies demonstrated that changes in cell volume in macula densa cells critically depend on the chosen experimental conditions. In spite of this finding, three major conclusions can be drawn from this work. First, increases in [NaCl]L under physiological relevant conditions result in macula densa cell shrinkage. Second, consistent with previous observations8.Kirk K.L. Bell P.D. Barfuss D.W. et al.Direct visualization of the isolated and perfused macula densa.Am J Physiol. 1985; 248: 890-894PubMed Google Scholar,11.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.Acta Physiol Scand. 1988; 133: 159-166Crossref PubMed Scopus (20) Google Scholar and in contrast to the adjacent cTAL cells, the apical membrane of macula densa cells is permeable to water. Third, macula densa cells lack effective cell volume regulatory mechanisms. As reported previously by others and also by our group,12.Liu R. Pittner J. Persson A.E. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration.J Am Soc Nephrol. 2002; 13: 2688-2696Crossref PubMed Scopus (59) Google Scholar, 13.Liu R. Persson A.E. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration.J Physiol. 2005; 563: 895-901Crossref PubMed Scopus (28) Google Scholar, 14.Peti-Peterdi J. Morishima S. Bell P.D. et al.Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.Am J Physiol Renal Physiol. 2002; 283: F197-F201Crossref PubMed Scopus (72) Google Scholar increases in [NaCl]L at constant or close-to-constant osmL (~300 mOsm/kg H2O) produce macula densa cell swelling. This has been reconfirmed in our current studies using both the volume-sensitive dye calcein and also by visualizing cell membranes of macula densa cells with multiphoton fluorescence microscopy. Increases in [NaCl]L at constant osmL also produced reversible decreases in calcein intensity in cTAL cells, suggesting cell swelling. In contrast, increasing [NaCl]L and osmL concomitantly caused increases in calcein fluorescence, indicating shrinkage of macula densa cells. In the presence of concomitant changes in [NaCl]L and osmL, there was a linear relationship between [NaCl]L and cell volume between 20 and 80 mmol/l [NaCl]L (Figure 2). Also, as shown in Figures 4 and 7 and in the Movie S1, Movie S2, concomitant increases in [NaCl]L and osmL led to modest decreases in cell height and a shrinkage of macula densa cells. Interestingly, in both calcein and membrane staining experiments, concomitant increases in [NaCl]L and osmL produced reversible swelling of cTAL cells. Thus, these studies report a fundamental difference in the way macula densa and cTAL cells respond when both [NaCl]L and osmL are altered concurrently.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2OWQ3MzMzNDY0ZGFmOTJkMzNhNWQ2MDI0MmJmYjg3MSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM0NTQ5OTc4fQ.gfx9BJzuTvrFaBXuiXSoC7dFb8aqlrQdtk8ZLRz_WLaBvMUU9OnowBLoIdTmy--GKd92WavYGWm5veO_bbjLPbS0tw3ZLU-rC9zb4bvZO9SJrXBncrRp009VM58eKaaUFBteWSsMROAlPwVyhYW-qddBfS3-Y8a89M_VuY0jGCkFMRrD7pW-TqfVf6hHy_htZ7J6-gFW2rSzR7xi6MJKtvOQwzs9Ihj6rdFxP1hEXu9PdSE9m4MkqQwZleYKh1cGfiQlEn6sK0mAgpt-A_eQZMDYeiF3x-UxAvn7fkxOdyk4epUOiBVuJMfL6QKNL77aTQpPbEXsdVLSPZk3tEAUTw Download .mp4 (1.35 MB) Help with .mp4 files Movie S1Cell membrane imaging of the macula densa.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhYjQzNmJiNzhkYzJlOWMxNWZjNTEyMjQ4N2Q3MmZkZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM0NTQ5OTc4fQ.C17l20nJWuP3T9Evxxi1TVsZGsbF4JRS2y6IPDiqF0grZ9Lv1EyGF9-ueAyf63VByhS2GdT6OmI3za1atokvTFij7Dcpb6eWhxl-akyNG6O8AO-YI1_71vwCcNu6HLV44v2IsdB1eJWvzynMGqtQhKCMrXokhKb5vz8tikwzjYUtRwQMX5bqGycGxWE2NjsLRUOzruMP7xkAB7DbIKKGUvtluyr59hRqMt1N-6OUwJFrfMUttTIxKZu99SKRUZhRqLL1P6rBlBBj5C0-6zaWTGzPqoTihPjRovzptEK5m4l44hVfiW4C-Ij0AL-OK4WkCsEYMll4iughlgNY902e8w Download .mp4 (0.62 MB) Help with .mp4 files Movie S2Four-dimensional imaging of macula densa cell volume changes. We interpret these data as follows: it is firmly established that both macula densa and cTAL cells possess the apical Na+:2Cl−:K+ cotransporter.19.Lapointe J.Y. Bell P.D. Cardinal J. Direct evidence for apical Na+:2Cl−:K+ cotransport in macula densa cells.Am J Physiol. 1990; 258: F1466-F1469PubMed Google Scholar Thus, increases in [NaCl]L will result in uptake of NaCl into both cell types. Increasing intracellular [NaCl] would then result in a relative increase in the osmotic gradient for water entry into the cell. Presumably, both cTAL and macula densa cells possess a finite basolateral water permeability so that water can enter across the basolateral membranes. Studies by Gonzalez et al.11.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.Acta Physiol Scand. 1988; 133: 159-166Crossref PubMed Scopus (20) Google Scholar have shown that the osmotic water permeability of the macula densa basolateral membrane is about 13 times higher than that of the apical membrane. Thus, a steady-state increase in NaCl entry would lead to water uptake and cell swelling, which would be blocked by furosemide, an inhibitor of the Na+:2Cl−:K+ cotransporter. Furosemide blocked the cell volume changes by 83%, a value that is in accordance with earlier studies indicating that ~80% of apical Na+ entry into the macula densa cells occurs via the Na+:2Cl−:K+ cotransporter, whereas the Na+:H+ exchanger is responsible for the remainder.20.Peti-Peterdi J. Bebok Z. Lapointe J.Y. et al.Novel regulation of cell [Na(+)] in macula densa cells: apical Na(+) recycling by H-K-ATPase.Am J Physiol Renal Physiol. 2002; 282: F324-F329Crossref PubMed Scopus (45) Google Scholar However, the difference between macula densa and cTAL cells appears to be that whereas the apical membrane of cTAL cells is impermeable to water, macula densa cells exhibit finite water permeability. Thus, when both [NaCl]L and osmL are increased, cTAL cells still swell, because there is no ‘effective change’ in the lumen to cell osmotic gradient in spite of changes in osmL, whereas in macula densa cells, the finite apical water permeability allows for an osmotic gradient to form across the apical membrane and, in the presence of elevated apical osmolality, the relative movement of water from cell to lumen. Although the present studies did not measure water permeability of macula densa cells, per se, the conclusion that can be reached is that the apical membrane of these cells is permeable enough to counteract the effects of enhanced NaCl entry. This is in accordance with earlier studies indicating that the water permeability of macula densa cells is ~12 times higher than that of the thick ascending limb.11.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.Acta Physiol Scand. 1988; 133: 159-166Crossref PubMed Scopus (20) Google Scholar,21.Burg M.B. Green N. Function of the thick ascending limb of Henle's loop.Am J Physiol. 1973; 224: 659-668Crossref PubMed Scopus (258) Google Scholar Thus, the overall effect of increases in [NaCl]L (in the absence of mannitol or other osmotic agents) is to elicit cell shrinkage in macula densa cells. Interestingly, the fractional change in calcein fluorescence intensity (5.5%; Figure 2) was smaller than the observed change in macula densa cross-sectional area (10%; Figure 5). Although there might exist several explanations for this, one possible reason is that changes in macula densa cell volume do not necessarily parallel changes in macula densa cell height. An example of this is an earlier study8.Kirk K.L. Bell P.D. Barfuss D.W. et al.Direct visualization of the isolated and perfused macula densa.Am J Physiol. 1985; 248: 890-894PubMed Google Scholar demonstrating concurrent heightening of macula densa cells with widening of the intercellular spaces. These results suggest that there is a finite permeability for water across the apical membrane of macula densa cells. Physiologically, as flow is increased through the cTAL, the augmented flow would produce elevations in osmL primarily owing to increases in [NaCl]L and [urea]L. Effective changes in the osmotic gradient across cells are related to the osmotic reflection coefficient of a particular solute. Thus, in spite of the fact that NaCl is transported into macula densa cells, its osmotic reflection coefficient across the apical membrane of macula densa cells is presumably high as it is in most cell types.22.Reuss L. General principles of water transport.in: Seldin D.W. Giebisch G. 3rd edn. The Kidney Physiology & Pathophysiology. 1. Lippincott Williams & Wilkins, Philadephia2000: 321-340Google Scholar This means that the osmotic driving force generated by Na+ and Cl− is almost equivalent to their concentration. However, despite some exceptions,21.Burg M.B. Green N. Function of the thick ascending limb of Henle's loop.Am J Physiol. 1973; 224: 659-668Crossref PubMed Scopus (258) Google Scholar most cells have a finite permeability to urea and therefore a lower reflection coefficient. This is entirely consistent with the results obtained in the current studies, where an equal osmotic concentration of urea was less effective in causing cell shrinkage than NaCl. One of the more interesting findings of the present studies is the lack of volume regulation in macula densa cells. Persson and his co-workers observed some signs of volume regulatory changes11.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.Acta Physiol Scand. 1988; 133: 159-166Crossref PubMed Scopus (20) Google Scholar, 12.Liu R. Pittner J. Persson A.E. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration.J Am Soc Nephrol. 2002; 13: 2688-2696Crossref PubMed Scopus (59) Google Scholar, 13.Liu R. Persson A.E. Simultaneous changes of cell volume and cytosolic calcium concentration in macula densa cells caused by alterations of luminal NaCl concentration.J Physiol. 2005; 563: 895-901Crossref PubMed Scopus (28) Google Scholar in macula densa cells upon alterations in osmolality, but also reported that increases in [NaCl]L lead to sustained changes in cell volume.11.Gonzalez E. Salomonsson M. Muller-Suur C. et al.Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.Acta Physiol Scand. 1988; 133: 159-166Crossref PubMed Scopus (20) Google Scholar Thus, there is conflicting information concerning the ability of macula densa cells to regulate cell volume. In response to cell swelling or shrinkage, most cells exhibit volume regulatory response. This is the case for cTAL cells (Figure 3b), where elevated [NaCl]L results in cell swelling (decrease in calcein fluorescence) followed by a time-dependent return toward control levels. In most cells, this regulatory volume decrease (RVD) is owing to the transport of osmotically active solutes from cell to extracellular fluid: chloride and potassium have both been shown to be involved in RVD in other cell types.23.Foskett J.K. Cell-volume control.in: Seldin D.W. Giebisch G. 3rd edn. The Kidney Physiology & Pathophysiology. 1. Lippincott Williams & Wilkins, Philadephia2000: 379-389Google Scholar However, macula densa cells (Figure 3b) show little tendency for volume regulation upon either cell shrinkage or cell swelling. One explanation for this is that previous work20.Peti-Peterdi J. Bebok Z. Lapointe J.Y. et al.Novel regulation of cell [Na(+)] in macula densa cells: apical Na(+) recycling by H-K-ATPase.Am J Physiol Renal Physiol. 2002; 282: F324-F329Crossref PubMed Scopus (45) Google Scholar indicated that intracellular [Na+] mirrored changes in [NaCl]L between 0 and 60 mmol/l. Thus, an elevation of [NaCl]L results in a sustained increase in intracellular [Na+]. If this also applies to other intracellular electrolytes such as chloride and potassium, then it would explain why there is a lack of volume regulation in macula densa cells, that is, no volume regulatory influx or efflux of osmotically active solutes. The finding that macula densa cells do not regulate volume is consistent with the role of this unique cell type as the sensor element for the TGF mechanism. In response to changes in the luminal environment, there are also sustained responses of basolateral membrane potential, intracellular [Na+], [Ca2+], and pH. Thus, a sustained response to alterations in the luminal environment appears to be a consistent characteristic of macula densa and may be an integral part of TGF signaling. However, a recent study failed to find a difference between the magnitude of in vitro afferent arteriole diameter responses to elevations in [NaCl]L whether the osmL was maintained constant or was increased concomitantly,24.Liu R. Carretero O.A. Ren Y. et al.Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback.Kidney Int. 2005; 67: 1837-1843Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar suggesting that changes in macula densa cell volume might not be directly involved in the TGF signaling process. As this study was performed using very low (~11 mmol/l) and high (~81 mmol/l) [NaCl]L, this finding does not eliminate the possibility that changes in cell volume can influence TGF responses over the physiological range of [NaCl]L and osmL. On the other hand, it is now known that macula densa cells are involved in other paracrine or signaling processes. For instance, macula densa cells produce both nitric oxide24.Liu R. Carretero O.A. Ren Y. et al.Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback.Kidney Int. 2005; 67: 1837-1843Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,25.Kovacs G. Komlosi P. Fuson A. et al.Neuronal nitric oxide synthase: its role and regulation in macula densa cells.J Am Soc Nephrol. 2003; 14: 2475-2483Crossref PubMed Scopus (43) Google Scholar and prostaglandin E2.17.Peti-Peterdi J. Komlosi P. Fuson A.L. et al.Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.J Clin Invest. 2003; 112: 76-82Crossref PubMed Scopus (121) Google Scholar Thus, it is possible that changes in cell volume may affect the production of these signaling molecules or may alter other, as yet to be identified, function(s) of macula densa. It is also possible that cell volume may be involved in TGF resetting.26.Schnermann J. Briggs J.P. Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion.in: Seldin D.W. Giebisch G. 3rd edn. The Kidney Physiology & Pathophysiology. 1. Lippincott Williams & Wilkins, Philadephia2000: 945-980Google Scholar We speculate that macula densa cell volume changes might play a role in TGF resetting under conditions where luminal electrolyte or non-electrolyte delivery from the medulla is altered, such as during salt restriction or water deprivation. Finally, it is well known that changes in cell volume can produce a wide range of alterations in cell function, including changes in protein synthesis and matrix synthesis.27.Lang F. Szabo I. Lepple-Wienhues A. et al.Cell volume in the regulation of metabolism, cell proliferation and apoptotic cell death.in: Okada Y. Cell Volume Regulation: The Molecular Mechanism and Volume Sensing Machinery. Elsevier Science BV, Amsterdam, The Netherlands1998: 49-58Crossref Scopus (14) Google Scholar, 28.Waldegger S. Lang F. Cell volume-regulated gene transcription.Kidney Blood Press Res. 1998; 21: 241-244Crossref PubMed Scopus (4) Google Scholar, 29.Tilly B.C. van der Wijk T. de Jonge H.R. Activation of cellular signalling pathways by hypotonicity.in: Okada Y. Cell Volume Regulation: The Molecular Mechanism and Volume Sensing Machinery. Elsevier Science BV, Amsterdam, The Netherlands1998: 59-66Google Scholar Therefore, it is possible that macula densa cell volume may influence not only the functional properties of macula densa cells, but also other elements within the juxtaglomerular apparatus. All materials were purchased from Sigma (St Louis, MO, USA) unless otherwise stated. Calcein/AM, TMA-DPH, and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) were obtained from Molecular Probes Inc. (Eugene, OR, USA). The protocol used was approved by the Institutional Animal Care & Use Committee, University of Alabama at Birmingham, Birmingham, AL, USA. Individual cTAL segments containing the macula densa plaque with attached glomeruli were dissected from rabbit kidneys (New Zealand White rabbits; 0.5–1.0 kg; Myrtle's Rabbitry, Thompson Station, TN, USA; n=32 animals in total) and perfused in vitro using methods similar to those described previously.30.Komlosi P. Frische S. Fuson A.L. et al.Characterization of basolateral chloride/bicarbonate exchange in macula densa cells.Am J Physiol Renal Physiol. 2005; 288: F380-F386Crossref PubMed Scopus (11) Google Scholar Dissection was performed at 4°C in an isosmotic, low-NaCl-containing Ringer dissection solution (Table 1). After transfer to a chamber that was mounted on the microscope, the tubule was cannulated and perfused with a perfusion solution. Tubules were bathed in a Ringer-like solution. All experiments were conducted at 37°C.Table 1Composition of experimental solutionsDissectionPerfusion 0 NaClaThe ‘0 NaCl’ solution contains ~4mmol/l of sodium but no chloride. low osmPerfusion 0 NaClaThe ‘0 NaCl’ solution contains ~4mmol/l of sodium but no chloride. high osmPerfusion 80 NaClPerfusion 160 ureaBathNaCl25——80—150KCl5————5Na2HPO41.61.61.61.61.61.6NaH2PO40.40.40.40.40.40.4CaCl21.5————1.5MgSO4111111Glucose555555K gluconate—5555—Ca gluconate—3333—NMDG cyclamate125—————Mannitol——145———Urea————160—HEPES102525252510Osmolality (mOsm/kg H2O)32560210210217305Concentrations are given in mmol/l. The solutions used in the dose–response measurements, shown in Figures 2, 3, 6, and 7, were obtained by mixing ‘perfusion 0 NaCl low osm’ or ‘perfusion 0 NaCl high osm’ solutions with ‘perfusion 80 NaCl’ or ‘perfusion 160 urea’ solutions. The pH of the dissection and bathing solutions was set to 7.4 with NaOH, whereas that of the perfusion solutions was adjusted to 7.2 with NMDG at 37°C. Osmolality of solutions was set with mannitol using a freezing-point depression osmometer.a The ‘0 NaCl’ solution contains ~4 mmol/l of sodium but no chloride. Open table in a new tab Concentrations are given in mmol/l. The solutions used in the dose–response measurements, shown in Figures 2, 3, 6, and 7, were obtained by mixing ‘perfusion 0 NaCl low osm’ or ‘perfusion 0 NaCl high osm’ solutions with ‘perfusion 80 NaCl’ or ‘perfusion 160 urea’ solutions. The pH of the dissection and bathing solutions was set to 7.4 with NaOH, whereas that of the perfusion solutions was adjusted to 7.2 with NMDG at 37°C. Osmolality of solutions was set with mannitol using a freezing-point depression osmometer. After having perfused with a control ‘perfusion 0 NaCl’ solution (Table 1) for 5 min, the perfusate was changed to ‘perfusion 80 NaCl’ solution for 5 min and then back to control. This procedure was performed at the beginning and at the end of each experiment, and the results of any test procedures performed between these periods were normalized to the average of these controls. To eliminate motion artifacts, a modified perfusion system was designed to provide for constant perfusion pressure during solution changes. Syringes supplying the perfusion solutions and the waste bottle were all pressurized. The perfusion flow is estimated to be ~30 nl/min.8.Kirk K.L. Bell P.D. Barfuss D.W. et al.Direct visualization of the isolated and perfused macula densa.Am J Physiol. 1985; 248: 890-894PubMed Google Scholar Exchange of solutions was achieved by a constant gravity-driven flow within this pressurized system. As the solution flow was ~15 μl/s and the volume in the pipette head is ~3 μl, the time constant of perfusion exchange was well below the temporal resolution of the image acquisition. Macula densa cells were loaded with volume-sensing dye calcein by adding calcein/AM (10−5 mol/l), dissolved in dimethyl sulfoxide containing 15% w/v pluronic acid, to the luminal perfusate. Loading required ~5 min. Calcein fluorescence intensity was measured inside the macula densa cells with fluorescence microscopy (PTI, Lawrenceville, NJ, USA) using a Nikon S Fluor × 40 objective, a Nikon TE2000 microscope, and a cooled SenSys charge-coupled camera (Photometrics, Tucson, AZ, USA). Fluorescence was measured at an emission wavelength of 530 nm in response to an excitation wavelength of 495 nm. The spontaneous decline in fluorescence intensity was corrected for by linear curve-fitting and normalization. In order to image plasma membranes to assess cell volume regulation, macula densa cells were stained for ~1 min with membrane-staining dye TMA-DPH (10−6 mol/l) in both the luminal perfusate and in the bath. TMA-DPH was excited at 800 nm using a diode-pumped, frequency-doubled Nd:vanadate pump laser (Verdi, 5 W) and a mode-locked titanium-sapphire femtosecond pulsed laser (Mira 900, both from Coherent, Santa Clara, CA, USA), coupled to a Leica DM IRBE microscope and Leica TCS SP confocal imaging system (Leica Microsystems, Heidelberg, Germany). The bandwidth at half-maximum intensity was ~7.4 nm. Fluorescence emission was detected using a Leica × 100 objective at 430 nm. In other experiments, the preparation was loaded with DiBAC4(3), a dye that labels the cytosol, and imaged in in xyzt dimensions with confocal microscopy at excitation and emission wavelengths of 488 and 530 nm, respectively. Volume rendering was performed with Voxx (Indiana Center for Biological Microscopy, Indianapolis, IN, USA) or Imaris (Bitplane, Zurich, Switzerland) software. Segmentation was performed based on intensity, supplemented with manual definition of the basolateral membrane. Tracking was performed using the connected components algorithm. Pseudolinescan images and annotated online movie were generated with ImageJ (National Institutes of Health, Bethesda, MD, USA) and Flash (Macromedia Inc., San Francisco, CA, USA) software, respectively. Data are expressed as means±s.e. Statistical analysis was performed with analysis of variance and Dunnett's or Bonferroni's test. This work was supported by Grant 32032 from the National Institute of Diabetes and Digestive and Kidney Diseases, Department of Health and Human Services, Bethesda, MD to Phillip Darwin Bell and Scientist Development Grant 0630096N from the American Heart Association, Dallas, TX to Peter Komlosi.