Effect of ammonium on the expression of osmosensitive genes in Madin–Darby canine kidney cells
1 Physiologisches Institut der Universitt, 80336 München, Germany
Abstract
The cells of the kidney medulla are exposed routinely to high extracellular concentrations of various solutes including NaCl, urea and ammonium (NH4+). Although it is well established that the expression of a variety of osmosensitive genes and proteins, which confer cytoprotection on renal medullary cells, is induced by high NaCl concentrations, the role of NH4+ in these cellular responses is unclear. This study thus addressed the effect of NH4+ on the expression of the betaine/GABA transporter (BGT-1), the sodium/myo-inositol cotransporter (SMIT), aldose reductase (AR), and heat shock protein 70 (HSP70) in Madin–Darby canine kidney (MDCK) cells, using Northern and Western blot analyses and enzyme-linked immunosorbent assay (ELISA). The incidence of apoptosis was monitored by determining caspase-3 activity and annexin V binding. Addition of NH4Cl (50 mM; total osmolality 400 mosmol (kg H2O)–1 to the medium was more effective than equiosmolar NaCl in increasing BGT-1 and HSP70 mRNA abundance, but less effective in enhancing BGT-1 and HSP70 expression at the protein level. Qualitatively similar results were obtained for SMIT and AR mRNAs. Exposure to both isotonic and hypertonic, NH4Cl-containing medium enhanced apoptosis compared with equiosmolar, NaCl-containing media. These results suggest that, in addition to NaCl, NH4Cl may play a role in regulating the intracellular accumulation of organic osmolytes, the abundance of HSP70 and cell turnover in the renal medulla in vivo.
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Introduction
Renal medullary cells are exposed routinely to high extracellular concentrations of various solutes including NaCl, urea and ammonium (NH4+) (Manitius et al. 1960; Jamison & Kriz, 1982; Stern et al. 1985; Packer et al. 1991). Intracellular accumulation of metabolically neutral, non-perturbing organic osmolytes, in particular the trimethylamines betaine and glycerophosphorylcholine (GPC) and the polyols myo-inositol and sorbitol, allows osmotic adaptation to the high extracellular NaCl concentrations (Law & Burg, 1991; Beck et al. 1998). While high intracellular concentrations of betaine and myo-inositol are achieved by increased uptake via the sodium and chloride dependent betaine/GABA transporter (BGT-1) and the sodium/myo-inositol cotransporter (SMIT) (Kwon & Handler, 1995), elevated cell sorbitol contents are accomplished by enhanced, aldose reductase- (AR) mediated production from glucose (Bagnasco et al. 1987; Sands & Schrader, 1990). The enhanced expression of BGT-1, SMIT and AR underlying hypertonicity-induced osmolyte-accumulation is due to enhanced transcription of the respective genes (Woo et al. 2002a). Binding of the tonicity-responsive enhancer (TonE) binding protein/nuclear factor of activated T cells (TonEBP/NFAT5) to multiple TonEs in the 5'-flanking region of these genes is a critical step in this process and one that stimulates transcription of TonEBP/NFAT5 target genes. Both redistribution from the cytoplasm to the nucleus and enhanced expression of TonEBP/NFAT5 participate in the upregulation of tonicity-sensitive genes (Woo et al. 2000). In addition to BGT-1, SMIT and AR, one of the genes encoding the inducible 70 kDa heat shock protein (HSP70) is a target for TonEBP/NFAT5 (Woo et al. 2002b). Following exposure to high extracellular concentrations of NaCl, but not of urea, HSP70 expression is induced vigorously (Cohen et al. 1991; Neuhofer et al. 1999). This molecular chaperone plays a decisive role in counteracting the deleterious effects of the excessive urea concentrations prevailing in the inner medulla of many mammals in antidiuresis (Müller et al. 1998; Beck et al. 2000; Neuhofer et al. 2001). Failure to adapt to these extreme conditions leads to apoptotic cell death (Kültz & Chakravarty, 2001; Neuhofer et al. 2001).
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In contrast to the considerable knowledge gathered over the last decade on the effect of elevated NaCl and urea concentrations on the function of renal epithelial cells, there is little information on the effect of high NH4+ concentrations. In view of tissue NH4+ concentrations exceeding 50 mmol l–1 in the papilla of antidiuretic mammals (Manitius et al. 1960; Valtin, 1966), the present study was undertaken to examine the effect of elevated NH4Cl concentrations on BGT-1, SMIT, AR, HSP70 and TonEBP/NFAT5 expression in MDCK cells using Northern and Western blot analyses and in situ enzyme-linked immunosorbent assay (ELISA). A further goal of this study was to assess parameters of apoptotic cell death after exposure of these cells to high NH4Cl concentrations.
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Methods
Culture and treatment of MDCK cells
Madin–Darby canine kidney (MDCK) cells obtained from the American Type Culture Collection (CCL-34; ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; low glucose) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and penicillin–streptomycin (100 u ml–1 and 100 μg ml–1; Invitrogen, Karlsruhe, Germany) at 37°C in a humidified atmosphere containing 5% CO2–95% air. Cells were seeded in 100 mm plastic dishes (4 x 105 cells dish–1; Greiner, Frickenhausen, Germany) or in 24-well plates (2 x 105 cells well–1; Costar, Cambridge, MA, USA), and cultured to confluence.
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For experiments, cells were incubated for the indicated periods in isotonic medium (osmolality 300 mosmol (kg H2O)–1), or in medium made hypertonic by the addition of 50 mM NaCl (hyperNaCl; total osmolality 400 mosmol (kg H2O)–1), or in medium made hypertonic by the addition of 5–50 mM NH4Cl as indicated (hyperNH4Cl; total osmolality 310–400 mosmol (kg H2O)–1), or in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (isoNH4Cl; total osmolality 300 mosmol (kg H2O)–1) for 72 h. The respective media were replaced daily.
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In situ cellular ELISA for quantification of BGT-1
The in situ ELISA allows a quantitative analysis of reaction products and reflects the amount of membrane-bound BGT-1. The main details of the methodology are based on standard immunoperoxidase staining. The cells in 24-well plates were washed three times with Dulbecco's PBS (DPBS) and then fixed at 4°C in 4% paraformaldehyde in DPBS for 20 min. After washing, the cells were permeabilized with Triton X-100 (Sigma, Taufkirchen, Germany), 0.1% in DPBS for 5 min. The endogenous peroxidase activity was blocked by a 30-min incubation with 0.2% H2O2 in methanol at room temperature. The non-specific protein binding sites were blocked by a 60-min incubation at room temperature with 10% FBS in DPBS. This step was followed by incubation with rabbit anti-BGT-1 antibody (Biotrend, Cologne, Germany) in 10% FBS in DPBS (1 : 1000) for 60 min at room temperature. Specificity of the signal was demonstrated by simultaneous incubation with the 15-amino acid peptide used for raising the antibody (Biotrend). This procedure diminished the signal significantly and concentration dependently, demonstrating that the antibody specifically detects BGT-1 in MDCK cells. The cells were washed three times with 0.1% Tween-20 in DPBS and incubated overnight at 4°C with peroxidase-conjugated goat antirabbit IgG (Dianova, Hamburg, Germany) in 10% FBS in DPBS. Finally, the peroxidase reaction was allowed to proceed for 20 min at room temperature with 3,3',5,5'-tetramethyl-benzidine (TMB) Liquid Substrate System (Sigma). Adding 0.5 M H2SO4 stopped the reaction, and the absorbances were measured at 450 nm (ref. 620 nm) in a BioSpec-1601 E spectrophotometer (Shimadzu, Duisburg, Germany). The absorbance at 450 nm is proportional to the amount of BGT-1 expressed in the MDCK cells.
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Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis
Following the treatments, cells were washed three times with chilled PBS and scraped into 8 M urea/PBS containing 0.1% Triton X-100, 1 μg ml–1 leupeptin, 10 ng ml–1 aprotinin, 100 μM phenylmethylsulphonyl fluoride (PMSF), 100 μM dithiothreitol (DTT), 200 μM sodium orthovanadate (Na3VO4) and 1 mM sodium fluoride (NaF), 100 μl (100-mm tissue culture dish)–1. Cells were lysed with three cycles of snap-freezing and thawing. The extracts were stored for 15 min at room temperature, vortexed vigorously, and centrifuged at 12 000 g for 15 min at 4°C. Protein concentrations in the supernatant were determined in duplicate, using a commercially available protein assay (Bio-Rad, Munich, Germany). Aliquots (40 μg) of total protein were subjected to SDS-PAGE and Western blot analysis using a mouse anti-Hsp70 (Stressgen, Canada) antibody or rabbit TonEBP/NFAT5 antiserum as described in detail elsewhere (Neuhofer et al. 2002a).
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Northern blot analysis
After the respective treatments, the cells were washed with ice-cold PBS and lysed by addition of 1 ml of TRI-reagent (PeqLab Biotech, Erlangen, Germany). Total RNA was recovered according to the recommendations of the manufacturer. Aliquots (20 μg) were electrophoresed through 1% agarose/formaldehyde gels, blotted onto nitrocellulose membrane and immobilized by ultraviolet-crosslinking. For Northern blot analysis, the blots were prehybridized for 2 h at 55°C in a solution containing 50% formamide, 5x saline–sodium citrate buffer (SSC), 0.1% SDS, and 10% blocking reagent (Roche, Mannheim, Germany) and were hybridized overnight in the same solution containing 20 ng ml–1 digoxigenin-labelled human AR (GenBank accession no. J05474), canine SMIT (M85068), canine BGT-1 (M80403) or human HSP70 (M11717) cDNA (Neuhofer et al. 2002a). After hybridization, the membranes were washed twice for 15 min each with 2x SSC/0.1% SDS at room temperature and twice for 15 min with 0.1% SSC/0.1% SDS at 68°C. Non-radioactive detection procedures were carried out using antidigoxigenin-AP (Roche, Mannheim, Germany), followed by incubation with CDP-star chemiluminescent substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2'-(5' chloro) tricyclo [3.3.1.13 7] decan}-4-yl)-1-phenyl phosphate for alkaline phosphatase (Roche) as previously described (Burger-Kentischer et al. 1999). To correct for differences in RNA loading, the membranes were stripped and rehybridized with digoxigenin-labelled cDNA specific for glyceraldehyde phosphate dehydrogenase (GAPDH; X011677). Signals were quantified by laser densitometry (Ultrascan XL; Pharmacia, Freiburg, Germany).
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Determination of caspase-3 activity
Relative caspase-3 activity was assessed using a commercially available colourimetric caspase-3 assay based on the hydrolysis of the synthetic caspase-3 substrate paranitroanilin-conjugated Asp-Glu-Val-Asp (pNA-DEVD; CaspACE; Promega, Madison, WI, USA). After the specific treatments, caspase-3 activity was determined as described by the manufacturer on 50 μg protein in 96-well plates. Specificity of the assay was demonstrated by parallel incubation of each extract in the presence of the caspase-3 inhibitor Z-VAD-FMK. Relative caspase-3 activity was determined as the increase in A405. (Spectrafluor, Tecan Group, Maennedorf, Switzerland).
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Flow cytometry
Cell viability was monitored further by labelling of cell surface-exposed phosphatidylserine by annexin V-FITC in combination with propidium iodide (PI) according to the manufacturer's recommendations (ApoAlert Annexin V Apoptosis Kit, Clontech, Heidelberg, Germany). Briefly, MDCK cells were trypsinized, washed once with serum-containing media, once with binding buffer and resuspended at a concentration of 106 cells (ml)–1 in binding buffer (Clontech). Aliquots of 100 μl were incubated for 15 min in the dark with annexin V-FITC (100 ng in TRIS-NaCl; Clontech) and/or PI (500 ng in binding buffer) and subsequently analysed by flow cytometry (FacsScan; Becton-Dickinson, Heidelberg, Germany).
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Statistical analysis
The data are presented as means ± S.E.M. The significance of differences between means was established by Students' t test for independent samples and by multivariate ANOVA for multiple samples. P < 0.05 was regarded as significant.
Results
Effect of NH4Cl on AR, BGT-1 and SMIT mRNA abundance in MDCK cells
Figure 1 demonstrates that following addition of either 50 mM NH4Cl (total medium osmolality 400 mosmol(kg H2O)–1 hyperNH4Cl) or 50 mM NaCl (medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl), the abundance of AR, BGT-1 and SMIT mRNAs increased significantly compared with isotonic controls (medium osmolality 300 mosmol (kg H2O)–1; isocontrol). Interestingly, the addition of NH4Cl increased the mRNA levels of the respective genes to a significantly greater extent than equiosmolar NaCl. Compared with isotonic controls (isocontrol), isotonic replacement of 50 mM NaCl by equiosmolar NH4Cl (final medium osmolality 300 mosmol (kg H2O)–1; isoNH4Cl) caused a significant increase in the abundance of AR and SMIT mRNAs but did not affect the abundance of BGT-1 mRNA.
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MDCK cells were kept for 72 h in isotonic medium (300 mosmol (kg H2O)–1; isocontrol), in medium made hypertonic by the addition of either 50 mM NaCl or 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNaCl, hyperNH4Cl) or 200 mM NaCl (700 mosmol (kg H2O)–1; +200 mM NaCl), or in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (isoNH4Cl). Subsequently, the abundance of the respective mRNAs was assessed by Northern blot analysis, normalized to GAPDH abundance and expressed as increase compared with isocontrol. Means ± S.E.M. for n = 3–4 independent experiments; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNaCl. Representative blots are shown below.
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As a positive control, mRNA from cells exposed to an additional 200 mM NaCl (final medium osmolality 700 mosmol (kg H2O)–1; +200 mM NaCl) was also loaded, since this manoeuvre is known to increase the expression of the respective genes strongly.
Effect of NH4Cl on BGT-1 expression in MDCK cells
Figure 2 shows that the addition of 50 mM NH4Cl (hyperNH4Cl) resulted in a time- and concentration-dependent increase in BGT-1 expression (Fig. 2A and B). Although the addition of NH4Cl at concentrations of 25 and 50 mM increased the expression of BGT-1 significantly compared with isotonic controls (Fig. 2B), NH4Cl was significantly less effective than equiosmolar NaCl (Fig. 2A and B).
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A, MDCK cells were kept in isotonic medium (), or exposed to medium made hypertonic by addition of either 50 mM NaCl () or 50 mM NH4Cl () for 48, 72 or 96 h. Thereafter, membrane-associated BGT-1 was determined as decribed in Methods. Means ± S.E.M. for three separate experiments with 3–6 replicates per experiment; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNH4Cl. B, MDCK cells were exposed to hypertonic, NH4Cl-containing media (addition of 5–50 mM NH4Cl, hyperNH4Cl) or to hypertonic NaCl-containing medium (addition of 50 mM NaCl, hyperNaCl) for 48 h. Thereafter, membrane-associated BGT-1 was determined as decribed in Methods. Means ± S.E.M. for three separate experiments with 3–6 replicates per experiment; *P < 0.05 versus isocontrol; #P < 0.05 versus +50 mM NH4Cl.
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Effect of NH4Cl on HSP70 expression in MDCK cells
Compared with isotonic controls (isocontrol), HSP70 mRNA abundance in MDCK cells exposed to medium made hypertonic by the addition of either 50 mM NH4Cl or 50 mM NaCl (final medium osmolality 400 mosmol (kg H2O)–1; hyperNH4Cl, hyperNaCl) was significantly increased (Fig. 3A). Incubation in isotonic, NH4Cl-containing medium (300 mosmol(kg H2O)–1; isoNH4Cl) also resulted in a significant upregulation of HSP70 mRNA (Fig. 3A) to levels comparable to that seen after addition of 50 mM NaCl (medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl). Although HSP70 mRNA abundance was elevated under the latter condition, HSP70 (protein) expression was not significantly different from isotonic controls (Fig. 3B). In contrast, after treatment with hypertonic, NH4Cl-containing medium (+50 mM NH4Cl; final medium osmolality 400 mosmol (kg H2O)–1; hyperNH4Cl), HSP70 expression was significantly higher than that in isotonic controls (isocontrol) but less than after equiosmolar addition of NaCl (hyperNaCl) (Fig. 3B). Expression of the constitutively expressed HSP27 was not affected after exposure of MDCK cells to either isotonic or hypertonic NH4Cl-containing media (Fig. 3B).
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MDCK cells were kept in isotonic medium (isocontrol; 300 mosmol (kg H2O)–1), or incubated in either medium made hypertonic by the addition of 50 mM NaCl (400 mosmol (kg H2O)–1; hyperNaCl), in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (300 mosmol (kg H2O)–1; isoNH4Cl), or in medium made hypertonic by the addition of 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNH4Cl) for 72 h. Subsequently, HSP70 mRNA and protein abundance were assessed by Northern (A) and Western (B) blot analysis, respectively. Means ± S.E.M. for n = 3; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNaCl.
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Effect of NH4Cl on TonEBP/NFAT5 expression in MDCK cells
As demonstrated in Fig. 4, exposure of MDCK cells to isotonic medium in which 50 mM NaCl had been replaced by 50 mM NH4Cl (isoNH4Cl) had no significant effect on TonEBP/NFAT5 abundance compared with MDCK cells kept in isotonic medium (isocontrol). However, Incubation in hypertonic, NH4Cl medium (400 mosmol (kg H2O)–1 by the addition of 50 mM NH4Cl; hyperNH4Cl) enhanced TonEBP/NFAT5 abundance strongly and significantly more than exposure to equiosmolar medium made hypertonic by the addition of 50 mM NaCl (hyperNaCl).
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MDCK cells were kept in isotonic medium (isocontrol; 300 mosmol(kg H2O)–1), or either incubated in medium made hypertonic by the addition of 50 mM NaCl (400 mosmol (kg H2O)–1; hyperNaCl), in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (300 mosmol (kg H2O)–1; isoNH4Cl), or in medium made hypertonic by the addition of 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNH4Cl) for 72 h. Subsequently, TonEBP/NFAT5 abundance was determined by Western blot ananlysis. Means ± S.E.M. for n = 3; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNaCl.
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NH4Cl-induced apoptosis in MDCK cells
As shown in Fig. 5A, MDCK cells exposed to 50 mM NH4Cl, either under isotonic (replacement of 50 mM NaCl by 50 mM NH4Cl; medium osmolality 300 mosmol (kg H2O)–1; isoNH4Cl) or hypertonic conditions (+50 mM NH4Cl; medium osmolality 400 mosmol (kg H2O)–1; hyperNH4Cl) for 72 h demonstrated significantly increased caspase-3 activity compared with MDCK cells exposed to hyertonic NaCl (+50 mM NaCl, final medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl). These observations were confirmed by flow cytometric analysis of annexin V-FITC binding. Translocation of phosphatidylserine to the outer leaflet of cell membranes is a characteristic feature of early stages of apoptosis. In later apoptotic stages PI, which penetrates only cells with disrupted membranes, serves as a marker for necrotic cells. Hence, annexin V-FITC-positive/PI-negative and annexin V-FITC-positive/PI-positive cells represent the early and apoptotic-lysed stages, respectively.
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MDCK cells were incubated for 3 days in either isotonic medium (300 mosmol(kg H2O)–1; iso), in medium made hypertonic by the addition of 50 mM NaCl (medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl), in isotonic medium in which 50 mM NaCl were replaced by 50 mM NH4Cl (medium osmolality 300 mosmol(kg H2O)–1; isoNH4Cl) or in medium made hypertonic by the addition of 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNH4Cl). A, thereafter, relative caspase-3 activity was determined as described in Methods. Specificity was demonstrated by addtion of the caspase-3 inhibitor Z-VAD-FMK as indicated (Z-VAD-FMK). Means ± S.E.M. for n = 3–4. P < 0.05 versus hyperNaCl. B, after the respective treatments, cells were treated with trypsin, washed and incubated with annexin V-FITC and propidium iodide (PI) and analysed by flow cytometry as described in Methods. Representative results of three independent experiments are shown.
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As shown in Fig. 5B, the number of apoptotic-lysed cells was higher in MDCK cells after exposure to 50 mM NH4Cl under either isotonic or hypertonic conditions, whereas hypertonic NaCl (400 mosmol (kg H2O)–1) was not associated with an increased incidence of apoptosis (Fig. 5A and B).
Discussion
In antidiuresis, the cells of the renal medulla are exposed to a uniquely harsh environment characterized by low oxygen tension and high extracellular concentrations of Na+, K+, NH4+, Cl– and urea (Valtin, 1966; Baumgartl et al. 1972; Sone et al. 1993; Brezis et al. 1994). During antidiuresis or acidosis, NH4+ concentrations in the inner medulla may reach values that impose osmoregulatory constraints on cells resident in this kidney region (Manitius et al. 1960; Valtin, 1966; Stern et al. 1985; Packer et al. 1991). This notion is supported by the observation that MDCK cells exposed to medium made hypertonic by NH4Cl addition exhibited a variety of responses with respect to the expression of osmosensitive genes, in general similar to those after raising the medium osmolality to equiosmolar levels by NaCl addition. Both the abundance of mRNA (for AR, BGT-1, SMIT and HSP70) and the level of protein expression (BGT, HSP70 and TonEBP/NFAT5) were increased in cells exposed to hypertonic media, regardless of whether NaCl or NH4Cl was used to elevate the medium tonicity. Although the addition of either solute was associated with qualitatively comparable effects, subtle quantitative differences were apparent. In general, NH4Cl increased the abundance of AR, BGT-1 and SMIT mRNAs more effectively than equiosmolar NaCl. This finding is in agreement with the observation that TonEBP/NFAT5 expression was higher after exposure to hypertonic NH4Cl than after exposure to hypertonic NaCl (Fig. 4). However, the abundance of several TonEBP/NFAT5 target gene products (i.e. BGT, HSP70) as assessed by in situ ELISA or immunoblot analysis, was lower in NH4Cl- compared with NaCl-treated cells (Figs 2 and 3). This divergence between mRNA and protein abundance may reflect reduced translation efficiency or/and accelerated protein degradation in NH4Cl-treated cells. Enhanced protein degradation, however, is unlikely, since studies by Ling et al. on renal tubular cells have shown that exposure to NH4Cl reduces rather than accelerates protein degradation (Ling et al. 1996). On the other hand, there is evidence for an inhibitory effect of elevated NH4Cl concentrations (20 mM) on protein synthesis in cultured renal epithelial cells (Jurkovitz et al. 1992). These and the present results suggest that hypertonic NH4Cl impairs translation of the respective mRNAs. Of interest, 72 h exposure to isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl was associated with increased abundance of most but not all mRNAs studied. Thus NH4Cl per se may affect the induction or/and stability of the respective mRNAs. In support of this notion, hypertonicity has been shown recently to affect the stability of HSP70 mRNA (Alfieri et al. 2002). Since TonEBP/NFAT5 abundance was not significantly different after incubation in isotonic, NH4Cl-containing medium, it appears unlikely that this transcription factor mediates the increase in the abundance of the respective mRNAs. The finding that not all tonicity-inducible mRNAs (BGT-1) respond identically to exposure to isotonic, NH4Cl-containing medium is not surprising, since evidence for differences in the regulation of the expression of various osmosensitive genes has been provided previously (Atta et al. 1999; Neuhofer et al. 2002b).
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The present observations of induction of both TonEBP/NFAT5 and TonEBP/NFAT5 target genes by hypertonic, NH4Cl-containing medium implies that this transcription factor plays a major role in enhancing the expression of osmosensitive genes observed under this condition. Additional influences, however, may also participate in regulating the expression of osmosensitive genes, thus explaining the subtle, albeit noticeable, differences in the effects elicited by hypertonic NH4Cl-containing media compared with those produced by hypertonic NaCl-containing media (Neuhofer et al. 2002a).
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It is well established that high NaCl concentrations induce cell death, preferentially by apoptosis (Santos et al. 1998; Michea et al. 2000; Horio et al. 2001). The present finding that neither the rate of apoptosis nor caspase-3 activity was significantly higher in cells exposed to hypertonic, NaCl-containing medium than in the isotonic controls is probably due to the only moderate degree of hypertonicity (400 mosmol (kg H2O)–1). However, exposure to NH4Cl-containing media consistently elicited higher rates of cell death associated with higher caspase-3 activities than incubation in the respective, equiosmolar NaCl-containing media. Since NH4Cl induces apoptosis not only in MDCK cells but also in gastric epithelial and C6 glioma cells (Buzanska et al. 2000; Suzuki et al. 2002), it is reasonable to assume that NH4Cl at high concentrations acts as a general cytotoxic agent. Renal medullary cells thus are exposed not only to apoptosis-inducing concentrations of NaCl and urea (Neuhofer et al. 1998, 2004; Santos et al. 1998) but also to apoptogenic concentrations of NH4Cl. As shown in Figs 2 and 3, NH4Cl is less effective than NaCl in inducing processes that confer protection against urea- or NaCl-induced apoptosis, i.e. HSP70 production and BGT-1-mediated betaine accumulation (Horio et al. 2001; Neuhofer et al. 2001). This may, at least in part, explain the finding that on a molar basis NH4Cl is more cytotoxic than NaCl.
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High concentrations of urea or K+ are known to modify the adaptive response of renal epithelial cells to elevated NaCl concentrations (Neuhofer & Beck, 2005). When considering the effects of high NH4+ concentrations on renal medullary cells in their natural environment in the intact kidney in vivo, one should keep in mind that adaptation of these cells to high NH4+ concentrations also may be influenced by high extracellular urea or K+ concentrations as they exist in the renal medulla.
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In summary, the response of MDCK cells to high extracellular NH4Cl concentrations shows many similarities to, but also subtle differences from that evoked by equiosmolar NaCl concentrations. This implies that special attention should be directed to the NH4Cl-induced effects when studying the adaptation of inner medullary cells in situ to their exceptional environment.
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Neuhofer W & Beck FX (2005). Cell survival in the hostile environment of the renal medulla. Annu Rev Physiol 67, in press.
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Woo SK, Lee SD & Kwon HM (2002a). TonEBP transcriptional activator in the cellular response to increased osmolality. Pflugers Arch 444, 579–585.
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Woo SK, Maouyo D, Handler JS & Kwon HM (2000). Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity. Am J Physiol Cell Physiol 278, C323–C330., 百拇医药(Wolfgang Neuhofer, Monika)
Abstract
The cells of the kidney medulla are exposed routinely to high extracellular concentrations of various solutes including NaCl, urea and ammonium (NH4+). Although it is well established that the expression of a variety of osmosensitive genes and proteins, which confer cytoprotection on renal medullary cells, is induced by high NaCl concentrations, the role of NH4+ in these cellular responses is unclear. This study thus addressed the effect of NH4+ on the expression of the betaine/GABA transporter (BGT-1), the sodium/myo-inositol cotransporter (SMIT), aldose reductase (AR), and heat shock protein 70 (HSP70) in Madin–Darby canine kidney (MDCK) cells, using Northern and Western blot analyses and enzyme-linked immunosorbent assay (ELISA). The incidence of apoptosis was monitored by determining caspase-3 activity and annexin V binding. Addition of NH4Cl (50 mM; total osmolality 400 mosmol (kg H2O)–1 to the medium was more effective than equiosmolar NaCl in increasing BGT-1 and HSP70 mRNA abundance, but less effective in enhancing BGT-1 and HSP70 expression at the protein level. Qualitatively similar results were obtained for SMIT and AR mRNAs. Exposure to both isotonic and hypertonic, NH4Cl-containing medium enhanced apoptosis compared with equiosmolar, NaCl-containing media. These results suggest that, in addition to NaCl, NH4Cl may play a role in regulating the intracellular accumulation of organic osmolytes, the abundance of HSP70 and cell turnover in the renal medulla in vivo.
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Introduction
Renal medullary cells are exposed routinely to high extracellular concentrations of various solutes including NaCl, urea and ammonium (NH4+) (Manitius et al. 1960; Jamison & Kriz, 1982; Stern et al. 1985; Packer et al. 1991). Intracellular accumulation of metabolically neutral, non-perturbing organic osmolytes, in particular the trimethylamines betaine and glycerophosphorylcholine (GPC) and the polyols myo-inositol and sorbitol, allows osmotic adaptation to the high extracellular NaCl concentrations (Law & Burg, 1991; Beck et al. 1998). While high intracellular concentrations of betaine and myo-inositol are achieved by increased uptake via the sodium and chloride dependent betaine/GABA transporter (BGT-1) and the sodium/myo-inositol cotransporter (SMIT) (Kwon & Handler, 1995), elevated cell sorbitol contents are accomplished by enhanced, aldose reductase- (AR) mediated production from glucose (Bagnasco et al. 1987; Sands & Schrader, 1990). The enhanced expression of BGT-1, SMIT and AR underlying hypertonicity-induced osmolyte-accumulation is due to enhanced transcription of the respective genes (Woo et al. 2002a). Binding of the tonicity-responsive enhancer (TonE) binding protein/nuclear factor of activated T cells (TonEBP/NFAT5) to multiple TonEs in the 5'-flanking region of these genes is a critical step in this process and one that stimulates transcription of TonEBP/NFAT5 target genes. Both redistribution from the cytoplasm to the nucleus and enhanced expression of TonEBP/NFAT5 participate in the upregulation of tonicity-sensitive genes (Woo et al. 2000). In addition to BGT-1, SMIT and AR, one of the genes encoding the inducible 70 kDa heat shock protein (HSP70) is a target for TonEBP/NFAT5 (Woo et al. 2002b). Following exposure to high extracellular concentrations of NaCl, but not of urea, HSP70 expression is induced vigorously (Cohen et al. 1991; Neuhofer et al. 1999). This molecular chaperone plays a decisive role in counteracting the deleterious effects of the excessive urea concentrations prevailing in the inner medulla of many mammals in antidiuresis (Müller et al. 1998; Beck et al. 2000; Neuhofer et al. 2001). Failure to adapt to these extreme conditions leads to apoptotic cell death (Kültz & Chakravarty, 2001; Neuhofer et al. 2001).
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In contrast to the considerable knowledge gathered over the last decade on the effect of elevated NaCl and urea concentrations on the function of renal epithelial cells, there is little information on the effect of high NH4+ concentrations. In view of tissue NH4+ concentrations exceeding 50 mmol l–1 in the papilla of antidiuretic mammals (Manitius et al. 1960; Valtin, 1966), the present study was undertaken to examine the effect of elevated NH4Cl concentrations on BGT-1, SMIT, AR, HSP70 and TonEBP/NFAT5 expression in MDCK cells using Northern and Western blot analyses and in situ enzyme-linked immunosorbent assay (ELISA). A further goal of this study was to assess parameters of apoptotic cell death after exposure of these cells to high NH4Cl concentrations.
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Methods
Culture and treatment of MDCK cells
Madin–Darby canine kidney (MDCK) cells obtained from the American Type Culture Collection (CCL-34; ATCC, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM; low glucose) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and penicillin–streptomycin (100 u ml–1 and 100 μg ml–1; Invitrogen, Karlsruhe, Germany) at 37°C in a humidified atmosphere containing 5% CO2–95% air. Cells were seeded in 100 mm plastic dishes (4 x 105 cells dish–1; Greiner, Frickenhausen, Germany) or in 24-well plates (2 x 105 cells well–1; Costar, Cambridge, MA, USA), and cultured to confluence.
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For experiments, cells were incubated for the indicated periods in isotonic medium (osmolality 300 mosmol (kg H2O)–1), or in medium made hypertonic by the addition of 50 mM NaCl (hyperNaCl; total osmolality 400 mosmol (kg H2O)–1), or in medium made hypertonic by the addition of 5–50 mM NH4Cl as indicated (hyperNH4Cl; total osmolality 310–400 mosmol (kg H2O)–1), or in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (isoNH4Cl; total osmolality 300 mosmol (kg H2O)–1) for 72 h. The respective media were replaced daily.
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In situ cellular ELISA for quantification of BGT-1
The in situ ELISA allows a quantitative analysis of reaction products and reflects the amount of membrane-bound BGT-1. The main details of the methodology are based on standard immunoperoxidase staining. The cells in 24-well plates were washed three times with Dulbecco's PBS (DPBS) and then fixed at 4°C in 4% paraformaldehyde in DPBS for 20 min. After washing, the cells were permeabilized with Triton X-100 (Sigma, Taufkirchen, Germany), 0.1% in DPBS for 5 min. The endogenous peroxidase activity was blocked by a 30-min incubation with 0.2% H2O2 in methanol at room temperature. The non-specific protein binding sites were blocked by a 60-min incubation at room temperature with 10% FBS in DPBS. This step was followed by incubation with rabbit anti-BGT-1 antibody (Biotrend, Cologne, Germany) in 10% FBS in DPBS (1 : 1000) for 60 min at room temperature. Specificity of the signal was demonstrated by simultaneous incubation with the 15-amino acid peptide used for raising the antibody (Biotrend). This procedure diminished the signal significantly and concentration dependently, demonstrating that the antibody specifically detects BGT-1 in MDCK cells. The cells were washed three times with 0.1% Tween-20 in DPBS and incubated overnight at 4°C with peroxidase-conjugated goat antirabbit IgG (Dianova, Hamburg, Germany) in 10% FBS in DPBS. Finally, the peroxidase reaction was allowed to proceed for 20 min at room temperature with 3,3',5,5'-tetramethyl-benzidine (TMB) Liquid Substrate System (Sigma). Adding 0.5 M H2SO4 stopped the reaction, and the absorbances were measured at 450 nm (ref. 620 nm) in a BioSpec-1601 E spectrophotometer (Shimadzu, Duisburg, Germany). The absorbance at 450 nm is proportional to the amount of BGT-1 expressed in the MDCK cells.
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Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis
Following the treatments, cells were washed three times with chilled PBS and scraped into 8 M urea/PBS containing 0.1% Triton X-100, 1 μg ml–1 leupeptin, 10 ng ml–1 aprotinin, 100 μM phenylmethylsulphonyl fluoride (PMSF), 100 μM dithiothreitol (DTT), 200 μM sodium orthovanadate (Na3VO4) and 1 mM sodium fluoride (NaF), 100 μl (100-mm tissue culture dish)–1. Cells were lysed with three cycles of snap-freezing and thawing. The extracts were stored for 15 min at room temperature, vortexed vigorously, and centrifuged at 12 000 g for 15 min at 4°C. Protein concentrations in the supernatant were determined in duplicate, using a commercially available protein assay (Bio-Rad, Munich, Germany). Aliquots (40 μg) of total protein were subjected to SDS-PAGE and Western blot analysis using a mouse anti-Hsp70 (Stressgen, Canada) antibody or rabbit TonEBP/NFAT5 antiserum as described in detail elsewhere (Neuhofer et al. 2002a).
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Northern blot analysis
After the respective treatments, the cells were washed with ice-cold PBS and lysed by addition of 1 ml of TRI-reagent (PeqLab Biotech, Erlangen, Germany). Total RNA was recovered according to the recommendations of the manufacturer. Aliquots (20 μg) were electrophoresed through 1% agarose/formaldehyde gels, blotted onto nitrocellulose membrane and immobilized by ultraviolet-crosslinking. For Northern blot analysis, the blots were prehybridized for 2 h at 55°C in a solution containing 50% formamide, 5x saline–sodium citrate buffer (SSC), 0.1% SDS, and 10% blocking reagent (Roche, Mannheim, Germany) and were hybridized overnight in the same solution containing 20 ng ml–1 digoxigenin-labelled human AR (GenBank accession no. J05474), canine SMIT (M85068), canine BGT-1 (M80403) or human HSP70 (M11717) cDNA (Neuhofer et al. 2002a). After hybridization, the membranes were washed twice for 15 min each with 2x SSC/0.1% SDS at room temperature and twice for 15 min with 0.1% SSC/0.1% SDS at 68°C. Non-radioactive detection procedures were carried out using antidigoxigenin-AP (Roche, Mannheim, Germany), followed by incubation with CDP-star chemiluminescent substrate disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2'-(5' chloro) tricyclo [3.3.1.13 7] decan}-4-yl)-1-phenyl phosphate for alkaline phosphatase (Roche) as previously described (Burger-Kentischer et al. 1999). To correct for differences in RNA loading, the membranes were stripped and rehybridized with digoxigenin-labelled cDNA specific for glyceraldehyde phosphate dehydrogenase (GAPDH; X011677). Signals were quantified by laser densitometry (Ultrascan XL; Pharmacia, Freiburg, Germany).
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Determination of caspase-3 activity
Relative caspase-3 activity was assessed using a commercially available colourimetric caspase-3 assay based on the hydrolysis of the synthetic caspase-3 substrate paranitroanilin-conjugated Asp-Glu-Val-Asp (pNA-DEVD; CaspACE; Promega, Madison, WI, USA). After the specific treatments, caspase-3 activity was determined as described by the manufacturer on 50 μg protein in 96-well plates. Specificity of the assay was demonstrated by parallel incubation of each extract in the presence of the caspase-3 inhibitor Z-VAD-FMK. Relative caspase-3 activity was determined as the increase in A405. (Spectrafluor, Tecan Group, Maennedorf, Switzerland).
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Flow cytometry
Cell viability was monitored further by labelling of cell surface-exposed phosphatidylserine by annexin V-FITC in combination with propidium iodide (PI) according to the manufacturer's recommendations (ApoAlert Annexin V Apoptosis Kit, Clontech, Heidelberg, Germany). Briefly, MDCK cells were trypsinized, washed once with serum-containing media, once with binding buffer and resuspended at a concentration of 106 cells (ml)–1 in binding buffer (Clontech). Aliquots of 100 μl were incubated for 15 min in the dark with annexin V-FITC (100 ng in TRIS-NaCl; Clontech) and/or PI (500 ng in binding buffer) and subsequently analysed by flow cytometry (FacsScan; Becton-Dickinson, Heidelberg, Germany).
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Statistical analysis
The data are presented as means ± S.E.M. The significance of differences between means was established by Students' t test for independent samples and by multivariate ANOVA for multiple samples. P < 0.05 was regarded as significant.
Results
Effect of NH4Cl on AR, BGT-1 and SMIT mRNA abundance in MDCK cells
Figure 1 demonstrates that following addition of either 50 mM NH4Cl (total medium osmolality 400 mosmol(kg H2O)–1 hyperNH4Cl) or 50 mM NaCl (medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl), the abundance of AR, BGT-1 and SMIT mRNAs increased significantly compared with isotonic controls (medium osmolality 300 mosmol (kg H2O)–1; isocontrol). Interestingly, the addition of NH4Cl increased the mRNA levels of the respective genes to a significantly greater extent than equiosmolar NaCl. Compared with isotonic controls (isocontrol), isotonic replacement of 50 mM NaCl by equiosmolar NH4Cl (final medium osmolality 300 mosmol (kg H2O)–1; isoNH4Cl) caused a significant increase in the abundance of AR and SMIT mRNAs but did not affect the abundance of BGT-1 mRNA.
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MDCK cells were kept for 72 h in isotonic medium (300 mosmol (kg H2O)–1; isocontrol), in medium made hypertonic by the addition of either 50 mM NaCl or 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNaCl, hyperNH4Cl) or 200 mM NaCl (700 mosmol (kg H2O)–1; +200 mM NaCl), or in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (isoNH4Cl). Subsequently, the abundance of the respective mRNAs was assessed by Northern blot analysis, normalized to GAPDH abundance and expressed as increase compared with isocontrol. Means ± S.E.M. for n = 3–4 independent experiments; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNaCl. Representative blots are shown below.
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As a positive control, mRNA from cells exposed to an additional 200 mM NaCl (final medium osmolality 700 mosmol (kg H2O)–1; +200 mM NaCl) was also loaded, since this manoeuvre is known to increase the expression of the respective genes strongly.
Effect of NH4Cl on BGT-1 expression in MDCK cells
Figure 2 shows that the addition of 50 mM NH4Cl (hyperNH4Cl) resulted in a time- and concentration-dependent increase in BGT-1 expression (Fig. 2A and B). Although the addition of NH4Cl at concentrations of 25 and 50 mM increased the expression of BGT-1 significantly compared with isotonic controls (Fig. 2B), NH4Cl was significantly less effective than equiosmolar NaCl (Fig. 2A and B).
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A, MDCK cells were kept in isotonic medium (), or exposed to medium made hypertonic by addition of either 50 mM NaCl () or 50 mM NH4Cl () for 48, 72 or 96 h. Thereafter, membrane-associated BGT-1 was determined as decribed in Methods. Means ± S.E.M. for three separate experiments with 3–6 replicates per experiment; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNH4Cl. B, MDCK cells were exposed to hypertonic, NH4Cl-containing media (addition of 5–50 mM NH4Cl, hyperNH4Cl) or to hypertonic NaCl-containing medium (addition of 50 mM NaCl, hyperNaCl) for 48 h. Thereafter, membrane-associated BGT-1 was determined as decribed in Methods. Means ± S.E.M. for three separate experiments with 3–6 replicates per experiment; *P < 0.05 versus isocontrol; #P < 0.05 versus +50 mM NH4Cl.
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Effect of NH4Cl on HSP70 expression in MDCK cells
Compared with isotonic controls (isocontrol), HSP70 mRNA abundance in MDCK cells exposed to medium made hypertonic by the addition of either 50 mM NH4Cl or 50 mM NaCl (final medium osmolality 400 mosmol (kg H2O)–1; hyperNH4Cl, hyperNaCl) was significantly increased (Fig. 3A). Incubation in isotonic, NH4Cl-containing medium (300 mosmol(kg H2O)–1; isoNH4Cl) also resulted in a significant upregulation of HSP70 mRNA (Fig. 3A) to levels comparable to that seen after addition of 50 mM NaCl (medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl). Although HSP70 mRNA abundance was elevated under the latter condition, HSP70 (protein) expression was not significantly different from isotonic controls (Fig. 3B). In contrast, after treatment with hypertonic, NH4Cl-containing medium (+50 mM NH4Cl; final medium osmolality 400 mosmol (kg H2O)–1; hyperNH4Cl), HSP70 expression was significantly higher than that in isotonic controls (isocontrol) but less than after equiosmolar addition of NaCl (hyperNaCl) (Fig. 3B). Expression of the constitutively expressed HSP27 was not affected after exposure of MDCK cells to either isotonic or hypertonic NH4Cl-containing media (Fig. 3B).
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MDCK cells were kept in isotonic medium (isocontrol; 300 mosmol (kg H2O)–1), or incubated in either medium made hypertonic by the addition of 50 mM NaCl (400 mosmol (kg H2O)–1; hyperNaCl), in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (300 mosmol (kg H2O)–1; isoNH4Cl), or in medium made hypertonic by the addition of 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNH4Cl) for 72 h. Subsequently, HSP70 mRNA and protein abundance were assessed by Northern (A) and Western (B) blot analysis, respectively. Means ± S.E.M. for n = 3; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNaCl.
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Effect of NH4Cl on TonEBP/NFAT5 expression in MDCK cells
As demonstrated in Fig. 4, exposure of MDCK cells to isotonic medium in which 50 mM NaCl had been replaced by 50 mM NH4Cl (isoNH4Cl) had no significant effect on TonEBP/NFAT5 abundance compared with MDCK cells kept in isotonic medium (isocontrol). However, Incubation in hypertonic, NH4Cl medium (400 mosmol (kg H2O)–1 by the addition of 50 mM NH4Cl; hyperNH4Cl) enhanced TonEBP/NFAT5 abundance strongly and significantly more than exposure to equiosmolar medium made hypertonic by the addition of 50 mM NaCl (hyperNaCl).
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MDCK cells were kept in isotonic medium (isocontrol; 300 mosmol(kg H2O)–1), or either incubated in medium made hypertonic by the addition of 50 mM NaCl (400 mosmol (kg H2O)–1; hyperNaCl), in isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl (300 mosmol (kg H2O)–1; isoNH4Cl), or in medium made hypertonic by the addition of 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNH4Cl) for 72 h. Subsequently, TonEBP/NFAT5 abundance was determined by Western blot ananlysis. Means ± S.E.M. for n = 3; *P < 0.05 versus isocontrol; #P < 0.05 versus hyperNaCl.
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NH4Cl-induced apoptosis in MDCK cells
As shown in Fig. 5A, MDCK cells exposed to 50 mM NH4Cl, either under isotonic (replacement of 50 mM NaCl by 50 mM NH4Cl; medium osmolality 300 mosmol (kg H2O)–1; isoNH4Cl) or hypertonic conditions (+50 mM NH4Cl; medium osmolality 400 mosmol (kg H2O)–1; hyperNH4Cl) for 72 h demonstrated significantly increased caspase-3 activity compared with MDCK cells exposed to hyertonic NaCl (+50 mM NaCl, final medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl). These observations were confirmed by flow cytometric analysis of annexin V-FITC binding. Translocation of phosphatidylserine to the outer leaflet of cell membranes is a characteristic feature of early stages of apoptosis. In later apoptotic stages PI, which penetrates only cells with disrupted membranes, serves as a marker for necrotic cells. Hence, annexin V-FITC-positive/PI-negative and annexin V-FITC-positive/PI-positive cells represent the early and apoptotic-lysed stages, respectively.
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MDCK cells were incubated for 3 days in either isotonic medium (300 mosmol(kg H2O)–1; iso), in medium made hypertonic by the addition of 50 mM NaCl (medium osmolality 400 mosmol (kg H2O)–1; hyperNaCl), in isotonic medium in which 50 mM NaCl were replaced by 50 mM NH4Cl (medium osmolality 300 mosmol(kg H2O)–1; isoNH4Cl) or in medium made hypertonic by the addition of 50 mM NH4Cl (400 mosmol (kg H2O)–1; hyperNH4Cl). A, thereafter, relative caspase-3 activity was determined as described in Methods. Specificity was demonstrated by addtion of the caspase-3 inhibitor Z-VAD-FMK as indicated (Z-VAD-FMK). Means ± S.E.M. for n = 3–4. P < 0.05 versus hyperNaCl. B, after the respective treatments, cells were treated with trypsin, washed and incubated with annexin V-FITC and propidium iodide (PI) and analysed by flow cytometry as described in Methods. Representative results of three independent experiments are shown.
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As shown in Fig. 5B, the number of apoptotic-lysed cells was higher in MDCK cells after exposure to 50 mM NH4Cl under either isotonic or hypertonic conditions, whereas hypertonic NaCl (400 mosmol (kg H2O)–1) was not associated with an increased incidence of apoptosis (Fig. 5A and B).
Discussion
In antidiuresis, the cells of the renal medulla are exposed to a uniquely harsh environment characterized by low oxygen tension and high extracellular concentrations of Na+, K+, NH4+, Cl– and urea (Valtin, 1966; Baumgartl et al. 1972; Sone et al. 1993; Brezis et al. 1994). During antidiuresis or acidosis, NH4+ concentrations in the inner medulla may reach values that impose osmoregulatory constraints on cells resident in this kidney region (Manitius et al. 1960; Valtin, 1966; Stern et al. 1985; Packer et al. 1991). This notion is supported by the observation that MDCK cells exposed to medium made hypertonic by NH4Cl addition exhibited a variety of responses with respect to the expression of osmosensitive genes, in general similar to those after raising the medium osmolality to equiosmolar levels by NaCl addition. Both the abundance of mRNA (for AR, BGT-1, SMIT and HSP70) and the level of protein expression (BGT, HSP70 and TonEBP/NFAT5) were increased in cells exposed to hypertonic media, regardless of whether NaCl or NH4Cl was used to elevate the medium tonicity. Although the addition of either solute was associated with qualitatively comparable effects, subtle quantitative differences were apparent. In general, NH4Cl increased the abundance of AR, BGT-1 and SMIT mRNAs more effectively than equiosmolar NaCl. This finding is in agreement with the observation that TonEBP/NFAT5 expression was higher after exposure to hypertonic NH4Cl than after exposure to hypertonic NaCl (Fig. 4). However, the abundance of several TonEBP/NFAT5 target gene products (i.e. BGT, HSP70) as assessed by in situ ELISA or immunoblot analysis, was lower in NH4Cl- compared with NaCl-treated cells (Figs 2 and 3). This divergence between mRNA and protein abundance may reflect reduced translation efficiency or/and accelerated protein degradation in NH4Cl-treated cells. Enhanced protein degradation, however, is unlikely, since studies by Ling et al. on renal tubular cells have shown that exposure to NH4Cl reduces rather than accelerates protein degradation (Ling et al. 1996). On the other hand, there is evidence for an inhibitory effect of elevated NH4Cl concentrations (20 mM) on protein synthesis in cultured renal epithelial cells (Jurkovitz et al. 1992). These and the present results suggest that hypertonic NH4Cl impairs translation of the respective mRNAs. Of interest, 72 h exposure to isotonic medium in which 50 mM NaCl was replaced by 50 mM NH4Cl was associated with increased abundance of most but not all mRNAs studied. Thus NH4Cl per se may affect the induction or/and stability of the respective mRNAs. In support of this notion, hypertonicity has been shown recently to affect the stability of HSP70 mRNA (Alfieri et al. 2002). Since TonEBP/NFAT5 abundance was not significantly different after incubation in isotonic, NH4Cl-containing medium, it appears unlikely that this transcription factor mediates the increase in the abundance of the respective mRNAs. The finding that not all tonicity-inducible mRNAs (BGT-1) respond identically to exposure to isotonic, NH4Cl-containing medium is not surprising, since evidence for differences in the regulation of the expression of various osmosensitive genes has been provided previously (Atta et al. 1999; Neuhofer et al. 2002b).
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The present observations of induction of both TonEBP/NFAT5 and TonEBP/NFAT5 target genes by hypertonic, NH4Cl-containing medium implies that this transcription factor plays a major role in enhancing the expression of osmosensitive genes observed under this condition. Additional influences, however, may also participate in regulating the expression of osmosensitive genes, thus explaining the subtle, albeit noticeable, differences in the effects elicited by hypertonic NH4Cl-containing media compared with those produced by hypertonic NaCl-containing media (Neuhofer et al. 2002a).
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It is well established that high NaCl concentrations induce cell death, preferentially by apoptosis (Santos et al. 1998; Michea et al. 2000; Horio et al. 2001). The present finding that neither the rate of apoptosis nor caspase-3 activity was significantly higher in cells exposed to hypertonic, NaCl-containing medium than in the isotonic controls is probably due to the only moderate degree of hypertonicity (400 mosmol (kg H2O)–1). However, exposure to NH4Cl-containing media consistently elicited higher rates of cell death associated with higher caspase-3 activities than incubation in the respective, equiosmolar NaCl-containing media. Since NH4Cl induces apoptosis not only in MDCK cells but also in gastric epithelial and C6 glioma cells (Buzanska et al. 2000; Suzuki et al. 2002), it is reasonable to assume that NH4Cl at high concentrations acts as a general cytotoxic agent. Renal medullary cells thus are exposed not only to apoptosis-inducing concentrations of NaCl and urea (Neuhofer et al. 1998, 2004; Santos et al. 1998) but also to apoptogenic concentrations of NH4Cl. As shown in Figs 2 and 3, NH4Cl is less effective than NaCl in inducing processes that confer protection against urea- or NaCl-induced apoptosis, i.e. HSP70 production and BGT-1-mediated betaine accumulation (Horio et al. 2001; Neuhofer et al. 2001). This may, at least in part, explain the finding that on a molar basis NH4Cl is more cytotoxic than NaCl.
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High concentrations of urea or K+ are known to modify the adaptive response of renal epithelial cells to elevated NaCl concentrations (Neuhofer & Beck, 2005). When considering the effects of high NH4+ concentrations on renal medullary cells in their natural environment in the intact kidney in vivo, one should keep in mind that adaptation of these cells to high NH4+ concentrations also may be influenced by high extracellular urea or K+ concentrations as they exist in the renal medulla.
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In summary, the response of MDCK cells to high extracellular NH4Cl concentrations shows many similarities to, but also subtle differences from that evoked by equiosmolar NaCl concentrations. This implies that special attention should be directed to the NH4Cl-induced effects when studying the adaptation of inner medullary cells in situ to their exceptional environment.
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