2007 JASN IMPACT FACTOR 7.111	HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP
advanced	CURRENT ISSUE	ARCHIVES	JASN Express	ONLINE SUBMISSION

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ÇCetinkaya, I. Articles by Kleta, R. Search for Related Content PubMed PubMed Citation Articles by ÇCetinkaya, I. Articles by Kleta, R.

Inhibition of Na⁺-Dependent Transporters in Cystine-Loaded Human Renal Cells: Electrophysiological Studies on the Fanconi Syndrome of Cystinosis

Ibrahim ÇCetinkaya^*,, Eberhard Schlatter, Jochen R. Hirsch, Peter Herter, Erik Harms^* and Robert Kleta^*

* Department of Pediatrics, University Children’s Hospital Muenster, Muenster, Germany; Department of Internal Medicine D, Experimental Nephrology, University Hospital Muenster, Muenster, Germany; Max-Planck-Institute for Molecular Physiology, Dartmund, Germany.

Correspondence to Dr. Eberhard Schlatter, Universitaetsklinikum Münster, Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Domagkstrasse 3A, 48149 Münster, Germany. Phone: 49-251-83-56991; 49-251-83-56973; E-mail: eberhard.schlatter{at}uni-muenster.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
ABSTRACT. Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V_m) of IHKE-1 cells were -30.7 ± 0.4 mV (n = 151). CDME concentration-dependently altered V_m with an initial depolarization (2.7 ± 0.2 mV;n = 76; 1 mM CDME) followed by a more pronounced hyperpolarization (-9.9 ± 1.0 mV;n = 49). Three Na⁺-dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 ± 0.7 mV (n = 59), and phosphate (1.8 mM) depolarized by 9.7 ± 1.1 mV (n = 18). Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V_m by 7.1 ± 0.3 mV (n = 21) followed by a repolarization by 2.9 ± 0.3 mV/min (n = 17), reflecting Na⁺/H⁺-exchanger activity. Acute addition of 1 mM CDME did not alter the alanine- and propionate-induced changes in V_m, but it reduced the phosphate-induced depolarization by 37 ± 9% (n = 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 ± 4% (n =30), 45 ± 9% (n = 9), and 78 ± 15% (n = 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V_m of IHKE-1 cells and that at least three Na⁺-dependent transporters are inhibited, the Na⁺-phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na⁺-gradient are involved in the development of the Fanconi syndrome of cystinosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cystinosis is an autosomal recessive disease in which a defect in the transport of the amino acid cystine out of the lysosomes leads to its intralysosomal accumulation (1–4) in multiple organs, including the kidney (5). The lysosomal cystine transporter has been identified and named cystinosin (6). First clinical symptoms early in childhood in the form of severe electrolyte disturbances, solute losses with the urine, and failure to thrive are due to the Fanconi syndrome (7). The pathogenesis of intralysosomal cystine accumulation leading to the dysfunction of the proximal tubule is not dissolved and continues to be a matter of investigation. There is no animal model for the Fanconi syndrome of cystinosis. Goldman et al. (8) and later Reeves (9) showed that lysosomes can be loaded with amino acids by incubation with the respective methyl esters. Finally, Foreman et al. (10) established an experimental model for cystinosis by incubating proximal tubular cells from rat kidney in cystine dimethyl ester (CDME), leading to high intracellular concentrations of cystine similar to that in cystinosis patients (5,11,12). Sakarcan et al. (11) showed that cystine in this model is mainly accumulated intralysosomally. So far, uptake measurements of radiolabeled substances by rat and rabbit proximal tubules and by porcine proximal tubular cells (LLC-PK₁) have been used to obtain insights into the pathophysiologic mechanisms of the Fanconi syndrome of cystinosis (7,10,13–15). Inhibition of volume reabsorption and a decrease in the lumen-negative transepithelial potential difference were shown under these conditions (13,14). Furthermore, inhibition of glucose, different amino acids, bicarbonate, and phosphate reabsorption in cystine-loaded proximal tubules was demonstrated (7,13). Inhibition of glucose transport was also shown in LLC-PK₁ cells (15). Corresponding to the decreased glucose uptake, cystine loading resulted in a reduced number of glucose transporters on the apical membrane of LLC-PK₁ cells (15) and in brush border membrane vesicles from rats after intraperitoneal CDME injections (16). Salmon et al. (13) used the CDME model to show that reduction in proximal tubular transport was due to an inhibition of active transport but not to an increase in proximal tubular permeability, as it was previously supposed for the Fanconi syndrome caused by maleic acid (17). Most of proximal tubular transport of solutes across the luminal membrane is secondarily coupled with active Na⁺-transport; studies were therefore performed to investigate the relationship between intracellular cystine accumulation and energy metabolism. Na⁺/K⁺-ATPase was not directly inhibited by CDME loading (14,18), but a decrease in intracellular phosphate and ATP-levels of cystine-loaded tubules with subsequent inhibition of Na⁺/K⁺-ATPase activity could be shown (14,15,18). According to these findings, there was an increase in intracellular Na⁺-concentration parallel to a decrease in intracellular K⁺-concentration in LLC-PK₁ cells (19) and in rat proximal tubules (18).

This study’s aim was to investigate potential effects of CDME on basal membrane voltages (V_m) of immortalized proximal tubular cells of human origin (IHKE-1 cells) (20) and to learn whether CDME differently affects acutely or after incubation some of the main Na⁺-dependent transporters that are responsible for solute uptake across the apical membrane of these human proximal tubule cells. Several studies on IHKE-1 cells have shown that these cells are typical proximal tubule cells. They express specific key enzymes (21), different Na⁺-dependent and -independent amino acid transporters (22,23), and organic cation transporters (24) and they are able to regulate transport by natriuretic peptides (25) or to take up albumin (26). Here we present evidence for the existence of proximal tubule–specific Na⁺/H⁺-exchanger (NHE-3) and the Na⁺-phosphate transporter (NaPi-IIa) in the apical membrane. Using the experimental model of CDME loading, we present the first electrophysiologic studies with the slow whole-cell patch clamp technique on IHKE-1 cells. We demonstrate acute effects of CDME loading on conductances, and thus V_m, of these cells and the inhibition of all three transporters studied, with the Na⁺-phosphate transporter being the most sensitive.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
IHKE-1 cells were cultured as described previously (20,25). In short, IHKE-1 cells were maintained in an atmosphere of 8% CO₂/92% air at 37°C in Dulbecco modified Eagle’s medium (DMEM) and Ham F-12 medium (1:1) containing 44 mM NaHCO₃, 15 mM HEPES (pH 7.3), 1.6 nM epidermal growth factor, 100 nM hydrocortisone, 65 nM transferrin, 0.84 µM insulin, 29 nM Na₂SeO₃, 0.5 mM pyruvic acid, 4 mM L-glutamine, 5 ml/L ciprofloxacin (Ciprobay 100), and 1% fetal calf serum (FCS). Three to ten days after trypsinization (0.05% trypsin, 0.02% ethylenediaminetetraacetate [EDTA] in Mg²⁺ and Ca²⁺-free phosphate buffer), IHKE-1 cells were used from passages 140 to 177. Cells were grown on glass cover slips until confluence. Under these conditions, cells grow in a polarized fashion, with the apical membrane facing upward and forming apical microvilli (see Figure 8). Using functional tests, we previously demonstrated this polarized geometry with correct sorting of transport systems to the apical and basolateral membrane (24).

View larger version (69K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Scanning electron micrographs of the apical surface membrane of IHKE-1 cells. The morphology of an IHKE-1 cell surface with numerous long microvilli is shown by secondary electrons (SE). The corresponding localization of NaPi is imaged by the material-dependent signal of back-scattered electrons (BE). Both signals are combined in a double exposure overlay (SE+BE).

V_m were measured with the slow whole-cell patch clamp technique (27). For this method, pipettes were filled with a solution containing 95 mM K⁺-gluconate, 30 mM KCl, 4.8 mM Na₂HPO₄, 1.2 mM NaH₂PO₄, 5 mM D-glucose, 0.73 mM Ca²⁺-gluconate, 1 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1.03 mM MgCl₂, 1 mM ATP, pH 7.2. To this solution 162 µM nystatin was added before use. Patch pipettes had an input resistance of 2.5 to 12.5 M. V_m was measured in the current clamp mode of a patch clamp amplifier (U. Fröbe, Physiologisches Institut, Universität Freiburg, Germany) and recorded continuously on a pen recorder (WeKaGraph WK-250R; WKK, Kaltbrunn, Switzerland).

RT-PCR Analyses
Total RNA was isolated using the RNeasy-kit (Qiagen, Hilden, Germany). Isolated total RNA was incubated with 10 U DNase I (Promega, Heidelberg, Germany) at 37°C for 1 h to digest isolated traces of genomic DNA. RNA and DNase I were then separated by an additional clean-up step using a new RNeasy column. cDNA first-strand synthesis was performed in a total reaction volume of 30 µl containing 5 µg of total RNA, 10 nM dNTP-Mix (Biometra, Göttingen, Germany), 1 nM p(dT)10 nucleotide primer (Boehringer, Mannheim, Germany), and 200 U of molony murine leukemia virus reverse transcriptase (MMLV-RT; Promega). One thirtieth of each cDNA first-strand reaction mixture was then subjected to a 50 µl of PCR reaction in a UNO II thermo cycler (Biometra) using 20 pmol of each primer and 1 unit of Taq DNA polymerase (Qiagen). Reaction conditions were as follows: 3 min at 94°C, 30 s at 59°C, and 1 min at 72°C for 1 cycle; 30 s at 94°C, 30 s at the optimal annealing temperature (OAT), and 1 min at 72°C for 30 cycles; 30 s at 94°C, 30 s at OAT, and 10 min at 72°C for 1 cycle. PCR reaction products were analyzed by agarose gel electrophoresis. Positive signals obtained from PCR experiments were sequenced by GATC (Konstanz, Germany). The following PCR primers for NHE-3 and NaP_i-IIa were used (listed in 5' to 3' direction). The sequence is followed by the expected fragment length for the respective sense and antisense primer:

NHE-3 sense: TGC CCT GGT GGT GCT TCT G

NHE-3 antisense: GAT GCT GCT GTT TCT CCG CTT CT

Fragment length = 823 bp; OAT = 63°C

NaPi-IIa sense: TGT CTG CTT CCT GCT GCT G

NaPi-IIa antisense: CAC CCT TAC TCC TGC CTA TCC TA

Fragment length = 719 bp; OAT = 61°C

GAPDH sense: CTG CCC CCT CTG CTG ATG

GAPDH antisense: GTC CAC CAC CCT GTT GCT GT

Fragment length = 614 bp; OAT = 61°C

Scanning Electron Microscopy and Immunogold Labeling
Cultured IHKE-1 cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h. For immunogold scanning electron microscopy, a rabbit polyclonal antibody directed against the NaP_i transporter of flounder kidney was employed (28). This antibody is directed against a portion of the protein that shows an 84% identity between the flounder and human NaP_i transporter and covers mostly the extracellular loop of the protein. Most of the differences between the flounder and human isoforms are localized in the transmembrane domains; crossreaction of this antibody with the human NaP_i must therefore be assumed. Immunogold labeling was performed according to the following incubation protocol: (1) blocking of free aldehyde sites with 50 mM glycine in PBS for 15 min;(2) blocking of unspecific binding sites with 5% normal goat serum diluted in 0.5% bovine serum albumin and 0.2% gelatin in PBS (PBG) for 30 min;(3) incubation with primary antibodies diluted 1:200 in PBG for 2 h (this step was omitted in control experiments); (4) after several PBS rinsing steps, a 1-h incubation with a 5 nm of gold-coupled goat anti-rabbit IgG antibody (British BioCell International, Cardiff, UK) diluted 1:50 in PBG was performed to detect binding sites of the primary antibody; (5) after several PBS washing steps, cells were postfixed with 2% glutaraldehyde in PBS for 5 min; (6) after twice rinsing with distilled water, a silver enhancement (British BioCell International) of the gold markers was performed to enable imaging of small gold conjugates at low magnification;(7) immunogold-labeled cells were then dehydrated in an ascending ethanol series and critical-point dried; (8) to provide conductivity and to avoid charging artifacts, dried cells were coated with 10 nm of carbon by electron gun evaporation. Labeled IHKE-1 cells were studied with a Hitachi S-800 scanning electron microscope (Nissei Sangyo, Ratingen, Germany). The cell surface morphology was imaged by secondary electrons (SE). The corresponding distribution of silver-enhanced gold conjugates was detected by the signal of backscattered electrons (BE). BE and SE images were recorded from the same scanned area at an accelerating voltage of 20 kV and at a working distance of 10 mm.

Biochemicals
DMEM and Ham F12 medium were obtained from Life Technologies (Karlsruhe, Germany). Glutamine, FCS, HEPES, and trypsin were purchased from Biochrom (Berlin, Germany). Ciprofloxacin was obtained from Bayer (Leverkusen, Germany). L-cystine dimethyl ester and L-alanine were purchased from Sigma (Taufkirchen, Germany). All other standard chemicals were supplied by Sigma and Merck (Darmstadt, Germany).

Statistical Analyses
Data are presented as original recordings from individual experiments or as mean values ± SEM, with the number of experiments given in brackets. For statistical analyses Student’s paired and unpaired two-sided t test were used with each effect compared to its own averaged pre- and postexperimental controls. A p-value < 0.05 was considered significant and is indicated by an asterisk.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Basal V_m of IHKE-1 cells were -30.7 ± 0.4 mV (n = 151). Increasing extracellular K⁺-concentration from 3.6 mM to 18.6 mM depolarized cells by 3.4 ± 0.1 mV (n = 101). The addition of 3 mM Ba²⁺, a blocker of K⁺-channels, depolarized V_m by 11.3 ± 0.7 mV (n = 14), again indicating the presence of a resting K⁺ conductance in these cells (23). Removal of extracellular Na⁺ led to a marked hyperpolarization due to the presence of a nonselective cation conductance, which also determines the relatively depolarized resting voltage of these cells (23,25). Removal of glucose did not result in a significant change in V_m (n =7).

We first investigated whether CDME acutely affects basal V_m of IHKE-1 cells. Addition of CDME to the bath solution led to an initial depolarization of V_m within seconds, mostly followed by a more pronounced hyperpolarization. Both effects were concentration-dependent between 0.1 and 5 mM (Figure 1) and complete within 30 s to 1 min. One millimolar CDME led to almost maximal effects on V_m of IHKE-1 cells; we therefore performed most of the following experiments with this concentration. To identify the conductance that is influenced by CDME loading CDME was added in the presence of 3 mM Ba²⁺ to block all K⁺ conductances. The CDME-induced initial depolarization of V_m was 2.5 ± 0.3 mV without Ba²⁺ and 3.3 ± 0.5 mV with Ba²⁺ (n = 14), a difference that is not significant (Figure 2). The secondary and usually more pronounced hyperpolarization induced by CDME was -7.6 ± 1.4 mV without Ba²⁺ and -1.1 ± 1.6 mV with Ba²⁺ (n = 8; Figure 2). The hyperpolarization induced by CDME could be blocked completely by Ba²⁺; it can therefore be concluded that CDME activated a K⁺-conductance.

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. (Upper panel) Original recording of a membrane voltage (Vm) measurement from an immortalized human kidney epithelial (IHKE-1) cell after addition of 1 mM cystine dimethyl ester (CDME) to the bath solution. CDME had in most experiments a biphasic effect on Vm with an initial depolarization followed by a more pronounced hyperpolarization. (Lower panel) Summary of CDME effects on basal Vm of IHKE-1 cells. Both the initial depolarization and the subsequent hyperpolarization were concentration-dependent. Numbers in brackets indicate the number of observations. * indicates statistical significance of the effect.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. (Upper panels) Effects of 3 mM Bs2+ on the CDME (1 mM)–induced changes in Vm of an IHKE-1 cell. The two panels show a continuing original recording. Ba2+ abolishes the CDME-induced hyperpolarization of Vm but not the initial depolarization. (Lower panel) Summary of experiments with Ba2+ (paired experiments). The two bar charts on the left show the effect of Ba2+ on the CDME-induced depolarization;those on the right show the effect on hyperpolarization. Numbers in brackets indicate the number of observations. * indicates statistical significance of the effect.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. (Upper panels) Two original recordings of alanine-induced changes in Vm of IHKE-1 cells. On the left, a control experiment with 1 mM alanine; on the right, the alanine-effect on Vm after 4.5 h of incubation with 1 mM CDME (unpaired experiments). (Lower panels) Summary of CDME effects on alanine-induced changes in Vm of IHKE-1 cells. Neither acute addition of CDME (1 or 5 mM) nor incubation for 30 min significantly reduced this effect. Numbers in brackets indicate the number of experiments. * indicates statistical significance of effect.

View larger version (23K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Summary of CDME effects on different Na+-dependent transporters of IHKE-1 cells. Data are given as percent inhibition compared with control. Numbers in brackets indicate the number of experiments. * indicates statistical significance of the effect.

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. (Upper panels) Original recordings of propionate-induced changes in Vm of IHKE-1 cells. On the left, a control experiment after addition of 20 mM propionate to the bath solution; on the right, the propionate effect on Vm after 4 h of incubation with 1 mM CDME. Note that repolarization after incubation with CDME is abolished compared with the control. (Lower panel) Summary of CDME effects on propionate-induced changes in Vm of IHKE-1 cells. On the left, the effects on initial depolarization after addition of 20 mM propionate; on the right, the effects on repolarization. Numbers in brackets indicate the number of experiments. * indicates statistical significance of the effect.

View larger version (53K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Detection of mRNA of the Na+/H+ isoform NHE-3, the Na+-phosphate transporter NaPi-IIa, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in IHKE-1 cells (and human kidney) by RT-PCR. The negative control does not include cDNA.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. (Upper panel) Original recording of phosphate-induced changes in Vm of IHKE-1 cells. In these experiments, the bath solution contained HEPES as a buffer. A paired experiment is shown with acute addition of 1 mM CDME to the bath solution (second part of the trace). Note the inhibition of phosphate-induced depolarization after acute addition of CDME. (Lower panel) Summary of CDME effects on phosphate-induced changes of Vm of IHKE-1 cells. The first bar pair summarizes the paired experiments with CDME given acutely. The second bar pair shows the summary of effects after incubation of IHKE-1 cells. Numbers in brackets indicate the number of experiments. *indicates statistical significance of the effect.

Incubation of the cells with CDME reduced the alanine-induced depolarization of V_m of IHKE-1 cells by 57 ± 4% (V_m for controls, 17.6 ±0.7 mV, n = 59;V_m for CDME, 7.6 ± 0.7 mV, n = 30; Figure 6). Incubation times varied between 30 min and 51 h, but there was no correlation between time and effect. To determine the minimal time needed to get an effect, we performed additional experiments with addition of 1 mM CDME to the bath solution during a time period of up to 30 min. During this time period, alanine-induced depolarizations in the presence of CDME (15.1 ± 1.2 mV) were not significantly different from those under control conditions before the addition of CDME (13.4 ± 0.9 mV; n = 8; Figure 3), indicating the onset of effects of CDME after at least 30 min of incubation.

Next we investigated the effect of CDME incubation on Na⁺/H⁺-exchanger activity. After addition of propionate, there was no significant difference in the initial depolarization of V_m between controls (7.1 ± 0.3 mV;n = 21) and CDME incubation (7.6 ± 1.6 mV; n = 8) (Figure 4), whereas the repolarization of V_m was inhibited by 78 ± 15% (controls, 2.9 ± 0.3 mV/min, n = 17; CDME, 0.6 ± 0.4 mV/min, n = 8;Figures 4 and 6).

Finally, we examined the phosphate-induced effect on V_m after incubation with 1 mM CDME. Similar to the effect after acute addition of CDME the inhibition was now 45 ±9% (controls, 9.7 ± 1.1 mV, n = 18; CDME, 5.3 ± 0.9 mV, n =9; Figures 5 and 6).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The pathophysiologic mechanisms that lead to the renal Fanconi syndrome in cystinosis are not resolved and continue to be a matter of investigation (7). Important insights were obtained in previous studies by using a model in which cells were loaded with CDME (7,11,13–16,18,19,30–33), which led to high intracellular cystine concentrations (10,30), predominantly in lysosomes (11,30). This model therefore mimics the biochemical hallmark of cystinosis (7). So far with this model uptake measurements with radiolabeled solutes were performed in rat (10,18,31,34) and rabbit (11,13,14,32,33) proximal tubules and with cultured porcine LLC-PK₁ cells (15,19,30). These studies showed an inhibition of volume absorption (13,14), reduction of the transepithelial potential difference (13), and inhibition of transport of different solutes (7,10,13–15). Most of proximal tubular transport is Na⁺-coupled; depending therefore on the Na⁺-gradient from the extra- to the intracellular space, the role of energy metabolism, Na⁺/K⁺-ATPase activity, intracellular phosphate, and ATP levels in this experimental model have been investigated (14,18,31,33,34). The activity of the Na⁺/K⁺-ATPase was not inhibited under V_max conditions (14,18), but a reduction in intracellular phosphate (32,35) and ATP content (14,18,34) was supposed to lead consecutively to an inhibition of Na⁺/K⁺-ATPase activity (14,18,34). According to these findings, an increase in the intracellular Na⁺-concentration parallel with a decrease in the intracellular K⁺-concentration in LLC-PK₁ cells (19) and for rat proximal tubules was reported (18). An increase in backflux due to an increase in proximal tubular permeability, as it was supposed previously for the maleic acid-induced Fanconi syndrome (17), could be excluded. This indicates that inhibition of transport was due to changes in active but not passive transport processes (13).

With the same experimental model, we performed for the first time electrophysiologic studies with the slow whole-cell patch clamp technique with immortalized human kidney epithelial cells derived from the proximal tubule (IHKE-1 cells) (23,25) and for which various morphologic (24,25), biochemical (20), and functional (22–24) characteristics of proximal tubule cells have been shown by several laboratories. This method allows real time and paired observations of CDME effects on V_m itself and on electrogenic transport systems such as the Na⁺-coupled alanine and phosphate transporter and indirectly via changes in cellular pH of the Na⁺/H⁺ exchanger. Despite the fact that we were able to detect the mRNA of an isoform of the Na⁺-glucose cotransporter (SGLT2) in these cells (unpublished observation), this transporter does not apparently contribute to the V_m. Consequently, effects of CDME on this transporter could not be studied electrophysiologically.

CDME acutely altered V_m of IHKE-1 cells in a concentration-dependent manner. An initial depolarization was mostly followed by a more pronounced hyperpolarization. By blocking K⁺-channels with Ba²⁺, we found that the secondary and more pronounced hyperpolarization was due to an activation of K⁺-channels, whereas the smaller initial depolarization was independent of changes in a K⁺-conductance. Further investigations are needed to learn if this transient depolarization is due to activation of nonselective cation channels or changes in the activity of Cl^--channels. The present experiments do not show whether CDME activated K⁺-channels directly or indirectly. These data do, however, show for the first time that CDME itself has an acute effect on V_m of human kidney epithelial cells. The mechanisms responsible for the acute effect on K⁺-channels in the proximal tubule need to be examined.

Next we investigated the acute effects of CDME loading on different Na⁺-dependent cotransporters of IHKE-1 cells. IHKE-1 cells possess different Na⁺-dependent amino acid transport systems in their apical membrane (22,23). We first investigated the effect of CDME on the Na⁺-alanine cotransporter, which leads to significant depolarization in these cells (23). Addition of 1 or 5 mM CDME or presence of 1 mM CDME for up to 30 min had no effect on the alanine-induced depolarization of V_m. Incubation of cells with 1 mM CDME for 30 min to 51 h (13 ± 3 h) resulted in an inhibition of the alanine-induced depolarization of V_m by 57%. These findings indicate that at least 30 min of CDME accumulation are necessary in these human proximal tubule cells to interfere with the Na⁺-alanine cotransporter. Previously a concentration- and time-dependent reduction of the uptake capacity of -methylglucoside, a nonmetabolized glucose analog, by LLC-PK₁ cells was shown for CDME (16). In earlier studies, different incubation times from 10 min (10) to 2 (19) or 5 d (15) and CDME-concentrations from 0.1 (15) to 2 mM (10,18,19,31) were used. In our experiments, incubation of IHKE-1 cells with 1 mM CDME for 3 h led to intracellular cystine contents of 20 nmol of cystine/mg protein similar to those found in cystinosis patients (36). The phenomenon of time- and concentration-dependent cell toxicity of CDME measured as lactate dehydrogenase (LDH) activity in the medium (30) or trypan blue staining (10) was described previously for cultured porcine cells (LLC-PK₁). The viability of the CDME-treated cells in our study was evident, as their V_m was not different or even slightly higher compared with control cells.

Foreman et al. (10) already showed excretion of different amino acids into the urine after CDME-treatment of rats in vivo and inhibition of uptake of the amino acids lysine, glycine, and taurine in in vitro experiments with rat proximal tubules. In contrast, Foreman et al. (31) showed in another study that the Na⁺-dependent uptake of proline was the same in brush border membranes prepared from rat proximal tubule vesicles in controls and after cystine loading. In that study, incubation times were in the range of minutes. This discrepancy fits with our findings that an incubation time of more than 30 min was necessary to affect the Na⁺-alanine transporter. Amino acids are nearly completely reabsorbed by the proximal tubule; inhibition of Na⁺-dependent amino acid transporters in this nephron segment therefore explains the urinary losses seen in cystinotic patients.

Comparable to the Na⁺-coupled alanine transport, CDME had no acute effect on Na⁺/H⁺-exchanger activity. Incubation with CDME for >30 min led, however, to an inhibition of Na⁺/H⁺-exchanger activity by 78%. An inhibition of the Na⁺/H⁺-exchanger was previously shown in cystine-loaded LLC-PK₁ cells (19). The Na⁺/H⁺-exchanger NHE-3 is responsible for a large fraction of the Na⁺ reabsorbed by the proximal tubule. Inhibition of this transporter is therefore an important factor for the urinary Na⁺-losses of cystinotic patients with Fanconi syndrome. As IHKE-1 cells express the NHE-3 mRNA (Figure 7), it seems highly likely that the observed decrease in Na⁺/H⁺ activity by CDME is due to interference with this isoform. The Na⁺/H⁺-exchanger, which functions in parallel with a Cl^--HCO₃^--exchanger, is also involved in the reabsorption of the filtered HCO₃^- in the proximal tubule. Thus, its inhibition also plays a major role in the severe HCO₃^- losses, leading to metabolic acidosis (proximal tubular acidosis type 2) seen in cystinotic patients.

As shown in Figures 7 and 8, the IHKE1-cells express the Na⁺-dependent phosphate transporter in their apical surface membrane, mostly at the microvilli. The existence of a high expression of this transporter, which is responsible for phosphate reabsorption and is typical for the proximal tubule but not other portions of the nephron, is further demonstrated by the large phosphate-dependent depolarization of the V_m. Such a contribution would not be expected if this transporter would only function as a housekeeping mechanism for phosphate uptake. Among the three investigated Na⁺-dependent transporters, this Na⁺-phosphate cotransporter was already inhibited by 37% after acute addition (1 to 2 min) of CDME. After incubation with CDME for >30 min, the inhibition of the phosphate-induced depolarization of V_m was 45%. In previous studies on rats, increased urinary excretion of phosphate after parenteral administration of CDME was demonstrated in vivo (10) and in vitro (unpublished observations of M. Baum) (7). In a recent in vivo study on the maleic acid-induced Fanconi syndrome, increased urinary excretion of phosphate was demonstrated as soon as 90 min after maleic acid injection in contrast to a decrease in NaP_i-2 mRNA levels only after 4 h and NaP_i-2 protein levels at 24 h, suggesting two different mechanisms for immediate and second-phase phosphaturia in that model (37).

Inhibition of the Na⁺-phosphate cotransporter is probably an important factor for the severe urinary phosphate losses in cystinotic patients, leading to the major problem of vitamin D-refractory rachitis and growth failure in addition to the chronic metabolic acidosis and dehydration due to NaHCO₃-losses and consequent reduced volume reabsorption. Fanconi syndromes develop in inborn as well as acquired disorders, and it is probable that different pathophysiologic mechanisms play a role in leading to the dysfunction of the proximal tubule (7). In the current study, we used the CDME model, which was used in most previous studies, on the pathogenesis of the Fanconi syndrome of cystinosis (7). This in vitro model might not duplicate the pathogenesis in vivo (7). However, most of proximal tubular solute transport across the luminal membrane is Na⁺-dependent; the effect of CDME on the above described substrate-induced changes in V_m in our experiments suggest therefore the involvement of two possible mechanisms: (1) Phosphate- and ATP-depletion with consecutive inhibition of Na⁺/K⁺-ATPase activity and dissipation of the Na⁺-gradient from the extra- to the intracellular space, which drives the Na⁺-coupled solute transport (7,13,14,18,32–34);(2) reduction in the number of luminal Na⁺-dependent transporters by hindered trafficking of transporters to the luminal membrane or direct inhibition of luminal transporters by a general unspecific or cytotoxic effect of CDME.

The current study cannot clearly differentiate between these possibilities. In all cases, changes in Na⁺-coupled electrogenic transport of solutes would lead to respective changes in V_m. Our findings suggest that it is probable that different fast and slow mechanisms are involved. The acute inhibition of the Na⁺-phosphate cotransporter by CDME in our experiments might suggest that this transporter is the most sensitive to cell loading with cystine. This might be due to a stronger dependence of the Na⁺-phosphate cotransporter on the Na⁺-gradient or to direct inhibition of the luminal transporter. A decrease in the number of luminal transporters is not to be expected during that short time period of minutes. In contrast, inhibition of all three Na⁺-dependent transporters after hours of incubation with CDME may be due to additional reduction in the number of luminal transporters, as it was previously described for Na⁺-dependent glucose transporters of LLC-PK₁ (15) cells and rat brush border membrane vesicles (16), or to a more general cytotoxicity of CDME with consecutive inhibition of multiple transporters.

In conclusion, we demonstrate for the first time that CDME itself has an acute effect on human proximal tubule cells by altering V_m through activation of K⁺ channels and that at least three Na⁺-dependent transporters of IHKE-1 cells are inhibited, the most sensitive apparently being the Na⁺-phosphate cotransporter. This suggests the involvement of phosphate depletion and dissipation of the Na⁺-gradient in the initial development of the Fanconi syndrome of cystinosis.

	Acknowledgments

 
We gratefully acknowledge the expert technical work of Ingrid Salwicek, Heike Stegemann, and Ulrich Siegel. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (KI 976/7–1).

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gahl WA, Bashan N, Tietze F, Bernardini I, Schulman JD: Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science 217: 1263–1265, 1982[Abstract/Free Full Text]
2. Steinherz R, Tietze F, Gahl WA, Triche TJ, Chiang H, Modesti A, Schulman JD: Cystine accumulation and clearance by normal and cystinotic leukocytes exposed to cystine dimethyl ester. Proc Natl Acad Sci USA 79: 4446–4450, 1982[Abstract/Free Full Text]
3. Gahl WA, Tietze F, Bashan N, Bernardini I, Raiford D, Schulman JD: Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions. Biochem J 216: 393–400, 1983[Medline]
4. Jonas AJ, Smith ML, Schneider JA: ATP-dependent lysosomal cystine efflux is defective in cystinosis. J Biol Chem 257: 13185–13188, 1982[Abstract/Free Full Text]
5. Schneider JA, Bradley K, Seegmiller JE: Increased cystine in leukocytes from individuals homozygous and heterozygous for cystinosis. Science 157: 1321–1322, 1967[Abstract/Free Full Text]
6. Town M, Jean G, Cherqui S, Attard M, Forestier L, Whitmore SA, Callen DF, Gribouval O, Broyer M, Bates GP, van’t Hoff W, Antignac C: A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet 18: 319–324, 1998[CrossRef][Medline]
7. Baum M: The Fanconi syndrome of cystinosis: Insights into the pathophysiology. Pediatr Nephrol 12: 492–497, 1998[CrossRef][Medline]
8. Goldman R, Kaplan A: Rupture of rat liver lysosomes mediated by L-amino acid esters. Biochim Biophys Acta 318: 205–216, 1973[Medline]
9. Reeves JP: Accumulation of amino acids by lysosomes incubated with amino acid methyl esters. J Biol Chem 254: 8914–8921, 1979[Abstract/Free Full Text]
10. Foreman JW, Bowring MA, Lee J, States B, Segal S: Effect of cystine dimethylester on renal solute handling and isolated renal tubule transport in the rat: a new model of the Fanconi syndrome. Metabolism 36: 1185–1191, 1987[CrossRef][Medline]
11. Sakarcan A, Timmons C, Baum M: Intracellular distribution of cystine in cystine-loaded proximal tubules. Pediatr Res 35: 447–450, 1994[Medline]
12. Gahl WA, Thoene JG, Schneider JA: Cystinosis: A disorder of lysosomal membrane transport. In: The Metabolic and Molecular Bases of Inherited Disease, 8th edition, edited by Scriver CR, Beaudet AL, Valle D, Sly WS, New York, McGraw Hill, 2001, pp 5085–5108
13. Salmon RF, Baum M: Intracellular cystine loading inhibits transport in the rabbit proximal convoluted tubule. J Clin Invest 85: 340–344, 1990
14. Coor C, Salmon RF, Quigley R, Marver D, Baum M: Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading. J Clin Invest 87: 955–961, 1991
15. Ben-Nun A, Bashan N, Potashnik R, Moran A: Cystine accumulation impairs glucose transport in LLC-PK1 cells. Cell Physiol Biochem 1: 294–304, 1991
16. Ben-Nun A, Bashan N, Potashnik R, Cohen-Luria R, and Moran, A: Cystine loading induces Fanconi’s syndrom in rats: In vivo and vesicle studies. Am J Physiol 265: F839–F844, 1993
17. Bergeron M, Dubord L, Hausser C, Schwab C: Membrane permeability as a cause of transport defects in experimental Fanconi syndrome. A new hypothesis. J Clin Invest 57: 1181–1189, 1976
18. Foreman JW, Benson LL, Wellons M, Avner ED, Sweeney W, Nissim I, Nissim I: Metabolic studies of rat renal tubule cells loaded with cystine: The cystine dimethylester model of cystinosis. J Am Soc Nephrol 6: 269–272, 1995[Abstract]
19. Ben-Nun A, Bashan N, Potashnik R, Cohen-Luria R, and Moran, A: Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK₁ cells. Am J Physiol 263: C516–C520, 1992
20. Tveito G, Hansteen IL, Dalen H, Haugen A: Immortalization of normal human kidney epithelial cells by nickel(II). Cancer Res 49: 1829–1835, 1989[Abstract/Free Full Text]
21. Jessen H, Roigaard H, Riahi-Esfahani S, Jacobsen C: A comparative study on the uptake of -aminoisobutyric acid by normal and immortalized human embryonic kidney cells from proximal tubule. Biochim Biophys Acta 1190: 279–288, 1994[Medline]
22. Jessen H, Roigaard H, Jacobsen C: Uptake of neutral - and -amino acids by human proximal tubular cells. Biochim Biophys Acta 1282: 225–232, 1996[Medline]
23. Hirsch JR, Gonska T, Waldegger S, Lang F, Schlatter E: Na⁺-dependent and -independent amino acid transport systems in immortalized human kidney epithelial cells derived from the proximal tubule. Kidney Blood Press Res 21: 50–58, 1998[CrossRef][Medline]
24. Stachon A, Hohage H, Feidt C, Schlatter E: Characterization of organic cation transport across the apical membrane of proximal tubular cells with the fluorescent dye 4-Di-1-ASP. Cell Physiol Biochem 7: 264–274, 1997[CrossRef]
25. Hirsch JR, Meyer M, Magert HJ, Forssmann WG, Mollerup S, Herter P, Weber G, Cermak R, Ankorina-Stark I, Schlatter E, Kruhoffer M: cGMP-dependent and -independent inhibition of a K⁺: conductance by natriuretic peptides: Molecular and functional studies in human proximal tubule cells. J Am Soc Nephrol 10: 472–480, 1999[Abstract/Free Full Text]
26. Drumm K, Gassner B, Silbernagl S, Gekle, M: Albumin in the mg/l-range activates NF-kappaB in renal proximal tubule-derived cell lines via tyrosine kinases and proteinkinase. C Eur J Med Res 6: 247–258, 2001
27. Kleta R, Mohrmann M, Schlatter E: Effects of cell differentiation on ion conductances and membrane voltage in LLC-PK1 cells. Pflügers Arch 429: 370–377, 1995[CrossRef][Medline]
28. Kohl B, Herter P, Hulseweh B, Elger M, Hentschel H, Kinne RK, Werner A: Na-Pi cotransport in flounder: Same transport system in kidney and intestine. Am J Physiol 270: F937–F944, 1996
29. Schlatter E, Ankorina-Stark I, Haxelmans S, Hohage H: Moxonidine inhibits Na⁺/H⁺ exchange in proximal tubule cells and cortical collecting duct. Kidney Int 52: 454–459, 1997[Medline]
30. Moran A, Ben Nun A, Potashnik R, Bashan N: Renal cells in culture as a model for cystinosis. J Basic Clin Physiol Pharmacol 1: 357–372, 1990[Medline]
31. Foreman JW, Benson LL: Effect of cystine loading on substrate oxidation by rat renal tubules. Pediatr Nephrol 4: 236–239, 1990[CrossRef][Medline]
32. Bajaj G, Baum M: Proximal tubule dysfunction in cystine-loaded tubules: Effect of phosphate and metabolic substrates. Am J Physiol 271: F717–F722, 1996
33. Sakarcan A, Aricheta R, Baum M: Intracellular cystine loading causes proximal tubule respiratory dysfunction: effect of glycine. Pediatr Res 32: 710–713, 1992[Medline]
34. Ben Nun A, Bashan N, Potashnik R, Cohen-Luria R, Moran A: Cystine loading induces Fanconi’s syndrome in rats: In vivo and vesicle studies. Am J Physiol 265: F839–F844, 1993
35. Al-Bander H, Etheredge SB, Paukert T, Humphreys MH, Morris RC Jr: Phosphate loading attenuates renal tubular dysfunction induced by maleic acid in the dog. Am J Physiol 248: F513–F521, 1985
36. Oude Elferink RP, Harms E, Strijland A, Tager JM: The intralysosomal pH in cultured human skin fibroblasts in relation to cystine accumulation in patients with cystinosis. Biochem Biophys Res Commun 116: 154–161, 1983[CrossRef][Medline]
37. Haviv YS, Wald H, Levi M, Dranitzki-Elhalel M, Popovtzer MM: Late-onset downregulation of NaPi-2 in experimental Fanconi syndrome. Pediatr Nephrol 16: 412–416, 2001[CrossRef][Medline]

This article has been cited by other articles:

				A. Thirumurugan, A. Thewles, R. D. Gilbert, S.-A. Hulton, D. V. Milford, C. J. Lote, and C. M. Taylor Urinary L-lactate excretion is increased in renal Fanconi syndrome Nephrol. Dial. Transplant., July 1, 2004; 19(7): 1767 - 1773. [Abstract] [Full Text] [PDF]

				M. Park, A. Helip-Wooley, and J. Thoene Lysosomal Cystine Storage Augments Apoptosis in Cultured Human Fibroblasts and Renal Tubular Epithelial Cells J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2878 - 2887. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ÇCetinkaya, I. Articles by Kleta, R. Search for Related Content PubMed PubMed Citation Articles by ÇCetinkaya, I. Articles by Kleta, R.