Characterization of a Na+–Ca2+ exchanger in podocytes

Karl-Georg Fischer1,, Nils Jonas1, Florian Poschenrieder1, Clemens Cohen2, Matthias Kretzler2, Stefan Greiber1 and Hermann Pavenstädt1

1 University Hospital Freiburg, Department of Medicine, Division of Nephrology and General Medicine, Freiburg, Germany and 2 Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University Munich, Munich, Germany



   Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Knowledge about Ca2+ extrusion mechanisms in podocytes is limited. The aim of the study was to test whether a Na+–Ca2+ exchanger (NCX) is present in differentiated podocytes and if so to examine its regulatory properties.

Methods. Intracellular Ca2+ concentration ([Ca2+]i) and intracellular pH were measured microspectrofluorometrically in single podocytes. Expression of NCX mRNA was studied by reverse transcription–polymerase chain reaction. NCX protein expression was investigated by immunocytochemistry.

Results. Substitution of extracellular Na+ (from 145 to 0, 5, 10, 20, and 30 mM) with N-methyl-D-glucamine resulted in a Na+ concentration-dependent, reversible increase of [Ca2+]i. Complete extracellular Na+ substitution (0 Na+) increased [Ca2+]i reversibly from 95±5 to 275±16 and back to 66±5 nM (n=205). Raising the intracellular Na+ concentration by application of 50 µM monensin increased [Ca2+]i from 105±22 to 192±45 nM (n=12). The [Ca2+]i response induced by a low Na+ concentration required extracellular Ca2+ and did not correlate with changes of intracellular pH. The effect was blocked by the NCX inhibitor benzamil (IC50~100 nM). Neither flufenamate (100 µM, n=6), a blocker of non-selective cation channels, nor Hoe 694 (1 µM, n=6), an inhibitor of the Na+–H+ exchanger, did significantly influence the [Ca2+]i response induced by extracellular Na+ depletion. Activation of protein kinase C (PKC) by short-term application (5 min) of phorbol 12-myristate-13-acetate (PMA; 10 nM, n=4; 100 nM, n=7) inhibited Na+–Ca2+ exchange, whereas PKC inhibition by long-term incubation (24 h) with PMA (100 nM, n=9) or bisindolylmaleimide I (100 nM, n=11) both increased Na+–Ca2+ exchange, respectively. Expression of NCX mRNA was detected both in cultured differentiated podocytes and in podocytes directly pulled off from glomeruli ex vivo. NCX protein expression was detected by immunocytochemistry. In a different series of experiments, we studied the potential involvement of the exchanger in podocyte injury induced by the aminonucleoside puromycin. Pre-treatment of podocytes with 0.3 mM puromycin for 24 h significantly reduced the [Ca2+]i response induced by extracellular Na+ depletion (n=56). Compared with mRNA expression of the housekeeping gene GAPDH, NCX mRNA expression was significantly reduced by puromycin.

Conclusion. Our results demonstrate the presence of a Na+–Ca2+ exchanger in podocytes and its regulation by PKC. Inhibition of Na+–Ca2+ exchange by puromycin may contribute to podocyte injury in PAN nephrosis.

Keywords: immunocytochemistry; intracellular Ca2+ concentration; intracellular pH; Na+–Ca2+ exchange; podocyte; protein kinase C; puromycin aminonucleoside



   Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The intracellular calcium concentration ([Ca2+]i) is an important second messenger of many physiological functions and is known to mediate hormonal signalling in a variety of cell types. In order to achieve this purpose, intracellular Ca2+ homeostasis is accomplished by mechanisms that control Ca2+ fluxes across the sarcoplasmatic reticulum and the cell plasma membrane, respectively. An increase of [Ca2+]i is induced by Ca2+ release from intracellular stores and by an influx of Ca2+ from the extracellular space through selective or non-selective Ca2+ permeable channels, whereas the storage of [Ca2+]i in intracellular organelles and extrusion of Ca2+ across the cell plasma membrane reduce [Ca2+]i [1]. The major Ca2+ extrusion pathways are the plasma membrane Ca2+-ATPase and the Na+–Ca2+ exchanger [1]. The Na+–Ca2+ exchanger is known to operate reversibly, i.e. Ca2+ can be transported in an inward or outward direction across the plasma membrane in exchange for Na+, depending on the electrochemical gradients for Na+ and Ca2+. It was first identified in excitable cells, but recent studies suggest that it also operates in non-excitable cells, such as tubular and pancreatic duct cells. Within the glomerulus, the presence of a Na+–Ca2+ exchanger has been reported in mesangial cells [2], whereas the exchanger does not seem to operate in glomerular endothelial cells in culture [3].

The knowledge about Ca2+ extrusion mechanisms in podocytes is limited. The podocyte is the most differentiated cell type in the glomerulus, which stabilizes the glomerular tuft architecture and might participate in the regulation of the ultrafiltration coefficient Kf. Recently, a number of proteins being expressed in podocytes have been shown to play an important role in maintaining the structure of the glomerular filtration barrier, the alteration of which is associated with proteinuria. The podocyte is the target cell of injury in acute proteinuric glomerular diseases, such as minimal change nephropathy, focal sclerosis, and membraneous nephropathy [4]. In addition, the development of diabetic nephropathy [5] and the progression of chronic renal failure are both associated with podocyte damage. Concerning the regulation of [Ca2+]i in podocytes, it has been shown that different vasoactive hormones modulate [Ca2+]i in these cells [68].

Addressing Ca2+ extrusion mechanisms, the aim of this study was to test whether podocytes with a differentiated morphology possess a functional Na+–Ca2+ exchanger and if so, to investigate its regulatory properties. In addition, we addressed the potential involvement of the exchanger in podocyte damage induced by the aminonucleoside puromycin [9].



   Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell culture of mouse podocytes
Cultivation of mouse podocytes was as reported previously [10]. In brief, podocytes were maintained in RPMI 1640 medium (Boehringer, Mannheim, Germany) supplemented with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. To propagate undifferentiated podocytes, the culture medium was supplemented with 10 U/ml mouse recombinant {gamma}-interferon (Sigma, Deisenhofen, Germany) to enhance expression of the T-antigen, and cells were cultivated at 33°C (permissive conditions). To induce differentiation, podocytes were cultured on type I-collagen at 37°C without {gamma}-interferon (non-permissive conditions) for 14 days [10]. Cells were used between passages 6 and 30 and showed an arborized morphology. Cells stained positive for the podocyte markers WT-1, synaptopodin, nephrin, and p57. In addition, mRNA expression of the podocyte markers CD2AP and synaptopodin was detected by reverse transcription–polymerase chain reaction (RT–PCR).

Measurements of [Ca2+]i and intracellular pH (pHi)
Measurements of [Ca2+]i with the Ca2+-sensitive dye fura-2 were performed in single podocytes with an inverted fluorescence microscope set up. The system allows fura-2 fluorescence measurements at the single cell level. In brief, fura-2 fluorescence (excitation wavelengths: 340, 360, 380 nm, emission wavelength: 510 nm, all filters Lys and Optiks, Lyngby, Denmark) was measured in podocytes mounted in a bath on a stage of an inverted fluorescence microscope (bath temperature: 37°C, bath solution changes with 1 Hz). The set up was based on a high-speed filter wheel and a single photon tube (Hamamatsu, Herrsching, Germany) allowing a time resolution of up to 200 Hz. The filter wheel was run with 10 cycles/s. Fluorescence signal was amplified, digitized, and averaged every second. The fluorescence signal measured at 360 nm excitation, the isosbestic point, allowed for the detection of major perturbances in the system. Podocytes were incubated with fura-2 acetoxymethyl ester (fura-2/AM, 5 µM; Sigma) for 45 min at room temperature. Thereafter, a single cell was positioned under the measuring diaphragm, with the aperture covering approximately three-quarters of the cell. The measured noise signal and autofluorescence of the cell did not exceed 1% of the fura-2 fluorescence and the average autofluorescence value of six experiments was subtracted before calculation of the fluorescence emission ratio at 340/380 nm excitation. The 340/380 nm ratio is an estimate of [Ca2+]i. In 18 out of 55 experiments, calibration of the fura-2 fluorescence signal was successful using the Ca2+ ionophore ionomycin (5 µM) and low and high Ca2+ buffers. [Ca2+]i was calculated from the fluorescence ratio according to the equation described by Grynkiewicz et al. [11]. A KD for the fura-2–Ca2+ complex of 224 nM (37°C) was assumed. Fluorescence data are mainly given as fluorescence ratio 340/380 nm.

In separate experiments, cells were incubated for 30 min with the acetoxymethyl ester form of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM, 1 µM; Sigma). The emission intensity was measured at 530 nm after excitation at 436 and 488 nm. The 488/436 ratio is an estimate of the intracellular pH (pHi). Calibrations were performed with 10 µM nigericin in bath solutions with 145 mM K+ and different pH. Fluorescence data are given mainly as fluorescence ratio 488/436 nm.

RT–PCR studies in cultured differentiated mouse podocyte
RNA preparation
Total cellular RNA was isolated from mouse podocytes by guanidinium–acid phenol–chloroform extraction. The amount of mRNA was evaluated by spectrophotometry at 260 nm. The integrity of the mRNA was analysed after electrophoresis with a 1% agarose gel, staining with ethidium bromide and visualization by UV irradiation.

Reverse transcription
For first strand synthesis, total RNA from podocytes was mixed in 1xRT buffer (3.0 mM MgCl2, 75 mM KCl, 50 mM Tris–HCl, pH 8.3) and completed with 0.5 mM dNTP, 10 µM random hexanucleotide primer, 10 mM dithiothreitol, 0.02 U/ng (total RNA) RNAse inhibitor, and 100 U/µg (total RNA) MMLV reverse transcriptase (reverse transcriptase was omitted in some experiments to control for amplification of contaminating DNA). The RT was performed at 42°C for 60 min, followed by denaturation at 95°C for 5 min.

PCR amplification
PCR was performed in duplicates in a total volume of 20 µl, each containing 4 µl of RT reaction mixture (with 40 ng of cDNA) and 13.8 µl of PCR master mixture (16 mM Tris–HCl, 40 mM KCl, 0.4 mM MgCl2). The mixture was overlaid with mineral oil and heated for 1 min at 94°C. The samples were kept at 80°C until 2.2 µl of the starter mixture, containing 50 µM each of sense and antisense primer and 1 U Taq DNA polymerase, were added. The PCR amplification was performed in a Perkin Elmer Thermocycler 480. The PCR cycle profile included denaturation for 1 min at 94°C, annealing for 1 min at 60°C, and extension for 1 min at 72°C. Thirty cycles were performed to amplify mRNA for the Na+–Ca2+ exchanger and synaptopodin (annealing at 62°C), 15–20 cycles were performed to amplify mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Na+–Ca2+ exchanger mRNA was demonstrated with two different primer pairs. We selected primers from the 5' and 3' region of the Na+–Ca2+ exchanger gene; one primer pair amplified a 301 base pair (bp) fragment (‘Ex1’) corresponding to bp 647–947 of the gene, whereas the other primer pair amplified a 489 bp fragment (‘Ex2’) corresponding to bp 2310–2798 of the gene. Quantitative expression of Na+–Ca2+ exchanger mRNA was analysed with primer pair ‘Ex2’. RT–PCR for synaptopodin was performed to demonstrate that podocyte mRNA was studied. To control for variations in the amount of mRNA used for RT–PCR analysis and the efficiency of the RT reaction, expression of the housekeeping gene GAPDH was also analysed. Primers were selected from mouse sequences, which either had been deposited in the NIH/NCBI database (accession number given in square brackets) or had already been published elsewhere (synaptopodin). The primers were: Na+–Ca2+ exchanger [U70033]: f-TGTGTTTACGTGGTCCCTGA; r-TGGAAGCTGGTCTGTCTCCT (expected product size: 301 bp, named ‘Ex1’) or f-GGACCAACAGCTGGAGAGAG; r-ACACTTTGAACTGTTCCCCG (expected product size: 489 bp, named ‘Ex2’); synaptopodin [12]: f-GTCAAGGAACCTGCCAAGG; r-AGAAGGAAGGCCTGGGAG (expected product size: 312 bp, named ‘Syn’); GAPDH [M32599]: f-ACCCAGAAGACTGTGGATGG; r-AGGTGGAAGAGTGGGAGTTG (product size: 334 bp, named ‘GAPDH’). PCR amplification of RT reactions without reverse transcriptase revealed no PCR product, thereby excluding any amplification of genomic DNA.

Quantitative analysis
The amplification products of 10 µl of each PCR reaction were separated on a 1.5% agarose gel, stained with ethidium bromide (0.5 µg/ml), visualized by UV irradiation, and photographed with a Polaroid film 667. The film was taken to evaluate the band densities by volume integration using a Hewlett Packard IIcx flatbed scanner and a computer-based imaging software (ImageQuant; Molecular Dynamics, Krefeld, Germany). The results are expressed either as arbitrary densitometry units or alternatively as the respective Na+–Ca2+ exchanger to GAPDH ratio.

Single cell RT–PCR studies on mouse podocytes isolated from freshly microdissected glomeruli
The isolation and RT–PCR of single podocytes was performed as described [13]. In brief, kidneys of CD-1 mice were removed after death and the cortical tissue minced. Five to ten microdissected glomeruli were neuraminidase (Sigma) treated and transferred onto the plate of an inverted microscope. One glomerulus was fixed via a holding pipette. Single podocytes were selectively harvested by aspiration of the cell into a micropipette. After one freeze–thaw step an in situ random primed RT was performed on the content of the micropipette. PCR was performed using the following sequence-specific oligonucleotide primers and 50 amplification cycles: Na+–Ca2+ exchanger [U70033]: f-TGTGTTTACGTGGTCCCTGA; r-TGGAAGCTGGTCTGTCTCCT (expected product size: 301 bp, named ‘NaCa’). ß-Actin [M12481]: f-TGTTACCAACTGGGACGACA, r-TCTCAGCTGTGGTGGTGAAG (expected product size: 392 bp, named, ‘ß-Act.’). To control for DNA yield, the housekeeping gene ß-actin was analysed in aliquots of each sample. The amplified cDNA was run on a 5% polyacrylamid gel, stained with VistraGreen (Amersham, Braunschweig, Germany) and visualized with ImageQuant software on a Storm Fluorophosphorimager (both Molecular Dynamics).

Immunocytochemistry
For immunocytochemistry, cells were grown on collagen A-coated glass coverslips and fixed using 1% formaldehyde (10 min, room temperature). Subsequently, cells were permeabilized with 0.1% NP-40 (Sigma) (20 min, room temperature). After rinsing with PBS, non-specific binding sites were blocked in blocking solution (2% FCS, 2% BSA, and 0.2% gelatine in PBS) for 20 min. Following rinsing with PBS, incubation with primary antibodies recognizing Na+–Ca2+ exchanger type NCX1 (Chemicon, Temecula, USA; mouse, dilution 1:100) was performed (60 min, room temperature). After washing with PBS, antigen–antibody complexes were visualized using fluorochrome-conjugated secondary anti-mouse antibodies (Alexa Fluor 488 goat anti-mouse IgG, Molecular Probes; dilution 1:100, 60 min, room temperature). Sections were washed with PBS, rinsed in H2O, and mounted using 15% Mowiol 4-88 (Calbiochem, San Diego, USA), 50% glycerol in PBS. After overnight drying, specimens were analysed using a Zeiss Axioscope photomicroscope (Zeiss, Göttingen, Germany) with appropriate fluorescence filters.

Statistical analyses
Data are given as mean±SEM, where n refers to the number of experiments. Dependent on the respective set of data, paired t-test, unpaired t-test, or Wilcoxon test, respectively, were used to compare mean values within one experimental series. A P value <=0.05 was considered statistically significant.



   Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Removal of extracellular Na+ increases the cytosolic Ca2+ concentration in podocytes
Exposure of podocytes to a nominally Na+-free bath solution (substitution of Na+ by the membrane-impermeable cation N-methyl-D-glucamine) resulted in a reversible increase of [Ca2+]i from 95±5 to 275±16 and back to 66±5 nm (n=205). Figure 1AGo shows an original recording of the effect of reducing extracellular Na+ from 145 to 0 mM on [Ca2+]i of a single differentiated podocyte. The magnitude of the [Ca2+]i increase was strongly dependent on the extracellular Na+ concentration (summary, Figure 1BGo).



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Fig. 1.  Removal of extracellular Na+ increases the cytosolic Ca2+ concentration in podocytes. (A) Original recording of the effect of a complete substitution of extracellular Na+ by N-methyl-D-glucamine (0 Na+) on the cytosolic Ca2+ concentration of a single differentiated podocyte. (B) Summary of the cytosolic Ca2+ response of podocytes to different extracellular Na+ concentrations.

 

The [Ca2+]i increase induced by Na+ depletion depends on extracellular Ca2+
Reduction of extracellular Ca2+ from 1 mM to 1 µM markedly reduced the [Ca2+]i increase induced by a Na+-free bath solution (n=13, data not shown).

An increase of intracellular Na+ elevates [Ca2+]i
If the [Ca2+]i increase induced by depletion of extracellular Na+ was due to an altered activity of the Na+–Ca2+ exchanger, an increase of intracellular Na+ should likewise result in elevation of [Ca2+]i. In this regard, addition of the Na+ ionophore monensin (50 µM) led to an increase of [Ca2+]i from 105±22 to 192±45 nM (n=12, data not shown).

The [Ca2+]i increase induced by Na+ depletion does not result from changes of pHi
The observed [Ca2+]i increase induced by removal of extracellular Na+ could have resulted from changes of intracellular pH, e.g. by inhibition of the Na+–H+ exchanger. To clarify this issue, we additionally performed fluorescent experiments with the pH-sensitive dye BCECF. The NH3/ pre-pulse technique was applied to induce marked intracellular pH changes. As estimated from the 488/436 nm BCECF fluorescence ratio in Figure 2AGo, addition of ammonium chloride (NH3/ 20 mM) induced a rapid alkalinization by 0.5 pH units (from pHi 7.16±0.16 to pHi 7.66±0.28), which was followed by a rapid acidification below the pre-pulse baseline after its removal (n=19). As shown in the corresponding Ca2+ experiments summarized in Figure 2BGo, the same manoeuvre led to a small decrease of [Ca2+]i, followed by a [Ca2+]i increase slightly above the pre-pulse level after removal of the compound (n=9). Removal of extracellular Na+ did not result in a significant change of pHi (Figure 2CGo, n=27), whereas it markedly elevated [Ca2+]i (Figure 2DGo, n=205). To further rule out a relevant participation of a Na+–H+ exchanger in the [Ca2+]i response to a Na+-free bath solution, we investigated the influence of an extracellular Na+ removal on [Ca2+]i in the presence of Hoe 694, a specific blocker of the Na+–H+ exchanger. Hoe 694 at a concentration of 1 µM did not inhibit the [Ca2+]i response to extracellular Na+ depletion (n=6, data not shown).



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Fig. 2.  The effect of extracellular Na+ depletion on cytosolic Ca2+ concentration is not mediated by changes in cytosolic pH. Summary of the effect of an addition of NH3/ (20 mM) on pHi (A, n=19) and [Ca2+]i (B, n=9). Summary of the effect of extracellular Na+ depletion (0 Na+) on pHi (C, n=27) and [Ca2+]i (D, n=205); *P<0.05.

 

Benzamil inhibits the [Ca2+]i response to extracellular Na+ depletion
Pre-treatment of podocytes with benzamil, a known inhibitor of the Na+–Ca2+ exchanger [14], for 5 min irreversibly inhibited the [Ca2+]i response induced by removal of extracellular Na+. Benzamil in a concentration of 25 µM inhibited the [Ca2+]i response to depletion of extracellular Na+ by 92±4% (n=4). The effect of benzamil was concentration-dependent with an IC50 of ~100 nM. Figure 3AGo shows an original recording, Figure 3BGo summarizes the data. Flufenamate (100 µM), a blocker of non-selective cation channels, did not inhibit the [Ca2+]i response to removal of extracellular Na+ (n=6, data not shown).



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Fig. 3.  The Na+–Ca2+ exchanger blocker benzamil inhibits the cytosolic Ca2+ response to extracellular Na+ depletion. (A) Original recording of the effect of benzamil (25 µM) on the cytosolic Ca2+ response to extracellular Na+ depletion. (B) Concentration response curve of the effect of benzamil (n=4–16; IC50~100 nM).

 

Podocytes express mRNA for the Na+–Ca2+ exchanger
As depicted in Figure 4AGo, mRNA for the Na+–Ca2+ exchanger could be detected by RT–PCR in cultured differentiated mouse podocytes. mRNA for synaptopodin, a specific marker located in the foot processes of differentiated podocytes [12], was likewise detected, confirming the template for amplification of NCX to be truly derived from podocytes. RT-negative PCR reactions did not show an amplification product, thus excluding the amplification of contaminating DNA. Likewise, Na+–Ca2+ exchanger cDNA was detected in four out of seven ß-actin-positive single podocytes isolated from freshly microdissected glomeruli (Figure 4BGo) and in microdissected glomeruli. PCR product identity was confirmed by restriction enzyme digestion. Control PCRs of 7 ng mouse cortex RNA (RT-), medium aspirated near to the glomeruli (Med, n=5), and water controls (H2O) were all negative for Na+–Ca2+ exchanger as well as for ß-actin (Figure 4BGo).



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Fig. 4.  Podocytes express Na+–Ca2+ exchanger mRNA. (A) Cultured differentiated podocytes. RT–PCR analysis showing a 301 bp (Ex1) and a 489 bp (Ex2) cDNA fragment for the Na+–Ca2+ exchanger and a 312 bp cDNA fragment (Syn) for synaptopodin. M, 100 bp ladder; RT, reverse transcriptase. (B) Podocytes freshly isolated from a microdissected glomerulus. Single-cell PCR analysis showing a 301 bp cDNA fragment (NaCa) for the Na+–Ca2+ exchanger and a 390 bp cDNA fragment (ß-Act.) for ß-actin amplified separately from the same podocyte. Medium aspirated near to a glomerulus (Med.), water (H2O) and 7 ng RNA (RT-) each were negative for the Na+–Ca2+ exchanger and ß-actin, respectively.

 

Podocytes express Na+–Ca2+ exchanger protein
Na+–Ca2+ exchanger protein expression on podocytes was demonstrated by immunocytochemistry (Figure 5Go).



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Fig. 5.  Podocytes express Na+–Ca2+ exchanger protein. Immunofluorescence staining for the Na+–Ca2+ exchanger in podocytes. (A) Primary antibody (mouse anti-NCX1, dilution 1/100). (B) Secondary antibody (Alexa Fluor 488 goat anti-mouse, dilution 1/100). (C) Primary and secondary antibody.

 

Activation of protein kinase C (PKC) inhibits the [Ca2+]i response induced by removal of extracellular Na+
Figure 6Go summarizes the impact of PKC activity on the [Ca2+]i response to removal of extracellular Na+. Stimulation of PKC by short-term application (5 min) of both 10 (n=4) and 100 nM (n=7) phorbol 12-myristate-13-acetate (PMA) significantly inhibited the [Ca2+]i response to extracellular Na+ depletion (Figure 6AGo). In contrast, inhibition of PKC by long-term incubation (24 h) of podocytes both with 100 nM PMA (n=9, Figure 6BGo) or with the PKC inhibitor bisindolylmaleimide I (BIM, 100 nM, n=11, Figure 6CGo) increased the [Ca2+]i response induced by extracellular Na+ removal, respectively.



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Fig. 6.  PKC modulates Na+–Ca2+ exchanger activity in podocytes. Stimulation of PKC by short-term application (5 min) of phorbol 12-myristate-13-acetate (PMA, A) reduces the cytosolic Ca2+ response to extracellular Na+ depletion. Inhibition of PKC by long-term application (24 h) of PMA (B) or BIM (C) increases the cytosolic Ca2+ response to extracellular Na+ depletion; *P<0.05.

 

The aminonucleoside puromycin alters function and expression of the Na+–Ca2+ exchanger
As revealed by interference contrast microscopy, incubation with puromycin (0.3 mM) induced marked morphological changes in podocytes. Compared with cells grown under control conditions, which showed the typical arborized phenotype, after >24 h of incubation with puromycin retraction of foot processes and first perinuclear vacuoles were observed. After 40 h of incubation and often earlier, ~70% of the cells had detached from the culture dish; the remaining cells showed markedly broadened foot processes and an increasing number of perinuclear vacuoles. Acute exposure to puromycin (0.3 mM) did not change the [Ca2+]i of podocytes (n=6, data not shown). After incubation with puromycin (0.3 mM) for 24 h, the [Ca2+]i response of podocytes induced by extracellular Na+ removal was significantly diminished by 57±4% compared with podocytes grown under control conditions (n=54 and n=56, respectively; summary, Figure 7AGo). To address whether puromycin impairs expression of the Na+–Ca2+ exchanger, mRNA of three different sets of experiments was analysed in duplicates by RT–PCR. Compared with mRNA expression of the housekeeping gene GAPDH, mRNA levels of the Na+–Ca2+ exchanger were significantly reduced by pre-treatment with puromycin (0.3 mM) for 24 h. Figure 7BGo shows a PCR analysis of one RNA sample, Table 1Go summarizes the data of the quantitative analysis.



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Fig. 7.  Puromycin alters function and expression of the Na+–Ca2+ exchanger in podocytes. (A) Puromycin diminishes the cytosolic Ca2+ response to removal of extracellular Na+. Podocytes were grown for 24 h either under control conditions (C) or in the presence of 0.3 mM puromycin (Puro). Incubation with puromycin significantly diminishes the cytosolic Ca2+ response to removal of extracellular Na+. Summary of the experiments (*P<0.05). (B) Puromycin reduces mRNA expression of the Na+–Ca2+ exchanger. RNA was extracted from mouse podocytes that had been cultured for 24 h either under control conditions (C) or in culture medium containing 0.3 mM puromycin (Puro). RT–PCR analysis showing a 489 bp cDNA fragment for the Na+–Ca2+ exchanger (NCX) and a 334 bp cDNA fragment for the housekeeping gene GAPDH (GAPDH). M, 100 bp ladder; RT, reverse transcriptase. Compared with mRNA expression of the housekeeping gene GAPDH, mRNA levels of the Na+–Ca2+ exchanger were significantly reduced by puromycin.

 

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Table 1.  Quantification of NCX and GAPDH mRNA in control and puromycin-treated podocytes by RT–PCR and scanning densitometry

 



   Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the past podocytes were regarded as a passive barrier of the glomerular filter. However, recent studies suggest that podocytes might play a regulatory role in glomerular haemodynamics and contribute to the pathogenesis of several glomerular diseases [4,5]. A number of podocyte proteins have been shown to play an important role in maintaining the structure of the glomerular filtration barrier, the alteration of which is associated with proteinuria. Moreover, podocytes are the target cells of injury in acute proteinuric glomerular diseases, such as minimal change nephropathy, focal sclerosis and membraneous nephropathy [4].

Recently, it has been reported that podocytes can be propagated in cell culture and that cultured podocytes with a cobblestone-like appearance are able to transform to a more differentiated phenotype with an arborized morphology [15]. Arborized podocyte phenotype forms processes and expresses synaptopodin, which is found in foot processes of differentiated podocytes in vivo [12]. Only arborized podocytes staining positive for synaptopodin were used in this study. Podocytes additionally stained positive for the podocyte markers WT-1, nephrin, and p57. Furthermore, mRNA expression of synaptopodin and the podocyte marker CD2AP was detected by RT–PCR. Compared with previous podocyte cell cultures expressing a cobblestone phenotype, the cells used in this study might therefore better mimic the situation of the podocyte in vivo.

In podocytes, vasoactive hormones such as noradrenaline, angiotensin II, extracellular ATP, and others increase [Ca2+]i by a release of Ca2+ from intracellular stores and by an influx of Ca2+ from the extracellular space [68]. The hormone-induced influx of Ca2+ is probably mediated by a non-selective cation channel, whereas an L-type Ca2+ channel is not involved. Addressing Ca2+ extrusion mechanisms, in the present study we examined the Na+–Ca2+ exchange capability of podocytes by measuring the effect of a reduction of the transmembrane Na+ gradient on [Ca2+]i. Reduction of extracellular Na+ led to an increase of [Ca2+]i suggesting that the fluxes of [Ca2+]i and Na+ are coupled. The magnitude of the [Ca2+]i increase induced by extracellular Na+ depletion was strongly dependent on the extracellular Na+ concentration.

The contribution of Na+–Ca2+ exchange to modulation of [Ca2+]i in podocytes was confirmed by the inhibition of the Na+ depletion-induced [Ca2+]i increase by low concentrations of benzamil, an amiloride analogue which has been reported to inhibit Na+–Ca2+ exchange [14]. In contrast to benzamil, flufenamate, a blocker of non-selective cation channels, did not inhibit the Na+ depletion-induced [Ca2+]i response, indicating that removal of extracellular Na+ did not induce [Ca2+]i increase by activation of non-selective cation channels.

If the [Ca2+]i response induced by removal of extracellular Na+ were mediated by an enhanced Na+ export in exchange for Ca2+, it would be expected that an increase of the intracellular Na+ by a Na+ ionophore also increased [Ca2+]i. Indeed the Na+ ionophore monensin increased [Ca2+]i, most probably by coupling the extrusion of intracellular Na+ to Ca2+ entry via the Na+–Ca2+ exchanger.

To examine whether Na+–Ca2+ exchange in podocytes is dependent on the transmembrane Ca2+ gradient, [Ca2+]i was measured in the absence of extracellular Ca2+ and Na+. Here, the [Ca2+]i increase induced by extracellular Na+ depletion was markedly reduced in the absence of extracellular Ca2+, suggesting that it was predominantly due to Na+-dependent Ca2+ influx. Na+–Ca2+ exchange has been observed in mesangial cells [2], whereas in fibroblasts removal of extracellular Na+ led to an inositol phosphate-dependent mobilization of [Ca2+]i from intracellular stores [16].

Intracellular acidification is known to influence [Ca2+]i. In this regard, the [Ca2+]i increase observed in a Na+-free bath solution thus might have resulted from an inhibition of Na+–H+ exchange leading to intracellular acidification. However, the microfluorescence measurements of intracellular pH do not support this hypothesis. (i) In the absence of extracellular Na+ no significant decrease of intracellular pH was observed. (ii) Treatment of the cells with /NH3 caused a marked cellular alkalinization followed by an acidification, but only a very small increase of [Ca2+]i. (iii) Hoe 694, an inhibitor of Na+–H+ exchange, did not abolish the [Ca2+]i increase induced by extracellular Na+ removal.

In many experiments, [Ca2+]i of podocytes in Na+-free medium decreased rapidly below resting [Ca2+]i upon re-addition of extracellular Na+, indicating that under these conditions the Na+–Ca2+ exchanger may operate in the forward mode, i.e. extruding Ca2+ by exchanging intracellular Ca2+ for extracellular Na+. The direction of the Ca2+ movement via the Na+–Ca2+ exchanger can be further considered by calculating its driving force. The net driving force for the Ca2+ movement mediated by the Na+–Ca2+ exchange is determined by the difference between membrane voltage (Vm) and the reversal potential (ENaCa) for the exchanger: {Delta}V=Vm-ENaCa [17]. If, for example, {Delta}V<0, the Na+–Ca2+ exchanger will extrude Ca2+ out of the cell. In most cell types studied the stoichiometry of the exchange is 3 Na+:1 Ca2+ [18]. The reversal potential of the Na+–Ca2+ exchange ENaCa is therefore: ENaCa=3 ENa–2ECa, where ENa=(RT/F) ln([Na]o/Na]i) and ECa=(RT/2F) ln([Ca]o/Ca]i). R, T, and F are the gas constant, absolute temperature, and Faraday's number, respectively. Taken resting [Ca2+]i as ~100 nM, extracellular Ca2+ as 1.3 mM, extracellular Na+ as 145 mM and assuming that the intracellular Na+ activity is ~10 mM, and resting membrane voltage is about -55 mV, as recently reported [6], it can be calculated that ENaCa is -40 mV and the net driving force for the Ca2+ movement via the Na+–Ca2+ exchanger is about -15 mV. Hence, under resting conditions the Na+–Ca2+ exchanger in podocytes most probably takes Ca2+ out of the cell.

As further evidence for the presence of the Na+–Ca2+ exchanger in podocytes, Na+–Ca2+ exchanger mRNA was detected both in cultured differentiated mouse podocytes (RT–PCR) and in podocytes isolated from freshly microdissected glomeruli (single-cell RT–PCR). Moreover, NCX1 protein expression was detected by immunocytochemistry.

PKC is known as an important regulator of cellular Ca2+ homeostasis. With regard to the influence of PKC activity on Na+–Ca2+ exchange, it has been shown that acute activation of PKC leads to an increase of Na+–Ca2+ exchange in smooth muscle cells [19]. The data presented here indicate that short-term stimulation of PKC decreased Na+–Ca2+ exchange activity in podocytes. In contrast, inhibition of PKC led to a potentiation of Na+–Ca2+ exchange. Similar inhibitory effects of PKC on Na+–Ca2+ exchange have been reported in mesangial cells [20]. The biological role for the influence of PKC on Na+–Ca2+ exchange is obscure, but it may be speculated that activation of PKC by vasoactive agonists might limit the Ca2+ extrusion via the Na+–Ca2+ exchanger in podocytes.

PAN nephrosis is an experimental animal model characterized by massive proteinuria and marked morphological changes in podocytes, resembling features of minimal change nephropathy in humans [9]. Little is known about the cellular events leading to podocyte damage by puromycin. The morphological alterations induced by puromycin were associated with a reduction of the activity of the Na+–Ca2+ exchanger and with an inhibition of its transcription. Accumulation of cytosolic Ca2+ is known to be involved in several forms of cellular injury. Thus, it may be speculated that the reduced ability of podocytes to extrude Ca2+ via the Na+–Ca2+ exchanger in the presence of puromycin may participate in podocyte injury.

In summary, the study demonstrates the presence of a Na+–Ca2+ exchanger in podocytes, which extrudes Ca2+ under resting conditions. The functional activity of the exchanger is inhibited by PKC. An alteration of Na+–Ca2+ exchange by puromycin may contribute to podocyte injury in PAN nephrosis.



   Acknowledgments
 
We thank Mrs C. Hupfer for her excellent technical assistance. Cultured mouse podocytes were a kind gift of Dr P. Mundel, Department of Medicine and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, which is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Fi 691/1-2 and Pa 483/1-5.



   Notes
 
Correspondence and offprint requests to: Dr Karl-Georg Fischer, MD, University Hospital Freiburg, Department of Medicine, Division of Nephrology and General Medicine, Hugstetter Str. 55, D-79106 Freiburg, Germany. Email: fischer{at}med1.ukl.uni\|[hyphen]\|freiburg.de Back



   References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 30.11.01
Accepted in revised form: 11. 6.02