Nephrology Dialysis Transplantation

NDT Advance Access published online on February 13, 2007
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfl817

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Ciclosporin reduces paracellin-1 expression and magnesium transport in thick ascending limb cells

Chiz-Tzung Chang^1,2, Cheng-Chieh Hung², Ya-Chung Tian², Chih-Wei Yang² and Mai-Szu Wu²

¹Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan and ²Kidney Research Institute, Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Taipei, Taiwan

Correspondence and offprint requests to: Mai-Szu Wu, Division of Nephrology, Chang Gung Memorial Hospital, 222, Mai-Chin Road, Keelung, Taiwan. Email: maxwu1{at}adm.cgmh.org.tw

	Abstract

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Background. Renal magnesium (Mg²⁺) wasting is one of the ciclosporin (CsA) tubular effects. The major site of Mg²⁺ transport is the thick ascending limb (TAL), where 70% of the ultrafiltrable Mg²⁺ is reabsorbed paracellularly. Paracellin-1 is a tight junction protein, which regulates the paracellular Mg²⁺ transport in the TAL. We hypothesize that CsA reduces the expression and function of paracellin-1 and accounts for the observed renal Mg²⁺ wasting.

Methods. We established an immortalized cultured cortical TAL (cTAL) cell line from L-PK/Tag1 transgenic mice by microdissection. The cultured cells expressed paracellin-1 and the characteristics of cTAL cells. Real-time PCR and western blotting were used to test the CsA effects on paracellin-1 expression of cultured cTAL cells. Cytosolic-free Mg²⁺ concentration [Mg²⁺]_i change with time in a single cTAL cell was used as an indicator of transcellular Mg²⁺ transport and assessed by using fluorescence dye Mag-fura-2 AM. Paracellular Mg²⁺ transport was measured by cells grown in porous filters.

Results. The results showed that CsA significantly reduced paracellin-1 mRNA and protein expression in a dose-dependent manner. CsA (100 ng/ml) incubation for 24 h induced a decrease of paracellin-1 mRNA by 89.4% and paracellin-1 protein by 75.4%. CsA (100 ng/ml) did not change transcellular Mg²⁺ transport, but paracellular Mg²⁺ transport was decreased in CsA-treated cTAL cells by 74.4%.

Conclusion. These results suggested that reduced PCLN-1 expression and paracellular Mg²⁺ transport might play a role in the renal Mg²⁺ wasting in the CsA tubular effect.

Keywords: ciclosporin; magnesium transport; paracellin-1; PCLN-1; thick ascending limb

	Introduction

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Magnesium (Mg²⁺) is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability or neuromuscular excitability. Mg²⁺ also mediates cellular energy metabolism, ribosomal and membrane integrity. In addition, Mg²⁺ modulates the activity of several membrane transport and signal transduction systems. Homeostatsis of Mg²⁺ is essential to life in humans and in many other animals. Extracellular Mg²⁺ concentration is tightly regulated by renal excretion [1]. Despite the critical role of Mg²⁺ handling, the exact mechanisms mediating transepithelial Mg²⁺ transport remained obscure. In the past few years, the genetic disclosure of inborn errors of Mg handling lead to the discovery of several new proteins included in renal epithelial Mg²⁺ transport [2]. Paracellin-1 (claudin-16), encoded by the PCLN-1 gene, was a key player in paracellular Mg²⁺ reabsorption in the thick ascending limb (TAL) and was found to play an important role in the Mg²⁺ renal handling [3].

Prolonged ciclosporin (CsA) usage is associated with the development of lower serum Mg²⁺ levels. The renal Mg²⁺ wasting is thought to be the cause [4]. However, the exact mechanism is still unclear. Previous study had indicated that CsA and loop diuretics had a similar target along the nephron [5]. TAL is one of the targeted segments of the CsA effect. PCLN-1 is abundantly expressed in the thick ascending segment. It would be very interesting to know if CsA alters the activity of paracellin-1 and possibly plays a role in the observed renal magnesium wasting.

To answer the question, we tested the effect of CsA on the expression and activity of paracellin-1 in a model of cultured cortical TAL (cTAL).

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Transgenic mice
The experiments were carried out on cultured cells derived from the isolated cTAL segment from the kidney of an L-Pyruvate kinase (L-PK)/Tag1 transgenic mouse as previously described [6]. All the animal studies followed the institutional regulations for animal breed and experiments. Briefly, a vector containing a 2.7 Kb BamHI-BclI fragment of SV40 early region including the sequences coding for transforming large (T) and small (t) antigens (Tag) was placed under the control of a 3.2 kb fragment of rat L-PK gene regulatory region in the 5' flanking region. L-PK/Tag transgene in ECoRI-PvuI fragments were microinjected into fertilized mouse eggs. One line of transgenic mice (L-PK/Tag1) expressing the transgene in a tissue specific manner, i.e. kidney, intestine and liver, was created [6].

Cell isolation and culture
Three-month-old male L-PK/Tag1 mice fed a high carbohydrate diet (75%) were used to establish cTAL cell lines with a prolonged life span by microdissection. Mice anaesthisized with Nembutal were killed and the kidneys were removed. The renal capsule was removed and cortical slices were digested with 0.1% (w/v) type-1 collagenase (Sigma) in DMEM: HAM's F12 medium (DMEM: HAM's F12, 1:1 vol/vol; 60 nM sodium selenate; 5 µg/ml transferin; 2 mM glutamine; 5 µg/ml insulin; 50 nM dexamethasone; 1 nM triiodothyronine; 10 ng/ml epidermal growth factor; 2% fetal calf serum; 20 mM HEPES, pH 7.4) at 37°C for 1 h. The cortical slices were then moved to fresh modified DMEM:HAM's F12 medium without collagenase and cTAL segments were isolated by microdissection using sterile fine needles under stereomicroscope at room temperature. The dissected tubule segments were then cultured and subcultured in a modified culture medium (DMEM: HAM's F12, 1:1 vol/vol; 60 nM sodium selenate; 5 µg/ml transferin; 2 mM glutamine; 5 µg/ml insulin; 50 nM dexamethasone; 1 nM triiodothyronine; 10 ng/ml epidermal growth factor; 20 mM D-glucose; 2% fetal calf serum; 20 mM HEPES, pH 7.4 – containing 0.67 mM Mg²⁺) to establish a cTAL cell line at 37°C in 5% C0₂–95% air atmosphere. Like that observed in vivo, where the expression of L-PK gene was activated by a high carbohydrate diet, D-glucose in a cultured medium controlled the expression of Tag and the immortalized cTAL cell line was established [6]. All experiments were performed between the 30th and 50th passages on sets of confluent cells grown on a porous filter or petri dishes.

Immunofluorescence study
Cultured cTAL cells seeded on 8-well chambers were washed with PBS buffer and blocked with 1% BSA (bovine serum albumin) for 30 min. Cells were then stained with 1:25 monoclonal mouse FITC-conjugated anti-SV40 large and small t antibody (BD Bioscience Pharmingen, NJ, USA) for 30 min. For the immunofluorescence study of paracellin-1, cells were cultured on Costar filters and fixed with cold methanol (–20°C). After blocking with 1% BSA, the cells were incubated with rabbit anti-paracellin-1 antibody (Zymed Laboratories, San Francisco, CA, USA) (1:50) and rat anti-ZO1 antibody (R26.C 1:2 dilution, Developmental Studies Hybridoma Bank, University of Iowa; provided by Dr. Daniel A. Goodenough, Harvard Medical School) 4°C overnight. FITC-conjugated goat anti-rabbit immunoglobulin antibody (1:100) and Rhodamine-conjugated goat anti-rat immunoglobulin antibody (1:100) (Sigma, St Louis, MO, USA) were then used as secondary antibody for 1h at room temperature. Immunostained cells were examined under Olympus BX 50 immunofluorescence microscope. The images were taken by SPOT camera and analysed with SPOT software (Diagnostic Instruments Inc. Sterling Heights, MI, USA).

RNA extraction and RT-PCR
RNA was extracted from isolated cTAL segments microdissected from an adult mouse kidney and confluent cTAL cells by the method of Chomczynski and Sacchi. Total RNA concentration was treated with RNase-free DNase I (Boehringer Mannheim, Germany) at 37°C for 30 min and RNA concentration was evaluated by spectrophotometry. RNA (100 µg) was reverse transcribed with avian myeloblastosis virus reverse transcriptase (RT AMV, Boehringer) at 42°C for 60 min. Then 150 ng cDNA and non-reverse transcribed RNA were amplified for 32 cycles in 100 µl total volume containing 50 mM KCl, 20 mM Tris–HCl pH 8.4, 10 mM dNTP, 1.5 mM MgCl₂, 1 unit Taq polymerase and 10 pmoles of PCLN-1 or NKCC2 primers. The thermal cycling programme was as follows: 94°C for 1 min 60°C for 1 min, and 7°C for 3 min. The two primers from the PCLN-1 gene were as follows: anti-sense strand 5' –TTT GGC TGT CTC TGT CCG AGG- 3', sense strand 5' –AAG GAT CTT CTT CAG TAC GCT GC -3' [3]. The primers for mouse Na⁺-K⁺-Cl^– co-transporter (NKCC2), encoding for the kidney-specific Na⁺–K⁺–Cl^– co-transporter isoform in TAL were as follows: anti-sense strand 5'-CTT ggC TTC GGT TTT AGA TGA CCC G-3', sense strand 5'-GCA ATG CTG GCA TTT AGA CCC TCC G-3'. To exclude the possible contamination of cTAL cells with mouse distal tubule cells (DCT), RT-PCR studies for DCT specific thiazide-sensitive Na-Cl co-transporter (NCC) were also done. The primers for NCC were as follows: anti-sense strand 5'-CAG AGC AGC ATC CCG AGA TAA TC-3', sense strand 5'-TCT CAC CCT CCT CAT CCC CTA TCT-3'. Amplification products (PCLN-1 678 bp, NKCC2 406 bp and NCC 394 bp) were run on a 4% agarose gel with ethidium bromide and photographed. As control, the identity of the amplified products of ß-actin (460 bp) was run in the same time. RT-PCR studies for other claudin family members, including claudin 4, 7, 8 and 10, were also performed to examine the effect of CsA on other claudins [7].

Real-time PCR experiments
Real-time PCR was performed to quantify the changes in PCLN-1 mRNA expression caused by CsA. The primer-probe sets for PCLN-1 for real-time RT-PCR were designed by using Primer Express software (PE Applied Biosystem, Foster City, CA) according to the manufacturer's guidelines. The set of PCLN-1 primers taken from the mouse PCLN-1 gene were: anti-sense strand 5'-aca gtg ata ttg gtg ttc tca atg aaa-3', sense strand 5'-cca ttg tcc acc cga tga a-3'. Real-time RT-PCR was performed according to the manual of the TagMan EZ RT-PCR kit (Applied Biosystems). The reaction mixture (total volume, 25 µl) consisted of 3 µl of RNA sample; 5 µl of 5x TagMan EZ buffer; 3 µl of 25 mM manganese acetate; 0.75 µl each of dATP, dCTP, dGTP and dUTP; 0.25 µl of each 100 µM primer; 1 µl of fluorogenic probe; 2.5 U of recombinant DNA polymerase; 0.25 U of AmpErase uracil-N-glycosylase and 8.25 µl of RNase-free water. The thermal cycling conditions were as follows: 50°C for 2 min, 60°C for 30 min, 95°C for 5 min, followed by 50 cycles at 95°C for 10 s and at 62°C for 45 s. Standard curves for PCLN-1 and GAPDH were constructed by the serial 5-fold dilution of mouse whole kidney RNA extract. The ABI Prism 7700 sequence detection system (Perkin–Elmer, Applied Biosystem Inc.) was employed for PCR cycling, real-time data collection and analysis.

Western blotting
Confluent cultured cells grown in porous filter, with or without CsA treatment, were lysed in triple detergent buffer ([50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% NaN₃, 0.1% SDS, 1 mM EDTA, 100 µg/ml polymethylsulfonyly fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotionin, 0.1% NP-40 and 0.5% sodium deoxycholate]) for 5 min at 0°C. The cell lysates were centrifuged at 600 g for 30 min at 4°C to remove debris. Equal amounts of protein (50 µg) were loaded in each lane, electrophoresed through 8% SDS-polyacrylamide gels, and blotted onto a polyvinylidene difluoride membrane (Bio-Rad). Membranes were then rinsed in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and blocked with 5% dried milk powder in TBST for 1–2 h. Rabbit anti-paracellin-1 antibody (1:1000) as primary antibody (Zymed Laboratories, San Francisco, CA, USA) and goat anti-rabbit as secondary antibody (1:15 000) (Amersham Pharmacia Biotech Piscataway, NJ, USA) were used for immuoblotting. ECL plus system (Amersham) was used as blot detecting agent. Resulting membranes were re-blotted with mouse Tubulin- antibody (Lab Vision, CA, USA) for internal control.

Cytoplasmic Mg²⁺ measurements
Cytoplasmic-free Mg²⁺ concentration ([Mg²⁺]_i) measurements were determined with the Mg-sensitive fluorescent dyes Mag-fura 2-AM (Molecular Probes, Eugene, OR, USA). The cell-permeant acetoxymethyl ester (AM) form of the dye was dissolved in DMSO to a stock concentration of 5 mM and then, with the aid of Pluronic F-127 (Molecular Probes), was diluted to 5 µM Mag-fura 2-AM in media for 20 min at 37°C. Cells cultured to confluence in a 35 mm glass bottom petri dish (MatTek Coporation, Ashland, MA, USA) were subsequently washed three times with buffered salt solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 Na₂HPO₄, 0.2 KH₂PO₄, 1.0 CaCl₂, 5 glucose and 20 HEPES/Tris, at pH 7.4. The cTAL cells were incubated for a further 20 min to allow for complete de-esterfication and washed once before measurement of fluorescence.

Epifluorescence microscopy equipped with Fluar 100X objective (Axiovert 200, Carl Zeiss, Germany) was used to monitor the change of intracellular Mag-fura-2 fluorescence within the single cTAL cells. The fluoresence was recorded at 1-s intervals using a dual-excitation wavelength spectrofluorometer (LAMBDA DG-4, Shutter Instrument, CA, USA) with excitation for Mag-fura 2-AM at 335 and 385, and emission at 505 nm [8]. The [Mg²⁺]_i corresponding to the fluorescence emitted by intracellular Mg-fura-2 was calculated using the following equation: [Mg²⁺]_i = 1.4 mM x (R – R_min)/(R_max –R) x ß, where 1.4 mM represents the dissociation constant of the Mg-fura-2-Mg²⁺ complex. R is the ratio of fluorescence at 335 and 385 nm. R_max and R_min are the ratios of fluorescence at 335 and 385 nm in the presence of saturating Mg²⁺ and zero Mg²⁺, respectively. ß is the ratio of fluorescence of Mag-fura-2 at 385 nm in zero and saturating magnesium [8].

To further characterize the possible transcellullar magnesium transport, the [Mg²⁺]_i change with time (d[Mg²⁺]_i/dt) was used to estimate the possible effect of CsA on magnesium transport across cell membranes [9, 10]. The cTAL cells cultured to confluence were deprived of magnesium overnight (16 h). The basal [Mg²⁺]_i was measured and 5 mM magnesium was then put into culture medium to see the fluorescence change. The d[Mg²⁺]_i/dt was determined by linear regression analysis of fluorescence tracing over a period of 30 min.

Paracellular magnesium transport study
Mg transport studies were performed on cells grown on collagen-coated filters in order to calculate the rate constant of Mg²⁺ transport from apical to basal bath of the filters via the paracellular pathway. Cells grown on filters were bathed with a medium containing 10 mM of MgCl₂ in the apical sides without Mg²⁺ in basal sides. We then collected the medium every minute and determined the Mg²⁺ concentration by flame atomic absorption spectrometry [11]. The Mg²⁺ transport from apical to basal sides of the cells was estimated by calculating the rate constant using the same formula as previously described [5]. The rate constant of Mg²⁺ transport from the apical side to basal side was calculated as [ln(R₁)–ln(R₂)]/(t₂–t₁). R_x corresponds to the percent Mg remaining in the apical medium at time tx [12].

Potassium transport studies
The potassium transporting capacities of the cells were studied by radiolabelled rubidium (⁸⁶Rb⁺), used as the tracer of potassium movements, influx and efflux studies. Confluent cells in 24-well trays were washed with 1 ml modified phosphate buffered saline (PBS, in mM: NaCl 136; KCl 5; Na₂HPO₄ 1; glucose 5; HEPES 10; pH 7.4) pre-warmed to 37°C. The medium was removed and ⁸⁶Rb⁺ influx was initiated by adding of 1 ml PBS containing 2 µCi/ml ⁸⁶Rb⁺. The reaction was stopped by removing the solution and rinsing with 1 ml ice-cold 100 mM MgCl₂ solution for three times. Cells were disrupted with 0.5 ml 1M NaOH and radioactivity was determined by scintillation counting. The K⁺ influx was measured by incubating cells without (total influx) or with 0.5 mM ouabain or ouabain plus 10^–4 M furosemide in order to determine the ouabain-sensitive influx (Os) mediated by the Na⁺–K⁺ ATPase pumps and the ouabain-resistant furosemide-sensitive influx (Or-Fs) mediated by the Na⁺–K⁺–Cl^– cotransport [5].

Cell viability
Cell viability was estimated by a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, to determine the non-specific cytotoxicity of the CsA to TAL cells. Cells were seeded in 96-well plates (Corning Co., Corning, NY, USA). After culturing for 3 days, cells were exposed to various concentrations (1–600 ng/ml) of CsA. Following a 48-h incubation, 40 µl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well. After 2 h at 37°C, the cells were lysed by adding 100 µl of 20% (w/v) SDS and 50% (v/v) N, N-dimethylformamide (pH 4.7) and incubated overnight at 37°C. The absorbance at 570 nm was measured for each well using a Dynex microplate reader. The reported cell viability was the percentage of viable cells in comparison with the control wells. Duplicate measurements were made for each on at least two separate occasions.

Statistical analysis
Results are expressed as means ± SD from (n) experiments performed in duplicate or triplicate. Significant differences between groups were analysed by Student's t-test, or Tukey's analysis of variance. The statistical analysis was performed using the Statview^TM program (Macintosh). A P-value < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Character of cultured cTAL cells
To do the experiment in a cultured cell system, it is very important to find an appropriate model, which expresses functional paracellin-1. The TAL cell is the major site of paracellin-1 and Mg⁺² transport [13]. In a previous study, TAL was shown to be one of the major targets of CsA tubular effects [5]. For these reasons, cultured cells derived from mouse cTAL cells were established and used for the experiments.

We microdissected the cTAL from an L-PK/Tag1 transgenic mouse [6]. This cell line had prolonged life and retained the characteristics of intact cTAL cells. Cultured cTAL cells seeded on 8-well chamber slides showed positive immunostaining for large and small t antigen (Figure 1A). Confluent cTAL cells grown on collagen-coated petri dishes formed a monolayer of cuboid-shaped cells and formed small domes (Figure 1B). We used RT-PCR to detect the kidney-specific NKCC2 mRNA. Substantial amounts of NKCC2 transcripts (406 bp) (Figure 2A) were detected in both microdissected cTAL and cultured cTAL cells. To exclude the contamination of the cTAL cell line by adjacent distal tubule cells, distal tubule-specific NCC transcript (394 bp) was also checked by RT-PCR. NCC transcript can be found in whole kidney extract, but not in cTAL (Figure 2B). The expression of PCLN-1 mRNA in cTAL cells was examined by RT-PCR. PCLN-1 transcript (678 bp) was found substantially in cultured cTAL cells (Figure 3A). To visualize the cellular localization of paracellin-1, we performed immunoflurorescence study for paracellin-1 and cell junctional protein ZO-1 on cultured cTAL cells grown on filters. Paracellin-1 was found to be colocalized with ZO-1. These studies confirmed that paracellin-1 was located mainly along the cell junction (Figure 3B). The results above indicated that the cultured cTAL retained the main histological characters of cTAL. We then further characterized the functional property of cTAL. The different components of ⁸⁶Rb⁺ influx measured in the absence or presence of ouabain and furosemide allowed us to distinguish the ouabain-sensitive component of ⁸⁶Rb⁺ influx mediated by the Na⁺–K⁺ ATPase pumps and ouabain-resistant furosemide-sensitive component of ⁸⁶Rb⁺ influx reflecting the Na⁺–K⁺–Cl^– cotransport activity (Figure 2C). All these results indicated that the cultured cTAL cell retained the major characteristics of its parent cells and was an appropriate model to test the effect of CsA on paracellin-1 in renal TAL cells.

View larger version (111K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. Establishment of immortalized cTAL cultured cell line. (A) cTAL cells grown on a chamber slide showed positive immunostaining of small (t) and large (T) antigen (Magnification 400x). (B) Confluent TAL cells grown on petri dishes formed layers of cuboid-shaped cells and formed small domes (250x).

 

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. Properties of cultured mouse TAL cells. (A) RT-PCR results showing the amplified products of NKCC2 in microdissected TAL (Lane 1) and cultured TAL cells (35th passage) (Lane 2). As controls, no band was detected using non-reverse-transcribed RNA from cultured cells (Lane 3) or by omitting cDNA (Lane 4). ß-actin was used as an internal control. M represented the marker ladder. (B) NCC transcript was absent in cTAL (Lane2) but present in whole kidney extract (Lane1). (C) Time course of 86Rb+ influx performed on cultured cTAL cells grown on petri dishes and incubated without (squares) or with ouabain (circles) and ouabain plus furosemide (triangles). Each point was the mean from five separate experiments performed in duplicate.

 

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3. Presenceof PCLN-1 transcript and paracellin-1 in cTAL. (A) RT-PCR results showing the amplified products of PCLN-1 in microdissected TAL (Lane 1) and cultured TAL cells (35th passage) (Lane 4). As controls, no band was detected using non-reverse-transcribed RNA from cultured cells (Lane 2) or by omitting cDNA (Lane 3). ß-actin was used as an internal control. M represented the marker ladder. (B) Immunofluorescence study showed positive staining of paracellin-1 (left panel) and ZO-1 (middle panel). Merged picture (right panel) confirmed the colocalization of paracelllin-1 and ZO-1 at cell junction. Green, paracellin-1 staining; red, ZO-1 staining; yellow to orange, co-localization of paracellin-1 and ZO-1 in the merged panel (Magnification 400x).

 
The CsA decreased PCLN-1 mRNA expression
The expressions of PCLN-1 and ß-actin mRNA were measured by RT-PCR. The results were expressed as percentage change of controls. Significant changes in mRNA levels were found in PCLN-1, but not in the control ß-actin mRNA. Twenty-four hours after adding a different concentration of CsA to cultured cTAL cells, the CsA induced a 79.6, 90.2 and 97.9% decrease in PCLN-1 mRNA at doses of 100, 300 and 600 ng/ml, respectively, when compared with the control (P < 0.01) (Figure 4A). The changes were compared by optical density obtained by scanning densitometer of the PCR products at the exponential phase. Real-time PCR experiments were then undertaken to better quantify the decrease in PCLN-1 mRNA expression induced by CsA. The expression of PCLN-1 mRNA measured by real-time PCR decreased 74.2, 89.4 and 96.7% (CsA 50, 100 and 300 ng/ml for 24 h, P < 0.01, n = 3) in CsA-treated cells as compared with untreated cells (Figure 5). The expression of other claudin members, including claudin-4, 7, 8 and 10, which might locate in the tight junction area in TAL cells [7], however, were not changed significantly in cTAL cells treated with CsA (Figure 4B).

View larger version (63K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4. CsA effect on PCLN-1 and other claudins. (A) Representative RT-PCR results of PCLN-1 and ß-actin with or without CsA pretreatment. The CsA (100, 300 and 600 ng/ml) induced a 79.6, 90.2 and 97.9% decrease in PCLN-1 mRNA at doses of 100, 300 and 600 ng/ml, respectively, when compared with the control. M: marker; Control (Lane 1); CsA 100 ng/ml (Lane 2); CsA 300 ng/ml (Lane 3); CsA 600 ng/ml (Lane 4). (B) RT-PCR results of CsA effects on claudin-4,7,8 and 10. Using whole mouse kidney extract as control (Lane K), CsA 100 ng/ml (Lane CsA) did not affect the expression of claudin-4 and claudin-8 in cTAL as compared with cTAL cells without CsA treatment (Lane C).Claudin-7 and Clandin-10 were present in whole kidney extract but cannot be found in cTAL cells with or without CsA treatment.

 

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5. Quantification of PCLN-1 mRNA in cTAL cells by real-time PCR. The PCR was run in triplicate for each sample. PCLN-1/GAPDH ratio of RNA in CsA-treated (CsA50, CsA100 and CsA300) cTAL cells was plotted comparing to untreated cTAL cells (control). CsA50, CsA100 and CsA300 indicated pre-incubation of 50 ng/ml, 100 ng/ml and 300 ng/ml CsA for 24 h (*P < 0.01, n = 3).

 
The CsA decreased paracellin-1 protein expression
Parallel with the RT-PCR and real-time RT-PCR studies, western blotting studies for paracellin-1 expression also showed that CsA decreases paracellin-1 protein expression in cTAL. Compared with untreated cells, CsA 100 and 300 ng/ml for 24 h could decrease paracellin-1 by 75.4% and 93.5%, respectively (P < 0.01, n = 4) (Figure 6).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6. CsA effect on paracellin-1 protein expression. Representative western blotting result of CsA effect on paracellin-1 expression and bar charts from results of four separated experiments revealed that CsA reduced paracellin-1 expression in a dose dependent manner. CsA50, CsA100 and CsA300 indicated pre-incubation of 50 ng/ml, 100 ng/ml and 300 ng/ml CsA for 24 h (*P < 0.01, n = 4).

 
CsA decreased paracellular magnesium transport in cTAL cells
To clarify, the decreased mRNA and protein expression were functionally related. Mg⁺² transport studies were performed on cells grown on collagen-coated filters in order to calculate the rate constant of Mg²⁺ transport from apical to basal bath of the filters via the paracellular pathways. The rate constant reached its maximum at 10 min (Figure 7A). Therefore, a 10 min duration was set for the following experiments to measure the Mg⁺² paracellular transport. CsA decreased Mg⁺² transport rate by 74.4% (CsA 100 ng/ml, 24 h, n = 6, P < 0.01) and 88.5% (CsA 300 ng/ml, 24 h, n = 6, P < 0.01) (Figure 7B). These results paralleled the PCR and western blotting results and suggested the dose-dependent inhibitory effect of CsA on paracellin-1 expression and Mg⁺² paracellular transport in the same time. To exclude the possible effect of CsA on the transcellular Mg⁺² transport, cytoplasmic-free Mg⁺² concentration was measured by Mg-fura-2 in cTAL cells, with or without CsA 100 ng/ml, in normal culture medium containing 0.67 nM Mg⁺². There was no difference between the cytoplasmic-free [Mg⁺²]_i in cTAL cells without (0.55 ± 0.11 mM, n = 24) and with 100 ng/ml CsA (0.54 ± 0.10 mM, n = 24) treatment. Cells cultured with or without CsA 100 ng/ml were deprived of magnesium for 16 h, then add 5 mM Mg⁺² was added, which did not show a change in dynamic cytosolic-free [Mg²⁺]_i change either (d[Mg²⁺]_i/dt 151 ± 37 nM/s vs 144 ± 43 nM/s, control vs CsA, n = 24, ns). The results indicated transcellular transport might not play a significant role in the CsA effect on Mg²⁺ transport.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7. Effect of CsA on the kinetics of magnesium transport in TAL cells grown on filters. (A) The time course of rate constant in control TAL cells. The rate constant reached its maximum at 10 min. Therefore, a 10 min duration was set for the following experiments to measure the Mg2+ paracellular transport. (B) CsA decreased Mg2+ transport rate constants by 74.4% (CsA 100 ng/ml, 24 h, n = 6, *P < 0.01) and 88.5% (CsA 300 ng/ml, 24 h, n = 6, *P < 0.01).

 
Cell viability
To ensure that the observed changes induced by CsA were not related to cell damage, cell viability, used as an index of cell injury, was measured on cultured cTAL cells incubated without or with various (5–600 ng/ml) concentrations of CsA for 24 h. There was no significant difference between the percentage of viable cells in CsA-treated and in untreated cells (5 ng/ml: 97 ± 4%; 10 ng/ml: 98 ± 5%; 50 ng/ml: 102 ± 8%; 100 ng/ml; 101 ± 12%; 600 ng/ml: 98 ± 8% of viable cells vs untreated cells, n = 6). These findings indicated that cultured cTAL cells remained viable after being incubated with CsA for 24 h, suggesting that the observed decreases in PCLN-1 and paracellin-1 expression were not related to cell damage.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Mg²⁺ is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability or neuromuscular excitability. Furthermore, renal Mg²⁺ wasting with Mg²⁺ deficiency is also found in essential hypertension, heart failure and other cardiovascular disease [14]. Extracellular magnesium concentration is tightly regulated by the extent of intestinal absorption and renal excretion. Despite the critical role of magnesium handling, the exact mechanisms mediating magnesium transport remained obscure. Positional cloning had identified that PCLN-1 gene mutations, a gene encoding paracellin-1 protein in TAL cells, could cause renal Mg²⁺ wasting [3]. Paracellin-1 (claudin-16), located in tight junctions of the TAL, belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium. These findings provided insight into Mg²⁺ homeostasis and suggested an essential component of a selective paracellular conductance of Mg²⁺ [15]. More than 70% of filtrated magnesium is reabsorbed in the TAL segment along the nephron. Cortical TAL is the main segment of TAL responsible for magnesium flux [13]. TAL is also a main target of CsA tubular effect [5]. We, therefore, chose cultured cTAL cells for our experiments.

Hypomagnesium is a frequent complication in patients taking CsA. The complication comes from renal Mg²⁺ wasting and is usually neglected and underestimated [16]. It is very interesting to know if CsA affects this tight junction protein and contributes to the clinical hypomagnesium.

Our experiments revealed CsA decreased expression of the paracellin-1 and paracellular Mg²⁺ transport. Simultaneous d[Mg²⁺]_i/dt measurement showed no difference between cells with or without CsA incubation and might indicate the minor role of transcellular Mg²⁺ transport in the observed changes. Previous studies also suggested the insignificant transcellular Mg²⁺ transport in cTAL [13]. The results suggested that CsA might cause the renal Mg²⁺ wasting paracellularly by affecting the paracellin-1 expression in the tight junction of cTAL. Kim et al. [10] had suggested that CsA and tacrolimus inhibited the hormone-stimulated transcellular Mg⁺² uptake into the cultured mouse distal convoluted tubule cells. The present study suggested the effects of calcineurin inhibitors in TAL cells, which is one of the major segments of Mg handling in the kidney. These results might both contribute to the observed Mg renal wasting effect of CsA. The therapeutic dosage of CsA is around 50–150 ng/ml. The concentrations of CsA used in the experiments were between 50 and 600 ng/ml and 100 ng/ml of CsA was sufficient to alter the expression and function of PCLN-1. Our preliminary study in mice fed with CsA 15 mg/kg/body weight [17] showed that PCLN-1 expression in renal cortical slices decreased up to 55.6% compared with that of control mice and further confirmed our in vitro study (data not shown).

Clinically, the severe renal Mg²⁺ wasting is not constantly present in patients taking CsA. Our in vitro experiments indicated a large inhibition of paracellular Mg²⁺transport in cTAL cells. CsA can alter renal haemodynamics, decrease glomerular filtration and increase tubular sodium reabsorption [5], which, in combination with paracellular transport of Mg in cTAL, contribute to the renal Mg²⁺excretion in patients taking CsA.

Our previous studies had revealed that CsA decreased nitric oxide (NO) production [18], increased Na⁺–K⁺–Cl^– cotransporter activity [5], increased angiotensin II receptor density [19] and decreased prostaglandin production [20] in renal epithelial cells. The results of the present study raised the questions about the possible link among sodium transport, local intra-renal hormones and this specific tight junction protein. It is of interest to know if the alternation of the cotransporter activity will affect the paracellular Mg transport and expression of PCLN-1. We had tested the Mg transport with the presence of furosemide (10^–5 M, loop diuretics), barium (10^–7 M, potassium channel blocker) or NPPB (10^–5 M, chloride channel blocker). The inhibition of all these transport mechanisms had significantly reduced the paracellular Mg transport in cultured cTAL cells (data not shown). We also performed the RT-PCR experiments to see the effect of these inhibitors on the expression of PCLN-1. The PCLN-1 expression did not change in the presence of these inhibitors (data not shown). The results suggest that CsA exerts its effect via alternation of PCLN-1 expression, which might be independent of the apical Na⁺–K⁺–Cl^– cotransporter, apical potassium channel and basolateral chloride channel. Further study and experiments are necessary to illuminate the interaction and link of these parameters in CsA renal effects. Clinical intervention based on this information could be developed to improve the graft function and solve the electrolyte disorders.

We concluded that CsA reduced PCLN-1 and paracellin-1 expression and paracellular Mg²⁺ transport in cultured TAL cells. The effect might contribute to the clinically observed renal Mg²⁺ wasting and hypomagnesium.

	Acknowledgements

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
This study was supported by grants from the National Science Council, Taiwan. The authors would like to thank Dr Alain Vandewalle (INSERM Unit 773, Paris, France), Dr Yu-Shien Ko, Chih-Chun Chen (Microscopic laboratory, Chang Gung Memorial Hospital), Yi-Ching Ko, Chung-Tseng Huang and Hsiau-Mai Yu for their excellent and dedicated technical supports.

Conflict of interest statement. None declared.

	References

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 

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Received for publication: 27. 6.06
Accepted in revised form: 18.12.06

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