Cyclosporin increases the density of angiotensin II subtype 1 (AT1) receptors in mouse medullary thick ascending limb cells

Mai-Szu Wu1, Chih-Wei Yang1, Chiz-Tzuang Chang1, Marcelle Bens2 and Alain Vandewalle2

1 Division of Nephrology, Chang Gung Memorial Hospital, Taipei, Taiwan and 2 Institut National de la Santé et de la Recherche Médicale, Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, Paris, France

Correspondence and offprint requests to: Dr Mai-Szu Wu, Division of Nephrology, Chang Gung Memorial Hospital, 199, Tun Hwa North Road, Taipei, Taiwan. Email: maisuwu{at}ms9.hinet.net



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Cyclosporin A (CsA), a potent immunosuppressive agent, can be nephrotoxic. Because clinical studies have suggested that the intrarenal renin–angiotensin system may be involved in the mechanism responsible for CsA nephrotoxicity, we have analysed the effects of CsA on angiotensin II (Ang II) receptors in medullary thick ascending limb (mTAL) cells known to be sensitive to the action of CsA.

Methods. Experiments were carried out on subcultured mouse mTAL cells. The expression of mRNA of Ang II subtype 1 and 2 (AT1 and AT2) receptors was investigated using reverse transcription–polymerase chain reaction (RT–PCR). [3H]Ang II was used for radioligand and binding studies. Fluorimetric recordings using the fluorescent dye fura-2/AM were performed to determine the effect of CsA on the intracellular calcium ([Ca2+]i) content of untreated and Ang II-treated mTAL cells.

Results. Subcultured mTAL cells expressed AT1 and AT2 Ang II receptor mRNAs, and binding studies revealed that the AT1 receptors were the predominant Ang II receptor subtype (~90%) in mTAL cells. CsA (100 ng/ml, 24 h) increased (1.7-fold) the number of Ang II receptors (untreated, 315.8; +CsA, 543.6 fmol/mg protein) without altering the KD (untreated, 7.16; +CsA, 7.06 nM). CsA also significantly increased the level of [Ca2+]i measured in cultured mTAL cells both in the basal state (–CSA, 72.2; +CsA, 93.4 nM/106 cells) and in the presence of Ang II (–CSA, 97.8; +CsA, 206.3 nM/106 cells).

Conclusions. These findings suggest that the increase in Ang II AT1 receptors and [Ca2+]i caused by CsA may be involved in the mechanism(s) responsible for CsA nephrotoxicity.

Keywords: angiotensin-converting enzyme; AT1 receptor; cyclosporin; thick ascending limb cells



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of the potent immunosuppressive agent cyclosporin A (CsA) in the treatment of transplanted patients has greatly improved renal transplant graft survival. However, CsA can be nephrotoxic and harmful to the transplanted kidney [1]. Although the exact mechanism(s) of CsA nephrotoxicity remains unknown, several studies have shown that this immunosuppressive agent enhances renal arterial vasoconstriction and reduces intrarenal blood flow [2]. It is likely that these vascular alterations could be due to an intrarenal imbalance between vasodilating and vasoconstricting mediators, such as endothelin [3] or nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) [4]. Renal tubule epithelial cells have also been shown to play a role in the regulation of renal haemodynamics in a paracrine fashion [5]. Changes in specific tubular cell functions can therefore affect vascular reactivity. CsA has been shown to decrease the production of NO in mTAL cells [4], a condition that may emphasize the CsA effect on endothelin in endothelial cells [3]. Studies have suggested that the activation of the renin–angiotensin system (RAS) also plays an important role in the process of CsA nephrotoxicity [6]. It has also been shown that the inhibition of RAS by angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists can prevent the onset and progression of the renal toxicity caused by CsA [6,7]. The question arises of whether CsA can affect some components of the RAS expressed in renal tubule epithelial cells.

Previous studies have shown that the intrarenal Ang II content per gram of tissue is five times greater in the medulla than the cortex of the rat kidney [8]. Using a cRNA probe and in situ hybridization, Meister et al. [9] have shown that the AT1 receptor mRNA is expressed in the S3 segment of the proximal tubule and in the outer medullary thick ascending limb (mTAL) from the rat kidney. Recently, Poumarat et al. [10] have shown that Ang II AT1 receptors are present in both apical and basolateral membranes from rat mTALs. Furthermore, Ang II induced an increase in [Ca2+]i when added to the basolateral side of microperfused rat mTALs. mTAL cells are particularly sensitive to the action of CsA [4,6,11]. Cultured mTAL cells should therefore represent a suitable ex vivo cell model for use in investigating the effects of CsA on local Ang II activity. In this study, we investigated the effects of CsA on Ang II activity in subcultured mouse mTAL cells, which had retained the main characteristics of the parental line from which they were derived [12]. The main results show that CsA stimulated the number of AT1 Ang II receptors and increased the intracellular level of calcium ([Ca2+]i), a downstream intracellular signal of Ang II [13], measured either in the basal state or after Ang II stimulation. These data indicate that CsA can stimulate Ang II activity in renal epithelial cells, which in turn suggests that it may be involved in CsA nephrotoxicity and progressive renal failure in recipients of kidney transplants.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
We used subcultured cells derived from mTALs microdissected from the kidney of a normal 1-month-old male mouse [12]. mTAL cells were cultured routinely in a modified hormonally defined medium [Dulbecco’s modified Eagle’s medium (DMEM): Ham’s F12, 1:1 (v/v); 60 nM sodium selenate; 5 µg/ml transferrin; 2 mM glutamine; 5 µg/ml insulin; 50 nM dexamethasone; 1 nM triiodothyronine; 10 ng/ml epidermal growth factor (EGF); 2% fetal calf serum; 20 mM HEPES pH 7.4] at 37°C in an atmosphere of 5% CO2–95% air. The medium was replenished every 2 days and the cells were passaged once a week. All experiments were performed on sets of cells from the same passages to avoid unexpected variability in the responses due to the different passages analysed. As previously observed [12], the increase in cell cAMP content induced by 10–6 M dDAVP remained identical in confluent mTAL cells from passage 6 (8.1-fold) and passage 15 (7.3-fold). We also checked that the [3H]Ang II bound did not vary in cells analysed between the sixth and fifteenth passage (see below). Accordingly, all experiments were performed on the same sets of untreated and CsA-treated cells grown on Petri dishes from the sixth to the fifteenth passage.

RNA extraction and RT–PCR experiments
Reverse transcription–polymerase chain reaction (RT–PCR) experiments were performed to detect mRNA transcripts in intact microdissected mouse mTALs and in cultured mTAL cells. Total RNA extracted from microdissected mTALs and confluent cultured mTAL cells was treated with RNase-free DNase I (Boehringer, Mannheim, Germany) at 37°C for 30 min. The RNA concentration of the cultured cells was determined by spectrophotometry. The RNA (100 µg) was reverse-transcribed with avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer, Mannheim) at 42°C for 60 min. Microdissected tubule cDNA or 150 ng of cultured cell cDNA and non-reverse-transcribed RNAs extracted from cultured cells were amplified for 30–36 cycles (36 cycles for AT1 receptor, 36 cycles for AT2 receptor and 30 cycles for ß-actin primers) in a total volume of 100 µl of PCR buffer (50 mM KCl, 20 mM Tris–HCl pH 8.4) containing 10 µM dNTP, 1.5 mM MgCl2, 1 U of Taq polymerase, and 10 pmol each of of AT1 receptor, AT2 receptor and ß-actin primers. The set of primers taken from the mouse AT1A receptor gene were antisense strand 5'-GCA TCA TCT TTG TGG TGG G-3' and sense strand 5'-GAA GAA AAG CAC AAT CGC C-3' [14]; those from the mouse AT2 receptor gene were antisense strand 5'-AAC ACA GCT GTT GGT GAA TCC-3' and sense strand 5'-ATG CTC AGT GGT CTG CTG G-3' [15]; and those from the mouse ß-actin gene were antisense strand 5'-TCA TGA GGT AGT CCG TCA GG-3' and sense strand 5'-TCT AGG CAC CAA GGT GTG-3' [11]. The RT–PCRs for AT1 receptor, AT2 receptor and ß-actin were performed on the same RNA samples extracted from cultured cells. The temperature cycling programme was as follows: 94°C for 1 min, 60°C for 1 min, and 72°C for 3 min. The amplification products were run on a 4% agarose gel with ethidium bromide and photographed. All the sets of primers used yielded amplified products of the expected size, and sequence analyses of the PCR products confirmed that they matched well with the expected cDNAs analysed (data not shown).

Real-time PCR experiments
Real-time PCR was performed to quantify the changes in AT1 mRNA expression caused by CsA. The primer–probe sets for AT1 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 AT1 primers taken from the mouse AT1 receptor gene were: antisense strand 5'-ACA GTG ATA TTG GTG TTC TCA ATG AAA-3' and 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 AT1 receptor and GAPDH were constructed by the serial 5-fold dilution of mouse whole kindey 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.

Ang II-binding studies
Ang II receptor-binding assay was performed on cultured cells as previously described [12]. A total of 5x104 cells were seeded into 24-well trays (Corning Co., NY) and grown for 5 days. mTAL cells were then incubated in normal medium or in 100 ng/ml CsA-supplemented medium for 24 h before binding studies. Cells were incubated with increasing concentrations (0.2–10 nM) of [3H]Ang II (SA: 50 Ci/mmol; NENTM Life Science, Boston, MA) in DMEM supplemented with 1% bovine serum albumin (BSA) in a shaking water bath with or without two Ang II receptor antagonists, losartan or PD 123319, at 22°C for 2 h. Afterwards, the cells were rinsed three times with ice-cold phosphate-buffered saline (PBS) to remove any unbound [3H]Ang II. The cells were then lysed in 100 µl of 1 M NaOH plus 0.1% Triton X-100, and their radioactivity was counted. As controls, we measured the binding of [3H]Ang II to cells incubated at 4°C. Competitive binding studies were performed on cells incubated with 10 nM [3H]Ang II with or without an excess of unlabelled Ang II (10–8–10–5 M). Non-specific [3H]Ang II binding, which was <10% of the total [3H]Ang II bound, was measured on cells incubated with a 1000-fold excess of unlabelled Ang II. The specific [3H]Ang II binding, expressed as fmol/mg protein, was calculated as the difference between the total and non-specific binding. Scatchard plot analyses were performed to calculate the dissociation constant (KD) and the maximum binding capacity (Bmax). Protein content was determined by the method of Lowry using BSA as standard.

Intracellular calcium assay
The concentration of intracellular calcium [Ca2+]i was determined using the calcium-sensitive fluorescent probe fura-2/AM according to the method described by Haller et al. [16], with slight modifications. Briefly, confluent cells grown on 60 mm diameter Petri dishes were scraped off carefully using a rubber policeman, rinsed twice, and re-suspended in calcium-free buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM Na2HPO4, 6 mM glucose, 10 mM HEPES). Suspended cells were then incubated in the buffer supplemented with 5 µM fura-2/AM for 20 min at room temperature. Next, the extracellular dye was removed by rinsing three times with a large excess of calcium-free buffer. The cells were then re-suspended (106 cells/ml) in the calcium-free buffer supplemented with 1.5 mM Ca2+. Fluorescence intensity (F ) was measured as a function of time in a thermostatted quartz cuvette at 37°C while stirring continuously in an Hitachi F-4000 spectrofluorometer at 340/380 nm excitation wavelength and 505 nm emission wavelength. After a 2 min equilibration period, recordings were performed before and after adding 10–7 M Ang II. At the end of the experiment, the level of [Ca2+]i was determined as follows: the maximum fluorescence (Fmax) was determined by adding an excess of 10 mM CaCl2, and the minimum fluorescence (Fmin) was measured by lysing the cells with Triton X-100 at pH >8.4 and adjusting the free calcium to <2 nM by adding 10 mM EGTA to the cell suspension. The [Ca2+]i corresponding to the fluorescence emitted by intracellular fura-2 (F) was calculated using the following equation: [Ca2+]i = 224 nM x (F - Fmin)/(Fmax - F), where 224 nM represents the dissociation constant of the fura-2–Ca2+ complex.

CsA resistance assay
To examine the potential non-specific cytotoxicity of CsA on cultured mTAL cells, the cell viability was determined by the MTT [(3–4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye assay. Cells were seeded and grown for 5 days on 96-well trays (Corning) and then incubated for the next 24 h in 100 µl of defined medium, with or without increasing concentrations of CsA (5–600 ng/ml). The medium was then removed and replaced by 50 µl of MTT (5 mg/ml) for 2 h at 37°C. The cells were then lysed by adding 50 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 determined for each well using a Dynex microplate reader. The cell viability found for each concentration of CsA tested was compared with that of the untreated cells, and the results were expressed as a percentage of viable cells. All measurements were performed in duplicate.

Statistical analysis
The results are expressed as mean ± SD from (n) experiments performed in duplicate or triplicate. Any significant differences between the groups were analysed using Student’s t-test and a one-way ANOVA. The statistical analysis was performed using the StatviewTM program (Macintosh). A P-value < 0.05 was considered significant.



   Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of Ang II receptor mRNAs in cultured mTAL cells
RT–PCR was used to detect the expression of Ang II receptor mRNA in confluent subcultured mTAL cells. As shown in Figure 1, substantial amounts of AT1 and AT2 receptor transcripts were detected in both isolated microdissected mTAL and cultured cells. The sets of specific primers yielded amplified products of the expected size (AT1, 629 bp; AT2, 328 bp). No amplified products were detected for the controls when non-reverse-transcribed RNA from cultured cells was used, or when cDNA was omitted (Figure 1). These results indicated that intact and cultured mouse mTAL cells expressed Ang II receptors, including the expression of AT1 and AT2 receptor mRNAs.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. AT1 and AT2 mRNA expression. Ethidium bromide-stained gel of AT1, AT2 and ß-actin (used as control) shows amplified products of the expected size (AT1, 629 bp, AT2, 328 bp, ß-actin, 460 bp) obtained by RT–PCR from microdissected mTAL (lane 1) and cultured mTAL cells (lane 2). No band was detected when non-reverse-transcribed RNA from cultured cells (lane 3) was used or when the cDNA was omitted (lane 4). Molecular weight standards (M) were the 1 kb ladder from Gibco-BRL.

 
Binding of Ang II in cultured mTAL cells
[3H]Ang II binding studies were performed to identify and quantify AT1 and AT2 Ang II receptors in cultured mTAL cells. We first analysed the kinetics of [3H]Ang II bound to untreated mTAL cells. Studies performed at 22°C provided optimum binding capacities, and almost no bound [3H]Ang II was detected when experiments were carried out at 4°C (Figure 2A). Competitive binding studies performed at equilibrium (2 h at 22°C) showed that the maximum binding produced in the presence of 10 nM [3H]Ang II alone was gradually completely displaced when the cells were incubated in the presence of a 10- to 1000-fold excess of unlabelled Ang II (Figure 2B). To characterize the subtype of Ang II receptor present in cultured mTAL cells, [3H]Ang II binding studies were performed in the absence and presence of two Ang II receptor antagonists: losartan, a specific AT1 receptor antagonist; and PD 123319, used as an AT2 receptor antagonist [17]. The specific [3H]Ang II binding was almost completely displaced (92%) by the AT1 receptor antagonist, losartan (10–5 M) (Figure 2C). In contrast, the AT2 receptor antagonist, PD123319 (10–5 M), only displaced 12.2% of the [3H]Ang II binding (Figure 2C). These results indicated that most of the Ang II receptors expressed in mTAL are AT1 receptors.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. Identification of Ang II receptors in mTAL cells. (A) Confluent mTAL cells were incubated with various concentration of [3H]Ang II at 22°C (open circles) or 4°C (filled circles, for non-specific binding) for 2 h. Points correspond to the mean ± SD from five separate experiments. (B) Cells were incubated for 2 h at 22°C with 10 nM [3H]Ang II without (open circles) or with increasing concentrations of unlabelled Ang II (filled circles). Values are the mean ± SD from 12 separate experiments. (C) The binding of [3H]Ang II (open bar) was slightly displaced (–12%) by the AT2 receptor antagonist, PD123319 (black bar), and almost completely displaced by the AT1 receptor antagonist, losartan (hatched bar). Adding PD123319 did not increase the displacement induced by losartan alone (cross-hatched bar). Values are the mean ± SD from eight separate experiments. **P < 0.01, ***P < 0.001 vs untreated cells (open bar).

 
Effects of CsA on Ang II receptors
We next investigated the effects of CsA on the expression and parameters of Ang II receptors present in cultured mTAL cells. Figure 3 illustrates the levels of AT1 and AT2 mRNA expression within untreated and CsA-treated cells as compared with the expression of ß-actin used as standard. The results from RT–PCR suggested that the 24 h incubation with 100 ng/ml CsA induced a concomitant decrease in the expression of AT2 receptor (–41%) mRNA and an increase in the expression of AT1 receptor (+21%) mRNA as compared with untreated cells. Because most (>90%) of the Ang II receptors present in mTAL cells were AT1 receptors (Figure 2C), real-time PCR experiments were then undertaken to quantify better the increase in AT1 receptor mRNA expression induced by CsA. As shown in Figure 4, the expression of AT1 receptor mRNA measured by real-time PCR increased by 84.5% in CsA-treated cells as compared with untreated cells. [3H]Ang II binding studies were then performed to find out whether CsA also affected the characteristics of the binding of [3H]Ang II to cultured mTAL cells. Consistent with the observed increase in AT1 mRNA expression caused by CsA, this immunosuppressive agent altered the specific binding of Ang II in a dose-dependent manner (control, 328.5 ± 31.8; +CsA 5 ng/ml, 330.1 ± 42.3; +CsA 50 ng/ml, 410.8 ± 49.7; +CsA 100 ng/ml, 554.8 ± 57.8; +CsA 600 ng/ml, 510.3 ± 60.3 fmol/mg protein, n = 5). The maximal increase in Ang II bound was achieved for 100 ng/ml, and therefore this concentration was used in all subsequent experiments. CsA (100 ng/ml) increased the specific [3H]Ang II binding at all concentrations of Ang II tested (measured at 22°C after 2 h incubation): for 10 nM [3H]Ang II, CsA increased the Ang II bound by 77 ± 12% (Figure 5A). The results from Scatchard plot analyses indicated that the number of Ang II-binding sites remained constant in untreated cells from passage 7 and passage 14 (335.8 ± 23.5 vs 345.9 ± 52.3 fmol/mg protein, n = 3, NS). Scatchard plot analyses (Figure 5B) also showed that CsA caused a significant increase in the numbers of binding sites (control, 315.8 ± 23.5 fmol/mg protein; +CsA, 543.6 ± 43.2 fmol/mg protein, n = 10, P < 0.001) without affecting the KD (control, 7.16 ± 0.14; +CsA, 7.06 ± 0.19 nM, n = 10, NS). These results thus suggested that CsA stimulated Ang II receptors mainly by increasing their number.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. Effect of CsA on AT1 and AT2 expression in mTAL cells. (A) Ethidium bromide-stained 4% agarose gel of RT–PCR showing the amounts of amplified products of AT1 and AT2 compared with that of ß-actin, used as standard, in cultured mTAL cells incubated without (1) or with 100 ng/ml CsA (2). Molecular weight standards (M) were the 1 kb ladder from Gibco-BRL. (B) The bars represent the amplified products ratio for AT1 and AT2 over ß-actin for untreated (control) and CsA-treated cells (CsA). Values are the mean ± SD from four separate experiments. ***P < 0.001 vs control values.

 


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4. Quantification of AT1 receptor mRNA in mTAL cells by real-time PCR. The PCR was run in triplicate for each sample. Standard curves for (A) the AT1 receptor and (B) GAPDH. The standard curve was expressed as log ng of RNA vs threshold cycle (Ct). (C) AT1/GAPDH ratio of ng of RNA in untreated (control) and CsA-treated (CsA) mTAL cells.

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. Effect of CsA on [3H]Ang II binding in mTAL cells. (A) Untreated (open circles) and CsA-treated (filled circles) mTAL cells were incubated with various concentrations (0.2–10 nM) of [3H]Ang II for 2 h at 22°C. Points are the mean ± SD from 10 separate experiments. (B) Scatchard plots of [3H]Ang II binding to untreated (open circles) and CsA-treated (filled circles) mTAL cells. Each point is the mean of 10 determinations performed in duplicate.

 
The results from RT–PCR experiments suggested that, concomitantly with the increase in AT1 receptor expression, CsA induced a decrease of AT2 receptor. AT2 receptor is thought to down-regulate AT1 receptor expression [14]. Although the AT2 receptor accounted for only ~10% of Ang II receptors in mTAL cells, the question arises as to whether the decrease in AT2 expression could interfere with the CsA-stimulated AT1 expression. We therefore measured [3H]Ang II binding in CsA-treated mTAL cells in the presence of losartan and PD123319. The CsA stimulatory effect on [3H]Ang II binding could be blocked by losartan, but not by PD123319 (control, 328.6 ± 43.7; +CsA 100 ng/ml, 543.6 ± 43.2; +CsA +losartan 10–5 M, 110.3 ± 15.3; +CsA +PD123319 10–5 M, 553.6 ± 76.2 fmol/mg protein, n = 5). These results indirectly suggested that the stimulatory effect of CsA on the AT1 receptor was not related to the decrease in AT2 receptor expression.

Effects of CsA on [Ca2+]
The data from binding studies, indicating that CsA caused an increase in the number of Ang II receptors, led us to test the effects of CsA on the levels of [Ca2+]i from cultured mTAL cells. [Ca2+]i has been shown to be one of the most important intracellular signals induced by Ang II [13]. Cultured mTAL cells were exposed to 100 ng/ml CsA for 24 h, and the level of [Ca2+]i was determined by fluorimetric recording, using the calcium-sensitive fluorescent probe fura-2/AM. As shown in Figure 5, the basal level of [Ca2+]i was slightly greater in CsA-treated cells than in untreated cells (control, 72.2 ± 3.5; +CsA, 93.4 ± 7.9 nM/106 cells, n = 6, P < 0.05). Ang II (10–7 M) induced an increase in [Ca2+]i in both untreated and CsA-treated cultured mTAL cells (Figure 6A and B), but the stimulant effect of Ang II (10–7 M) was much more pronounced on CsA-treated cells than on untreated cells (control, 97.8 ± 8.4; +CsA, 206.3 ± 10.6 nM/106 cells, n = 6, P < 0.05) (Figure 6C). Consistent with the increase in Ang II AT1 receptors, CsA considerably enhanced the Ang II pathway [Ca2+]i signal in mouse mTAL cells.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 6. Effect of CsA on [Ca2+]i. Representative traces of the changes in [Ca2+]i induced by 10–7 M Ang II (arrows) in untreated (A) and 100 ng/ml CsA-treated (B) mTAL cells. (C) The bars represent the changes in [Ca2+]i measured before (open bars) and after adding Ang II (black bars) in untreated (control) and CsA-treated (CsA) cells. Values are the mean ± SD from six separate experiments. *P < 0.05 vs control values.

 
Effect of CsA on cell viability
To ensure that the changes induced by CsA were not attributable to cell damage, cell viability, used as an index of cell injury, was measured in cultured mTAL cells incubated for 24 h with or without a range of concentrations (5–600 ng/ml) of CsA. There was no significant difference between the percentage of viable cells in CsA-treated and untreated cells (5 ng/ml, 98 ± 4%; 10 ng/ml, 99 ± 5%; 50 ng/ml, 101 ± 8%; 100 ng/ml, 102 ± 12%; 600 ng/ml, 97 ± 8% of viable cells vs untreated cells, n = 5). These findings indicated that cultured TAL cells remained viable after being incubated with CsA for 24 h, suggesting that the observed decreases in AT2 receptor expression were not related to cell damage.



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results from this study show that CsA increases the number of Ang II receptors responsible for a major increase in [Ca2+]i in our model of cultured mouse mTAL cells. Ang II induces a variety of effects in the kidney, including potent vasoconstriction of the renal microcirculation, stimulation of sodium reabsorption through the release of aldosterone, alterations in the glomerular filtration and possible induction of mesangial cell hypertrophy. Specific Ang II receptors have been identified on the cell surface of a large number of target tissues, including vascular smooth muscle, adrenal glands, liver, lung, brain and kidneys [8]. Two main subtypes of Ang II receptors, AT1 and AT2, have been identified [17]. These receptors operate via different signal transduction pathways. The binding of Ang II to the AT1 receptor results in phosphatidylinositol hydrolysis, intracellular Ca2+ mobilization [13] and inhibition of adenylate cyclase [17], whereas activation of the AT2 receptor appears to cause a decrease in cGMP levels [17]. Using a cRNA probe and in situ hybridization, Meister et al. [9] have shown that the AT1 receptor mRNA is expressed in the S3 segment of the proximal tubule and in outer medullary TAL from the rat kidney. AT2 receptors are also expressed in the medullary area in fetal rats [8]. AT1 receptors have been detected in both apical and basolateral membranes of rat mTALs [10] but, in contrast to the basolateral AT1 receptors, the apical membrane-located AT1 receptors appear not to be coupled to Ca2+ signalling. Here we show that intact mouse mTAL segment and cultured mTAL cells express both AT1 and AT2 subtype Ang II receptors, and the results from binding studies using losartan indicate that most of the [3H]Ang II is bound specifically to AT1 receptors.

The interplay between CsA and the intrarenal RAS and its exact mechanism in the pathogenesis of hypertension and nephrotoxicity caused by CsA are complex and still remain not fully elucidated. In vitro studies have shown that CsA up-regulates Ang II receptors in human vascular smooth muscle cells [13]. Pharmacological studies using ACE inhibitors in both experimental models and humans to block RAS activity have also provided lines of evidence that the RAS certainly plays an important role in the progression of renal failure [18]. Burdmann et al. [18] have demonstrated that the interstitial fibrosis caused by CsA could be prevented by losartan and enalapril. Another study using human proximal tubule cells and renal fibroblasts has also shown that ACE inhibitors are able to prevent CsA-induced interstitial fibrosis directly independently of haemodynamic and systemic RAS effects. Previous studies have provided apparent divergent results concerning the effects of CsA on AT1 Ang II receptors. Tufro-McReddie et al. [19] have reported that the chronic administration of CsA to rats stimulated the renin activity and concomitantly reduced AT1 Ang II receptor mRNA expression in whole kidney. In contrast, Regitz-Zagrosek et al. [20] found that rats chronically treated with CsA exhibited increased numbers of total Ang II receptors in adrenal glands, liver and kidneys, and that the AT1 receptor antagonist DUP 753 abolished the increase in Ang II receptor density in these organs. Our study indicated that CsA increased Ang II, mostly AT1, receptor activity in isolated mTAL cells.

mTALs expressing Ang II receptors are very sensitive to the action of CsA. We have shown previously that CsA may alter NaCl absorption in cultured mouse mTAL cells [11]. It also impairs the production of NO in this model of cultured mTAL cells [4]. These findings strongly suggest that CsA may have pleiotropic effects affecting various specific tubular cell functions and the intrarenal microcirculation adjacent to the epithelial cells. Pre-incubating subcultured mTALs cells with non-toxic concentrations of CsA stimulates predominantly the expression of AT1 receptor, and the data from the Ang II binding studies indicate that CsA induces an increase in Bmax without affecting KD. As the increase in [Ca2+]i is one of the main intracellular signals induced by Ang II [13], CsA also amplifies the stimulant effect of Ang II on [Ca2+]i. The combination of these findings indicates that CsA increases the density of Ang II receptors, mainly AT1 receptors, in mTAL cells, which in turn become more sensitive to the action of Ang II. These results suggest that CsA, via the up-regulation of Ang II AT1 receptor, may directly or indirectly induce renal interstitial fibrosis. Interestingly, we found that CsA induces a decrease of AT2 receptor expression in mTAL cells. Tanaka et al. [14] have demonstrated that vascular AT1 receptor expression increases in AT2 knockout mice, suggesting that AT2 counteracts the AT1-mediated effect of Ang II through down-regulation of AT1 receptors. Here we show that blockade of AT2 receptors does not alter the stimulatory effect of CsA on AT1 receptors. These results suggest thus that the stimulatory effect of CsA on AT1 receptors seems not to be related directly to a decrease in AT2 receptor expression in this model of cultured mTAL cells.

It has been shown that the blockage of Ang II receptors by losartan or enalapril decreases the expression of transforming growth factor-ß1 and extracellular matrix proteins in a salt-depleted rat model of CsA-induced tubulointerstitial fibrosis. Yang et al. [6] have also shown in rat kidneys that the interstitial fibrosis induced by CsA via the decrease in renal EGF expression can be corrected by losartan. These results thus suggested that AT1 stimulation is associated with enhanced fibrosis and impaired healing. Consistent with these in vivo experiments, the results from this study show that CsA can directly stimulate Ang II AT1 receptors and thereby an increase in the level of [Ca2+]i in mTALs, thus suggesting that this pathway may be involved in the mechanism CsA nephrotoxicity.



   Acknowledgments
 
This work was funded by a grant from the Taiwan NMRP272 and NMRP513 and in part by the INSERM (France).

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Myers BD, Sibley R, Newton L et al. The long-term course of cyclosporine-associated chronic nephropathy. Kidney Int 1988; 33: 590–600[ISI][Medline]
  2. Garr MD, Paller MS. Cyclosporine augments renal but not systemic vascular reactivity. Am J Physiol 1990; 258: F211–F217[ISI][Medline]
  3. Bunchman TE, Brookshire CA. Cyclosporine-induced synthesis of endothelin by cultured human endothelial cells. J Clin Invest 1991; 88: 310–334[ISI][Medline]
  4. Wu MS, Yang CW, Bens M et al. Cyclosporine inhibits nitric oxide production in medullary ascending limb cultured cells. Nephrol Dial Transplant 1998; 13: 2814–2820[Abstract]
  5. Navar LG, Inscho EW, Majid SA et al. Paracrine regulation of the renal microcirculation. Physiol Rev 1996; 76: 425–532[Abstract/Free Full Text]
  6. Yang CW, Ahn HJ, Kim WY et al. Influence of the renin–angiotensin system on epidermal growth factor expression in normal and cyclosporine-treated rat kidney. Kidney Int 2001; 60: 847–857[CrossRef][ISI][Medline]
  7. Hannedouche TP, Natov S, Boitard C, Lacour B, Grunfeld JP. Angiotensin converting enzyme inhibition and chronic cyclosporine-induced renal dysfunction in type 1 diabetes. Nephrol Dial Transplant 1996; 11: 673–678[Abstract]
  8. Navar LG, Imig JD, Zou L, Wang CT. Intrarenal production of angiotensin II. Semin Nephrol 1997; 17: 412–422[ISI][Medline]
  9. Meister B, Lippoldt A, Bunnemann B, Inagami T, Ganten D, Fuxe K. Cellular expression of angiotensin type-1 receptor mRNA in the kidney. Kidney Int 1993; 44: 331–336[ISI][Medline]
  10. Poumarat JS, Houillier P, Rismondo C, Roques B, Lazar G, Paillard M, Blanchard A. The luminal membrane of rat thick limb expresses AT1 receptor and aminopeptidase activities. Kidney Int 2002; 62: 434–445[CrossRef][ISI][Medline]
  11. Wu MS, Yang CW, Bens M, Peng KC, Yu HM, Vandewalle A. Cyclosporine stimulates Na+–K+-Cl cotransport activity in cultured mouse medullary thick ascending limb cells. Kidney Int 2000; 58: 1652–1663[CrossRef][ISI][Medline]
  12. Wu MS, Bens M, Cluzeaud F, Vandewalle A. Role of F-actin in the activation of Na+–K+-Cl cotransport by forskolin and vasopressin in mouse kidney cultured thick ascending limb cells. J Membr Biol 1994; 142: 323–336[ISI][Medline]
  13. Avdonin PV, Cottet-Maire F, Afanasjeva GV et al. Cyclosporine A up-regulates angiotensin II receptors and calcium responses in human vascular smooth muscle cells. Kidney Int 1999; 55: 2407–2414[CrossRef][ISI][Medline]
  14. Tanaka M, Tsuchida S, Imai T et al. Vascular response to angiotensin II is exaggerated through an upregulation of AT1 receptor in AT2 knockout mice. Biochem Biophys Res Commun 1999; 258: 194–198[CrossRef][ISI][Medline]
  15. Mentzel S, Van Son JP, De Jong AS et al. Mouse glomerular epithelial cells in culture with features of podocytes in vivo express aminopeptidase A and angiotensinogen but not other components of the renin–angiotensin system. J Am Soc Nephrol 1997; 8: 706–719[Abstract]
  16. Haller H, Schaberg T, Lindschau C, Lode H, Distler A. Endothelin increases [Ca2+]i, protein phosphorylation, and O2– production in human alveolar macrophages. Am J Physiol 1991; 261: L478–L484[ISI][Medline]
  17. Timmermans PB, Wong PC, Chiu AT et al. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 1993; 45: 205–251[ISI][Medline]
  18. Burdmann EA, Andoh TF, Nast CC et al. Prevention of experimental cyclosporin-induced interstitial fibrosis by losartan and enalapril. Am J Physiol 1995; 269: F491–F499[ISI][Medline]
  19. Tufro-McReddie A, Gomez RA, Norling LL, Omar AA, Moore LC, Kaskel FJ. Effect of CsA on the expression of renin and angiotensin type 1 receptor genes in the rat kidney. Kidney Int 1993; 43: 615–622[ISI][Medline]
  20. Regitz-Zagrosek V, Auch-Schwelk W, Hess B et al. Tissue- and subtype-specific modulation of angiotensin II receptors by chronic treatment with cyclosporin A, angiotensin-converting enzyme inhibitors and AT1 antagonists. J Cardiovasc Pharmacol 1995; 42: 66–72
Received for publication: 23.10.02
Accepted in revised form: 12. 2.03