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NDT Advance Access published online on February 14, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn020
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Induction of NO synthase 2 in ventricular cardiomyocytes incubated with a conventional bicarbonate dialysis bath

Eleonora Grandi1,2,5, Marco Govoni1,3,5, Simone Furini1,2,5, Stefano Severi1,2, Emanuele Giordano1,3, Antonio Santoro4 and Silvio Cavalcanti1,2

1 Laboratory of Cellular and Molecular Engineering 2 Department of Electronics, Computer Science and Systems, University of Bologna 3 Department of Biochemistry ‘G.Moruzzi’, University of Bologna 4 Malpighi Nephrology Division, Policlinico S. Orsola-Malpighi, Bologna, Italy

Correspondence and offprint requests to: Emanuele Giordano, Laboratorio di Ingegneria Cellulare e Molecolare, Via Venezia, 52, I-43027, Cesena (FC), Italy. Tel: +39-0547-339243; Fax: +39-0547-339208, E-mail: emanuele.giordano{at}unibo.it



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. In hypotension-prone patients, conventional bicarbonate dialysis (BD) causes a reduced cardiovascular tolerance to the treatment with respect to acetate-free biofiltration (AFB). One possible explanation is an overproduction of endogenous NO (nitric oxide) due to the residual quote of acetate (4 mM) in the BD dialysate formulation. NO overload might cause the impairment of cardiovascular reactivity observed during BD. In this study, a potential direct impact of the residual quote of acetate on the cardiac cells was investigated.

Methods. Ventricular cardiac myocytes isolated from adult rat hearts were treated with three different dialysis baths with or without acetate: BD, AFB and AFB + 4 mM of acetate (AFB+). Corresponding levels of expression of the inducible NO synthase 2 (NOS2) were assessed after the treatments along with the measurement of single-cell action potential (AP).

Results. Incubation with acetate-containing dialysis solutions significantly enhanced (P < 0.05) the expression of NOS2 mRNA (BD: 1.11 ± 0.31; AFB+: 0.73 ± 0.04, NOS2/GAPDH intensitometric ratio) with respect to the acetate-free bath (AFB: 0.03 ± 0.01). Accordingly, protein translation was also enhanced (BD: 0.176 ± 0.021; AFB+: 0.135 ± 0.009, NOS2/{alpha}-tubuline intensitometric ratio) with respect to AFB (0.002 ± 0.001, P < 0.05). Measurement of the AP indicates that acetate-containing solutions determine a shortening of the repolarization phase as compared to treatment with AFB (BD: 95 ± 13; AFB+: 76 ± 10; AFB: 162 ± 16 ms).

Conclusion. These findings show that the residual quote of acetate of the BD bath formulation affects the expression of NOS2 and the duration of AP in cardiac cells. This might cause the cardiac contractile impairment in unstable patients during BD treatment.

Keywords: acetate; cardiomyocyte; haemodialysis; hypotension; nitric oxide



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Hypotension is a common intra-treatment complication of haemodialysis (HD) [1] and dialysate composition is known to play a key role in determining patient's tendency to hypotension. For instance, the substitution of acetate-buffer with bicarbonate in dialysate resulted in a significant reduction of intradialytic hypotensive episodes [2–4] although in conventional bicarbonate dialysis (BD), a residual amount of acetate (3–4 mM) is still present for reasons of chemical stability [5]. Substitution of BD with acetate-free dialysis (AFB) results in a further improvement of cardiovascular tolerance with reduction of dialysis-related hypotension episodes [6–11]. One possible explanation for the different pressure response to BD and AFB is that the residual acetate in BD enhances the production of endogenous NO (nitric oxide) [12]. In fact, it is well known that NO overload limits the vascular tone and the contractile force of cardiac muscle cell [13,14], which are essential to maintain a stable arterial pressure during the HD-induced hypovolaemia.

Beasley and Brenner [15] proposed that acute hypotension during HD may be sustained by inflammatory cytokines driving to an excess of NO production in vascular smooth muscle cells. BD has been shown to cause more monocyte activation and cytokine release than AFB [16], and to be more effective in activating endothelial and smooth muscle cells to synthesize/release pro-inflammatory and pro-apoptotic mediators [17]. Comparing in vitro the effects of different dialysate solutions on endothelial cells, Noris et al. [18] found that the inducible isoform of NO synthase (NOS2) was transcriptionally activated when the cells were exposed to acetate-containing dialysates. A similar result was also obtained ex vivo by incubating endothelial cells with blood samples from patients treated with BD [12].

We recently focused our attention on the different impacts of BD and AFB treatments on cardiac activity [11,19]. We showed that a higher risk of hypotension in BD versus AFB was consistent with a BD-induced blunted heart contractility [11], and that the QT interval on the ECG was shorter in patients undergoing BD with respect to AFB [19].

On these bases we formulated the hypothesis that BD treatment might affect cardiac activity possibly via a local overproduction of NO in muscle cardiac cells, as a result of NO2 induction driven by the residual quote of acetate. Indeed, cardiac myocytes are known to activate the transcription of their NOS2 gene upon stressful stimuli [20], although a direct evidence of this event as a response to acetate-containing HD has not been yet demonstrated.

Accordingly, the aim of the present study was to test different solutions (BD, AFB and AFB + 4 mM Na-acetate) as potential transcriptional inducers of the expression of NOS2 in adult rat ventricular cardiac myocytes. In addition, we performed single-cell action potential (AP) recordings to assess whether the three solutions have a differential functional impact on the cardiac muscle cell.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Materials
Dialysis baths (BD = Na+ 140 mM, K+ 2.5 mM, HCO3– 34 mM, acetate 3 mM, Cl– 109 mM, Ca2+ 1.5 mM, Mg2+ 0.5 mM, D-glucose 5.6 mM and AFB = Na+ 139 mM, K+ 3 mM, Cl– 147.5 mM, Ca2+ 2 mM, Mg2+ 0.5 mM D-glucose 5.6 mM) were from Hospal SpA, Bologna, Italy. Rats were purchased from Charles River Laboratories, Calco (LC), Italy. All the reagents, unless otherwise specified, were from Sigma, Milan, Italy.

Adult rat cardiac myocyte isolation
Ventricular myocytes were obtained by retrograde perfusion of hearts—isolated from adult male Sprague-Dawley rats weighing 150–175 g—with type II collagenase (Warthington Biochemical Corp., NJ, USA) and protease according to a previously described technique [21]. The investigation conforms to the NIH Guide for the Care and Use of Laboratory Animals.

Cardiomyocyte treatments
Isolated adult rat cardiac myocytes were divided in four aliquots and incubated for 8 h with different dialysis fluids with or without acetate: (1) BD (acetate = 3 mM); (2) AFB (acetate-free) and (3) AFB+ (AFB + 4 mM Na-acetate, chosen as the highest level of acetate conventionally used in BD). A further group was incubated in EBSS (NaCl 1.16 M, D-glucose 55.2 mM, NaH2PO4 10 mM, KCl 53 mM and NaHCO3 261 mM) added with 10 µg/mL E. coli lipopolysaccharide (LPS) and 400 U/mL tumour necrosis factor-{alpha} (TNF) corresponding to a reference stimulus for massive NOS2 transcriptional induction. All groups were incubated at 37°C for 8 h.

Total RNA extraction and evaluation of NOS2 transcriptional activation
Total RNA was extracted using the Trizol Reagent (Invitrogen, Milano, Italy). Reverse transcription was performed in 50 µL of a solution containing 0.1 U/µL AMV reverse transcriptase, 0.1 U/µL DNA polymerase, dNTPs mix (0.2 mM each dNTP), 1 mM MgSO4, 100 pmol upstream and downstream primers, and AMV reaction buffer and nuclease-free water to the final volume (Promega Corporation, WI, USA). NOS2 sense and antisense primers used for DNA amplification in the following polymerase chain reaction (PCR) were 5'-TGTCAGCCCTCAGAGTACA-3' and 5'-GCTGATGGAGTAGTAGCGG-3', respectively (TIB MOLBIO CBA, Genua, Italy); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense and antisense primers were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3', respectively (Invitrogen).

Ten microlitres of each PCR amplification product were separated by electrophoresis at 120 V for 1 h in 2% agarose gel in a TBE buffer (45 mmol/L Tris borate, 1 mmol/L EDTA, pH 8). Gel photographs were acquired under UV transillumination (Foto/Phoresis I, Hartland, WI, USA), digitalized and intensitometrically quantified (Quantiscan, Biosoft, Cambridge, UK) to obtain the ratio between NOS2 and GAPDH transcripts for each treatment group.

Western blot
Rat cardiac myocytes were homogenized in 5 mM DTT, 2 mM EDTA, 0.1% CHAPS, 0.1% Triton X-100 and protease inhibitors in 20 mM HEPES (pH 7.5). The protein extract was thus submitted to SDS–PAGE and western blotting as previously described [22] using specific anti NOS2 and anti-{alpha}-tubuline primary antibodies (Santa Cruz, CA, USA).

AP measurements
After treatment, cardiac myocytes were transferred into extracellular bath (in mM: 140 NaCl, 6 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, 5 HEPES; pH 7.4 with NaOH). Ruptured-patch whole-cell current-clamp procedure was used to measure the membrane potential. Signals were recorded with an Axopatch 200B amplifier using pCLAMP 9 (Axon Instruments Inc., Union City, CA, USA). Membrane potential signal was filtered at 5 kHz and sampled at 10 kHz. Borosilicate microelectrodes had a resistance ranging between 4 and 5 M{Omega} when filled with the pipette solution (in mM: 140 K aspartate, 8 KCl, 7 NaCl, 1 MgCl2, 10 HEPES, 5 Mg-ATP, 0.3 Na-GTP; pH 7.2 with KOH). Myocytes with an access resistance <15 M{Omega} were used for the AP analysis. APs were triggered at 1 Hz by square supra-threshold current pulses lasting 1 ms. The AP duration (APD) was measured at 90% of repolarization (APD90) and averaged over a >30-s window after steady APs were reached (at least after a train of 60 simulation pulses).

Statistical analysis
Values are reported as mean ± SE. Repeated measures (Geisser–Greenhouse correction) ANOVA and Tukey–Kramer multiple-comparison test were applied to APD90 data, for which normal distributions were found (Skewness, Kurtosis and Omnibus tests). The nonparametric Kruskal–Wallis ANOVA on ranks and multiple-comparison Z-test were applied to mRNA and protein expression data, for which the normal distribution assumption was not met. Statistical analyses were made using the NCSS 2001 package (NCSS Statistical Software, Kaysville, UT, USA).



   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
NOS2 gene expression
Our standard RT-PCR protocol produced a clear band of the predicted size (100 bp) for the NOS2 gene transcript (Figure 1A). The ratiometric analysis of the intensitograms against the respective GAPDH (453 bp) control showed a differential transcriptional expression of NOS2 gene, according to the specific cell treatment (Figure 1B). Namely, NOS2/GAPDH gene expression intensitometric ratios were 1.11 ± 0.31, 0.03 ± 0.01 and 0.73 ± 0.04, respectively upon incubation in BD, AFB and AFB+ solutions. NOS2 gene transcription was significantly enhanced (P < 0.05, N = 4) in cardiac myocytes incubated in both acetate-containing dialysates (BD and AFB+) with respect to AFB (Figure 1B). Remarkably, NOS2 gene expression levels after BD and AFB+ treatments were of the same magnitude of the one obtained when cardiac myocytes were challenged with LPS/TNF (0.99 ± 0.15), that was expected to highly induce NOS2 expression. On the other hand, incubation with AFB produced a substantial absence of effect on NOS2 transcript expression.


Figure 1
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  		Fig. 1 Expression of mRNA encoding for NO synthase 2 in rat cardiac myocytes after an 8-h incubation with LPS/TNF, BD, AFB and AFB+. (A) Lane 1: EBSS added with 10 µg/mL LPS and 400 U/mL TNF-{alpha}; lane 2: BD (containing 3 mM acetate); lane 3: AFB; lane 4: molecular weight marker (Mw) and lane 5: AFB+ (with 4 mM acetate added). (B) Results from N = 4 experiments (*P < 0.05) are shown in arbitrary units (NOS2/GAPDH mRNA ratios).

 
Protein expression
Western blot analysis revealed that translation followed the transcription of NOS2 gene in cardiac myocytes (Figure 2). Consistently with RT-PCR results, cardiac myocytes challenged with acetate-containing solutions (BD and AFB+) showed a clear signal revealing the induction of NOS2 protein, whereas virtual absence of signal was detected in the sample treated with AFB (Figure 2A). Namely, NOS2/{alpha}-tubuline protein expression intensitometric ratios were 0.176 ± 0.021, 0.002 ± 0.001 and 0.135 ± 0.009, respectively, upon incubation in BD, AFB and AFB+ solutions. In both acetate-containing dialysates, the protein expression was significantly enhanced (P < 0.05, N = 5) with respect to AFB (Figure 2B). As in the case of gene expression, also NOS2 protein expression levels after BD and AFB+ treatments were similar to that with the classical LPS/TNF stimulus (0.188 ± 0.087).


Figure 2
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  		Fig. 2 Western blot displays NO synthase 2 protein in rat cardiac myocytes after an 8-h incubation with different dialysis baths. (A) Lane 1: EBSS added with 10 µg/mL LPS and 400 U/mL TNF-{alpha}; lane 2: BD (containing 3 mM acetate); lane 3: AFB; lane 4: Mw; lane 5: AFB+ (with 4 mM acetate added). (B) Results from N = 5 experiments (*P < 0.05) are shown in arbitrary units (NOS2/{alpha}-tubuline protein ratios).

 
AP measurements
AP recordings showed distinct responses to the differential treatment, consistently with NOS2 expression results. The cells incubated with acetate-containing dialysates (BD and AFB+) showed significantly reduced APD with respect to the AFB-treated ones (Figure 3). APD90 was not significantly different between cardiac myocytes treated with the two acetate-containing solutions (BD 95 ± 13 ms, N = 7 animals, n = 20 cells; AFB+ 76 ± 10, N = 7 n = 11). Both proved to be significantly shorter (P < 0.001) than the one measured upon incubation with AFB (162 ± 16 ms, N = 7, n = 12).


Figure 3
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  		Fig. 3 Action potential duration at 90% of repolarization (APD90) in rat cardiac myocytes treated with BD, AFB and AFB+ (N = 7 animals; n = 20, 12 and 11 cells, respectively, *P < 0.001).

 
No differences among cell groups were found with respect to the membrane resting potential and the AP amplitude (–60 ± 0.65 mV and 69 ± 16 mV, respectively, N = 7, n = 43).



   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Transcriptional upregulation of the inducible isoform of NOS2 was shown in endothelial and smooth muscle cells in response to their challenge with acetate-containing dialysis baths [12,16,18]. The massive amount of NO produced by this enzyme in vascular cells has been proposed as a main player in the genesis of hypotension during HD. Actually, stressful stimuli are known to trigger the induction of NOS2 also in the cardiac muscle cell, where the resulting NO overload is responsible for cardiac contractile impairment, as observed in a number of pathological conditions [23,24]. A previous study [11] showed that a relevant reduction of cardiac contractile force occurred when patients prone to hypotension were treated with BD, where a residual amount of acetate is still present. This evidence prompted us to check the potential effect of acetate as an inducer of NOS2 in cardiac myocytes.

By measuring NOS2 mRNA and protein levels, we show here that acetate-containing BD and AFB+ solutions—but not AFB—induce NOS2 expression in adult rat cardiac myocytes. The extent of this induction is comparable with the response to the classical TNF/LPS stimulus (Figures 1 and 2), this would imply a strong pro-inflammatory activity of acetate.

As a critical molecule for cardiac myocyte homeostasis, intracellular NO production is normally tightly controlled by the regulated activity, at specific subcellular compartments, of the two constitutively expressed NOS1 and NOS3 isoforms [23]. The induction of NOS2 gene expression within the cardiac cell leads to the loss of this balance.

It has been proposed that acetate can induce NOS2 by triggering the release of proinflammatory mediators (such as TNF-{alpha}, IL-1β and IL-6) from both endothelial and smooth muscle cells [12,16,17]. Evidence is available that these molecules can be synthesized also by cardiac myocytes themselves [25]. It is worth noting that once induced, NOS2 constitutively synthesizes high (micromolar) levels of NO, without the need of further stimuli and over a long-lasting time [26]. The increase in NO2 concentration could be reinforced by repeated treatments with acetate-containing dialysis baths.

We furthermore performed AP recordings to assess whether NOS2 expression would be accompanied by a distinct electrical profile in cardiac myocytes. Reduced APD was observed in those cells where enhanced NOS2 expression was induced (Figure 3). Emerging evidences [27–30] suggest that NO has a key role in mediating alterations in APD. Expression of inducible NOS2 and consequent NO overload in single cardiac myocytes isolated from LPS-treated animals is accompanied by APD reduction, that is reversed by administration of specific NOS2 inhibitors [27–30]. A role for NO overload in this respect was also evidenced in vivo, being the increased NO level accompanied by shortening of QT interval, that is the ECG counterpart of the ventricular cell APD [31]. Remarkably, the shorter QT interval duration that we previously reported [19] in patients undergoing BD versus AFB is in agreement with the present finding of a shorter APD after incubation of cardiac myocytes with the BD bath. The clinical significance of the abbreviation of QT in the genesis of cardiac arrhythmias is not as clear as in the long QT syndromes. A familial syndrome of an abnormally shortened QT interval and arrhythmias has been described [32]. However, QTc intervals in our patients were within the normality range [19]. Thus, we rule out a NO-induced electrical derangement except in sporadic cases when other cofactors may play a role (e.g. high plasma calcium concentration, hyperkalaemia, etc.).

In the present study, following the approach pioneered by Amore et al. [12] in endothelial cells, we directly exposed cardiac myocytes to different dialysis baths. This heavy stressful stimulus to the cells might lead to a mild change in the permeability properties of the cell membrane as indicated by the observation that our measured values of membrane resting potential and AP amplitude, slightly deviate from what is reported in freshly isolated cells by our group (data not shown) or other groups [33]. In addition, regarding the specific instance of comparing different dialysis baths, other differences, but acetate, in the dialysate composition may play a role in the different outcomes of BD and AFB. As an example, BD has an alkaline pH whereas AFB is an acidic solution although a role for a differential pH when comparing the two solutions as inducer of endothelial cell activation was ruled out by Todeschini et al. [16]. However, the comparison between AFB versus AFB+ partly overcomes this limitation since the two baths only differ for either the presence (AFB+) or the absence (AFB) of acetate, so that the different response can unequivocally be attributed to acetate only. The presented set of experiments was carried out by adding to the AFB solution 4 mM of acetate—the usual reported concentration in the standard BD. However, we might have overestimated the actual acetate concentration reaching the patients’ cardiomyocytes in vivo. As our BD formula contains only 3 mM acetate, sample NOS2 WB were also performed adding the latter concentration to AFB. According to these analyses, 3mM and 4mM of acetate cause similar NO2 expressions (data not shown).

Taken together our findings support the hypothesis that the residual quote of acetate in BD solution produces an endogenous NO overload, which is functionally relevant for the cardiac cell. Indeed, high NO levels are known to depress cardiac electromechanical activity and to produce contractile impairment, as it is observed in endotoxaemia. Accordingly, left ventricular function improvement [34,35], haemodynamic stabilization [36,37] and overall survival benefit [38] were achieved in this condition by administration of NOS inhibitors. In addition, it was shown that exposure of isolated adult rat myocytes to endotoxin attenuated the inotropic responses to isoproterenol, and this effect was abolished by pretreatment with NOS inhibitors [39].

At this stage, our results point out a direct impact of a residual quote of acetate in the dialysis bath on NOS2 expression and AP duration in the cardiac cells. These findings are coherent with the hypothesis that BD treatment affects cardiac activity via a local overproduction of NO in muscle cardiac cells and support the indication that acetate-free dialysis might represent a treatment of choice in patients at risk for hypotension during BD, where selective NOS2 inhibitors could represent a novel therapeutic option.



   Acknowledgments
 
M.G. was the recipient of a fellowship from the National Institute for Cardiovascular Research (INRC). Financial support from Italian Ministry of University and Scientific Research (EF 2004) and INRC (to Emanuele Giordano) is gratefully acknowledged. We are indebted to Dr Marco Carboni for his critical suggestions in setting the RT-PCR protocol.

Conflict of interest statement. Part of this work was presented in poster format at the XIII (21–23 September 2006) National Congress of the Italian Society for Cardiovascular Research held in Imola (BO, Italy). The authors declare that they do not have any conflict of interest.



   Notes
 
5 Equally contributed to this work. Back



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 6. 9.07
Accepted in revised form: 10. 1.08


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