Modulation of gene expression by moxonidine in rats with chronic renal failure
Oliver Vonend1,*,
Thomas Apel1,*,
Kerstin Amann2,
Lorenz Sellin1,
Johannes Stegbauer1,
Eberhard Ritz2 and
Lars Christian Rump1
1 Department of Internal Medicine, Marienhospital Herne, University of Bochum, 2 Department of Pathology,University of Erlangen and 3 Department of Nephrology, University of Heidelberg, Germany
Correspondence and offprint requests to: Professor Dr L. C. Rump, Marienhospital Herne, Ruhr-Universität Bochum, Hölkeskampring 40, D-44625 Herne, Germany. Email: christian.rump{at}ruhr-uni-bochum.de
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Abstract
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Background. Sympathetic overactivity is a hallmark of chronic renal failure. In a previous experimental study, the sympatholytic drug moxonidine (MOX) had beneficial effects on progression of chronic renal failure. The present study investigates whether moxonidine influences the expression of genes associated with adaptive changes in kidneys of subtotally nephrectomized rats.
Methods. RNA was isolated from remnant kidneys of sham-operated, subtotally nephrectomized (SNX) and moxonidine-treated SNX (SNX-M) rats 12 weeks after operation. Genes that might play a role in renal adaptation processes after subtotal nephrectomy were selected and their expression was analysed by real-time reverse transcriptionpolymerase chain reaction (RTPCR).
Results. After subtotal nephrectomy, there was an increase in gene expression of cysteine protease cathepsin (H + L), ATP receptor subtypes P2Y2 and P2Y6, cell cycle regulator p21 and transforming growth factor-ß1 (TGF-ß1), and a decrease of the metalloprotease aminopeptidase-M (APM), membrane transporter megalin, ageing-related klotho, type I TGF-ß receptor, mitochondrial cytochrome oxidase-1, kallikrein, leucine zipper-1, matrix-degrading metalloprotease meprin, the organic anion transporter and the P2 receptor subtypes P2Y1 and P2Y4. In SNX-M rats, mRNA levels of APM, megalin, klotho, TGF-ß1, type I TGF-ß receptor, p21, P2Y1 and P2Y2 were shifted back towards control levels.
Conclusions. Several genes showing altered expression levels after subtotal nephrectomy were identified in remnant kidneys. These genes might act as candidates to promote disease progression. The sympatholytic drug moxonidine, at a concentration devoid of blood pressure effects, regulates the renal expression of some of these genes back towards control levels. To what extent sympathetic neurotransmitters directly alter expression of these genes in cultured renal cells currently is under investigation.
Keywords: 5/6 nephrectomy; chronic renal failure; gene expression; moxonidine; P2 receptors; sympathetic activity
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Introduction
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Chronic renal failure is characterized by sympathetic overactivity and contributes to the genesis of hypertension and progression of renal disease [1]. This has been well documented by microneurographic recording techniques in patients with chronic and end-stage renal failure as well as in renal transplant recipients [2]. The sympathetic neurotransmitters noradrenaline and ATP are released in excess from renal sympathetic nerve endings and possibly induce mitogenic effects, accelerating progression of renal disease [35]. Accordingly, in our previous study on subtotally nephrectomized rats, moxonidine, a drug that reduces sympathetic nerve activity, diminishes glomerulosclerosis and proteinuria independently of its effects on blood pressure [6].
The aim of the present study was to elucidate whether moxonidine is able to influence the expression of genes which might be associated with adaptive changes observed in renal tissue of rats with chronic renal failure [6]. Twenty genes, including three housekeeping genes, were selected for real-time reverse transcriptionpolymerase chain reaction (RTPCR) analysis. The selection of genes was based on one of the following criteria: genes previously analysed by in situ hybridization [6]; genes related to the sympathetic nervous system and its co-transmitter ATP [4,5]; or genes that have been described in the literature to be linked to adaptive changes within renal tissue [710]. This might help to understand the molecular mechanisms through which moxonidine, either directly by inhibition of the sympathetic nervous system or indirectly by modulating various other pathways, might ameliorate the progression of chronic renal failure.
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Materials and methods
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Animals
Male SpragueDawley rats (200 g) were housed in single cages at constant room temperature (20°C) and humidity (75%) under a controlled light/dark cycle and fed a diet containing 40 g of protein and 0.6 g of NaCl/100 g (Altromin, Lage/Lippe, Germany). After a 3 day adaptation period, the animals were randomly allocated to three groups. (i) Group 1: sham-operated control group (SHAM); (n = 6). (ii) Group 2: subtotally nephrectomized group, no treatment (SNX); (n = 9). (iii) Group 3: subtotally nephrectomized (5/6) group, moxonidine 1.5 mg/kg per day (SNX-M); (n = 9). The remnant and control kidneys were harvested and snap-frozen in liquid nitrogen 12 weeks after the operation.
RNA preparation
Cryopreserved kidney cortex of previously studied rats was analysed [6] for RNA expression. The kidney tissue was homogenized in liquid nitrogen with a dismembranator (Braun). RNA preparation was performed using the guanidiniumthiocyanatephenolchloroform extraction method. The quality of the RNA was controlled using gel electrophoresis and PCR with GAPDH primer to exclude genomic DNA contamination.
Real-time PCR
Specific primer pairs for the different genes were designed (Table 1) and synthesized by Genescan (Germany). Following DNA digestion (RNase-free DNase, Invitrogen, Germany), cDNA synthesis was performed with 3 µg of individual renal RNA extracts, 100 ng of hexanucleotide random primer and 1 U of superscript reverse transcriptase (Invitrogene, Germany) according to the manufacturer's manual. For real-time PCR, an ABI 7700 was used and the following composition: 1x buffer, 2.5 mM MgCl2, 60 nM SYBR-Green (Molecular Probes, Germany) 1 mM dNTPs, 1 mM primer, 100 ng of cDNA and 1 U of Taq polymerase (Invitrogene, Germany). The cycle profile was 3 min at 94°C followed by 40 times for 30 s at 94°C, 30 s at 60 or 66°C, and 20 s at 72°C. After the cycles, a melting curve and an agarose gel analysis was carried out for each primer. The results are presented as the
ct (difference in threshold cycle) of target gene vs housekeeping gene GAPDH [11].
ct values for target gene vs housekeeping gene are presented for all three groups (SHAM, SNX and SNX-Mrats). All
ct values were multiplied by 1 to correlate a positive shift in the
ct value with an increase in the amount of RNA. Differences of (n)
ct values between the groups 
ct represent a change in gene expression of 2(n). All real-time PCR experiments were repeated eight times to reduce technical variations maximally.
Statistics
Differences (
ct) in threshold cycles target gene vs housekeeping gene (
ct) were analysed by one-way ANOVA with Bonferroni's post hoc test (Analyze-It/general + clinical laboratory statistics version 1.71/UK). The confidence interval was 95%. P-values of <0.05 were considered statistically significant.
Quantitative competition PCR
Competition fragments were constructed for GPADH (296 bp) and megalin (275 bp) according to the method described by Scheuermann and Bauer [12]. PCR was performed as described above but adding competitor DNA and no SYBR-Green. Using different concentrations of 1000, 500, 100, 20, 4 and 1 amol, the megalin expression could be determined competitively. The band intensity on ethidium bromide-stained agarose gel was measured densiometrically under UV light with an alpha imager (alpha-inotech CA, USA).
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Results and Discussion
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Clinical and experimental studies had demonstrated that renal disease is associated with sympathetic overactivity [1,2,13]. Moreover, efforts to downregulate sympathetic activity seemed to have beneficial effects on structural and functional markers of renal disease in rats [6] and humans [14]. Specifically, subtotally nephrectomized rats, treated with the sympatholytic drug moxonidine, had less glomerulosclerosis, less vascular damage and less albuminuria than untreated rats 12 weeks after nephrectomy [6].
This study was conducted to elucidate whether moxonidine influences the expression of genes associated with adaptive changes observed in subtotally nephrectomized rats. Twenty genes, including three housekeeping genes, were selected (Table 1) for real-time RTPCR analyses. The mRNA was extracted from kidneys of rats 12 weeks after sham operation, subtotal nephrectomy as well as subtotal nephrectomy plus moxonidine treatment. Most of these genes have been described in the literature to be linked to adaptive changes within renal tissue [710]. The analysis of transforming growth factor-ß1 (TGF-ß1) and its receptor was added to verify the methodological approach of the actual study [6]. Since the effect of moxonidine on progression of renal failure was blood pressure independent, we hypothesized that its protective effect could partially be due to inhibition of the release of the mitogenic sympathetic neurotransmitters norepinephrine and ATP in the kidney [36]. Therefore, we also analysed the modulation of ATP (P2Y) receptor expression. These receptors have the potential to modulate mitogenic effects in vitro [4,5].
Modulated gene expression in experimental chronic renal failure
Three housekeeping genes (actin, GAPDH and HPRT) (Table 1) were selected, and threshold cycles (ct) of analysed target genes were compared with threshold cycles (ct) of GAPDH (
ct). The expression levels of the housekeeping genes actin (Fig. 1) and HPRT (data not shown) did not show any differences throughout the groups, thus documenting the efficacy of the methodology used.
Two different gene expression profiles could be observed. Nine genes had an increased or decreased expression after subtotal nephrectomy when compared with control animals (Fig. 1). These were genes coding for the cysteine proteases cathepsin H and L, mitochondrial cytochrome oxidase-1, kallikrein, leucine zipper-1, matrix-degrading metalloendopeptidase meprin ß, organic anion transporter and the nucleotide receptors P2Y4 and P2Y6. Moxonidine, however, had no effect on expression of this group of genes, so that it is unlikely that these genes are related to the beneficial effects observed in the treated animals.
Moxonidine-modulated gene expression
Figure 2 shows genes possibly involved in disease progression as they were up- or downregulated after subtotal nephrectomy when compared with sham-operated control animals. Moreover, since these genes were also shifted towards baseline control levels by the sympatholytic treatment, they might also contribute to the beneficial effect of the drug moxonidine. These were sequences coding for aminopeptidase M, endocytotic receptor megalin, ageing-related protein klotho, TGF-ß1, the type 1 TGF-ß receptor, cell cycle regulator p21 and the nucleotide receptors P2Y1 and P2Y2.
In accordance with previous observations using in situ hybridization [6], increased expression of TGF-ß has been confirmed by real-time RTPCR, showing that this method is a reliable approach to quantify gene expression possibly related to progression of renal disease. TGF-ß is a primary signal able to modulate a variety of factors that amplify renal fibrosis [15]. Even though the reduction in TGF-ß1 expression by moxonidine was not statistically significant when compared with untreated animals, a tendency was detectable. In line with this, the type 1 TGF-ß receptor expression showed a reversed image. For these and other genes, however, we cannot differentiate whether reversal of gene expression by moxonidine indicates that the expression of the respective genes had been stimulated primarily by sympathetic overactivity or whether the reversal was only a secondary event associated with better renal function after moxonidine treatment.
Another important gene in cell growth regulation and protection of cells from apoptotic stimuli is the cyclin G1 kinase inhibitor p21. Accordingly, p21 is able to modulate podocyte proliferation and maturation [16]. Moreover, p21 knock-out mice do not develop chronic renal failure after renal ablation [7]. In line with these findings, an increase of p21 expression was observed after subtotal nephrectomy, which was reversed by moxonidine treatment. Klotho is a ß-glucosidase-like protein, predominantly expressed in kidney, and involved in suppression of several phenotypes resembling ageing and premature death [17]. In patients and rats with chronic renal failure, expression of klotho is reduced [8]. Although klotho gene expression was only slightly reduced in subtotally nephrectomized rats, moxonidine treatment reversed its expression in our study.
Proteinuria is a key parameter in chronic renal failure. Due to glomerular injury and hyperfiltration, more albumin enters the tubular system. Endocytotic receptors such as megalin reabsorb and rescue filtered ligands, including Salbumin [9]. To confirm the real-time RTPCR results, a quantitative competition PCR was performed for megalin (Fig. 3). Due to the fact that competitor cDNA has to be constructed for each gene individually, this method is unsuitable for comparing a large number of genes. Artificial cDNA products for GAPDH and megalin were constructed. In comparison with 1000 amol GAPDH cDNA, the megalin cDNA quantity for the different groups was 228 amol in sham-operated rats, 11 amol in untreated and 328 amol in moxonidine-treated subtotally nephrectomized rats. Thus, this competitive analysis demonstrates, similar to the results of real-time RTPCR, a downregulation of renal megalin expression in chronic renal failure rats, which was reversed by moxonidine treatment. A link between albuminuria and the observed reduction of megalin expression is plausible, especially with the normalized expression levels after moxonidine treatment. However, whether these changes are a result or the cause of improved albumin excretion remains to be elucidated.

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Fig. 3. Quantitative competition PCR with GAPDH and megalin is shown. Competitor DNA at 1000, 500, 100, 20 or 4 amol is added to the PCR. The competitor DNA produces longer PCR products. GAPDH and megalin reactions were prepared with the same amount of cDNA. When both bands are visible, the amount of tissue cDNA equals the competitor DNA. Ma = marker.
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The expression level of aminopeptidase M (APM) is also dramatically changed after subtotal nephrectomy. APM converts T-kinin to the vasodilating peptide bradykinin. It is one of the major peptides degrading vasoactive angiotensin metabolites in the kidney [10]. APM inactivates angiotensin III, which can act as an agonist at the AT1 receptor [10]. The effects of AT1 receptor activation are numerous, including vasoconstriction and cell proliferation.
ATP activates P2Y receptors to induce proliferation of many cell types including human renal cells in culture [4,5]. mRNA coding for P2Y1 and P2Y4 receptors was found to be downregulated, even though not statistically significantly, whereas mRNA for P2Y2 and P2Y6 receptors was upregulated in subtotally nephrectomized rats when compared with control animals. In addition the expression profile for P2Y1 and P2Y2 was shifted towards control levels by moxonidine treatment. ATP is an excitatory co-transmitter of noradrenaline in the kidney and can be released in large amounts after all kinds of cell stress [4]. Thus P2 receptors might play a role in injury and repair mechanisms occurring in chronic renal failure. Increased P2Y2 expression has also been observed in congestive heart failure [18], ageing and after cytokine stimulation [19].
These data demonstrate altered expression of several genes in an experimental model of chronic renal failure. Even though analysis of mRNA does not always reliably predict the changes in protein expression and function, these genes could act as promoters of disease progression. Since subtotally nephrectomized animals have a significant increase in blood pressure as well as an impaired renal function and an increased albuminuria, we cannot distinguish whether changes in expression levels are a secondary phenomenon due to pressure- or albuminuria-related effects.
In addition, whole kidney cortex preparations consist of numerous cell types, glomeruli, blood vessels, tubules and, in the case of subtotally nephrectomized animals, scar tissue. Scar tissue clearly influences the expression level, in particular when nephrectomized rats were compared with sham-operated animals. However, this limitation interferes less with data obtained in treated and untreated animals, since both groups underwent the same surgical procedure. The more interesting question of our study was whether moxonidine influences gene expression. Since the sympatholytic drug moxonidine was used in a non-hypotensive dose, the pressure-independent drug effects could be analysed. Indeed, a non-hypotensive dose of moxonidine at least partially normalized the expression of several of the above-mentioned genes apparently involved in chronic renal failure rats. We acknowledge, however, that moxonidine also interacts with imidazoline (I2) receptors in the isolated perfused rat kidney and in cultured renal cells [20], therefore a change in expression levels by direct peripheral I2 receptor activation has to be considered. Moreover, beneficial effects of moxonidine on renal function, proteinuria and renal inflammation surely induce secondary alterations which again might modulate renal gene expression. To what extent sympathetic neurotransmitters are able to alter the expression of some of these genes directly is unknown and has to be tested outside of a complex organism. In this regard, studies on cultured renal cells currently are under investigation in our laboratory.
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Acknowledgments
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The invaluable technical help of Ms Petra Stunz and Bettina Priesch is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Ru 401/5-6).
Conflict of interest statement. None declared.
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Notes
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*These authors contributed equally to this work. 
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Received for publication: 5. 9.03
Accepted in revised form: 19. 5.04