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Clinical Science (2003) 104, 389–395 (Printed in Great Britain) Epithelial sodium channel (ENaC) subunit mRNA and protein expression in rats with puromycin aminonucleoside-induced nephrotic syndrome A. AUDIGE; , Z. R. YU, B. M. FREY, D. E. UEHLINGER, F. J. FREY and B. VOGT Division of Nephrology and Hypertension, Inselspital, University of Berne, Freiburgstrasse 3, 3010 Berne, Switzerland A B S T R A C T In experimental nephrotic syndrome, urinary sodium excretion is decreased during the early phase of the disease. The molecular mechanism(s) leading to salt retention has not been completely elucidated. The rate-limiting constituent of collecting duct sodium transport is the epithelial sodium channel (ENaC). We examined the abundance of ENaC subunit mRNAs and proteins in puromycin aminonucleoside (PAN)-induced nephrotic syndrome. The time courses of urinary sodium excretion, plasma aldosterone concentration and proteinuria were studied in male Sprague–Dawley rats treated with a single dose of either PAN or vehicle. The relative amounts of αENaC, βENaC and γENaC mRNAs were determined in kidneys from these rats by real-time quantitative TaqMan PCR, and the amounts of proteins by Western blot. The kinetics of urinary sodium excretion and the appearance of proteinuria were comparable with those reported previously. Sodium retention occurred on days 2, 3 and 6 after PAN injection. A significant up-regulation of αENaC and βENaC mRNA abundance on days 1 and 2 preceded sodium retention on days 2 and 3. Conversely, down-regulation of αENaC, βENaC and γENaC mRNA expression on day 3 occurred in the presence of high aldosterone concentrations, and was followed by a return of sodium excretion to control values. The amounts of αENaC, βENaC and γENaC proteins were not increased during PAN-induced sodium retention. In conclusion, ENaC mRNA expression, especially αENaC, is increased in the very early phase of the experimental model of PAN-induced nephrotic syndrome in rats, but appears to escape from the regulation by aldosterone after day 3. INTRODUCTION Nephrotic syndrome is characterized by proteinuria, hypoalbuminaemia and sodium retention with oedema formation [1]. Traditionally, salt retention in nephrotic syndrome has been explained by the hypovolaemia concept. This setting implies increased renal sodium reabsorption in response to the decreased oncotic press- ure following proteinuria, fluid loss into the interstitium, hypovolaemia, and subsequent hyperaldosteronism. However, several observations argue against a primary role of hypovolaemia and hyperaldosteronism. Plasma volume is often normal or even increased in the nephrotic patient, and hypervolaemia can be seen in some animal models of the syndrome [2–4]. Furthermore, sodium retention is observed prior to proteinuria during the Key words : aldosterone, epithelial sodium channel, nephrotic syndrome, puromycin aminonucleoside, real-time PCR, Western blot. Abbreviations : CCD, cortical collecting duct ; C , threshold cycle ; ENaC, epithelial sodium channel ; GAPDH, glyceraldehyde-3T phosphate dehydrogenase ; PAN, puromycin aminonucleoside ; RT, reverse transcription. Correspondence : Dr Bruno Vogt (e-mail bruno.vogt!insel.ch). # 2003 The Biochemical Society and the Medical Research Society 389 390 A. Audige! and others initial stages of relapse in children [3]. In addition, nephrotic syndrome can be induced in adrenalectomized rats [5,6]. Alternatively, salt retention can be explained by an intrarenal defect. In accordance with this hypothesis, the kidney taken from a rat rendered nephrotic by exposure to puromycin aminonucleoside (PAN) retains sodium abnormally when perfused in isolation [7,8]. Micropuncture studies in a unilateral model of PAN-induced nephrotic syndrome in rats have demonstrated that the site of sodium retention is beyond the distal convoluted tubule [9,10]. During the period of positive sodium balance, Na+,K+-ATPase activity has been shown to be enhanced in the cortical collecting duct (CCD) in several experimental models of nephrotic syndrome [6,11–13]. The rate-limiting factor for trans-epithelial sodium transport in the CCD is the activity of the amiloridesensitive epithelial sodium channel (ENaC). This channel is expressed primarily at the apical membrane of cells involved in the maintenance of extracellular fluid volume and blood pressure, such as in the distal nephron, the distal colon and the respiratory airways [14,15]. ENaCs are heteromultimeric proteins formed by the association of three homologous subunits, α, β and γ [16,17]. Regulation of ENaC activity can occur at different levels, i.e. transcription, translation, translocation and degradation, as well as single-channel open probability and conductance. ENaC subunits have been shown to be transcriptionally up-regulated by aldosterone, a lowsodium diet or dexamethasone in the kidney, colon and lung, in a tissue- and subunit-specific fashion [18]. Recently, in vitro microperfusion studies showed that amiloride, a collecting duct diuretic that blocks ENaCs, was able to restore sodium balance in CCDs from nephrotic animals [9]. The aim of the present study was to establish whether ENaC is transcriptionally regulated in nephrotic syndrome, and whether expression of ENaC subunit mRNAs and\or protein expression correlates with the profile of urinary sodium excretion. For this purpose, the experimental model of PAN-induced nephrotic syndrome in the rat was used. METHODS Corp., Lausanne, Switzerland) at a dose of 150 mg\kg body weight, diluted to 15 mg\ml in 0.9 % NaCl as described previously [6,11,12,19]. Control rats received only the solvent. Sodium, protein and creatinine levels were measured in 24 h urine samples. Urinary sodium and protein excretion was calculated as a function of urinary creatinine excretion. On the experimental days, animals were anaesthetized with sodium pentobarbital (50 mg\kg body weight ; intraperitoneal), and the kidneys were removed, frozen immediately in liquid nitrogen and kept at k80 mC until further use. Tissues were powdered with a mortar and pestle in a mixture of solid CO and acetone before being used for RNA extraction. # Plasma aldosterone concentrations were determined using a commercially available RIA (Diagnostic Products Corp., Los Angeles, CA, U.S.A. ; Iodine-125 tracer, Coat-A-Count). Approx. 0.2 ml of plasma was used for one measurement. RNA extraction Kidneys were homogenized in RNA extraction buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5 % sarcosyl and 0.1 M 2-mercaptoethanol [20]) using a Polytron. Total RNA was extracted according to the method of Chomczynski and Sacchi [20] with minor modifications. Briefly, the RNA pellet resulting from the first precipitation was resuspended in diethyl pyrocarbonate-treated water, and 0.1 vol. of 2 M sodium acetate and 1 vol. of phenol, pH 4.0, were added sequentially [20]. The suspension was vortexed and centrifuged at 10 000 g for 20 min. The upper phase was precipitated with propan-2-ol at k20 mC for 1 h. Reverse transcription (RT) RT was performed using 2 µg of total RNA in a final volume of 20 µl. RNA was incubated with 500 ng of oligo(dT) primer at 65 mC for 5 min, followed by incubation at room temperature for 15 min. Samples were then incubated with 250 µM of each dNTP, 20 units of RNase inhibitor and 25 units of avian myeloblastosis virus reverse transcriptase (Roche, Rotkreuz, Switzerland) in 1ibuffer (50 mM Tris\HCl, 40 mM MgCl , # 30 mM KCl, 1 mM dithiothreitol, pH 8.5) at 42 mC for 1 h. For control reactions (RT−), enzymes were replaced by diethyl pyrocarbonate-treated water. Experimental animals Male Sprague–Dawley rats (170–210 g body weight) were housed in metabolic cages with free access to normal rat chow diet and water. The animal work was undertaken according to Swiss national legislation governing the use of animals, and the committee on animal research at the University of Berne approved the protocol. After an adaptation period of 5 days so that the rats were in sodium balance, PAN nephrotic syndrome was induced by a single intraperitoneal injection of PAN (Alexis # 2003 The Biochemical Society and the Medical Research Society Real-time PCR Primers (Microsynth, Balgach, Switzerland) and TaqMan probes (Applied Biosystems, Rotkreuz, Switzerland) unique for each rat isoform were designed (Table 1), optimized and validated. Real-time quantitative TaqMan PCR using an ABI Prism 7700 Sequence Detection System and TaqMan Universal PCR Master Mix (Applied Biosystems) was performed according to the manufacturer’s protocol. Diluted RT samples (10 ng of Epithelial sodium channel and nephrotic syndrome Table 1 Primers and probes unique for each rat ENaC isoform used in real-time quantitative TaqMan PCR Gene Primer/probe Sequence αENaC Forward Reverse Probe CAGGGTGATGGTGCATGGT CCACGCCAGGCCTCAAG TGAAGCCACCATCATCCATAAAGGCAG βENaC Forward Reverse Probe CATAATCCTAGCCTGTCTGTTTGGA CAGTTGCCATAATCAGGGTAGAAGA CCCTGCAGTCATCGGAACTTCACACCT γENaC Forward Reverse Probe TGCAAGCAATCCTGTAGCTTTAAG GAAGCCTCAGACGGCCATT AATGGACACTGACCACCAGCTTGGC GAPDH Forward Reverse Probe CTGCCAAGTATGATGACATCAAGAA AGCCCAGGATGCCCTTTAGT TCGGCCGCCTGCTTCACCA total RNA) were amplified in a final volume of 25 µl. Primers were used at a concentration of 200 nM and probes (FAM\TAMRA-labelled) at a concentration of 100 nM, except for γENaC, for which the probe concentration was 200 nM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene and was amplified in separate reactions. RT+ reactions were performed in triplicate, and RT− (control) reactions in duplicate. The thermal cycling conditions were as follows : 50 mC for 2 min and 95 mC for 10 min, followed by 40 cycles of 95 mC for 15 s and 60 mC for 1 min. Relative quantification of gene expression Relative quantification of ENaC expression was carried out using the arithmetic formula method (Applied Biosystems User Bulletin 2, 1997). The average threshold cycle (CT) value for GAPDH was subtracted from the average CT values for αENaC, βENaC and γENaC for each RT sample to obtain the ∆CT values. The CT value represents the PCR cycle at which a statistically significant increase in reporter fluorescence is first detected. For each group of experimental animals, the average ∆CT values for αENaC, βENaC and γENaC of the control animals were determined and used as the calibrators. For each nephrotic animal, the ∆∆CT values for αENaC, βENaC and γENaC were calculated by subtracting the respective calibrator from the individual ∆CT value. The relative amounts of mRNA for αENaC, βENaC and γENaC of the animals with nephrotic syndrome compared with the controls was calculated by using the formula 2−∆∆CT. prepared. Frozen powdered kidney tissue was homogenized in cold sucrose buffer using FastPROTEIN GREEN tubes (Lysing Matrix D ; Q-Biogene, Alexis Corp.) and a BIO 101 Savant FastPrep FP120 device (QBiogene) at a speed of 4.0 units for 20 s. The homogenized tissue was centrifuged at 2100 g for 10 min at 4 mC. The supernatant was kept on ice and the pellet, dissolved in sucrose buffer, was centrifuged again at the same speed. The second supernatant was pooled with the first supernatant and the protein concentration was determined using a BCA Protein Assay Reagent Kit (Pierce). From each sample, 30 µg of protein was incubated with Laemmli buffer at 37 mC for 30 min and then loaded immediately on to 10 % (v\v) polyacrylamide gels. Proteins were transferred from the gels to PVDF membranes (Immobilon ; Millipore Corp., Bedford, MA, U.S.A.). Blots were blocked with 5 % (w\v) non-fat milk at room temperature for 90 min and incubated with the appropriate affinity-purified polyclonal antibody overnight at 4 mC (see below). The secondary antibody conjugated to horseradish peroxidase was a rabbit antichicken\turkey IgG for βENaC (Zymed, San Francisco, CA, U.S.A.) protein and a swine anti-rabbit IgG (DAKO, Glostrup, Denmark) for αENaC and γENaC. Antibody binding was visualized by enhanced chemiluminescence (ECL2 ; Amersham Pharmacia Biotech). Densitometry of individual bands was performed using the Scion Image software. Affinity-purified rabbit antibody against αENaC was a gift from Dr L. Dijkink (Universiteit Nijmegen, The Netherlands), and rabbit antibody against γENaC was from Dr Nicolette Farman (INSERM U478, Paris, France). The antibody against βENaC was raised in chickens using the peptide (amino acids 46–68) NH # CNYDSLRLQPLDTMESDSEVEAI (Davids Biotechnology, Regensburg, Germany) [21]. Statistics Statistical analyses were performed with SYSTAT 9.0 for Windows (SPSS Inc., Chicago, IL, U.S.A.). Metabolic data were compared using the two-sample t test. ENaC expression data were compared using the two-sample t test on the average ∆CT values of each animal. Signals for RT− of 40 cycles were observed in 11 % of all RT− reactions (results not shown). These signals constituted on average 0.01 % of those of the respective RT+ reactions, and were therefore considered as negligible. Results are presented as meanspS.D. RESULTS Western blots Metabolic studies Sucrose buffer containing 250 mM sucrose, 1 µg\ml leupeptin (Sigma), 10 mM triethanolamine (Merck) and 0.1 mg\ml PMSF (Calbiochem), pH 7.6, was freshly Urinary sodium excretion increased on day 1 in rats after PAN injection (PAN, 29.4p0.75 mmol\mmol of creatinine ; control, 19.5p0.5 mmol\mmol ; P 0.001), and # 2003 The Biochemical Society and the Medical Research Society 391 392 A. Audige! and others Figure 2 Expression of ENaC subunit mRNAs in kidneys from rats with PAN-induced nephrotic syndrome Figure 1 Renal excretion of sodium and protein by rats with PAN-induced nephrotic syndrome The time courses of urinary sodium excretion (A) and proteinuria (B) after a single intraperitoneal injection of PAN ( , >) or vehicle (, =) into male Sprague–Dawley rats are shown. Results on day 0 correspond to the 24 h urine sample collected the day before induction of nephrotic syndrome. Urinary sodium excretion and proteinuria values are meanspS.D. from five groups of animals for days 0 and 1, from four groups of animals for days 2 and 3, from two groups of animals for days 4–6, and from one group of animals for days 7–12. Each group consisted of six PAN-treated and six control rats. Significant differences between PAN-treated and control rats : *P 0.001. decreased on days 2 and 3 to levels that were lower than control levels [day 2 : PAN, 10.2p0.5 mmol\mmol ; control, 16.5p0.4 mmol\mmol (P 0.01) ; day 3 : PAN, 8.3p0.6 mmol\mmol ; control, 13.8p0.7 mmol\mmol (P 0.001)] (Figure 1A). Sodium excretion returned to control levels on day 4, decreased again on day 5, and reached the lowest level on day 6 (PAN, 7.5p1.3 mmol\ mmol ; control, 15.1p0.5 mmol\mmol ; P 0.001). This retention period was followed by increased urinary sodium excretion from days 9 to 11. Following injection of PAN, proteinuria was first evident on day 4, reached the highest level on day 8, and remained high until the end of the experimental period (Figure 1B). The mean daily urinary creatinine excretion was not different in PAN-treated rats and controls (results not shown). # 2003 The Biochemical Society and the Medical Research Society For each time point, the relative amount of mRNAs for αENaC, βENaC and γENaC in kidneys from six nephrotic animals (five on day 1) ( ) compared with six control animals () is given. Plots comprise results from two TaqMan PCR experiments (four experiments for αENaC on day 1). Results are meanspS.D. Significant differences between PAN-treated and control rats : *P 0.05, **P 0.01, ***P 0.001. Expression of ENaC mRNAs and plasma aldosterone concentrations Expression of αENaC and βENaC mRNAs in the kidneys of rats exposed to PAN was markedly increased relative to controls on day 1 (αENaC, 2.5-fold ; βENaC, 1.9-fold ; P 0.001) (Figure 2). Expression of αENaC and βENaC mRNAs on day 2 was still significantly higher than control levels (αENaC, 1.4-fold, P 0.01 ; βENaC, 1.5-fold, P 0.05). On day 3, expression of all three subunit mRNAs was decreased to very low levels compared with controls (αENaC, decreased to 20 %, P 0.01 ; βENaC, decreased to 40 %, P 0.05 ; γENaC, decreased to 30 %, P 0.05). On days 6 and 12, alterations in ENaC subunit mRNA levels were less pronounced compared with the preceding time points. Differences reached statistical significance for αENaC on day 6 (reduction to 80 % ; P 0.05), as well as for αENaC and γENaC on day 12 (reduction to 70 % ; P 0.05). On days 1 and 2, the increased abundance of αENaC and βENaC mRNAs in the kidneys of rats exposed to PAN occurred in the presence of increased plasma Epithelial sodium channel and nephrotic syndrome decreased on day 3. Furthermore, on day 6 and 12, aldosterone concentrations remained elevated, whereas the levels of ENaC subunit mRNAs were either decreased or unchanged (Figures 2 and 3). ENaC subunit protein expression Figure 3 Plasma aldosterone concentrations in rats with PAN-induced nephrotic syndrome For each time point, the plasma aldosterone concentration was measured in six nephrotic animals (4) and compared with that in six control animals (5). Results for each day are from an independent group of animals. Plasma aldosterone levels were increased in animals with PAN-induced nephrotic syndrome when compared with control animals during the whole study period (day 1, P 0.01 ; days 2, 3 and 6, P 0.002 ; day 12, P 0.0001). The abundance of αENaC protein in proteinuric rats was decreased on day 2 (P 0.01) and increased on day 6 (P 0.05) when compared with that in control animals (Figure 4). Despite the presence of increased plasma aldosterone concentrations (Figure 3), the abundance of βENaC or γENaC protein was not significantly changed throughout the study (Figure 4). When sodium excretion reached its lowest level, on day 6 (Figure 1), the abundance of αENaC protein was increased in PANtreated animals when compared with control animals (P 0.05) ; similarly, the amounts of βENaC and γENaC protein tended to be non-significantly increased (Figure 4). DISCUSSION Figure 4 Expression of ENaC subunit proteins in kidneys from rats with PAN-induced nephrotic syndrome (densitometry) For each time point, the protein expression of αENaC, βENaC and γENaC in kidneys from six nephrotic animals (five on day 1) ( ) compared with that in six control animals () is shown. Results are meanspS.D. Significant differences between PAN-treated and control rats : *P 0.05, **P 0.01. aldosterone concentrations (Figure 3). However, despite continued increased aldosterone concentrations, the expression of αENaC, βENaC and γENaC mRNAs was PAN is a xenobiotic that induces a nephrotic syndrome with proteinuria and sodium retention in rats [22]. The time course and profile of urinary sodium excretion and proteinuria following exposure to PAN have been studied in detail [11]. In line with that study, sodium retention occurred on days 2–3 and 5–7 in our PANtreated animals (Figure 1A), and proteinuria started on day 5 (Figure 1B). The present investigation demonstrates that, following PAN administration, (i) aldosterone concentrations increase, (ii) mRNA levels of ENaC subunits change, and (iii) these changes in mRNA levels are not paralleled by the amount of ENaC subunit protein expression. The renal site of salt retention in experimental nephrotic syndrome has been determined by micropuncture and clearance studies to be beyond the distal convoluted tubule, most likely in the CCD [10,23]. Two proteins constitute the main determinants of distal tubular sodium transport : the Na+,K+-ATPase pump in the basolateral membrane and the apically located ENaC. Na+,K+ATPase activity in the CCD correlates with urinary sodium excretion in three experimental models of nephrotic syndrome in rats [11]. The activity of the pump is regulated by several mechanisms, including an increase in intracellular sodium concentration through activation of the ENaC [24]. In in vitro microperfusion studies with amiloride, a collecting duct diuretic that blocks the ENaC, sodium balance could be restored in CCDs from nephrotic animals [9]. In our present study, sodium excretion was decreased on days 2 and 3, but returned to control values on days 4 and 8, in rats treated with PAN (Figure 1A). Whether the increase in urinary sodium excretion on day 1, an # 2003 The Biochemical Society and the Medical Research Society 393 394 A. Audige! and others observation made by other investigators previously, represents a non-specific toxic effect of the agent on tubular sodium transport is unknown [11]. The stimulation of Na+,K+-ATPase activity in PAN-treated animals might explain renal sodium retention during the early phase of this model of nephrotic syndrome [6,11,12]. Aldosterone controls sodium reabsorption in the CCD by acting on the basolateral cell surface expression of Na+,K+-ATPase and by regulating the apical cell surface expression of ENaC [25–27]. In our nephrotic rats, αENaC and βENaC mRNA expression was upregulated 1 and 2 days after PAN injection ; however, mRNA expression of all three ENaC subunits was strongly down-regulated on day 3, despite the presence of very high plasma aldosterone levels (Figures 2 and 3). The increased expression of αENaC and βENaC mRNAs on days 1 and 2 occurred in the presence of high aldosterone levels, and the pattern of this increase in the three different ENaC subunit mRNAs is comparable with the effect of aldosterone on αENaC, βENaC and γENaC mRNA expression in A6 cells [28]. Conversely, the decreased ENaC mRNA expression on day 3, an effect compatible with the observed transitory enhancement of sodium excretion on day 4, also occurred in the presence of high plasma aldosterone levels (Figures 2 and 3). The mechanism for this escape of ENaC mRNA expression from regulation by aldosterone is unknown. A speculative mechanism for the strong sodium retention on day 3, despite markedly down-regulated ENaC subunit mRNA levels, could be a diminished clearance of ENaC from the cell membrane, leading to elevated ENaC cell surface expression, mediated by Nedd4-2 or N4WBP5A, novel Nedd4\Nedd4-2-interacting protein [29–31]. It is conceivable that proteins that are abnormally filtered in proteinuric disease states activate such signalling pathways. Significant sodium retention occurred from day 5 to day 7 in the presence of high plasma aldosterone concentrations (Figures 1 and 3). During this period of sodium retenion, ENaC mRNAs normalized and protein levels of ENaC subunits remained unchanged (Figures 2 and 4). Although we did not measure relative mRNA abundance in defined tubular segments, but only in whole kidneys, it is tempting to speculate that this regulation occurred in the CCD. ENaC mRNA expression was not accompanied by corresponding changes in protein concentration. Thus it is not possible to link the amount of ENaC protein with transcription. A possible explanation could be increased trafficking of ENaC subunits from intracellular stores to the cell membrane [32]. In the present investigation, we measured the total amount of ENaC protein by Western blot. It is conceivable that the intracellular localization of ENaC subunits is altered in the PAN-induced disease state, an effect that might have been missed by our analyses. # 2003 The Biochemical Society and the Medical Research Society From day 8 to day 12, increased sodium loss despite high plasma aldosterone levels was observed in the PANtreated animals. The simplest explanation for this increased sodium loss is a dysregulation of protein synthesis and\or of functional incorporation of ENaC into the plasma membrane. This would be in line with the stable amounts of ENaC subunit proteins measured during this study period (Figure 4). An alternative mechanism for the stimulation of ENaC in experimental nephrotic syndromes might involve vasopressin. First, plasma vasopressin levels are elevated during the early phase of PAN-induced nephrotic syndrome. Secondly, in a rat model of acute and chronic elevation of circulating levels of vasopressin, increases in the levels of αENaC, βENaC and γENaC subunits were shown in the kidney by quantitative immunoblotting [33–35]. However, the pattern of αENaC, βENaC and γENaC subunits reported was different from our observations (Figure 2), in that all three subunits were increased in the vasopressin model, with a 2-fold increase for βENaC and 3-fold increase for γENaC measured at day 7 [35]. Furthermore, in A6 cells incubated with vasopressin, a selective elevation in the level of βENaC mRNA was observed [36]. The different patterns of αENaC, βENaC and γENaC subunit mRNAs observed in the PAN-induced nephrotic syndrome in our animal model suggests that other, as yet undefined, mechanisms regulate ENaC expression in this disease. Interestingly, PAN-induced nephrotic syndrome with renal sodium retention appears to be independent of vasopressin activity, as shown in Brattleboro rats, which genetically lack vasopressin secretion [11]. In conclusion, our results show that levels of ENaC subunit mRNAs, especially αENaC, are up-regulated during the early phase of PAN-induced nephrotic syndrome. This stimulation of ENaC expression is associated with sodium retention. ENaC mRNA levels appear to escape from regulation by aldosterone after day 3. No direct relationship between ENaC mRNA expression and ENaC protein amount was observed, indicating that additional mechanism(s) for the enhanced ENaC activity have to be considered. ACKNOWLEDGMENTS This work was supported by two grants from the Swiss National Foundation for Scientific Research (no. 3257205.99 and no. 31-061505.00). We thank Dr L. 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