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and 
ENaC
Department of Physiology and Biophysics and the Center for Excellence in Cardiovascular Renal Research, University of Mississippi Medical Center, Jackson, Mississippi
Submitted 23 May 2006 ; accepted in final form 12 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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or 
ENaC, using dominant-negative (DN) or small-interference RNA (siRNA) molecules, inhibits pressure-induced vasoconstriction in mouse renal interlobar arteries. To address this goal, isolated arteries were transiently transfected with or ENaC DN-cDNA or siRNA molecules. After 24 h, vessels were either 1) cannulated and pressurized for pressure-diameter response curves or 2) dissociated and immunolabeled to determine VSM cell endogenous ENaC protein expression. We found that transfection of ENaC DN-cDNA or siRNA suppresses -, but not ENaC protein expression. Similarly, ENaC DN-cDNA or siRNA suppresses -, but not ENaC protein expression. In addition, transfection of - or ENaC DN-cDNA or siRNA molecules inhibits pressure-induced vasoconstriction, but does not block agonist-induced vasoconstriction. Our results provide the first direct evidence that and ENaC proteins are essential in mediating myogenic vasoconstriction in mouse renal interlobar arteries. mechanotransduction; transfected isolated renal vessel; stretch-activated cation channel; siRNA; dominant negative
Members of the Degenerin/Epithelial Na+ Channel (DEG/ENaC) family of proteins are candidates for mechanosensitive ion channels in vascular smooth muscle. Evidence suggests DEG/ENaC proteins may act as mechanosensors. First, members of this evolutionarily conserved family form mechanosensors in the nematode, Caenorhabditis elegans (26). Second, ENaC channels can be activated by shear stress, a mechanical factor (5, 28). Third, ENaC proteins are required for normal mechanosensation in a variety of mammalian cell types (3, 10, 13, 21, 25, 32). Through selective pharmacological inhibition of DEG/ENaC channels, our laboratory has recently provided evidence that DEG/ENaC channels play an important role in myogenic constriction in isolated, pressurized rat middle cerebral arteries (9) and mouse renal interlobar arteries (18). These pharmacological inhibitors have provided a basic tool to screen for DEG/ENaC channel involvement because they block a broad spectrum of DEG/ENaC channels. While we detected and , but not 
, ENaC transcripts and protein in freshly dispersed VSMCs from rat cerebral and mouse renal vessels (9, 18), direct evidence supporting a role of and/or ENaC in myogenic constriction is still lacking. The goal of this investigation was to determine whether the presence of and/or ENaC is important in eliciting myogenic constriction in renal interlobar arteries. To achieve this goal, we used novel approaches, dominant-negative (DN) and small-interference RNA (siRNA), to silence endogenous ENaC expression. Our findings provide the first direct evidence that - and ENaC are essential for myogenic vasoconstriction in mouse renal interlobar arteries.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Preparation of dominant negative (DN) expression vectors and siRNA. The generation of our DN expression vectors has been previously described (8). Briefly, premature stop codons (X) were engineered into nucleotide bases encoding for amino acid I41 for ENaC (EGFP-I41X) and L160 for ENaC (EGFP-L160X) and ligated into Enhanced Green Fluorescent Protein C-terminal expression vector (pEGFP-C1). The EGFP expression vector was used as a negative control and EGFP fluorescence was used to identify transfection efficiency of the arteries (Fig. 1) and VSMC (Fig. 2).
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ENaC (SCNN_1b; ID # 69163) and ENaC (SCNN_1g; ID # 151576) were obtained from Ambion (Austin, TX). As a control, we used a non-targeting siRNA (Dharmacon, Chicago, IL) which activates the RNA-induced silencing complex (RISC).
Transfection of renal interlobar arteries.
To transfect renal interlobar arteries with DN-cDNA or siRNA, we used a modified protocol previously published by Kaide et al. (19). Briefly, male C57BL/6J mice (6–8 wks; Jackson Laboratory, Bar Harbor, ME) were anesthetized with halothane and decapitated. Kidneys were excised and placed in ice cold physiological saline solution [(PSS, pH adjusted to 7.4 with NaOH) containing (in mM) 130 NaCl, 4 KC1, 1.2 MgSO4, 4 NaHCO3, 1.8 CaC12, 10 HEPES, 1.18 KH2PO4, 6 glucose, 0.03 EDTA]. Interlobar arteries were dissected from hemi-sected kidneys and transferred to a 35 mm culture dish and transiently transfected with the DN-cDNA (10 µg of EGFP, EGFP-I41X, or EGFP-L160x) or siRNA (10 µg of non-targeting, ENaC, or ENaC) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Additional vessels were incubated with Lipofectamine (Lfx) alone as a vehicle control. After 4 hrs, a 1:1 ratio of Dulbecco’s Modified Eagle Medium (DMEM):F-12 supplement (GIBCO Laboratories) containing 100U/ml penicillin/streptomycin was added to the vessels. The vessels were maintained in organ culture for 20–24 hrs in a humidified incubator (95% air-5% CO2) at 37°C. At the end of the culture period, vessels were either used for vascular reactivity studies or were further dissociated for quantitative immunofluorescence studies.
ENaC immunofluorescence in dispersed VSMCs.
To determine endogenous expression of - and ENaC following vessel transfection with - and ENaC DN-cDNA or siRNA, VSMCs were enzymatically dissociated from the isolated vessels and fixed as described previously (18). Anti-ENaC antibodies were raised in rabbits [beta mouse ENaC (617–638): NH2-CNYDSLRLQPLDTMESDSEVEAI-COOH, gamma mouse ENaC (618–639): NH2-CPAPEAPVPGTPPPRYNTLRLD-COOH, Sigma Genosys, Woodlands TX]. The ENaC antigenic sequence has 100% identity with rat and 90% identity with human isoforms. The ENaC antigenic sequence has 84% identity with rat and 79% identity with human isoforms. Antibodies were affinity purified against the original antigenic sequence (Sigma Genosys) and screened by ELISA, immunolabeling in COS-7 cells transfected with and ENaC cDNA and immunolabeling in VSMCs with antigen competition. Samples were labeled with rabbit anti- - or ENaC antibodies (1:100) and mouse anti-smooth muscle -actin (1:200) to identify VSMC and normalize immunofluorescence. Samples were examined using fluorescence confocal microscopy (TCS-SP2, Leica Microsystems, Exton, PA). To normalize data, - and ENaC fluorescence was divided by -actin fluorescence for each cell. All -actin-labeled cells were used for analysis independent of EGFP fluorescence. Data were averaged from 3 animals in each group.
Vascular reactivity.
To determine vascular reactivity following transfection with - and/or ENaC DN-cDNA or siRNA, mouse renal interlobar arteries were cannulated and studied in a vessel chamber (CH/1/SH, Living Systems, Burlington, VT) and analyzed using MetaMorph software (Universal Imaging, Dowingtown, PA) as described previously (18). Following an initial incubation period (30 min; 75 mmHg), a concentration-response curve to phenylephrine (PE; 10–9-10–5 M) was generated in each vessel to determine vessel viability. Vessels with less than 60% maximal response (calculated as percent of baseline inner diameter) were excluded. After washing and re-equilibrating the vessel, a pressure-diameter curve was generated by exposing the interlobar arteries to step-wise increases in intraluminal pressure from 25 to 150 mmHg (25 mmHg steps, 5 min each). Then, arteries were equilibrated for 30 min with Ca2+-free PSS (same as above PSS plus 2 mM EGTA and omit 1.8 mM CaCl2) to determine the passive pressure-diameter curve as described above. Change in diameter was calculated as the difference between the active (PSS) and passive (Ca2+-free PSS) inner diameter at each pressure. Data were averaged from 5 animals in each group. When more than one treated artery was used from a single animal, the results of the multiple experiments were averaged and included as a single value.
Statistics. All data are expressed as means ± SE. A one-way ANOVA and two-way ANOVA with repeated measures were used to make comparisons where appropriate. Differences among groups were compared using the Student-Newman-Keuls post hoc test. Statistical significance was considered at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and ENaC protein expression in renal interlobar arteries.
We used quantitative immunofluorescence to determine if or ENaC DN isoforms or siRNA suppresses ENaC protein. Representative images and quantitative group data are shown in Figs. 2 and 3. Compared with the Lfx and EGFP controls, transfection of EGFP-I41X reduced endogenous ENaC expression 
60% (Fig. 2, A and B); and EGFP-L160X reduced endogenous ENaC expression 50% (Fig. 2, C and D). In addition, transfection with EGFP-I41X did not suppress endogenous ENaC expression (Fig. 2, C and D) nor did EGFP-L160X alter endogenous ENaC expression (Fig. 2, A and B). Similarly, compared with non-targeting siRNA controls, ENaC-siRNA suppressed 70% endogenous - (Fig. 3, A and B), but not ENaC (Fig. 3, C and D). In addition, ENaC-siRNA suppressed 60% endogenous - (Fig. 3, C and D), but not ENaC (Fig. 3, A and B). These data show DN and siRNA molecules selectively silence or ENaC expression in renal interlobar arteries.
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and ENaC DN-cDNA and siRNA molecules dramatically inhibited pressure-induced constriction (Fig. 5 and 6). Transfection with EGFP-L160X maximally inhibited 70% the myogenic response, while transfection with EGFP-I41X blocked 80% (Fig. 5, C-E). Compared with the Lipofectamine (Lfx) vehicle control, EGFP alone did not significantly alter myogenic tone (Fig. 5, B and E). As shown in Fig. 6, ENaC-siRNA and ENaC-siRNA significantly reduced myogenic constriction by 65 and 40%, respectively, in renal interlobar arteries compared with the non-targeting siRNA control.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and ENaC expression in mouse renal interlobar arteries is required for myogenic constriction.
Until recently, mammalian ENaC proteins have been described mainly in kidney epithelial cells where they form constitutively active channels that play a rate limiting role in sodium absorption, and therefore, an essential role in the control of sodium balance, blood volume, and blood pressure (14, 20). However, our findings suggest that ENaC proteins in VSMCs may contribute to local blood flow regulation via myogenic constriction. This is a novel finding and one that supports the unique function of this protein family as mechanosensors. The involvement of DEG/ENaC in mechanotransduction has been shown in many other species and cell types (2, 12, 14, 20, 24); including mammalian dorsal root ganglion, arterial baroreflex sensory neurons, osteoblasts, keratinocytes, pain and touch receptors, and renal tubular cells (3, 10, 13, 21, 25, 28). Here, we provide evidence and ENaC proteins are also involved in mechanotransduction in VSMCs.
Similiar to previous findings from our lab, we found (9, 18), and ENaC expression is concentrated at or near the membrane, as indicated by colocalization with -actin. Previous work by ourselves and others show -actin staining is quite different in VSMCs that have been freshly dissociated compared with VSMCs maintained in culture (9, 18, 33). In VSMCs maintained in culture, -actin staining is filamentous and distributed through out the cell. However, in freshly dispersed VSMCs, -actin is concentrated near the membrane, making it an excellent marker of near-membrane proteins. In contrast to our previous findings, in the current study we detected more cytoplasmic localization of and ENaC protein, suggesting some redistribution of ENaC towards the cytoplasm. However, it is important to note that despite the presence of cytoplasmic ENaC expression, most ENaC expression is still localized to the membrane region. The factors accounting for the cytoplasmic localization of ENaC are unknown, but are most likely due to the overnight incubation period since this was the only methodological difference between the previous (18) and current studies. We speculate that substances present in the culturing media and/or the loss of transmural pressure in the arteries are contributing factors. In previous reports, we have shown a pronounced cytoplasmic staining pattern in renal VSMCs maintained in culture for a longer period (1–3 wk) (18). Taken together, these findings suggest that localization of ENaC proteins within the VSMC, i.e., cytoplasmic vs. membrane, is not "hard-wired", but can be influenced by the cells’ environment. However, whether loss of transmural pressure and supplements in the culturing media are responsible for regulating ENaC localization remains to be determined.
Two approaches were used to determine if ENaC proteins are required for myogenic constriction; siRNA and dominant-negative ENaC isoforms. As expected, siRNA, a known posttranscriptional gene silencer, reduced protein levels of ENaC and ENaC by 50% 24 h after transfection. The effect of siRNA on ENaC channel activity per se cannot be determined from our investigation since electrophysiological measurements of channel activity were not made. However, our findings indicate the 50% reduction in immunoreactive protein levels of ENaC or ENaC resulted in an almost complete loss of the myogenic constrictor response. This indicates normal levels of both ENaC and ENaC protein are required for a normal myogenic constrictor response and further suggests they may interact or associate.
In addition to siRNA, we inhibited ENaC channel function using a DN approach. DN ENaC isoforms also inhibited and ENaC protein levels to a similar extent as siRNA, however, most likely by a different mechanism. The NH2-terminal fragment of a related degenerin family member, Mec-4, has been shown to inhibit the response to touch in C. elegans (17). A similar NH2-terminal fragment of ENaC has been shown to inhibit ENaC channel activity, most likely by associating with other full-length subunits to form nonfunctional channels (1). The improperly formed channels are thought to be unstable and degraded, the basis for a loss of channel activity. Based on this finding, we anticipated either DN-cDNA would inhibit protein levels of and ENaC. There are at least two possible explanations for these findings. First, and ENaC may form homomeric channels. However, we think this is unlikely given our finding that siRNA towards either subunit abolished 70–80% of the response. A second possibility is that the DN (EGFP-I41X) interacts with its full-length ENaC counterpart prior to assembly or association with ENaC. Further studies are required to determine the mechanisms underlying DN inhibition of ENaC channel function.
A critical issue is whether ENaC and ENaC can form an ion-conducting pore in the absence of ENaC. In heterologous expression systems, evidence suggests ENaC and ENaC are capable of associating in the absence of ENaC. When coexpressed without ENaC, ENaC immunoprecipitates with ENaC, suggesting a biochemical association between ENaC and ENaC that persists in the absence ENaC. Using the Xenopus oocyte expression system, a report by Bonny et al. (2a) demonstrates ENaC can generate amiloride-sensitive macroscopic currents after 6 days of incubation. A delayed trafficking of ENaC channels in the ooctye system may be the basis for difficulty in identifying currents generated by ENaC channels by others. Bonny et al.’s finding is significant because it demonstrates ENaC and ENaC are sufficient to form a pore. Alternatively, it is possible that another degenerin protein, such as an ASIC protein, also associates with ENaC and ENaC to form the pore of a channel in VSMCs.
Presently, there is no electrophysiological evidence that ENaC proteins form a channel in VSMCs. However, the presence of a non-voltage-gated, epithelial-like Na+ channel in VSMCs was reported by Renterghem and Lazdunski 15 years ago (30). Similar to ENaC channels, the channel reported by Renterghem and Lazdunski exhibited high Na+ selectivity, a 10-pS conductance and was not voltaged gated. Unlike ENaC channels, the VSMC channel was insensitive to amiloride, butsensitive to higher concentrations of phenamil, an amiloride analog. Since that time, the presence and importance of these channels have received little attention. Electrophysiologic evidence confirming the presence of ENaC channels in VSMCs remains an important area of future investigation.
In comparison to responses in freshly isolated renal interlobar arteries (18), a small loss of the myogenic constrictor response was observed in the transfection control experiments (Lfx, EGFP, NT-siRNA), particularly at higher pressures (
100 mmHg). However, this is not surprising given the 24-h incubation period required to induce gene silencing. Although it may be coincidental, we speculate the depression of the myogenic constrictor response following the 24-h incubation may be related to the redistribution of and ENaC towards the cytoplasm noted previously. A redistribution of ENaC proteins towards the cytoplasm may reflect a reduction in the amount of active, membrane-associated ENaC channels contributing to signal transduction, which might be expected to lead to a reduction in myogenic tone development.
It is generally accepted that pressure-induced VSM membrane depolarization (6, 15, 23) and subsequent Ca2+ influx via voltage-gated Ca2+ channels largely mediates myogenic constriction (6, 22, 31). Many studies have followed this initial finding to determine a common signaling mechanism initiating the myogenic response. However, it has been difficult to ascribe myogenic constriction to a single mechanism because increased pressure activates multiple factors that influence VSM depolarization and their relative contribution varies immensely between, and even within, vascular beds (16). Potential mechanisms include pressure-induced activation of membrane-associated enzyme systems, ion transportors and mechanosensitive ion channels (7).
Although we do not know the precise function of ENaC proteins or whether their function is consistent across vascular beds, we speculate that ENaC subunits are part of a large complex, previously modeled in C. elegans (24). ENaC subunits form the pore of the channel in VSMCs and interact with extracellular matrix and cytoskeletal proteins to form a larger mechanosensitive complex. This speculation is supported by findings that demonstrate cytoskeletal and extracellular matrix proteins interact with certain DEG/ENaC proteins (2, 21, 24, 27). Furthermore, at least one cytoskeletal protein may be required to mechanically gate a mechanosensitive channel formed by DEG proteins in C. elegans (26). We further speculate that upon mechanical activation, the channel opens and allows influx of Na+ and/or Ca2+ ions, presumably leading to depolarization. While our laboratory has shown that inhibition of DEG/ENaC abolishes pressure-induced increases in cytosolic Na+ and Ca2+ (18), we have not yet correlated this to pressure-induced depolarization.
In the current investigation, we provide evidence ENaC proteins are involved in the myogenic response in the renal circulation. However, ENaCs may not contribute to myogenic responses in every vascular bed. The involvement of other mechanosensitive proteins, such as transient receptor potential channels (11, 29) or acid-sensing ion channels, may play a role in myogenic constriction in other beds. Results from these experiments identify a new group of proteins involved in vascular function; however, many fundamental mechanistic questions remain. How is the channel gated by mechanical stimuli? Does the channel interact with extracellular matrix or other degenerin proteins? How is the channel regulated by hormonal and autocrine factors?
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-subunit of the epithelial sodium channel cloned from osteoblasts. Proc Natl Acad Sci USA 94: 1013–1018, 1997.This article has been cited by other articles:
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  J. L. McCord, S. G. Hayes, and M. P. Kaufman Acid-sensing ion and epithelial sodium channels do not contribute to the mechanoreceptor component of the exercise pressor reflex Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1017 - H1024. [Abstract] [Full Text] [PDF]
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 X. Wang, K. Takeya, P. I. Aaronson, K. Loutzenhiser, and R. Loutzenhiser Effects of amiloride, benzamil, and alterations in extracellular Na+ on the rat afferent arteriole and its myogenic response Am J Physiol Renal Physiol, July 1, 2008; 295(1): F272 - F282. [Abstract] [Full Text] [PDF]
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S. C. Grifoni, S. E. McKey, and H. A. Drummond Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2022 - H2030. [Abstract] [Full Text] [PDF]
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 H. A. Drummond, N. L. Jernigan, and S. C. Grifoni Sensing Tension: Epithelial Sodium Channel/Acid-Sensing Ion Channel Proteins in Cardiovascular Homeostasis Hypertension, May 1, 2008; 51(5): 1265 - 1271. [Full Text] [PDF]
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K. P. Gannon, L. G. VanLandingham, N. L. Jernigan, S. C. Grifoni, G. Hamilton, and H. A. Drummond Impaired pressure-induced constriction in mouse middle cerebral arteries of ASIC2 knockout mice Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1793 - H1803. [Abstract] [Full Text] [PDF]
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 H. A. Drummond, S. C. Grifoni, and N. L. Jernigan A New Trick for an Old Dogma: ENaC Proteins as Mechanotransducers in Vascular Smooth Muscle Physiology, February 1, 2008; 23(1): 23 - 31. [Abstract] [Full Text] [PDF]
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 R. Schubert, D. Lidington, and S.-S. Bolz The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction Cardiovasc Res, January 1, 2008; 77(1): 8 - 18. [Abstract] [Full Text] [PDF]
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N. L. Jernigan, B. LaMarca, J. Speed, L. Galmiche, J. P. Granger, and H. A. Drummond Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H409 - H420. [Abstract] [Full Text] [PDF]
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H. A. Coleman and H. C. Parkington Endothelin enhances activity of mechanosensitive channels: A mechanism for ET augmentation of the myogenic response Cardiovasc Res, November 1, 2007; 76(2): 197 - 198. [Full Text] [PDF]
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C. J. Busst, K. J. Scurrah, J. A. Ellis, and S. B. Harrap Selective Genotyping Reveals Association Between the Epithelial Sodium Channel {gamma}-Subunit and Systolic Blood Pressure Hypertension, October 1, 2007; 50(4): 672 - 678. [Abstract] [Full Text] [PDF]
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Significantly different from Lfx control and EFGP, P < 0.05.