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Introduction: Degenerin proteins, such as bENaC and ASIC2, have been implicated in cardiovascular function. However, their role in metabolic syndrome have not been studied. To begin to assess this interaction, we evaluated the impact of a high fat diet (HFD) on mice lacking normal levels of ASIC2 (ASIC2-/-) and βENaC (βENaCm/m). Methods: Twenty-week-old male and female mice were placed on a 60% HFD for 12 weeks. Body weight was measured weekly, and body composition by noninvasive ECHO MRI and fasting blood glucose were measured at 0, 4, 8 and 12 weeks. A glucose tolerance test was administered after 12 weeks. Differences between ASIC2-/-/βENaCm/m and WT groups were compared using independent t-tests or ANOVA where appropriate within each sex. Data are presented as mean ± SEM and ASIC2-/-/βENaCm/m vs. WT. Results: At 20 weeks of age, ASIC2-/-/βENaCm/m mice (n=9F/10M) weighed less and gained less weight thanWT (n=12F/16M). Total body fat and lean body masses were reduced in female and male ASIC2-/-/bENaCm/m mice. Total body fat and lean body masses as % control were identical at the end of 12 weeks. Fasting blood glucoses were lower in female and male ASIC2-/-/βENaCm/m vs. WT mice after 12 weeks HFD. The area under the curve for the glucose tolerance test was reduced in female and tended (p=.079) to decrease in male ASIC2-/-/βENaCm/m. Plasma leptin and insulin were reduced in female and male ASIC2-/-/βENaCm/m vs. WT mice. Plasma insulin in female ASIC2-/-/βENaCm/m mice remained unchanged throughout the HFD period. Liver and liver fat masses, as well as percent liver fat, were reduced in both female and male ASIC2-/-/bENaCm/m mice after HFD. Plasma triglycerides, cholesterol, LDL- and HDL-cholesterols were markedly improved in male and/or female ASIC2-/-/βENaCm/m following the HFD. Discussion: These novel findings suggest that loss of ASIC2 and βENaC offer a significant protection against HFD-induced metabolic syndrome. [ABSTRACT FROM AUTHOR]

The monocyte–macrophage system plays an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. We examined ENaC/ASIC subunit transcript expression and the importance of select subunits in migration of bone marrow derived monocytes (freshly isolated) and macrophages (monocytes differentiated in culture). We also examined the effect of select subunit deletion on macrophage phenotype. BM monocytes were harvested from the femurs of male and female WT and KO mice (6–12 weeks of age). Our results show that α, β, γENaC, and ASIC1‐5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, βENaC, and ASIC2 subunits contribute to chemotactic migration responses in BM monocyte–macrophages. Polarization markers (CD86, soluble TNFα) in BM macrophages from mice lacking ASIC2a plus βENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of βENaC or ASIC2. Taken together, these data suggest that βENaC and ASIC2 play an important role in BM macrophage migration and loss of βENaC and/or ASIC2 partially polarizes macrophages to the M1 phenotype. Thus, targeting ENaC/ASIC expression in BM macrophages may regulate their ability to migrate to sites of injury. [ABSTRACT FROM AUTHOR]

Recent studies suggest that certain acid-sensing ion channels (ASIC) are expressed in vascular smooth muscle cells (VSMCs) and are required for VSMC functions. However, electrophysiological evidence of ASIC channels in VSMCs is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMCs express ASIC-like channels. To address this hypothesis, we used RT-PCR, Western blotting, immunolabeling, and conventional whole cell patch-clamp technique. We found extracellular [H.sup.+]-induced inward currents in 46% of cells tested (n = 58 of 126 VSMCs, pH 6.5-5.0). The percentage of responsive cells and the current amplitude increased as the external [H.sup.+] concentration increased ([pH.sub.6.0], n = 28/65 VSMCs responsive, mean current density = 8.1 [+ or -] 1.2 pA/pF). Extracellular acidosis ([pH.sub.6.0]) shifted the whole cell reversal potential toward the Nernst potential of [Na.sup.+] (n = 6) and substitution of extracellular [Na.sup.+] by N-methyl-D-glucamine abolished the inward current (n = 6), indicating that [Na.sup.+] is a major charge carrier. The broad-spectrum ASIC blocker amiloride (20 [micro]M) inhibited proton-induced currents to 16.5 [+ or -] 8.7% of control (n = 6, pH6.0). Psalmotoxin 1 (PcTx1), an ASIC1a inhibitor and ASIC1b activator, had mixed effects: PcTxl either 1) abolished [H.sup.+]-induced currents (11% of VSMCs, 5/45), 2) enhanced or promoted activation of [H.sup.+]-induced currents (76%, 34/45), or 3) failed to promote [H.sup.+] activation in nonresponsive VSMCs (13%, 6/45). These findings suggest that freshly dissociated cerebral artery VSMCs express ASIC-like channels, which are predominantly formed by ASIC1b. amiloride; proton; acid-sensing ion channel doi: 10.1152/aj pcell.00511.2009.

Renal blood flow (RBF) autoregulation is mediated by at least two mechanisms, the fast acting myogenic response (-5 s) and slow acting tubuloglomerular feedback (TGF; ~25 s). Previous studies suggest epithelial [Na.sup.+] channel (ENaC) family proteins, [beta]-ENaC in particular, mediate myogenic constriction in isolated renal interlobar arteries. However, it is unknown whether [beta]-ENaC-mediated myogenic constriction contributes to RBF autoregulation in vivo. Therefore, the goal of this investigation was to determine whether the myogenic mediated RBF autoregulation is inhibited in a mouse model of reduced [beta]-ENaC (m/m). To address this goal, we evaluated the temporal response of RBF and renal vascular resistance (RVR) to a 2-min step increase in mean arterial pressure (MAP). Pressure-induced changes in RBF and RVR at 0-5, 6-25, and 110-120 s after step increase in MAP were used to assess the contribution of myogenic and TGF mechanisms and steady-state autoregulation, respectively. The rate of the initial increase in RVR, attributed to the myogenic mechanism, was reduced by ~50% in m/m mice, indicating the speed of the myogenic response was inhibited. Steady-state autoregulation was similar between [beta]-ENaC +/+ and m/m mice. Although the rate of the secondary increase in RVR, attributed to TGF, was similar in [beta]-ENaC +/+ and m/m mice, however, it occurred over a longer period (+ 10 s), which may have allowed TGF to compensate for a loss in myogenic autoregulation. Our findings suggest [beta]-ENaC is an important mediator of renal myogenic constriction-mediated RBF autoregulation in vivo. epithelial [Na.sup.+] channel; ion channel; degenerin; renal blood flow autoregulation; renal injury; myogenic constriction doi: 10.1152/ajprenal.00496.2009

Induction of heme oxygenase-1 (HO-1) in the renal medulla increases carbon monoxide and bilirubin production and decreases ANG II-mediated superoxide production. The goal of this study was to determine the importance of increases in bilirubin to the antioxidant effects of HO-1 induction in cultured mouse thick ascending loop of Henle (TALH) and inner medullary collecting duct (IMCD3) cells. Bilirubin levels were decreased by using small interfering RNAs (siRNAs) targeted to biliverdin reductase (BVR), which is the cellular enzyme responsible for the conversion of biliverdin to bilirubin. Treatment of cultured TALH or IMCD-3 cells with BVR siRNA (50 or 100 nM) resulted in an 80% decrease in the level of BVR protein and decreased cellular bilirubin levels from 46 [+ or -] 5 to 23 [+ or -] 4 nM (n = 4). We then determined the effects of inhibition of BVR on ANG II-mediated superoxide production. Superoxide production induced by ANG II ([10.sup.-9] M) significantly increased in both TALH and IMCD-3 cells. Treatment of TALH cells with BVR siRNA resulted in a significant increase in ouabain-sensitive rubidium uptake from 95 [+ or -] 6 to 122 [+ or -] 5% control (n = 4, P < 0.05). Lastly, inhibition of BVR with siRNA did not prevent the decrease in superoxide levels observed in cells pretreated with the HO-1 inducer, hemin. We conclude that decreased levels of cellular bilirubin increase ANG II-mediated superoxide production and sodium transport; however, increases in bilirubin are not necessary for HO-1 induction to attenuate ANG II-mediated superoxide production. bilirubin; renal medulla; heme oxygenase; oxidative stress; small interfering RNA; sodium transport; rubidium uptake doi: 10.1152/ajpregu.90933.2008.

Recent studies suggest certain epithelial [Na.sup.+] channel (ENaC) proteins may be components of mechanosensitive ion channel complexes in vascular smooth muscle cells that contribute to pressure-induced constriction in middle cerebral arteries (MCA). However, the role of a specific ENaC protein, [beta]ENaC, in pressure-induced constriction of MCAs has not been determined. The goal of this study was to determine whether pressure-induced constriction in the MCA is altered in a mouse model with reduced levels of [beta]ENaC. Using quantitative immunofluorescence, we found whole cell [beta]ENaC labeling in cerebral vascular smooth muscle cells (VSMCs) was suppressed 46% in [beta]ENaC homozygous mutant (m/m) mice compared with wild-type littermates (+/+). MCAs from [beta]ENaC +/+ and m/m mice were isolated and placed in a vessel chamber for myographic analysis. Arteries from [beta]ENaC+/+ mice constricted to stepwise increases in perfusion pressure and developed maximal tone of 10 [+ or -] 2% at 90 mmHg (n = 5). In contrast, MCAs from [beta]ENaC m/m mice developed significantly less tone (4 [+ or -] 1% at 90 mmHg, n = 5). Vasoconstrictor responses to KCl (4-80 mM) were identical between genotypes and responses to phenylephrine ([10.sup.-7] [-10.sup.-4] M) were marginally altered, suggesting that reduced levels of VSMC [beta]ENaC specifically inhibit pressure-induced constriction. Our findings indicate [beta]ENaC is required for normal pressure-induced constriction in the MCA and provide further support for the hypothesis that [beta]ENaC proteins are components of a mechanosensor in VSMCs. epithelial [Na.sup.+] channel; ion channel; degenerin; myogenic constriction; middle cerebral artery

Heme oxygenase (HO)- l induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 [micro]M) or heroin (50 [micro]M) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with [10.sup.-9] M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5 [+ or -] 5 to 136 [+ or -] 18 relative fluorescence units (RFU)/[micro][m.sup.2]. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64 [+ or -] 5, 64 [+ or -] 8, and 41 [+ or -] 4 RFU/[micro][m.sup.2], respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 [micro]M) before exposure to ANG II. DHE fluorescence averaged 80 [+ or -] 7 RFU/[micro][m.sup.2] after incubation with ANG II and was significantly decreased to 55 [+ or -] 7 and 53 [+ or -] 4 RFU/[micro][m.sup.2] after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO. bilirubin; carbon monoxide; carbon monoxide-releasing molecule A1

Recent studies suggest members of the degenerin (DEG)/epithelial [Na.sup.+] channel (ENaC)/acid-sensing ion channel (ASIC) protein family play an important role in vascular smooth muscle cell (VSMC) migration. In a previous investigation, we found suppression of a certain DEG/ENaC/ASIC member, ASIC2, increased VSMC chemotactic migration, raising the possibility that ASIC2 may play an inhibitory role. Because ASIC2 protein was retained in the cytoplasm, we reasoned increasing surface expression of ASIC2 might unmask the inhibitory role of ASIC2 in VSMC migration so we could test the hypothesis that ASIC2 inhibits VSMC migration. Therefore, we used the chemical chaperone glycerol to enhance ASIC2 expression. Glycerol 1) increased cytoplasm ASIC2 expression, 2) permitted detection of ASIC2 at the cell surface, and 3) inhibited platelet-derived growth factor (PDGF)-bb mediated VSMC migration. Furthermore, ASIC2 silencing completely abolished the inhibitory effect of glycerol on migration, suggesting upregulation of ASIC2 is responsible for glycerol-induced inhibition of VSMC migration. Because other investigators have shown that glycerol regulates ENaC/ASIC via interactions with a certain heat shock protein, heat shock protein 70 (Hsc70), we wanted to determine the importance of Hsc70 on ASIC2 expression in VSMCs. We found that Hsc70 silencing increases ASIC2 cell surface expression and inhibits VSMC migration, which is abolished by cosilencing ASIC2. These data demonstrate that Hsc70 inhibits ASIC2 expression, and, when the inhibitory effect of Hsc70 is removed, ASIC2 expression increases, resulting in reduced VSMC migration. Because VSMC migration contributes to vasculogenesis and remodeling following vascular injury, our findings raise the possibility that ASIC2-Hsc70 interactions may play a role in these processes. vascular smooth muscle cell migration; ion channel; degenerin proteins; acid-sensing ion channel; heat shock protein 70; interference ribonucleic acid

Recent studies from our laboratory demonstrated the importance of mechanosensitive epithelial [Na.sup.+] channel (ENaC) proteins in pressure-induced constriction in renal and cerebral arteries. ENaC proteins are closely related to acid-sensing ion channel 2 (ASIC2), a protein known to be required for normal mechanotransduction in certain sensory neurons. However, the role of the ASIC2 protein in pressure-induced constriction has never been addressed. The goal of the current study was to investigate the role of ASIC2 proteins in pressure-induced, or myogenic, constriction in the mouse middle cerebral arteries (MCAs) from ASIC2 wild-type (+/+), heterozygous (+/-), and null (-/-) mice. Constrictor responses to KC1 (20-80 mM) and phenylephrine ([10.sup.-7]-[10.sup.-4] M) were not different among groups. However, vasoconstrictor responses to increases in intraluminal pressure (15-90 mmHg) were impaired in MCAs from [ASIC2.sup.-/-] and [sup.+/-] mice. At 60 and 90 mmHg, MCAs from [ASIC2.sup.+/+] mice generated 13.7 [+ or -] 2.1% and 15.8 [+ or -] 2.0% tone and [ASIC2.sup.-/-] mice generated 7.4 [+ or -] 2.8% and 12.5 [+ or -] 2.4% tone, respectively. Surprisingly, MCAs from [ASIC2.sup.+/-] mice generated 1.2 [+ or -] 2.2% and 3.9 [+ or -] 1.8% tone at 60 and 90 mmHg. The reason underlying the total loss of myogenic tone in the [ASIC2.sup.+/-] is not clear, although the loss of mechanosensitive [b[ET.sub.A]]- and [gamma]-ENaC proteins may be a contributing factor. These results demonstrate that normal ASIC2 expression is required for normal pressureinduced constriction in the MCA. Furthermore, ASIC2 may be involved in establishing the basal level of myogenic tone. mechanotransduction; myogenic constriction; degenerin; acid-sensing ion channel

Recent work from our laboratory indicates that epithelial [Na.sup.+] channel (ENaC) function plays an important role in modulating myogenic vascular reactivity. Increases in dietary sodium are known to affect vascular reactivity. Although previous studies have demonstrated that dietary salt intake regulates ENaC expression and activity in epithelial tissue, the importance of dietary salt on ENaC expression in vascular smooth muscle cells (VSMCs) and its role in myogenic constriction is unknown. Therefore, the goal of the present study was to determine whether dietary salt modulates ENaC expression and function in myogenic vasoconstriction. To accomplish this goal, we examined ENaC expression in freshly dispersed VSMCs and pressure-induced vasoconstrictor responses in isolated mesenteric resistance arteries from normotensive Sprague-Dawley rats fed a normal-salt (NS; 0.4% NaCl) or high-salt (HS; 8% NaCl for 2 wk) diet. VSMCs from the mesenteric arteries of NS-fed animals express [alpha]-, [beta]-, and [gamma]-ENaC. The HS diet reduced whole cell [alpha]- and [gamma]-ENaC and induced a pronounced translocation of [beta]-ENaC from intracellular regions toward the VSMC membrane (~336 nm). Associated with this change in expression was a change in the importance of ENaC in pressure-induced constriction. Pressure-induced constriction in NS-fed animals was insensitive to ENaC inhibition with 1 [micro]M benzamil, suggesting that ENaC proteins do not contribute to myogenic constriction in mesenteric arteries under NS intake. In contrast, ENaC inhibition blocked pressure-induced constriction in HS-fed animals. These data suggest that dietary sodium regulates ENaC expression and the quantitative importance of the vascular ENaC signaling pathway contributing to myogenic constriction. mechanotransduction; pressure-induced constriction

Mechanosensitive ion channels are thought to initiate pressure-induced vasoconstriction, however, the molecular identity of these channels is unknown. Recent work from our laboratory suggests that members of the Degenerin/Epithelial [Na.sup.+] Channel (DEG/ENaC) family may be components of the mechanosensitive ion channel complex in vascular smooth muscle (VSM); however, the specific DEG/ENaC proteins mediating myogenic constriction are unknown. The goal of this study is to determine if specific knockdown of [beta] or [gamma]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 [beta] or [gamma]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 [beta]ENaC DN-cDNA or siRNA suppresses [beta]-, but not [gamma]ENaC protein expression. Similarly, [gamma]ENaC DN-cDNA or siRNA suppresses [gamma]-, but not [beta]ENaC protein expression. In addition, transfection of [beta]- or [gamma]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 [beta] and [gamma]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

Vascular smooth muscle cell (VSMC) migration plays a key role in tissue repair after arterial wall injury. VSMC migration requires integration of chemical and mechanical signaling mechanisms. Recently, we showed that epithelial [Na.sup.+] channel (ENaC) proteins are expressed in VSMCs and that ENaC inhibition abolishes pressure-induced constriction in isolated artery segments. However, whether ENaC proteins play a role in VSMC migration is unknown. The goal of this study was to determine whether ENaC molecules are required for VSMC migration. Using RT-PCR, immunoblotting, and immunolabeling, we detected expression of [alpha], [beta]-, and [gamma]ENaC transcripts and proteins in cultured VSMCs (SV40-LT and A10 cells). Of the three proteins, [beta]ENaC was the most readily detected in both cell lines by immunolocalization and Western blotting. Inhibition of ENaC activity with 1 [micro]M benzamil blunted VSMC migration associated with wound healing (40.3% at 8 h and 26.2% at 24 h) and in response to the chemotactic stimulant platelet-derived growth factor-BB (38.1%). Furthermore, silencing ENaC gene expression with small interfering RNA blunted VSMC migration. These data indicate that expression of ENaC proteins is required for normal VSMC migration and suggest a potential new role for ENaC proteins in vascular tissue repair. amiloride; vascular remodeling; wound healing; degenerin

Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial N[a.sup.+] channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of [beta]- and [gamma]-subunits of ENaC, but not [alpha]-ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced [beta]- and [gamma]-ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation. mechanosensation; degenerins; neurotrophins; tyrosine kinase A; pheochromocytoma cells

The myogenic response is an essential component of renal blood flow autoregulation and is the inherent ability of vascular smooth muscle cells (VSMCs) to contract in response to increases in intraluminal pressure. Although mechano-sensitive ion channels are thought to initiate VSMC stretch-induced contraction, their molecular identity is unknown. Recent reports suggest degenerin/epithelial [Na.sup.+] channels (DEG/ENaC) may form mechanotransducers in sensory neurons and VSMCs; however, the role of DEG/ENaC proteins in myogenic constriction of mouse renal arteries has not been established. To test the hypothesis that DEG/ ENaC proteins are required for myogenic constriction in renal vessels, we first determined expression of ENaC transcripts and proteins in mouse renal VSMCs. Then, we determined pressure- and agonist-induced constriction and changes in vascular smooth muscle cytosolic [Ca.sup.2+] and [Na.sup.+] in isolated mouse renal interlobar arteries following DEG/ENaC inhibition with amiloride and benzamil. We detect [alpha]-, [beta]-, and [gamma]ENaC transcript and protein expression in cultured mouse renal VSMC. In contrast, we detect only [beta]- and [gamma]- but not [alpha]ENaC protein in freshly dispersed mrVMSC. Selective DEGfENaC inhibition, with low doses of amiloride and benzamil, abolishes pressure-induced constriction and increases in cytosolic [Ca.sup.2+] and [Na.sup.+] without diminishing agonist-induced responses in isolated mouse interlobar arteries. Our findings indicate that DEG/ENaC proteins are required for myogenic constriction in mouse interlobar arteries and are consistent with our hypothesis that DEG/ENaC proteins may be components of mechanosensitive ion channel complexes required for myogenic vasoconstriction. mechanotransduction; renal blood flow autoregulation; amiloride; benzamil; isolated renal vessel; stretch-activated cation channel; calcium; sodium

Author(s): Margaret P. Price [1, 2]; Gary R. Lewin [1, 3]; Sabrina L. McIlwrath [3]; Chun Cheng [2]; Jinghui Xie [2]; Paul A. Heppenstall [3]; Cheryl L. Stucky [3]; Anne [...]

In independent studies, our laboratory has shown the importance of the degenerin proteins β-epithelial Na+ channel (βENaC) and acid-sensing ion channel 2 (ASIC2) in pressure-induced constriction (PIC) in renal interlobar arteries. Most, but not all, of the PIC response is abolished in mice lacking normal levels of βENaC or in ASIC2-null mice, indicating that the functions of βENaC and ASIC2 cannot fully compensate for the loss of the other. Degenerin proteins are known to associate and form heteromeric channels in expression systems, but whether they interact biochemically and functionally in vascular smooth muscle cells is unknown. We hypothesized that βENaC and ASIC2 interact to mediate PIC responses in renal vessels. To address this possibility, we 1) used biochemical approaches to show that βENaC associates into high-molecular-weight complexes and immunoprecipitants with ASIC2 in vascular smooth muscle cells and then 2) examined PIC in renal afferent arterioles in mice lacking normal levels of βENaC (βENaCm/m) or/and ASIC2 (ASIC2-/-) using the isolated afferent arteriole-attached glomerulus preparation. We found that the sensitivity of the PIC response (slope of the relationship between intraluminal pressure and percent myogenic tone) decreased to 26%, 27%, and -8% of wild-type controls in ASIC2-/-, βENaCm/m, and ASIC2-/-/βENaCm/m groups, respectively, suggesting that the PIC response was totally abolished in mice deficient in both ASIC2 and βENaC. Surprisingly, we found that resting internal diameters were 20-30% lower (60 mmHg, Ca2+ free) in ASIC2-/-/βENaCm/m (11.3 ± 0.5 µm) mice compared with control (14.4 ± 0.6 µm, P = 0.0007, independent two-tailed t test) or singly modified (15.7 ± 1.0 to 16.3 ± 1.1 µm) mice, suggesting compensatory vasoconstriction or remodeling. We then examined mean arterial blood pressure (MAP) using radiotelemetry and glomerular injury using histological examination of renal sections. We found that 24-h MAP was mildly elevated ( + 8 mmHg) in ASIC2-/-/βENaCm/m mice versus wild-type controls and the glomerular injury score was modestly increased by 38%. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of βENaC and ASIC2 and that mice lacking normal levels of ASIC2 and βENaC have mild renal injury and increased MAP. [ABSTRACT FROM AUTHOR]

The epithelial [Na.sup.+] channel (ENaC) is composed of three homologous subunits: [Alpha], [Beta] and [Gamma]. We used gene targeting to disrupt the [Beta] subunit gene of ENaC in mice. The [Beta]ENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the -/- mice, we found an increased urine [Na.sup.+] concentration despite hyponatremia and a decreased urine [K.sup.+] concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to [Alpha]ENaC-deficient mice, which die because of defective lung liquid clearance, neonatal [Beta]ENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo, the [Beta] subunit is required for ENaC function in the renal collecting duct, but, in contrast to the [Alpha] subunit, the [Beta] subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the [Beta]ENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.