What Evolutionary Evidence Implies About the Identity of the Mechanoelectrical Couplers in Vascular Smooth Muscle Cells.

Loss of pressure-induced vasoconstriction increases susceptibility to renal and cerebral vascular injury. Favored paradigms underlying initiation of the response include transient receptor potential channels coupled to G protein-coupled receptors or integrins as transducers. Degenerin channels may also mediate the response. This review addresses the 1) evolutionary role of these molecules in mechanosensing, 2) limitations to identifying mechanosensitive molecules, and 3) paradigm shifting molecular model for a VSMC mechanosensor.

ßENaC and ASIC2 associate in VSMCs to mediate pressure-induced constriction in the renal afferent arteriole.

In independent studies, our laboratory has shown the importance of the degenerin proteins ß-epithelial Na+ channel (ßENaC) 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 ßENaC or in ASIC2-null mice, indicating that the functions of ßENaC 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 ßENaC and ASIC2 interact to mediate PIC responses in renal vessels. To address this possibility, we 1) used biochemical approaches to show that ßENaC 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 ßENaC (ßENaCm/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-/-, ßENaCm/m, and ASIC2-/-/ßENaCm/m groups, respectively, suggesting that the PIC response was totally abolished in mice deficient in both ASIC2 and ßENaC. Surprisingly, we found that resting internal diameters were 20-30% lower (60 mmHg, Ca2+ free) in ASIC2-/-/ßENaCm/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-/-/ßENaCm/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 ßENaC and ASIC2 and that mice lacking normal levels of ASIC2 and ßENaC have mild renal injury and increased MAP.NEW & NOTEWORTHY Transmission of systemic blood pressure to delicate renal microvessels is a primary determinant of vascular injury in chronic kidney disease progression to end-stage renal disease. Here, we identified two degenerin family members, with an evolutionary link to mechanosensing, that interact biochemically and functionally to regulate systemic blood pressure and renal injury. Thus, degenerin proteins may serve as a target for the development of therapies to prevent or delay renal disease progression.

Epithelial sodium channels in macrophage migration and polarization: role of proinflammatory cytokines TNFα and IFNγ.

Migration of monocytes-macrophages plays an important role in phagocytosis of pathogens and cellular debris in a variety of pathophysiological conditions. Although epithelial Na+ channels (ENaCs) are required for normal migratory responses in other cell types, their role in macrophage migration signaling is unknown. To address this possibility, we determined whether ENaC message is present in several peripheral blood monocyte cell populations and tissue-resident macrophages in healthy humans using the Human Protein Atlas database (www.proteinatlas.org) and the mouse monocyte cell line RAW 264.7 using RT-PCR. We then determined that selective ENaC inhibition with amiloride inhibited chemotactic migration (∼50%), but not phagocytosis, of the mouse monocyte-macrophage cell line RAW 264.7. Furthermore, we generated a cell line stably expressing an NH2-terminal truncated αENaC to interrupt normal channel trafficking and found it suppressed migration. Prolonged exposure (48 h) of RAW 264.7 cells to proinflammatory cytokines interferon γ (IFNγ) and/or tumor necrosis factor α (TNFα) inhibited RAW 264.7 migration and abolished the amiloride (1 µM)-sensitive component of migration, a finding consistent with ENaC downregulation. To determine if proinflammatory cytokines regulate αENaC protein expression, cells were exposed to proinflammatory cytokines IFNγ (10 ng/mL, last 48 h) and TNFα (10 ng/mL, last 24 h). By Western blot analysis, we found whole cell αENaC protein is reduced ≥50%. Immunofluorescence demonstrated heterogeneous αENaC inhibition. Finally, we found that overnight exposure to amiloride stimulated morphological changes and increased polarization marker expression. Our findings suggest that ENaC may be a critical molecule in macrophage migration and polarization.

Pressure-induced constriction of the middle cerebral artery is abolished in TrpC6 knockout mice.

Pressure-induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. While TrpC6 has been suggested to contribute to PIC signaling, direct supporting evidence is contradictory. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20-90 mmHg), depolarization (4-80 mM KCl)-, and adrenergic receptor (phenylephrine; PE 10-7-10-4 M)-mediated constriction of isolated middle cerebral artery (MCA) segments from 9-wk-old male wild-type (TrpC6+/+, n = 7) and homozygous null (TrpC6-/-, n = 9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). Vasoconstrictor responses to KCl and PE were identical in TrpC6-/- and TrpC6+/+ mice. In contrast, PIC responses were totally abolished in TrpC6-/- mice. At 90 mmHg, the calculated myogenic tone was -0.8 ± 0.5 vs. 10.7 ± 1.7%, P = 0.0002 in TrpC6-/- and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain and stress or morphological properties of wall thickness and wall-to-lumen ratio at 50 mmHg between TrpPC6-/- and TrpC6+/+ mice. Although these results demonstrate that TrpC6 is critical for the integrated PIC response, they do not identify whether TrpC6 acts as a mechanosensor or a downstream signaling component.NEW & NOTEWORTHY Pressure-induced, but not agonist-induced, vasoconstriction is abolished in the middle cerebral artery (MCA) of TrpC6 null mice. TrpC6 localization in dissociated cerebral vascular smooth muscle cells is primarily cytoplasmic and not associated with the surface membrane where a mechanoelectrical coupler might be expected. These findings suggest that TrpC6 is required for transduction of pressure-induced constriction in the MCA; however, its role as a mechanoelectrical coupler or downstream signal amplifier remains unresolved.

Tumor necrosis factor-α impairs cerebral blood flow in pregnant rats: role of vascular ß-epithelial Na+ channel.

Preeclampsia is a pregnancy-related disorder characterized by hypertension, vascular dysfunction and an increase in circulating inflammatory factors including the cytokine, tumor necrosis factor-α (TNF-α). Studies have shown that placental ischemia is associated with 1) increased circulating TNF-α, 2) attenuated pressure-induced cerebral vascular tone, and 3) suppression of ß-epithelial Na+ channel (ßENaC) protein in cerebral vessels. In addition to its role in epithelial Na+ and water transport, ßENaC is an essential signaling element in transduction of pressure-induced (aka "myogenic") constriction, a critical mechanism of blood flow autoregulation. While cytokines inhibit expression of certain ENaC proteins in epithelial tissue, it is unknown if the increased circulating TNF-α associated with placental ischemia mediates the loss of cerebrovascular ßENaC and cerebral blood flow regulation. Therefore, the purpose of this study was to test the hypothesis that increasing plasma TNF-α in normal pregnant rats reduces cerebrovascular ßENaC expression and impairs cerebral blood flow (CBF) regulation. In vivo TNF-α infusion (200 ng/day, 5 days) inhibited cerebrovascular expression of ßENaC and impaired CBF regulation in pregnant rats. To determine the direct effects of TNF-α and underlying pathways mediating vascular smooth muscle cell ßENaC reduction, we exposed cultured VSMCs (A10 cell line) to TNF-α (1-100 ng/mL) for 16-24 h. TNF-α reduced ßENaC protein expression in a concentration-dependent fashion from 0.1 to 100 ng/mL, without affecting cell death. To assess the role of canonical MAPK signaling in this response, VSMCs were treated with p38MAPK or c-Jun kinase (JNK) inhibitors in the presence of TNF-α. We found that both p38MAPK and JNK blockade prevented TNF-α-mediated ßENaC protein suppression. These data provide evidence that disorders associated with increased circulating TNF-α could lead to impaired cerebrovascular regulation, possibly due to reduced ßENaC-mediated vascular function.NEW & NOTEWORTHY This manuscript identifies TNF-α as a possible placental-derived cytokine that could be involved in declining cerebrovascular health observed in preeclampsia. We found that infusion of TNF-α during pregnancy impaired cerebral blood flow control in rats at high arterial pressures. We further discovered that cerebrovascular ß-epithelial sodium channel (ßENaC) protein, a degenerin protein involved in mechanotransduction, was reduced by TNF-α in pregnant rats, indicating a potential link between impaired blood flow and this myogenic player. We next examined this effect in vitro using a rat vascular smooth muscle cell line. TNF-α reduced ßENaC through canonical MAPK-signaling pathways and was not dependent on cell death. This study demonstrates the pejorative effects of TNF-α on cerebrovascular function during pregnancy and warrants future investigations to study the role of cytokines on vascular function during pregnancy.

Interleukin-17 Reduces ßENaC via MAPK Signaling in Vascular Smooth Muscle Cells.

Degenerin proteins, such as the beta epithelial Na+ channel (ßENaC), are essential in the intracellular signaling of pressure-induced constriction, an important vascular smooth muscle cell (VSMC) function. While certain cytokines reduce ENaC protein in epithelial tissue, it is unknown if interleukin-17 (IL-17), a potent pro-inflammatory cytokine, directly mediates changes in membrane-associated ßENaC in VSMCs. Therefore, we tested the hypothesis that exposure to IL-17 reduces ßENaC in VSMCs through canonical mitogen-activated protein kinase (MAPK) signaling pathways. We treated cultured rat VSMCs (A10 cell line) with IL-17 (1-100 ng/mL) for 15 min to 16 h and measured expression of ßENaC, p38MAPK, c-jun kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). IL-17 reduced ßENaC protein expression in a concentration-dependent fashion and increased phosphorylation of p38MAPK by 15 min and JNK by 8 h. NFκB was unaffected by IL-17 in VSMCs. IL-17 treatment reduced VSMC viability but had no effect on cell death. To determine the underlying signaling pathway involved in this response, VSMCs were treated before and during IL-17 exposure with p38MAPK or JNK inhibitors. We found that JNK blockade prevented IL-17-mediated ßENaC protein suppression. These data demonstrate that the pro-inflammatory cytokine IL-17 regulates VSMC ßENaC via canonical MAPK signaling pathways, raising the possibility that ßENaC-mediated loss of VSMC function may occur in inflammatory disorders.

Role of PTP1B in POMC neurons during chronic high-fat diet: sex differences in regulation of liver lipids and glucose tolerance.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of leptin receptor signaling and may contribute to leptin resistance in diet-induced obesity. Although PTP1B inhibition has been suggested as a potential weight loss therapy, the role of specific neuronal PTP1B signaling in cardiovascular and metabolic regulation and the importance of sex differences in this regulation are still unclear. In this study, we investigated the impact of proopiomelanocortin (POMC) neuronal PTP1B deficiency in cardiometabolic regulation in male and female mice fed a high-fat diet (HFD). When compared with control mice (PTP1B flox/flox), male and female mice deficient in POMC neuronal PTP1B (PTP1B flox/flox/POMC-Cre) had attenuated body weight gain (males: -18%; females: -16%) and fat mass (males: -33%; female: -29%) in response to HFD. Glucose tolerance was improved by 40%, and liver lipid accumulation was reduced by 40% in PTP1B/POMC-Cre males but not in females. When compared with control mice, deficiency of POMC neuronal PTP1B did not alter mean arterial pressure (MAP) in male or female mice (males: 112 ± 1 vs. 112 ± 1 mmHg in controls; females: 106 ± 3 vs. 109 ± 3 mmHg in controls). Deficiency of POMC neuronal PTP1B also did not alter MAP response to acute stress in males or females compared with control mice (males: Δ32 ± 0 vs. Δ29 ± 4 mmHg; females: Δ22 ± 2 vs. Δ27 ± 4 mmHg). These data demonstrate that POMC-specific PTP1B deficiency improved glucose tolerance and attenuated diet-induced fatty liver only in male mice and attenuated weight gain in males and females but did not enhance the MAP and HR responses to a HFD or to acute stress.

Biliverdin Reductase A Attenuates Hepatic Steatosis by Inhibition of Glycogen Synthase Kinase (GSK) 3ß Phosphorylation of Serine 73 of Peroxisome Proliferator-activated Receptor (PPAR) α.

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3ß (GSK3ß) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3ß phosphorylates serine 73 (Ser(P)73) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3ß activity and Ser(P)73 of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKß-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

Luminal ANG II is internalized as a complex with AT1R/AT2R heterodimers to target endoplasmic reticulum in LLC-PK1 cells.

ANG II has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of ß-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of ß-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.

Altered myogenic vasoconstriction and regulation of whole kidney blood flow in the ASIC2 knockout mouse.

Previous studies from our laboratory have suggested that degenerin proteins contribute to myogenic constriction, a mechanism of blood flow regulation and protection against pressure-dependent organ injury, in renal vessels. The goal of the present study was to determine the importance of one family member, acid-sensing ion channel 2 (ASIC2), in myogenic constriction of renal interlobar arteries, myogenic regulation of whole kidney blood flow, renal injury, and blood pressure using ASIC2(+/+), ASIC2(+/-), and ASIC2(-/-) mice. Myogenic constriction in renal interlobar arteries was impaired in ASIC2(+/-) and ASIC2(-/-) mice, whereas constriction to KCl/phenylephrine was unchanged. Correction of whole kidney renal vascular resistance (RVR) during the first 5 s after a 10- to 20-mmHg step increase in perfusion pressure, a timeframe associated with myogenic-mediated correction of RVR, was slowed (4.2 ± 0.9, 0.3 ± 0.7, and 2.4 ± 0.3 resistance units/s in ASIC2(+/+), ASIC2(+/-), and ASIC2(-/-) mice). Although modest reductions in function were observed in ASIC2(-/-) mice, greater reductions were observed in ASIC2(+/-) mice, which may be explained by protein-protein interactions of ASIC2 with other degenerins. Isolated glomeruli from ASIC2(+/-) and ASIC2(-/-) mice had modest alterations in the expression of inflammation and injury markers (transforming growth factor-ß, mouse anti-target of antiproliferative antibody-1, and nephrin), whereas ASIC2(+/-) mice had an increase in the remodeling marker collagen type III. Consistent with a more severe loss of function, mean arterial pressure was increased in ASIC2(+/-) mice (131 ± 3 mmHg) but not in ASIC2(-/-) mice (122 ± 3 vs. 117 ± 2 mmHg in ASIC2(+/+) mice). These results suggest that ASIC2 contributes to transduction of the renal myogenic response and are consistent with the protective role of myogenic constriction against renal injury and hypertension.

Placental ischemia-induced increases in brain water content and cerebrovascular permeability: role of TNF-α.

Cerebrovascular complications and increased risk of encephalopathies are characteristic of preeclampsia and contribute to 40% of preeclampsia/eclampsia-related deaths. Circulating tumor necrosis factor-α (TNF-α) is elevated in preeclamptic women, and infusion of TNF-α into pregnant rats mimics characteristics of preeclampsia. While this suggests that TNF-α has a mechanistic role to promote preeclampsia, the impact of TNF-α on the cerebral vasculature during pregnancy remains unclear. We tested the hypothesis that TNF-α contributes to cerebrovascular abnormalities during placental ischemia by first infusing TNF-α in pregnant rats (200 ng/day ip, from gestational day 14 to 19) at levels to mimic those reported in preeclamptic women. TNF-α increased mean arterial pressure (MAP, P < 0.05) and brain water content in the anterior cerebrum (P < 0.05); however, TNF-α infusion had no effect on blood-brain barrier (BBB) permeability in the anterior cerebrum or posterior cerebrum. We then assessed the role of endogenous TNF-α in mediating these abnormalities in a model of placental ischemia induced by reducing uterine perfusion pressure followed by treatment with the soluble TNF-α receptor (etanercept, 0.8 mg/kg sc) on gestational day 18. Etanercept reduced placental ischemia-mediated increases in MAP, anterior brain water content (P < 0.05), and BBB permeability (202 ± 44% in placental ischemic rats to 101 ± 28% of normal pregnant rats). Our results indicate that TNF-α mechanistically contributes to cerebral edema by increasing BBB permeability and is an underlying factor in the development of cerebrovascular abnormalities associated with preeclampsia complicated by placental ischemia.

Reduced uterine perfusion pressure induces hypertension in the pregnant mouse.

Despite preeclampsia being one of the leading causes of maternal death and a major contributor of maternal and perinatal morbidity, the mechanisms responsible for its pathogenesis have yet to be fully elucidated. Growing evidence indicates that reduced uteroplacental perfusion and the resulting placental ischemia triggers the cascade of events leading to this maternal disorder. While the well-established rat model of reduced uterine perfusion pressure (RUPP) is providing invaluable insight into the etiology of preeclampsia, the aim of this study was to develop a mouse model of reduced uterine perfusion to expand mechanistic investigation by incorporation with novel gene-targeted mice. To accomplish this aim, a sham surgical procedure or a restriction of blood flow at the abdominal aorta and the ovarian arteries was initiated at day 13 of gestation in C57BL/6J mice. Mean arterial pressure measured in conscious, chronically instrumented mice was significantly elevated in the RUPP (120 ± 4 mmHg) compared with the sham (104 ± 4 mmHg) mice at day 18 of gestation (P < 0.01). Placental ischemia reduced fetal weights (0.95 ± 0.04 and 0.80 ± 0.02 g; RUPP vs. Sham, respectively; P < 0.02) and increased circulating levels of antiangiogenic soluble fms-related tyrosine kinases (sFlt)-1 (P < 0.05) in the RUPP at day 18 of gestation. Plasma concentrations of sFlt-1 are increased in preeclamptic patients and in response to reduced uterine perfusion in the rat. Thus, these results suggest that the mouse model of reduced uterine perfusion is applicable to facilitate novel mechanistic investigation into the etiology of hypertension that results from placental ischemia during pregnancy.

Heme oxygenase-1 promotes migration and ß-epithelial Na+ channel expression in cytotrophoblasts and ischemic placentas.

Preeclampsia is thought to arise from inadequate cytotrophoblast migration and invasion of the maternal spiral arteries, resulting in placental ischemia and hypertension. Evidence suggests that altered expression of epithelial Na(+) channel (ENaC) proteins may be a contributing mechanism for impaired cytotrophoblast migration. ENaC activity is required for normal cytotrophoblast migration. Moreover, ß-ENaC, the most robustly expressed placental ENaC message, is reduced in placentas from preeclamptic women. We recently demonstrated that heme oxygenase-1 (HO-1) protects against hypertension in a rat model of placental ischemia; however, whether HO-1 regulation of ß-ENaC contributes to the beneficial effects of HO-1 is unknown. The purpose of this study was to determine whether ß-ENaC mediates cytotrophoblast migration and whether HO-1 enhances ENaC-mediated migration. We showed that placental ischemia, induced by reducing uterine perfusion suppressed, and HO-1 induction restored, ß-ENaC expression in ischemic placentas. Using an in vitro model, we found that HO-1 induction, using cobalt protoporphyrin, stimulates cytotrophoblast ß-ENaC expression by 1.5- and 1.8-fold (10 and 50 µM). We then showed that silencing of ß-ENaC in cultured cytotrophoblasts (BeWo cells), by expression of dominant-negative constructs, reduced migration to 56 ± 13% (P < 0.05) of control. Importantly, HO-1 induction enhanced migration (43 ± 5% of control, P < 0.05), but the enhanced migratory response was entirely blocked by ENaC inhibition with amiloride (10 µM). Taken together, our results suggest that ß-ENaC mediates cytotrophoblast migration and increasing ß-ENaC expression by HO-1 induction enhances migration. HO-1 regulation of cytotrophoblast ß-ENaC expression and migration may be a potential therapeutic target in preeclamptic patients.

Bone marrow monocytes and macrophages from mice lacking ßENaC and ASIC2 have a reduced chemotactic migration response and polarization.

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, ß, γENaC, and ASIC1-5 transcripts are expressed in BM macrophages and monocytes to varying degrees. At least αENaC, ßENaC, 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 ßENaC were shifted towards the M1 phenotype. Furthermore, select M1 phenotypic markers were recovered with rescue of ßENaC or ASIC2. Taken together, these data suggest that ßENaC and ASIC2 play an important role in BM macrophage migration and loss of ßENaC 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.

ßENaC is required for whole cell mechanically gated currents in renal vascular smooth muscle cells.

Myogenic constrictor responses in small renal arteries and afferent arterioles are suppressed in mice with reduced levels of ß-epithelial Na⁺ channel (ßENaC(m/m)). The underlying mechanism is unclear. Decreased activity of voltage-gated calcium channels (VGCC) or mechanically gated ion channels and increased activity of large conductance calcium-activated potassium (BK) channels are a few possible mechanisms. The purpose of this study was to determine if VGCC, BK, or mechanically gated ion channel activity was altered in renal vascular smooth muscle cell (VSMC) from ßENaC(m/m) mice. To address this, we used whole cell patch-clamp electrophysiological approaches in freshly isolated renal VSMCs. Compared with ßENaC(+/+) controls, the current-voltage relationships for VGCC and BK activity are similar in ßENaC(m/m) mice. These findings suggest neither VGCC nor BK channel dysfunction accounts for reduced myogenic constriction in ßENaC(m/m) mice. We then examined mechanically gated currents using a novel in vitro assay where VSMCs are mechanically activated by stretching an underlying elastomer. We found the mechanically gated currents, predominantly carried by Na⁺, are observed with less frequency (87 vs. 43%) and have smaller magnitude (-54.1 ± 12.5 vs. -20.9 ± 4.9 pA) in renal VSMCs from ßENaC(m/m) mice. Residual currents are expected in this model since VSMC ßENaC expression is reduced by 50%. These findings suggest ßENaC is required for normal mechanically gated currents in renal VSMCs and their disruption may account for the reduced myogenic constriction in the ßENaC(m/m) model. Our findings are consistent with the role of ßENaC as a VSMC mechanosensor and function of evolutionarily related nematode degenerin proteins.

Expression of heme oxygenase-1 in thick ascending loop of henle attenuates angiotensin II-dependent hypertension.

Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.

Impaired myogenic constriction of the renal afferent arteriole in a mouse model of reduced ßENaC expression.

Previous studies demonstrate a role for ß epithelial Na(+) channel (ßENaC) protein as a mediator of myogenic constriction in renal interlobar arteries. However, the importance of ßENaC as a mediator of myogenic constriction in renal afferent arterioles, the primary site of development of renal vascular resistance, has not been determined. We colocalized ßENaC with smooth muscle α-actin in vascular smooth muscle cells in renal arterioles using immunofluorescence. To determine the importance of ßENaC in myogenic constriction in renal afferent arterioles, we used a mouse model of reduced ßENaC (ßENaC m/m) and examined pressure-induced constrictor responses in the isolated afferent arteriole-attached glomerulus preparation. We found that, in response to a step increase in perfusion pressure from 60 to 120 mmHg, the myogenic tone increased from 4.5 ± 3.7 to 27.3 ± 5.2% in +/+ mice. In contrast, myogenic tone failed to increase with the pressure step in m/m mice (3.9 ± 0.8 to 6.9 ± 1.4%). To determine the importance of ßENaC in myogenic renal blood flow (RBF) regulation, we examined the rate of change in renal vascular resistance following a step increase in perfusion pressure in volume-expanded animals. We found that, following a step increase in pressure, the rate of myogenic correction of RBF is inhibited by 75% in ßENaC m/m mice. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of ßENaC.

Is there a role of proinflammatory cytokines on degenerin-mediated cerebrovascular function in preeclampsia?

Preeclampsia (PE) is associated with adverse cerebrovascular effects during and following parturition including stroke, small vessel disease, and vascular dementia. A potential contributing factor to the cerebrovascular dysfunction is the loss of cerebral blood flow (CBF) autoregulation. Autoregulation is the maintenance of CBF to meet local demands with changes in perfusion pressure. When perfusion pressure rises, vasoconstriction of cerebral arteries and arterioles maintains flow and prevents the transfer of higher systemic pressure to downstream microvasculature. In the face of concurrent hypertension, loss of autoregulatory control exposes small delicate microvessels to injury from elevated systemic blood pressure. While placental ischemia is considered the initiating event in the preeclamptic cascade, the factor(s) mediating cerebrovascular dysfunction are poorly understood. Elevated plasma proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-17 (IL-17), are potential mediators of autoregulatory loss. Impaired CBF responses to increases in systemic pressure are attributed to the impaired pressure-induced (myogenic) constriction of small cerebral arteries and arterioles in PE. Myogenic vasoconstriction is initiated by pressure-induced vascular smooth muscle cell (VSMC) stretch. Recent studies from our laboratory group indicate that proinflammatory cytokines impair the myogenic mechanism of CBF autoregulation via inhibition of vascular degenerin proteins, putative mediators of myogenic constriction in VSMCs. This brief review links studies showing the effect of proinflammatory cytokines on degenerin expression and CBF autoregulation to the pathological cerebral consequences of preeclampsia.

Renal inflammation and elevated blood pressure in a mouse model of reduced {beta}-ENaC.

Previous studies suggest ß-epithelial Na(+) channel protein (ß-ENaC) may mediate myogenic constriction, a mechanism of blood flow autoregulation. A recent study demonstrated that mice with reduced levels of ß-ENaC (ß-ENaC m/m) have delayed correction of whole kidney blood flow responses, suggesting defective myogenic autoregulatory capacity. Reduced renal autoregulatory capacity is linked to renal inflammation, injury, and hypertension. However, it is unknown whether ß-ENaC m/m mice have any complications associated with reductions in autoregulatory capacity such as renal inflammation, injury, or hypertension. To determine whether the previously observed altered autoregulatory control was associated with indicators of renal injury, we evaluated ß-ENaC m/m mice for signs of renal inflammation and tissue remodeling using marker expression. We found that inflammatory and remodeling markers, such as IL-1ß, IL-6, TNF-α, collagen III and transforming growth factor-ß, were significantly upregulated in ß-ENaC m/m mice. To determine whether renal changes were associated with changes in long-term control of blood pressure, we used radiotelemetry and found that 5-day mean arterial blood pressure (MAP) was significantly elevated in ß-ENaC m/m (120 ± 3 vs. 105 ± 2 mmHg, P = 0.016). Our findings suggest loss of ß-ENaC is associated with early signs of renal injury and increased MAP.

ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity.

BACKGROUND/AIMS: The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. METHODS: We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. RESULTS: Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 µM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. CONCLUSION: These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase.