Paraoxonase 2 is an ER chaperone that regulates the epithelial Na+ channel.

The mammalian paraoxonases (PONs) have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2, and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in fisher rat thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts before ENaC subunit ubiquitination. As a result of enhanced ENaC subunit ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirects the channel for ER-associated degradation.

γENaC/CD9 in urinary extracellular vesicles as a potential biomarker of MR activity.

Primary aldosteronism (PA) is caused by autonomous overproduction of aldosterone, which induces organ damage directly via activation of the mineralocorticoid receptor (MR); however, no specific or sensitive biomarkers are able to reflect MR activity. Recently, it is found that urinary extracellular vesicles (uEVs) are secreted by multiple cell types in the kidney and are an enriched source of kidney-specific proteins. Here, we evaluate sodium transporters in uEVs as candidates of biomarkers of MR activity in the clinical setting. Sixteen patients were examined to determine their plasma aldosterone concentration (PAC) and renin activity, and their morning urine was collected. The protein levels of two sodium transporters in uEVs, γ-epithelial sodium channel (γENaC) and thiazide-sensitive sodium chloride cotransporter (NCC), were quantified by Western blot analysis, and their clinical correlation with PAC was determined. Consequently, we found PAC was significantly correlated with the γENaC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.71). PAC was also correlated with the NCC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.61). In two PA patients, treatment with an MR antagonist or adrenalectomy reduced γENaC/CD9 in uEVs. In conclusion, γENaC/CD9 in uEVs is a valuable biomarker of MR activity in PA patients and may be a useful biomarker for other MR-associated diseases.

An optical-based biosensor of the epithelial sodium channel as a tool for diagnosing hypertension.

Arterial hypertension (HTN) is a world health concern presenting difficulties for its early detection. It leads to cardiovascular and kidney complications that increase morbidity in adults. Overexpression in the epithelial sodium channel (ENaC) in membrane platelets can be related with the presence of HTN and thus can be used as a biomarker to detect this medical condition. Here, we propose a method for HTN diagnosis based on gold nanoparticles (GNPs) conjugated to an antibody against the ENaC present on platelets. These functionalized GNPs were analyzed by Zeta potential, dynamic light scattering, electron microscopy, and other spectroscopic techniques. To verify that the GNPs and α-ENaC antibodies formed conjugates (GNPs-antiENaC) that maintained their specificity to the target, we carried out an indirect immunofluorescence detection assay of GNPs-antiENaC bound to a secondary antibody labeled with a fluorophore. Our results show that the presence of GNPs increase the fluorescence intensity in platelets treated with GNPs-antiENaC conjugates. It is also observed a clear tendency of the fluorescence signal in platelets treated with the conjugates that could be used for discrimination between normotensive and hypertensive samples. The proposed assay can be implemented as a very sensitive routine test to diagnose HTN.

Distribution of aquaporins and sodium transporters in the gastrointestinal tract of a desert hare, Lepus yarkandensis.

Lepus yarkandensis is a desert hare of the Tarim Basin in western China, and it has strong adaptability to arid environments. Aquaporins (AQPs) are a family of water channel proteins that facilitate transmembrane water transport. Gastrointestinal tract AQPs are involved in fluid absorption in the small intestine and colon. This study aimed to determine the distribution of AQPs and sodium transporters in the gastrointestinal tract of L. yarkandensis and to compare the expression of these proteins with that in Oryctolagus cuniculus. Immunohistochemistry was performed to analyse the cellular distribution of these proteins, and the acquired images were analysed with IpWin32 software. Our results revealed that AQP1 was located in the colonic epithelium, central lacteal cells, fundic gland parietal cells, and capillary endothelial cells; AQP3 was located in the colonic epithelium, small intestinal villus epithelium, gastric pit and fundic gland; AQP4 was located in the fundic gland, small intestinal gland and colonic epithelium; and epithelial sodium channel (ENaC) and Na+-K+-ATPase were located in the epithelial cells, respectively. The higher expression levels of AQP1, AQP3, ENaC and Na+-K+-ATPase in the colon of L. yarkandensis compared to those in O. cuniculus suggested that L. yarkandensis has a higher capacity for faecal dehydration.

Estrogen negatively regulates the renal epithelial sodium channel (ENaC) by promoting Derlin-1 expression and AMPK activation.

The main functions of the epithelial sodium channel (ENaC) in the kidney distal nephron are mediation of sodium and water balance and stabilization of blood pressure. Estrogen has important effects on sodium and water balance and on premenopausal blood pressure, but its role in the regulation of ENaC function is not fully understood. Female Sprague-Dawley rats were treated with 17ß-estradiol for 6 weeks following bilateral ovariectomy. Plasma estrogen, aldosterone, creatinine, and electrolytes were analyzed, and α-ENaC and derlin-1 protein expression in the kidney was determined by immunohistochemistry and western blotting. The expression levels of α-ENaC, derlin-1, AMPK, and related molecules were also examined by western blotting and real-time PCR in cultured mouse renal collecting duct (mpkCCDc14) epithelial cells following estrogen treatment. Immunofluorescence and coimmunoprecipitation were performed to detect α-ENaC binding with derlin-1 and α-ENaC ubiquitination. The results demonstrated that the loss of estrogen elevated systolic blood pressure in ovariectomized (OVX) rats. OVX rat kidneys showed increased α-ENaC expression but decreased derlin-1 expression. In contrast, estrogen treatment decreased α-ENaC expression but increased derlin-1 expression in mpkCCDc14 cells. Moreover, estrogen induced α-ENaC ubiquitination by promoting the interaction of α-ENaC with derlin-1 and evoked phosphorylation of AMPK in mpkCCDc14 cells. Our study indicates that estrogen reduces ENaC expression and blood pressure in OVX rats through derlin-1 upregulation and AMPK activation.

MST3 is involved in ENaC-mediated hypertension.

Liddle syndrome is an inherited form of human hypertension caused by increasing epithelial Na+ channel (ENaC) expression. Increased Na+ retention through ENaC with subsequent volume expansion causes hypertension. In addition to ENaC, the Na+-K+-Cl- cotransporter (NKCC) and Na+-Cl- symporter (NCC) are responsible for Na+ reabsorption in the kidneys. Several Na+ transporters are evolutionarily regulated by the Ste20 kinase family. Ste20-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 phosphorylate downstream NKCC2 and NCC to maintain Na+ and blood pressure (BP) homeostasis. Mammalian Ste20 kinase 3 (MST3) is another member of the Ste20 family. We previously reported that reduced MST3 levels were found in the kidneys in spontaneously hypertensive rats and that MST3 was involved in Na+ regulation. To determine whether MST3 is involved in BP stability through Na+ regulation, we generated a MST3 hypomorphic mutation and designated MST3+/- and MST3-/- mice to examine BP and serum Na+ and K+ concentrations. MST3-/- mice exhibited hypernatremia, hypokalemia, and hypertension. The increased ENaC in the kidney played roles in hypernatremia. The reabsorption of more Na+ promoted more K+ secretion in the kidney and caused hypokalemia. The hypernatremia and hypokalemia in MST3-/- mice were significantly reversed by the ENaC inhibitor amiloride, indicating that MST3-/- mice reabsorbed more Na+ through ENaC. Furthermore, Madin-Darby canine kidney cells stably expressing kinase-dead MST3 displayed elevated ENaC currents. Both the in vivo and in vitro results indicated that MST3 maintained Na+ homeostasis through ENaC regulation. We are the first to report that MST3 maintains BP stability through ENaC regulation.

Mapping the sites of localization of epithelial sodium channel (ENaC) and CFTR in segments of the mammalian epididymis.

The sperm produced in the seminiferous tubules pass through the rete testis, efferent ducts, and epididymis. The epididymis has three distinct regions known as caput, corpus, and cauda. The transit through the epididymis is an essential process in sperm maturation. The lumen of each epididymal region has a unique fluid composition regulated by many ion channels and transporters in the epithelial cells. The objective of this study was to map the sites of localization of ion channels ENaC and CFTR along the length of the mouse and rat epididymis using confocal microscopic imaging. The integrity of the fine structure of the tissues was verified by fluorescent phalloidin staining of actin filaments visualized by high-resolution confocal microscopy. The 2D and 3D images showed preservation of the stereocilia. Based on these images we determined morphometric parameters of the epithelial cells and ducts. ENaC and CFTR immunofluorescence appeared almost continuously on the apical membrane of caput and in smooth muscle myoid cells. In cauda, CFTR expression was observed continuously in long stretches of epithelium interrupted by clusters of cells that showed no CFTR expression. Similar patterns of localization were observed in both mouse and rat samples. Mutations in the CFTR gene are known to result in male infertility. Based on the widespread presence of ENaC along the epididymis we suggest that mutations in ENaC subunits may also be associated with male infertility. The diverse phenotypes associated with CFTR mutations may be due to malfunction of CFTR at specific subcellular locations in the male reproductive system.

Localization of epithelial sodium channel (ENaC) and CFTR in the germinal epithelium of the testis, Sertoli cells, and spermatozoa.

Spermatogenesis starts within the seminiferous tubules of the testis by mitotic division of spermatogonia that produces spermatocytes. Meiotic division of these spermatocytes produces haploid spermatids that differentiate into spermatozoa. In this study, we examined the expression of ENaC and CFTR (a Cl- channel) in rat testicular sections using confocal microscopic immunofluorescence. The structural integrity of the seminiferous tubule sections was verified by precise phalloidin staining of the actin fibers located abundantly at both basal and adluminal tight junctions. The acrosome forming regions in the round spermatids were stained using an FITC coupled lectin (wheat germ agglutinin). In all phases of the germ cells (spermatogonia, spermatocytes, and spermatids) ENaC was localized in cytoplasmic pools. Prior to spermiation, ENaC immunofluorescence appeared along the tails of the spermatids. In spermatozoa isolated from the epididymis, ENaC was localized at the acrosome and a central region of the sperm flagellum. The mature sperm are transcriptionally silent. Hence, we suggest that ENaC subunits in cytoplasmic pools in germ cells serve as the source of ENaC subunits located along the tail of spermatozoa. The locations of ENaC is compatible with a possible role in the acrosomal reaction and sperm mobility. In contrast to ENaC, CFTR immunofluorescence was most strongly observed specifically within the Sertoli cell nuclei. Based on the nuclear localization of CFTR we suggest that, in addition to its role as an ion channel, CFTR may have an independent role in gene regulation within the nuclei.

Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice.

PURPOSE: The concentration-sensitive sodium channel (NaC) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that NaC contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether NaC is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between NaC gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. METHODS: LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. NaC protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of NaC and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. RESULTS: The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the NaC protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of NaC decreased in the lung, as well as those for α-, ß-, and γ-ENaC during ALI. Thus, NaC expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. CONCLUSION: Our results suggest that suppression of the gene expression of NaC is involved in the development of pulmonary edema in ALI.

Male Sex is Associated with a Reduced Alveolar Epithelial Sodium Transport.

Respiratory distress syndrome (RDS) is the most frequent pulmonary complication in preterm infants. RDS incidence differs between genders, which has been called the male disadvantage. Besides maturation of the surfactant system, Na+ transport driven alveolar fluid clearance is crucial for the prevention of RDS. Na+ transport is mediated by the epithelial Na+ channel (ENaC) and the Na,K-ATPase, therefore potential differences in their expression or activity possibly contribute to the gender imbalance observed in RDS. Fetal distal lung epithelial (FDLE) cells of rat fetuses were separated by sex and analyzed regarding expression and activity of the Na+ transporters. Ussing chamber experiments showed a higher baseline short-circuit current (ISC) and amiloride-sensitive ΔISC in FDLE cells of female origin. In addition, maximal amiloride-sensitive ΔISC and maximal ouabain-sensitive ΔISC of female cells were higher when measured in the presence of a permeabilized basolateral or apical membrane, respectively. The number of FDLE cells per fetus recoverable during cell isolation was also significantly higher in females. In addition, lung wet-to-dry weight ratio was lower in fetal and newborn female pups. Female derived FDLE cells had higher mRNA levels of the ENaC- and Na,K-ATPase subunits. Furthermore, estrogen (ER) and progesterone receptor (PR) mRNA levels were higher in female cells, which might render female cells more responsive, while concentrations of placenta-derived sex steroids do not differ between both genders during fetal life. Inhibition of ER-ß abolished the sex differences in Na+ transport and female cells were more responsive to estradiol stimulation. In conclusion, a higher alveolar Na+ transport, possibly attributable to a higher expression of hormone receptors in female FDLE cells, provides an explanation for the well known sex-related difference in RDS occurrence and outcome.

Effects of atorvastatin on systemic and renal NO dependency in patients with non-diabetic stage II-III chronic kidney disease.

AIMS: Clinical trials suggest that statins have beneficial effects on the cardiovascular system independent from their cholesterol lowering properties. In patients with chronic kidney disease stage II-III, we tested the hypothesis that atorvastatin increased systemic and renal nitric oxide (NO) availability using L-N(G) -monomethyl arginine (L-NMMA) as an inhibitor of NO production. METHODS: In a randomized, placebo-controlled, crossover study patients were treated with atorvastatin for 5 days with standardized diet and fluid intake. Glomerular filtration reate (GFR), fractional excretions of sodium (FENa ), urinary excretion of aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ ), vasoactive hormones (renin, angiotensin II, aldosterone, arginine vasopressin, endothelin-1 and brain natriuretic peptide) and central blood pressure (BP) estimated by applanation tonometry were measured before and after systemic administration of the NO inhibitor L-NMMA. RESULTS: Atorvastatin caused a significant reduction in U-ENaCγ , but sodium excretion, C H 2 O , FENa and u-AQP2 were not changed by atorvastatin. L-NMMA reduced renal effect variables, including GFR, FENa and u-ENaCγ and increased brachial BP and central BP to a similar extent during both treatments. Vasoactive hormones were changed in the same way by L-NMMA during atorvastatin and placebo treatment. CONCLUSION: During, atorvastatin and placebo treatment, inhibition of nitric oxide synthesis induced the same response in brachial and central blood pressure, GFR, renal tubular function and vasoactive hormones. Thus, the data do not support that atorvastatin changes nitric oxide availability in patients with mild nephropathy. The reduced u-ENaC may reflect changes in sodium absorption in the nephron induced by atorvastatin.

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin.

The epithelial sodium channel (ENaC) is involved in Na(+) responses such as Na(+) absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.

Effect of estradiol on the expression of renal sodium transporters in rats.

BACKGROUND: Although estradiol has been thought to perform an important role in blood pressure regulation, the effects of estradiol on the expression of renal sodium transporters are not fully understood. METHODS: Female Sprague-Dawley rats were treated with 17ß-estradiol or vehicle for 10 days after ovariectomy, and after both ovariectomy and adrenalectomy to eliminate the effect of aldosterone. RESULTS: In the ovariectomized (OVX) rats, estradiol decreased the abundance of the Na-K-2Cl cotransporter (NKCC2) (31.5% of control (OVX), p < 0.01), Na-Cl cotransporter (NCC) proteins (40.5% of control (OVX), p < 0.01) and α- and γ-subunits of the epithelial sodium channel (ENaC) (44.7% and 11.0% of control (OVX), p < 0.01). Estradiol also reduced plasma aldosterone levels (OVX + 17ß-estradiol vs. OVX, 116.3 ± 44.4 vs. 184.2 ± 33.4 pmol/l, p < 0.05) and systolic blood pressure (OVX + 17ß-estradiol vs. OVX, 115 ± 4 vs. 132 ± 2 mmHg, p < 0.05). In rats having undergone adrenalectomy and ovariectomy, estradiol did not reduce systolic blood pressure, or the expression of sodium transporters. CONCLUSION: Estradiol decreased systolic blood pressure, plasma aldosterone levels, and the expression of renal sodium transporters. After aldosterone was eliminated, estradiol did not affect blood pressure or the expression of sodium transporters, which indicates that the effect of estradiol on the renal sodium transporters is at least partly influenced by aldosterone.

Functions of aquaporin 1 and α-epithelial Na+ channel in rat acute lung injury induced by acute ischemic kidney injury.

PURPOSE: To establish a rat model of acute ischemic kidney injury by continually occluding the bilateral renal artery and renal veins, the functions of α-epithelial Na(+) channel (α-ENaC) and aquaporin (AQP1) in lung injury induced by acute kidney injury (AKI) were examined and compared with lung injury induced by endotoxin. METHODS: Male Wistar rats were randomly divided into three groups: control group, AKI group, and sepsis group. The concentrations of AQP1 and α-ENaC in the lung tissue were detected. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum and bronchoalveolar lavage fluid were also detected. RESULTS: The arterial blood pH in AKI group and PaO2 in sepsis group decreased 2 h after the experiment. A significant pulmonary interstitial and alveolar space edema, which showed a typical pathological change in acute lung injury, was found in AKI and sepsis group 8 h after the experiment. Two hours after the experiment, the concentration of TNF-α and IL-6 in the serum and bronchoalveolar lavage fluid (BALF) in AKI and sepsis group increased, whereas the pulmonary expression of AQP1 and α-ENaC decreased. The pulmonary AQP1 and α-ENaC of the rats were negatively correlated with TNF-α and IL-6 in BALF. The relevance among AQP1, α-ENaC, TNF-α, and IL-6 in sepsis group was higher than that in AKI group. CONCLUSION: The TNF-α and IL-6 levels increased significantly and the pulmonary expression of AQP1 and α-ENaC declined at the early stage of AKI.

Immunohistological classification of ionocytes in the external gills of larval Japanese black salamander, Hynobius nigrescens Stejneger.

In this cytological and immunohistological study, we clarified the localization of the membrane transporters Na(+) , K(+) -ATPase (NKA), vacuolar-type H(+) -ATPase (VHA), and epithelial sodium channel (ENaC) and distinguished ionocyte subtypes in the gill of the Japanese salamander (Hynobius nigrescens). In larvae (IY stages 43-65), NKA immunoreactivity was observed on the basolateral plasma membrane in more than 60% cells and less than 20% cells in the primary filaments and secondary lamellae of the external gills, respectively. VHA immunoreactivity was observed on the apical membrane of some epithelial cells in the secondary lamellae of the external gills. High ENaCα immunoreactivity was widely observed on the apical cell membrane of a population of squamous cells, presumably pavement cells (PVCs), and mitochondria-rich cells (MRCs), in the primary filaments and secondary lamellae of the external gills. Using double immunofluorescence microscopy, epithelial cell types involved in ionic regulation were characterized and divided into three ionocyte types: NKA-, NKA- and ENaC-, and VHA-positive cells. VHA-immunoreactive cells as well as NKA-positive cells were observed during IY stages 43-65 of the salamander larvae. During late stages of metamorphosis, NKA, VHA, and ENaCα immunoreactivities in the external gills decreased and finally disappeared during the completion of metamorphosis (IY stage 68). PVCs and MRCs in the external gills are probably involved in acid-base balance regulation and osmoregulation in urodele amphibian larvae. The results are discussed in relation to the ionocytes previously reported in fish gills and the frog skin epithelium.

Deletion of the ubiquitin ligase Nedd4L in lung epithelia causes cystic fibrosis-like disease.

Cystic fibrosis is caused by impaired ion transport due to mutated cystic fibrosis transmembrane conductance regulator, accompanied by elevated activity of the amiloride-sensitive epithelial Na(+) channel (ENaC). Here we show that knockout of the ubiquitin ligase Nedd4L (Nedd4-2) specifically in lung epithelia (surfactant protein C-expressing type II and Clara cells) causes cystic fibrosis-like lung disease, with airway mucus obstruction, goblet cell hyperplasia, massive inflammation, fibrosis, and death by three weeks of age. These effects of Nedd4L loss are likely caused by enhanced ENaC function, as reflected by increased ENaC protein levels, increased lung dryness at birth, amiloride-sensitive dehydration of lung explants, and elevated ENaC currents in primary alveolar type II cells analyzed by patch clamp recordings. Moreover, the lung defects were rescued with administration of amiloride into the lungs of young knockout pups via nasal instillation. Our results therefore suggest that the ubiquitin ligase Nedd4L can suppress the onset of cystic fibrosis symptoms by inhibiting ENaC in lung epithelia.

Inactivation of Bardet-Biedl syndrome genes causes kidney defects.

Bardet-Biedl syndrome (BBS) is a rare hereditary autosomal recessive disease associated with several features including obesity, hypertension, and renal abnormalities. The underlying mechanisms of renal defects associated with BBS remain poorly defined. We examined the histological, molecular, and functional renal changes in BBS mouse models that have features of the human disorder. Interestingly, obese hypertensive Bbs4(-/-) mice exhibited inflammatory infiltration and renal cysts, whereas the obese normotensive Bbs2(-/-) mice had only minor inflammatory infiltration. Accordingly, the expression level of inducible nitric oxide synthase was elevated in the kidney of both BBS mice with a more marked increase in Bbs4(-/-) mice. In contrast, endothelial nitric oxide synthase expression was decreased in Bbs4(-/-), but not Bbs2(-/-), mice. Similarly, the expression levels of transient receptor potential vanilloid 1 and 4 channels as well as ß- and γ-subunits of epithelial Na channel were significantly reduced only in the kidney of Bbs4(-/-) mice. Metabolic studies revealed changes in urine output and urinary concentrations of creatinine, blood urea nitrogen, sodium, and potassium with a more pronounced effect in Bbs4(-/-) mice. Finally, we found that calorie restriction which prevented obesity in BBS mice reversed the morphological and molecular changes found in Bbs2(-/-) and Bbs4(-/-) mice, indicating the kidney abnormalities associated with BBS are obesity related. These findings extend our understanding of the function of BBS proteins and emphasize the importance of these proteins in renal physiology.

Low pulmonary expression of epithelial Na(+) channel and Na(+), K(+)-ATPase in newborn infants with congenital diaphragmatic hernia.

BACKGROUND: It has been suggested from several animal studies and clinical observations that congenital diaphragmatic hernia (CDH) with pulmonary hypoplasia is accompanied by a disturbed perinatal ion transport. This could lead to respiratory distress due to slower clearance of fetal lung fluid at birth. OBJECTIVES: The purpose of this study was to determine whether CDH is related to changes in the expression of three rate-limiting transporter proteins in lung epithelium at birth. METHODS: Tracheal aspirate was collected from 12 newborn infants with CDH and from 8 newborn control patients. Sampling was performed at postnatal age 18 and at 43 h in the CDH group and at 18 h in the control group. The protein abundance of α-, ß- and γ-epithelial Na(+) channel (ENaC), aquaporin 5 and Na(+), K(+)-ATPase α(1) was analyzed using semiquantitative immunoblotting. RESULTS: The levels of ß-ENaC, γ-ENaC and Na(+), K(+)-ATPase α(1) collected at 18 h postnatally were significantly lower in CDH infants compared to control infants. In the CDH group, no significant difference in the expression of the ENaC subunits, Na(+), K(+)-ATPase α(1) or aquaporin 5 could be detected between the two sampling time points. CONCLUSIONS: This downregulation may result in an abnormal lung fluid absorption which could be an important mechanism behind the respiratory distress seen in newborn CDH patients.

Tissue engineering the mechanosensory circuit of the stretch reflex arc: sensory neuron innervation of intrafusal muscle fibers.

The sensory circuit of the stretch reflex arc, composed of specialized intrafusal muscle fibers and type Ia proprioceptive sensory neurons, converts mechanical information regarding muscle length and stretch to electrical action potentials and relays them to the central nervous system. Utilizing a non-biological substrate, surface patterning photolithography and a serum-free medium formulation a co-culture system was developed that facilitated functional interactions between intrafusal muscle fibers and sensory neurons. The presence of annulospiral wrappings (ASWs) and flower-spray endings (FSEs), both physiologically relevant morphologies in sensory neuron-intrafusal fiber interactions, were demonstrated and quantified using immunocytochemistry. Furthermore, two proposed components of the mammalian mechanosensory transduction system, BNaC1 and PICK1, were both identified at the ASWs and FSEs. To verify functionality of the mechanoreceptor elements the system was integrated with a MEMS cantilever device, and Ca(2+) currents were imaged along the length of an axon innervating an intrafusal fiber when stretched by cantilever deflection. This system provides a platform for examining the role of this mechanosensory complex in the pathology of myotonic and muscular dystrophies, peripheral neuropathy, and spasticity inducing diseases like Parkinson's. These studies will also assist in engineering fine motor control for prosthetic devices by improving our understanding of mechanosensitive feedback.

Defining an inhibitory domain in the gamma subunit of the epithelial sodium channel.

Proteases activate the epithelial sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186). To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We found that partial or complete deletion of a short segment in the γ-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract, R158-F168 (RFLNLIPLLVF), which inhibited wild-type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the γ inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel.