Epithelial Sodium Channel Protein Research Articles

TREATMENT WITH HUMAN UMBILICAL CORD-DERIVED MESENCHYMAL STEM CELLS IN A PIG MODEL OF SEPSIS-INDUCED ACUTE KIDNEY INJURY: EFFECTS ON MICROVASCULAR ENDOTHELIAL CELLS AND TUBULAR CELLS IN THE KIDNEY.

Background: Approximately 50% of patients with sepsis develop acute kidney injury (AKI), which is predictive of poor outcomes, with mortality rates of up to 70%. The endothelium is a major target for treatments aimed at preventing the complications of sepsis. We hypothesized that human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) could attenuate tubular and endothelial injury in a porcine model of sepsis-induced AKI. Methods: Anesthetized pigs were induced to fecal peritonitis, resulting in septic shock, and were randomized to treatment with fluids, vasopressors, and antibiotics (sepsis group; n = 11) or to that same treatment plus infusion of 1 × 10 6 cells/kg of hUC-MSCs (sepsis+MSC group; n = 11). Results: At 24 h after sepsis induction, changes in serum creatinine and mean arterial pressure were comparable between the two groups, as was mortality. However, the sepsis+MSC group showed some significant differences in comparison with the sepsis group: lower fractional excretions of sodium and potassium; greater epithelial sodium channel protein expression; and lower protein expression of the Na-K-2Cl cotransporter and aquaporin 2 in the renal medulla. Expression of P-selectin, thrombomodulin, and vascular endothelial growth factor was significantly lower in the sepsis+MSC group than in the sepsis group, whereas that of Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) was lower in the former. Conclusion: Treatment with hUC-MSCs seems to protect endothelial and tubular cells in sepsis-induced AKI, possibly via the TLR4/NF-κB signaling pathway. Therefore, it might be an effective treatment for sepsis-induced AKI.

Collagen Glycopeptides from Transglutaminase-Induced Glycosylation Exhibit a Significant Salt Taste-Enhancing Effect.

This study aimed to prepare collagen glycopeptides by transglutaminase-induced glycosylation and to explore their salt taste-enhancing effects and mechanism. Collagen glycopeptides were obtained by Flavourzyme-catalyzed hydrolysis, followed by transglutaminase-induced glycosylation. The salt taste-enhancing effects of collagen glycopeptides were evaluated by sensory evaluation and an electronic tongue. LC-MS/MS and molecular docking technologies were employed to investigate the underlying mechanism responsible for the salt taste-enhancing effect. The optimal conditions were 5 h for enzymatic hydrolysis, 3 h for enzymatic glycosylation, and 1.0% (E/S, w/w) for transglutaminase. The grafting degree of collagen glycopeptides was 26.9 mg/g, and the salt taste-enhancing rate was 59.0%. LC-MS/MS analysis revealed that Gln was the glycosylation modification site. Molecular docking confirmed that collagen glycopeptides can bind to salt taste receptors epithelial sodium channel protein and transient receptor potential vanilloid 1 through hydrogen bonds and hydrophobic interaction. Overall, collagen glycopeptides have a significant salt taste-enhancing effect, which contributes to the application of collagen glycopeptides for salt reduction without compromising taste in the food industry.

An aptasensor using ceria electrodeposited-screen-printed carbon electrode for detection of epithelial sodium channel protein as a hypertension biomarker.

Epithelial sodium channel (ENaC) is a transmembrane protein that has an essential role in maintaining the levels of sodium in blood plasma. A person with a family history of hypertension has a high enough amount of ENaC protein in the kidneys or other organs, so that the ENaC protein acts as a marker that a person is susceptible to hypertension. An aptasensor involves aptamers, which are oligonucleotides that function similar to antibodies, as sensing elements. An electrochemical aptasensor for the detection of ENaC was developed using a screen-printed carbon electrode (SPCE) which was modified by electrodeposition of cerium oxide (CeO2). The aptamer immobilization was via the streptavidin–biotin system. The measurement of changes in current of the active redox [Fe(CN)6]3−/4− was carried out by differential pulse voltammetry. The surfaces of SPCE and SPCE/CeO2 were characterized using scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The Box–Behnken experimental optimization design revealed the streptavidin incubation time, aptamer incubation time and streptavidin concentrations were 30 min, 30 min and 10.8 µg ml−1, respectively. Various concentrations of ENaC were used to obtain the linearity range of 0.05–3.0 ng ml−1, and the limits of detection and quantification were 0.012 ng ml−1 and 0.038 ng ml−1, respectively. This aptasensor method has the potential to measure the ENaC protein levels in urine samples as well as to be a point-of-care device.

The roles of cytokines and water sodium channel proteins in acute kidney injury-induced acute lung injury rats

Objective To observe the physiopathologic changes of lung in rats with acute ischemic kidney injury,and to study the roles of cytokine,epithelial sodium channel protein (ENaC) and aquaporin 1 (AQP1) in acute lung injury brought on by acute ischemic kidney injury in rats.Methods A total of 60 healthy male Wistar rats (300-320 g) were randomly (random number) divided into control groups (group A,n =30) and acute kidney injury group (group B,n =30).The model of acute ischemic kidney injury in rats was made by bilateral renal arteiovenous blockage with clamps.Six rats of each group were sacrificed at 0,2,4,6 and 8 hours after modeling.Lung tissue of rats was harvested and stained with hematoxylin-eosin (HE) staining method,and the pathological changes of lung were observed under microscope.The ratio of wet and dry weight (W/D) of lung was calculated.The levels of protein in bronchoalveolar lavage fluid (BALF) were measured.The levels of IL-6 and TNF-α both in serum and BALF were tested.The concentrations of AQP1 and α-ENaC in lung were measured.Results At six hours after modeling,the pH value of arterial blood of rats in group B began to get lowered compared to group A.There was no difference in partial pressure of oxygen in arterial blood between two groups during entire period of experiment (P ＞0.05).Protein level in BALF and W/D of lung increased significantly two hours after modeling in rats of group B (P ＜ 0.05).The histopathological changes of acute lung injury including swollen aleolar epithelium,widened interalveolar septum,edema of alveoli and alveolar interstitium,alveolar neurophil sequestration,erythrocytes and protein in exudates were observed.The levels of TNF-α and IL-6 in serum and in BALF began to increase at two hours after modeling.The levels of AQP1 and α-ENaC of lung in rats with acute kidney injury decreased gradually and were lower than those in rats of group A (P ＜0.05).Conclusions Aleolar epithelial-endothelial barrier function was already compromised at the beginning of AKI,suggesting the acute lung injury was already brought on.The levels of TNF-α,IL-6 in serum and in BALF increased after the occurrence of acute kidney injury.The decreases in lung AQP1 and α-ENaC might contribute to the lung injury caused by early acute kidney injury. Key words: Acute kidney injury; Acute lung injury; Cytokine; Aquaporin 1; Epithelial sodium channel protein

Investigation of transmembrane protein fused in lipid bilayer membranes supported on porous silicon

This article investigates a device made from a porous silicon structure supporting a lipid bilayer membrane (LBM)fused with Epithelial Sodium Channel protein. The electrochemically-fabricated porous silicon template had pore diameters in the range 0.2~2 µm. Membranes were composed of two synthetic phospholipids: 1,2-diphytanoyl-sn-glycero-3-phosphoserine and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine. The LBMwas formed by means of the Langmuir-Blodgett and Langmuir-Schaefer techniques, at a monolayer surface tension of 26 m Nm−1 in room temperature and on a deionized water subphase, which resulted in an average molecular area of 0.68–0.73 nm2. Fusion of transmembrane protein was investigated using Atomic Force Microscopy. Initial atomic force microscopy results demonstrate the ability to support lipid bilayers fused with transmembrane proteins across a porous silicon substrate. However, more control of the membrane’s surface tension using traditional membrane fusion techniques is required to optimize protein incorporation.

Genetic Variations in the Sodium Balance-Regulating Genes ENaC, NEDD4L, NDFIP2 and USP2 Influence Blood Pressure and Hypertension

Background/Aims: In humans, the kidneys regulate blood pressure by balancing sodium concentrations. Fine-tuning of renal sodium reabsorption and excretion depends on the epithelial sodium channel protein (ENaC: protein complex of SCNN1A, SCNN1B, and SCNN1G). The surface expression of ENaC components is directed by the ubiquitination of ENaC by NEDD4L, an ENaC-specific E3 ubiquitin ligase, and is regulated by the deubiquitination of ENaC by USP2. The activity of NEDD4L in turn is regulated by phosphorylation by SGK1 and also through interaction with NDFIP2. Methods: We analyzed 91 SNPs in 7 genes using the genotype data of 8,842 individuals from the Korea Association REsource subject pool for their correlation with blood pressure and hypertension. Results: 25 SNPs in the SCNN1A, SCNN1B, SCNN1G, NEDD4L, NDFIP2, and USP2 loci were found to be associated with blood pressure. An additional hypertension case-control study identified 13 SNPs in SCNN1B, SCNN1G, and NEDD4L that were linked to hypertension. Conclusion: These results support our hypothesis that individual variations in blood pressure are attributed to variants of the genes that regulate renal sodium reabsorption and excretion. Our data also suggest that it would be meaningful to investigate the role of NEDD4L-mediated ubiquitination in the pathogenesis of hypertension.

Ibuprofen-Induced Patent Ductus Arteriosus Closure: Physiologic, Histologic, and Biochemical Effects on the Premature Lung

The goal was to study the pulmonary, biochemical, and morphologic effects of a persistent patent ductus arteriosus in a preterm baboon model of bronchopulmonary dysplasia. Preterm baboons (treated prenatally with glucocorticoids) were delivered at 125 days of gestation (term: 185 days), given surfactant, and ventilated for 14 days. Twenty-four hours after birth, newborns were randomly assigned to receive either ibuprofen (to close the patent ductus arteriosus; n = 8) or no drug (control; n = 13). After treatment was started, the ibuprofen group had significantly lower pulmonary/systemic flow ratio, higher systemic blood pressure, and lower left ventricular end diastolic diameter, compared with the control group. There were no differences in cardiac performance indices between the groups. Ventilation index and dynamic compliance were significantly improved with ibuprofen. The improved pulmonary mechanics in ibuprofen-treated newborns were not attributable to changes in levels of surfactant protein B, C, or D, saturated phosphatidylcholine, or surfactant inhibitory proteins. There were no differences in tracheal concentrations of cytokines commonly associated with the development of bronchopulmonary dysplasia. The groups had similar messenger RNA expression of genes that regulate inflammation and remodeling in the lung. Lungs from ibuprofen-treated newborns were significantly drier (lower wet/dry ratio) and expressed 2.5 times more epithelial sodium channel protein than did control lungs. By 14 days after delivery, control newborns had morphologic features of arrested alveolar development (decreased alveolar surface area and complexity), compared with age-matched fetuses. In contrast, there was no evidence of alveolar arrest in the ibuprofen-treated newborns. Ibuprofen-induced patent ductus arteriosus closure improved pulmonary mechanics, decreased total lung water, increased epithelial sodium channel expression, and decreased the detrimental effects of preterm birth on alveolarization.

Reply to Eisenhut

REPLY: Leptospirosis is a zoonotic disease of global importance and can be caused by any one of more than 11 species and 200 serovars of spirochetes of the genus Leptospira (2). In recent years, a severe form of leptospirosis, involving pulmonary hemorrhage, has emerged as a serious clinical syndrome (8, 9). The mechanisms of pathogenesis, host defense, and protective immunity remain poorly understood. Understanding the mechanisms of immunity to leptospirosis is of major importance in developing a successful vaccine against leptospirosis as well as in gaining insight into the pathogenesis of natural or induced infection. In an article recently published in AJP-Renal Physiology, we investigated epithelial sodium transporter protein expression in an animal model of leptospirosis with the objective of determining the mechanisms involved in the pathogenesis of pulmonary edema in this condition (1). We found that, in pulmonary cells, expression of epithelial sodium channel protein was reduced, and NKCC1 protein expression was increased. In his Letter to the Editor regarding that same article, Eisenhut (6) hypothesized that the most likely mechanism explaining these changes in epithelial membrane ion transporter protein expression is the effect of the cytokines tumor necrosis factor (TNF) and interleukin (IL)-1. This is a plausible explanation, given that Eisenhut et al. (4, 5) have shown that, in other models of infectious disease, including meningococcemia, such a mechanism is involved. The authors showed that, in meningococcal septicemia patients with pulmonary edema, levels of TNF and IL-1 are high, and systemic epithelial ion transport is impaired, leading to greater fractional excretion of sodium, as well as to elevated levels of sodium in sweat and saliva. In a kinetic study, Vernel-Pauillac and Merien (10) compared the mRNA expression levels of various cytokines in the peripheral blood mononuclear cells of Leptospira interrogansinoculated hamsters. The authors observed pronounced mRNA expression of the Th1 cytokines TNF, interferon(IFN), and IL-12, with transcripts being detected as early as 1 h postinfection. In addition, the expression of anti-inflammatory cytokines, such as IL-4 and IL-10, was prominent from 1 to 4 days postinfection in response to infection with L. interrogans. It has also been demonstrated that levels of soluble IL-2R, IL-6, TNF, IFN, and IL-12 are elevated in sera obtained from patients treated for acute leptospirosis (3, 7). Eisenhut (6) has raised a very interesting point that merits further consideration and investigation.

Antibodies against purified epithelial sodium channel protein from bovine renal papilla.

Monoclonal and polyclonal antibodies against amiloride-sensitive sodium channel protein purified from bovine renal papilla have been produced. These antibodies show considerable specificity in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays and can immunoprecipitate radiolabeled channel protein. The polyclonal antibodies bind two sodium channel subunits on Western blots, namely, the 300- and 110-kDa polypeptides, and cross react with channel protein isolated from the amphibian A6 renal epithelial cell line. They can be used to immunoaffinity purify the channel in relatively high yield from a crude, detergent-solubilized bovine kidney homogenate. These antibodies should be useful in isolating the sodium channel gene, in studying the channel protein structure, in studying immunocytochemical localization, and in allowing purification of the channel from other tissue sources.

The epithelial sodium channel. Subunit number and location of the amiloride binding site.

Sodium dodecyl sulfate gel electrophoresis of the radioiodinated native amiloride-sensitive epithelial sodium channel protein isolated from bovine renal papilla and cultured amphibian A6 cells under denatured and nonreduced conditions revealed an 125I-labeled protein band of Mr approximately 730,000. Upon reduction, this protein was resolved into five major polypeptide bands with apparent average Mr values of 315,000, 149,000, 95,000, 71,000, and 55,000. The amiloride analog [3H]methylbromoamiloride has been used as a photoaffinity label to determine the location of the binding site for amiloride on the epithelial sodium channel protein. [3H]Methylbromoamiloride binds covalently to the sodium channel at high affinity binding sites with a half-maximal binding concentration of 0.2 microM. [3H]Methylbromoamiloride was specifically photoincorporated into the Mr approximately 150,000 polypeptide and this incorporation was blocked by addition of excess amiloride. These data suggest that the epithelial sodium channel protein is composed of at least five nonidentical polypeptide subunits, only one of which specifically binds amiloride.