BACKGROUND: Urea transporters (UTs) are important in urine concentration and in urea recycling, and UT-B has been implicated in both. In kidney, UT-B was originally localized to outer medullary descending vasa recta, and more recently detected in inner medullary descending vasa recta. Endogenously produced microRNAs (miRs) bind to the 3'UTR of genes and generally inhibit their translation, thus playing a pivotal role gene regulation. METHODS: Mice were dehydrated for 24 hours then sacrificed. Inner and outer medullas were analyzed by polymerase chain reaction (PCR) and quantitative PCR for miRNA expression and analyzed by western blotting for protein abundance. RESULTS: MiRNA sequencing analysis of mouse inner medullas showed a 40% increase in miRNA-200c in dehydrated mice compared with controls. An in silico analysis of the targets for miR-200c revealed that miRNA-200c could directly target the gene for UT-B. PCR confirmed that miR-200c is up-regulated in the inner medullas of dehydrated mice while western blot showed that UT-B protein abundance was down-regulated in the same portion of the kidney. However, in the outer medulla, miR-200c was reduced and UT-B protein was increased in dehydrated mice. CONCLUSIONS: This is the first indication that UT-B protein and miR-200c may each be differentially regulated by dehydration within the kidney outer and inner medulla. The inverse correlation between the direction of change in miR-200c and UT-B protein abundance in both the inner and outer medulla suggests that miR-200c may be associated with the change in UT-B protein in these 2 portions of the kidney medulla.
Chloroquine, a widely used anti-malaria drug, has gained popularity for the treatment of rheumatoid arthritis, systemic lupus erythematosus (SLE), and human immunodeficiency virus (HIV). Unfortunately, chloroquine may also negatively impact renal function for patients whose fluid and electrolyte homeostasis is already compromised by diseases. Chronic administration of chloroquine also results in polyuria, which may be explained by suppression of the antidiuretic response of vasopressin. Several of the transporters responsible for concentrating urine are vasopressin-sensitive including the urea transporters UT-A1 and UT-A3, the water channel aquaporin-2 (AQP2), and the Na+-K+-2Cl− cotransporter (NKCC2). To examine the effect of chloroquine on these transporters, Sprague-Dawley rats received daily subcutaneous injections of 80 mg·kg−1·day−1 of chloroquine for 4 days. Twenty-four hour urine output was twofold higher, and urine osmolality was decreased by twofold in chloroquine-treated rats compared with controls. Urine analysis of treated rats detected the presence chloroquine as well as decreased urine urea and cAMP levels compared with control rats. Western blot analysis showed a downregulation of AQP2 and NKCC2 transporters; however, UT-A1 and UT-A3 abundances were unaffected by chloroquine treatment. Immunohistochemistry showed a marked reduction of UT-A1 and AQP2 in the apical membrane in inner medullary collecting ducts of chloroquine-treated rats. In conclusion, chloroquine-induced polyuria likely occurs as a result of lowered cAMP production. These findings suggest that chronic chloroquine treatment would exacerbate the already compromised fluid homeostasis observed in diseases like chronic kidney disease.
Amiloride-sensitive epithelial sodium (Na+) channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends upon a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits, α, β, and γ. The C-terminal domain of all three subunits is intracellular and contains a proline rich motif (PPxY). Mutations or deletion of this PPxY motif in the β and γ subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein (Nedd4-2) to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when MDCK cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected epithelial cells (A6) expressing endogenous ENaC, ENaC appears to be degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by the rate of Nedd4-2–mediated ENaC ubiquitination. Controlling the rate of degradation is apparently important enough to have multiple, redundant pathways to control Nedd4-2 and ENaC ubiquitination.
Background: Epithelial sodium channels (ENaC) are activated by proteolytic cleavage. Several proteases including furin and prostasin cleave ENaC.
Results: Cathepsin B also cleaves and activates ENaC. Cathepsin B cleaves ENaC α but not β or γ subunits.
Conclusion: Cathepsin B is a secreted protease, so it may cleave ENaC at the cell surface.
Significance: Cathepsin B cleavage represents a novel ENaC regulatory mechanism.
The intercalated cell Cl−/HCO3− exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl− absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle’s syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle’s variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle’s mutation increased total Cl− absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl− absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.
Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.
Aim: Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. Methods: Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. Results: PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. Conclusion: Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.
Alcohol affects total body sodium balance, but the molecular mechanism of its effect remains unclear. We used single-channel methods to examine how ethanol affects epithelial sodium channels (ENaC) in A6 distal nephron cells. The data showed that ethanol significantly increased both ENaC open probability (Po) and the number of active ENaC in patches (N). 1-Propanol and 1-butanol also increased ENaC activity, but iso-alcohols did not. The effects of ethanol were mimicked by acetaldehyde, the first metabolic product of ethanol, but not by acetone, the metabolic product of 2-propanol. Besides increasing open probability and apparent density of active channels, confocal microscopy and surface biotinylation showed that ethanol significantly increased α-ENaC protein in the apical membrane. The effects of ethanol on ENaC Po and N were abolished by a superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) and blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Consistent with an effect of ethanol-induced reactive oxygen species (ROS) on ENaC, primary alcohols and acetaldehyde elevated intracellular ROS, but secondary alcohols did not. Taken together with our previous finding that ROS stimulate ENaC, the current results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde.
Phosphatidylinositol phosphates (PIPs) are known to regulate epithelial sodium channels (ENaC). Lipid binding assays and coimmunoprecipitation showed that the amino-terminal domain of the β- and γ-subunits of Xenopus ENaC can directly bind to phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidic acid (PA). Similar assays demonstrated various PIPs can bind strongly to a native myristoylated alanine-rich C-kinase substrate (MARCKS), but weakly or not at all to a mutant form of MARCKS. Confocal microscopy demonstrated colocalization between MARCKS and PIP2. Confocal microscopy also showed that MARCKS redistributes from the apical membrane to the cytoplasm after PMA-induced MARCKS phosphorylation or ionomycin-induced intracellular calcium increases. Fluorescence resonance energy transfer studies revealed ENaC and MARCKS in close proximity in 2F3 cells when PKC activity and intracellular calcium concentrations are low. Transepithelial current measurements from Xenopus 2F3 cells treated with PMA and single-channel patch-clamp studies of Xenopus 2F3 cells treated with a PKC inhibitor altered Xenopus ENaC activity, which suggest an essential role for MARCKS in the regulation of Xenopus ENaC activity.