Interaction between Epithelial Sodium Channel γ-Subunit and Claudin-8 Modulates Paracellular Sodium Permeability in Renal Collecting Duct.

BACKGROUND: Water and solute transport across epithelia can occur via the transcellular or paracellular pathways. Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. In the renal collecting duct, which is a typical absorptive tight epithelium, coordination between transcellular sodium reabsorption and paracellular permeability may prevent the backflow of reabsorbed sodium to the tubular lumen along a steep electrochemical gradient. METHODS: To investigate whether transcellular sodium transport controls tight-junction composition and paracellular permeability via modulating expression of the transmembrane protein claudin-8, we used cultured mouse cortical collecting duct cells to see how overexpression or silencing of epithelial sodium channel (ENaC) subunits and claudin-8 affect paracellular permeability. We also used conditional kidney tubule-specific knockout mice lacking ENaC subunits to assess the ENaC's effect on claudin-8 expression. RESULTS: Overexpression or silencing of the ENaC γ-subunit was associated with parallel and specific changes in claudin-8 abundance. Increased claudin-8 abundance was associated with a reduction in paracellular permeability to sodium, whereas decreased claudin-8 abundance was associated with the opposite effect. Claudin-8 overexpression and silencing reproduced these functional effects on paracellular ion permeability. Conditional kidney tubule-specific ENaC γ-subunit knockout mice displayed decreased claudin-8 expression, confirming the cell culture experiments' findings. Importantly, ENaC ß-subunit or α-subunit silencing or kidney tubule-specific ß-ENaC or α-ENaC knockout mice did not alter claudin-8 abundance. CONCLUSIONS: Our data reveal the specific coupling between ENaC γ-subunit and claudin-8 expression. This coupling may play an important role in preventing the backflow of reabsorbed solutes and water to the tubular lumen, as well as in coupling paracellular and transcellular sodium permeability.

YjbH Solubility Controls Spx in Staphylococcus aureus: Implication for MazEF Toxin-Antitoxin System Regulation.

Bacterial cells respond to environmental stresses by modulating their gene expression and adjusting their proteome. In Staphylococcus aureus, selective degradation by ClpP protease eliminates damaged proteins and regulates the abundance of functional proteins such as many important stress-induced transcriptional regulators. Degradation by ClpP requires the unfolding activity of partner Clp ATPases, such as ClpX and ClpC, and assistance of substrate-specific adaptor proteins such as YjbH and TrfA. Herein, we demonstrated that YjbH is aggregated in response to growth stress stimuli, such as oxidative and antibiotic stresses. In consequence, its function as an adaptor protein is compromised. YjbH controls the degradation of the stress-induced transcriptional regulator, Spx. Aggregated YjbH cannot assist Spx degradation, which results in Spx accumulation. We discovered that depending on the stress stimulus, Spx can be soluble or insoluble, and, consequently, transcriptionally active or inactive. Therefore, Spx accumulation and solubility are key components governing activation of Spx-dependent genes. Spx positively regulates expression of a ClpCP adaptor protein TrfA. TrfA in turn is required for degradation of MazE antitoxin, the unstable component of the MazEF toxin-antitoxin system, that neutralizes the endoribonuclease activity of MazF toxin. Bacterial toxin-antitoxin systems are associated with dormancy and tolerance to antibiotics that are related to chronic and relapsing infections, and it is at present a key unresolved problem in medicine. MazF activity was linked to growth stasis, yet the precise environmental signals that trigger MazE degradation and MazF activation are poorly understood. Here we propose a model where YjbH serves as a sensor of environmental stresses for downstream regulation of MazEF activity. YjbH aggregation, soluble Spx, and TrfA, coordinately control MazE antitoxin levels and consequently MazF toxin activity. This model implies that certain stress conditions culminate in modulation of MazF activity resulting in growth stasis during in vivo infections.

Time-course of sodium transport along the nephron in nephrotic syndrome: The role of potassium.

The mechanism of sodium retention and its location in kidney tubules may vary with time in nephrotic syndrome (NS). We studied the mechanisms of sodium retention in transgenic POD-ATTAC mice, which display an inducible podocyte-specific apoptosis. At day 2 after the induction of NS, the increased abundance of NHE3 and phosphorylated NCC in nephrotic mice compared with controls suggest that early sodium retention occurs mainly in the proximal and distal tubules. At day 3, the abundance of NHE3 normalized, phosphorylated NCC levels decreased, and cleavage and apical localization of γ-ENaC increased in nephrotic mice. These findings indicate that sodium retention shifted from the proximal and distal tubules to the collecting system. Increased cleavage and apical localization of γ-ENaC persisted at day 5 in nephrotic mice when hypovolemia resolved and steady-state was reached. Sodium retention and γ-ENaC cleavage were independent of the increased plasma levels of aldosterone. Nephrotic mice displayed decreased glomerular filtration rate and urinary potassium excretion associated with hyperkaliemia at day 3. Feeding nephrotic mice with a low potassium diet prevented hyperkaliemia, γ-ENaC cleavage, and led to persistent increased phosphorylation of NCC. These results suggest that potassium homeostasis is a major determinant of the tubular site of sodium retention in nephrotic mice.

Aldosterone controls primary cilium length and cell size in renal collecting duct principal cells.

Primary cilia are nonmotile sensory organelles found on the surface of almost all kidney tubule epithelial cells. Being exposed to the tubular lumen, primary cilia are thought to be chemo- and mechanosensors of luminal composition and flux, respectively. We hypothesized that, Na+ transport and primary cilia exist in a sensory functional connection in mature renal tubule epithelial cells. Our results demonstrate that primary cilium length is reduced in mineralocorticoid receptor (MR) knockout (KO) mice in a cell autonomous manner along the aldosterone-sensitive distal nephron (ADSN) compared with wild type (as µm ± SEM; 3.1 ± 0.2 vs 4.0 ± 0.1). In mouse cortical collecting duct (mCCD)cl1 cells, which are a model of collecting duct (CD) principal cells, changes in Na+ transport intensity were found to mediate primary cilium length in response to aldosterone (as µm ± SEM: control: 2.7 ± 0.9 vs aldosterone treated: 3.8 ± 0.8). Cilium length was positively correlated with the availability of IFT88, a major intraflagellar anterograde transport complex B component, which is stabilized in response to exposure to aldosterone treatment. This suggests that the abundance of IFT88 is a regulated, rate limiting factor in the elongation of primary cilia. As previously observed in vivo, aldosterone treatment increased cell volume of cultured CD principal cells. Knockdown of IFT88 prevents ciliogenesis and inhibits the adaptive increase in cell size that was observed in response to aldosterone treatment. In conclusion, our results reveal a functional connection between Na+ transport, primary cilia, and cell size, which may play a key role in the morphological and functional adaptation of the CD to sustained changes in active Na+ reabsorption due to variations in aldosterone secretion.

PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 Interacts with Polycomb-Group Proteins and Histones and Regulates Arabidopsis Flowering and Development.

Polycomb-group (PcG) proteins mediate epigenetic gene regulation by setting H3K27me3 via Polycomb Repressive Complex 2 (PRC2). In plants, it is largely unclear how PcG proteins are recruited to their target genes. Here, we identified the PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) protein, which interacts with all three Arabidopsis thaliana PRC2 histone methyltransferases and is required for maintaining full H3 occupancy at several Arabidopsis genes. PWO1 localizes and recruits CURLY LEAF to nuclear speckles in Nicotiana benthamiana nuclei, suggesting a role in spatial organization of PcG regulation. PWO1 belongs to a gene family with three members having overlapping activities: pwo1 pwo2 pwo3 triple mutants are seedling lethal and show shoot and root meristem arrest, while pwo1 single mutants are early flowering. Interestingly, the PWWP domain of PWO1 confers binding to histones, which is reduced by a point mutation in a highly conserved residue of this domain and blocked by phosphorylation of H3S28. PWO1 carrying this mutation is not able to fully complement the pwo1 pwo2 pwo3 triple mutant, indicating the requirement of this domain for PWO1 in vivo activity. Thus, the PWO family may present a novel class of histone readers that are involved in recruiting PcG proteins to subnuclear domains and in promoting Arabidopsis development.

Characterization of the Polycomb-Group Mark H3K27me3 in Unicellular Algae.

Polycomb Group (PcG) proteins mediate chromatin repression in plants and animals by catalyzing H3K27 methylation and H2AK118/119 mono-ubiquitination through the activity of the Polycomb repressive complex 2 (PRC2) and PRC1, respectively. PcG proteins were extensively studied in higher plants, but their function and target genes in unicellular branches of the green lineage remain largely unknown. To shed light on PcG function and modus operandi in a broad evolutionary context, we demonstrate phylogenetic relationship of core PRC1 and PRC2 proteins and H3K27me3 biochemical presence in several unicellular algae of different phylogenetic subclades. We focus then on one of the species, the model red alga Cyanidioschizon merolae, and show that H3K27me3 occupies both, genes and repetitive elements, and mediates the strength of repression depending on the differential occupancy over gene bodies. Furthermore, we report that H3K27me3 in C. merolae is enriched in telomeric and subtelomeric regions of the chromosomes and has unique preferential binding toward intein-containing genes involved in protein splicing. Thus, our study gives important insight for Polycomb-mediated repression in lower eukaryotes, uncovering a previously unknown link between H3K27me3 targets and protein splicing.

Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium.

The key role of the primary cilium in developmental processes is illustrated by ciliopathies resulting from genetic defects of its components. Ciliopathies include a large variety of dysmorphic syndromes that share in common the presence of multiple kidney cysts. These observations suggest that primary cilia may control morphogenetic processes in the developing kidney. In this study, we assessed the role of primary cilium in branching tubulogenesis and/or lumen development using kidney collecting duct-derived mCCDN21 cells that display spontaneous tubulogenic properties when grown in collagen-Matrigel matrix. Tubulogenesis and branching were not altered when cilium body growth was inhibited by Kif3A or Ift88 silencing. In agreement with the absence of a morphogenetic effect, proliferation and wound-healing assay revealed that neither cell proliferation nor migration were altered by cilium body disruption. The absence of cilium following Kif3A or Ift88 silencing in mCCDN21 cells did not alter the initial stages of tubular lumen generation while lumen maturation and enlargement were delayed. This delay in tubular lumen maturation was not observed after Pkd1 knockdown in mCCDN21 cells. The delayed lumen maturation was explained by neither defective secretion or increased reabsorption of luminal fluid. Our results indicate that primary cilia do not control early morphogenetic processes in renal epithelium. Rather, primary cilia modulate tubular lumen maturation and enlargement resulting from luminal fluid accumulation in tubular structures derived from collecting duct cells.