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Ubiquitination influences the expression of the epithelial Na+ channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na+ could stimulate ubiquitination. Administration of amiloride to block Na+ entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na+ produced by monensin in FRT cells or acute Na+ repletion in rats increased ubiquitination and decreased overall ENaC expression. NEW & NOTEWORTHY: We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na+ channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na+ can stimulate ENaC ubiquitination. [ABSTRACT FROM AUTHOR]

The pore-forming a-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channelmediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney. NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+. [ABSTRACT FROM AUTHOR]

Paraoxonase 3 (PON3) is expressed in the aldosterone‐sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+‐coupled co‐transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride‐induced natriuresis and lower plasma [K+] at baseline. Single channel recordings in split‐open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA‐mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+], sodium chloride co‐transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3 has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. Key points: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems.We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model.At baseline, Pon3 KO mice had lower blood [K+], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride‐induced natriuresis, and enhanced sodium chloride co‐transporter (NCC) phosphorylation.Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+] and ‐NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls.Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron. [ABSTRACT FROM AUTHOR]

Mineralogy and separation experiments were carried out for a low-grade linnaeite ore (0.052%), which belonged to limonite-hematite-pyrite type complex mineral. Under the grinding fineness of 80% -0.074 mm, linnaeite concentrate which contained cobalt grade of 0.51%, recovery rate of 80.99%, sulfur grade of 23.79%, recovery of 88.03% was obtained by closed-circuit processes of one roughing, two scaenging and one cleaning, which used sulfate acid (4500 g/t) and copper sulfate (300 g/t) as activator, so-dium silicate (1000 g/t) and CMC (30 g/t) as inhibitor, ethyl xanthate（100 g/t）and butyl xanthate (100 g/t) as collector, 2# oil (40 g/t) as forther in roughing, no agent in cleaing and first scavenging, used ethyl xan-thate（50 g/t）and butyl xanthate (50 g/t) as collector, 2# oil (20 g/t) as forther in second scavenging.

The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non‐canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope‐tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti‐ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well‐characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non‐epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti‐α subunit antibody recognizes an intense non‐specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone‐induced full length α‐subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the β‐ and γ‐subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti‐ENaC antibodies. [ABSTRACT FROM AUTHOR]

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the γ subunit. Here, we investigated the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in Fisher rat thyroid cells by coexpressing the apical membrane-bound protease channel-activating protease 1 (CAP1; prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney, we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest COOH-terminal fragment of γENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in Fisher rat thyroid cells. Similar cleaved peptides were observed in total and cell surface fractions of the rat kidney. This outcome suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using nonreducing gels, in which the NH2- and COOH-terminal fragments of γENaC are linked by a disulfide bond. Under these conditions, the major cleaved form in the rat kidney had an apparent molecular mass of 56 kDa, ∼4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant γENaC species in the apical membrane of rat and mouse kidneys on a low-Na diet is the twice-cleaved, presumably activated form.NEW & NOTEWORTHY We have identified the major aldosterone-dependent cleaved form of the epithelial Na channel (ENaC) γ subunit in the kidney as a twice-cleaved peptide. This form appears to be identical in size with a subunit cleaved in vitro by the extracellular protease channel-activating protease 1 (prostasin). In the absence of reducing agents, it has an overall molecular mass less than that of the intact subunit, consistent with the excision of an inhibitory domain. [ABSTRACT FROM AUTHOR]