Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P.bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P.bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P.bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na⁺-K⁺-2Cl⁻ Cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aß isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aß in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aß. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aß and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aß and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.

Demonstration of the functional impact of vasopressin signaling in the thick ascending limb by a targeted transgenic rat approach.

The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gs-mediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na(+)-K(+)-2Cl(-)-cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissue-specific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications.

Vasopressin lowers renal epoxyeicosatrienoic acid levels by activating soluble epoxide hydrolase.

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 µM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVP-sensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis.

Impact of Soil Inoculation with Bacillus amyloliquefaciens FZB42 on the Phytoaccumulation of Germanium, Rare Earth Elements, and Potentially Toxic Elements.

Bioaugmentation promises benefits for agricultural production as well as for remediation and phytomining approaches. Thus, this study investigated the effect of soil inoculation with the commercially available product RhizoVital®42, which contains Bacillus amyloliquefaciens FZB42, on nutrient uptake and plant biomass production as well as on the phytoaccumulation of potentially toxic elements, germanium, and rare earth elements (REEs). Zea mays and Fagopyrum esculentum were selected as model plants, and after harvest, the element uptake was compared between plants grown on inoculated versus reference soil. The results indicate an enrichment of B. amyloliquefaciens in inoculated soils as well as no significant impact on the inherent bacterial community composition. For F. esculentum, inoculation increased the accumulation of most nutrients and As, Cu, Pb, Co, and REEs (significant for Ca, Cu, and Co with 40%, 2042%, and 383%, respectively), while it slightly decreased the uptake of Ge, Cr, and Fe. For Z. mays, soil inoculation decreased the accumulation of Cr, Pb, Co, Ge, and REEs (significant for Co with 57%) but showed an insignificant increased uptake of Cu, As, and nutrient elements. Summarily, the results suggest that bioaugmentation with B. amyloliquefaciens is safe and has the potential to enhance/reduce the phytoaccumulation of some elements and the effects of inoculation are plant specific.

Effect of Sodium Chloride on Pyrite Bioleaching and Initial Attachment by Sulfobacillus thermosulfidooxidans.

Biomining applies microorganisms to extract valuable metals from usually sulfidic ores. However, acidophilic iron (Fe)-oxidizing bacteria tend to be sensitive to chloride ions which may be present in biomining operations. This study investigates the bioleaching of pyrite (FeS2), as well as the attachment to FeS2 by Sulfobacillus thermosulfidooxidans DSM 9293T in the presence of elevated sodium chloride (NaCl) concentrations. The bacteria were still able to oxidize iron in the presence of up to 0.6M NaCl (35 g/L), and the addition of NaCl in concentrations up to 0.2M (~12 g/L) did not inhibit iron oxidation and growth of S. thermosulfidooxidans in leaching cultures within the first 7 days. However, after approximately 7 days of incubation, ferrous iron (Fe2+) concentrations were gradually increased in leaching assays with NaCl, indicating that iron oxidation activity over time was reduced in those assays. Although the inhibition by 0.1M NaCl (~6 g/L) of bacterial growth and iron oxidation activity was not evident at the beginning of the experiment, over extended leaching duration NaCl was likely to have an inhibitory effect. Thus, after 36 days of the experiment, bioleaching of FeS2 with 0.1M NaCl was reduced significantly in comparison to control assays without NaCl. Pyrite dissolution decreased with the increase of NaCl. Nevertheless, pyrite bioleaching by S. thermosulfidooxidans was still possible at NaCl concentrations as high as 0.4M (~23 g/L NaCl). Besides, cell attachment in the presence of different concentrations of NaCl was investigated. Cells of S. thermosulfidooxidans attached heterogeneously on pyrite surfaces regardless of NaCl concentration. Noticeably, bacteria were able to adhere to pyrite surfaces in the presence of NaCl as high as 0.4M. Although NaCl addition inhibited iron oxidation activity and bioleaching of FeS2, the presence of 0.2M seemed to enhance bacterial attachment of S. thermosulfidooxidans on pyrite surfaces in comparison to attachment without NaCl.