Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1, 2. Interruption of IL18 action reduces atherosclerosis in mice3, 4. Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney5. NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or from Apoe−/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe−/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis

Functionomics of novel mutations in NCC and their relevance in development of Gitelman syndrome

<p>Gitelman syndrome is a genetic disease characterized by low blood pressure and salt wasting. In most cases, Gitelman syndrome results from loss-of-function mutations in the solute carrier family 12 (SLC12A3) gene, which encodes the thiazide-sensitive sodium chloride co-transporter (NCC). At present, more than 250 distinct loss-of-function mutations have been identified in patients with Gitelman syndrome. Functional analysis has been limited by the use of only Xenopus laevis oocytes as a model system. The aim of the present study is to understand the functional consequences of these NCC mutations in mammalian cell line systems. Recently, our group published a new technique to isolate primary cells using Complex Object Parametric Analyzer and Sorter (COPAS). We have pioneered with this COPAS sorting platform to isolate and culture distal convoluted tubules (DCT) based on parvalbumin-eGFP transgenic mice model. These primary cultures exhibit several characteristics of the original epithelium including thiazide-sensitive transepithelial NaCl transport. By generating crossbred of parvalbumin-eGFP mice with NCC knockout we aim to obtain DCT primary cell line without endogenous expression of wild type NCC, which in turn will be substituted by ectopic expression of mutated NCC. Generation of this model will create an excellent platform for further functional analysis of mutant NCC proteins. Currently, out of several patients with Gitelman syndrome symptoms eight new mutations were selected. The majority of these mutations exhibited a disturbed phosphorylation and glycosylation pattern as well as diminished expression of NCC. However, functional consequences of these mutations remain to be elucidated. Overall, functional screening of many NCC mutations in DCT cultures are now possible and will also allow to unravel a complex system of NCC regulatory mechanisms.</p

Do premotor interneurons act in parallel on spinal motoneurons and on dorsal horn spinocerebellar and spinocervical tract neurons in the cat?

It has previously been established that ventral spinocerebellar tract (VSCT) neurons and dorsal spinocerebellar tract neurons located in Clarke's column (CC DSCT neurons) forward information on actions of premotor interneurons in reflex pathways from muscle afferents on α-motoneurons. Whether DSCT neurons located in the dorsal horn (dh DSCT neurons) and spinocervical tract (SCT) neurons are involved in forwarding similar feedback information has not yet been investigated. The aim of the present study was therefore to examine the input from premotor interneurons to these neurons. Electrical stimuli were applied within major hindlimb motor nuclei to activate axon-collaterals of interneurons projecting to these nuclei, and intracellular records were obtained from dh DSCT and SCT neurons. Direct actions of the stimulated interneurons were differentiated from indirect actions by latencies of postsynaptic potentials evoked by intraspinal stimuli and by the absence or presence of temporal facilitation. Direct actions of premotor interneurons were found in a smaller proportion of dh DSCT than of CC DSCT neurons. However, they were evoked by both excitatory and inhibitory interneurons, whereas only inhibitory premotor interneurons were previously found to affect CC DSCT neurons [as indicated by monosynaptic excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in dh DSCT and only IPSPs in CC DSCT neurons]. No effects of premotor interneurons were found in SCT neurons, since monosynaptic EPSPs or IPSPs were only evoked in them by stimuli applied outside motor nuclei. The study thus reveals a considerable differentiation of feedback information provided by different populations of ascending tract neurons

AQP9 mRNA expression levels in four types of glioblastoma.

<p>Expression levels of AQP9 in 173 patients were retrieved from the TCGA database and compared to patient descriptors that have previously been associated with four types of glioblastoma: classical, mesenchymal, neural and proneural <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075764#pone.0075764-Verhaak1" target="_blank">[25]</a>. Chi-square analysis suggests uneven AQP9 expression levels among these four tumor types (P<0.0001). High AQP9 expression (>200%) appears to be associated primarily with mesenchymal tumors. X-axis labels refer to <i>AQP9</i> expression in specific patients compared to average <i>AQP9</i> expression in TCGA glioma samples.</p

Double-immunofluorescence labeling of human glioma tissue.

<p>Robust anti-CD15 (red) and anti-AQP9 co-labeling was observed in myelomonocytic cells (asterisk). In 4 patient-biopsies weaker CD15 immunolabeling was also observed in a number of larger cells, likely identifying these cells as BTSCs (examples marked by arrowheads). Non-myelomonocytic AQP9 positive cells (examples marked by arrows) were located near putative BTSCs, but no clear examples of AQP9 and CD15 co-expression were observed in these cells. Two areas are shown as overlay images (left), green channel (center) and red channel (right). Nuclei (blue). Scale bar  = 20 µm.</p

Aquaporin-9 Protein Is the Primary Route of Hepatocyte Glycerol Uptake for Glycerol Gluconeogenesis in Mice

It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9(-/-) knock-out mice remained normoglycemic. The reason for this improvement in AQP9(-/-) mice was not established before. Here, we show increased glucose output (by 123% +/- 36% S. E.) in primary hepatocyte culture when 0.5 mM extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9(-/-) cells. Likewise, the increase was abolished by 25 mu M HTS13286 (IC(50) similar to 2 mu M), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate-and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9(-/-) mice