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Chronic inflammation is postulated to be involved in the development of end-stage renal disease in diabetes, but which specific circulating inflammatory proteins contribute to this risk remain unknown. To study this, we examined 194 circulating inflammatory proteins in subjects from three independent cohorts with type 1 and type 2 diabetes. In each cohort, we identified an extremely robust kidney risk inflammatory signature (KRIS), consisting of 17 proteins enriched in tumor necrosis factor-receptor superfamily members, that was associated with a 10-year risk of end-stage renal disease. All these proteins had a systemic, non-kidney source. Our prospective study findings provide strong evidence that KRIS proteins contribute to the inflammatory process underlying end-stage renal disease development in both types of diabetes. These proteins point to new therapeutic targets and new prognostic tests to identify subjects at risk of end-stage renal disease, as well as biomarkers to measure responses to treatment of diabetic kidney disease.A robust signature of circulating inflammatory proteins is causally associated with 10-year risk of progression to end-stage renal disease in diabetic patients

Background Stearoyl-CoA desaturases (SCDs) are key enzymes involved in de novo monounsaturated fatty acid synthesis. They catalyze the desaturation of saturated fatty acyl-CoA substrates at the delta-9 position, generating essential components of phospholipids, triglycerides, cholesterol esters and wax esters. Despite being crucial for interpreting SCDs roles across species, the evolutionary history of the SCD gene family in vertebrates has yet to be elucidated, in particular their isoform diversity, origin and function. This work aims to contribute to this fundamental effort. Results We show here, through comparative genomics and phylogenetics that the SCD gene family underwent an unexpectedly complex history of duplication and loss events. Paralogy analysis hints that SCD1 and SCD5 genes emerged as part of the whole genome duplications (2R) that occurred at the stem of the vertebrate lineage. The SCD1 gene family expanded in rodents with the parallel loss of SCD5 in the Muridae family. The SCD1 gene expansion is also observed in the Lagomorpha although without the SCD5 loss. In the amphibian Xenopus tropicalis we find a single SCD1 gene but not SCD5, though this could be due to genome incompleteness. In the analysed teleost species no SCD5 is found, while the surrounding SCD5-less locus is conserved in comparison to tetrapods. In addition, the teleost SCD1 gene repertoire expanded to two copies as a result of the teleost specific genome duplication (3R). Finally, we describe clear orthologues of SCD1 and SCD5 in the chondrichthian, Scyliorhinus canicula , a representative of the oldest extant jawed vertebrate clade. Expression analysis in S. canicula shows that whilst SCD1 is ubiquitous, SCD5 is mainly expressed in the brain, a pattern which might indicate an evolutionary conserved function. Conclusion We conclude that the SCD1 and SCD5 genes emerged as part of the 2R genome duplications. We propose that the evolutionary conserved gene expression between distinct lineages underpins the importance of SCD activity in the brain (and probably the pancreas), in a yet to be defined role. We argue that an expression independent of an external stimulus, such as diet induced activity, emerged as a novel function in vertebrate ancestry allocated to the SCD5 isoform in various tissues (e.g. brain and pancreas), and it was selectively maintained throughout vertebrate evolution.

Unique amongst the teleost, Plotosidae catfish possess a dendritic organ (DO) as a purported salt secreting organ, whereas other marine teleosts rely on their gill ionocytes for active NaCl excretion. To address the role of the DO in ionregulation, ligation experiments were conducted in brackish water (BW) 3‰ and seawater (SW) 34‰ acclimated Plotosus lineatus and compared to sham operated fish. Ligation in SW resulted in an osmoregulatory impairment in blood (elevated ions and hematocrit) and muscle (dehydration). However, SW ligation did not elicit compensatory changes in gill or kidney Na+/K+-ATPase (NKA) activity and/or protein expression while a decrease in anterior intestine and increased in posterior intestine were observed but this was not reflected at the protein level. Following ligation in SW, protein levels of carbonic anhydrase (CA) and V-ATPase B subunit (VHAB) were higher in kidney but either lower (CA) or unchanged (VHAB) in other tissues. Taken together, the osmotic disturbance in ligated SW fish indicates the central role of the DO in salt secretion and the absence of a compensatory response from the gill.

The ability of euryhaline Mozambique tilapia to tolerate extreme environmental salinities makes it an excellent model for investigating iono-regulation. This study aimed to characterize and fill important information gap of the expression levels of key ion transporters for Na(+) and Cl(-) in the gill and esophageal-gastrointestinal tract of Mozambique tilapia acclimated to freshwater (0 ppt), seawater (30 ppt) and hypersaline (70 ppt) environments. Among the seven genes studied, it was found that nkcc2, nkcc1a, cftr, nka-α1 and nka-α3, were more responsive to salinity challenge than nkcc1b and ncc within the investigated tissues. The ncc expression was restricted to gills of freshwater-acclimated fish while nkcc2 expression was restricted to intestinal segments irrespective of salinity challenge. Among the tissues investigated, gill and posterior intestine were found to be highly responsive to salinity changes, followed by anterior and middle intestine. Both esophagus and stomach displayed significant up-regulation of nka-α1 and nka-α3, but not nkcc isoforms and cftr, in hypersaline-acclimated fish suggesting a response to hypersalinity challenge and involvement of other forms of transporters in iono-regulation. Changes in gene expression levels were partly corroborated by immunohistochemical localization of transport proteins. Apical expression of Ncc was found in Nka-immunoreactive cells in freshwater-acclimated gills while Nkcc co-localized with Nka-immunoreactive cells expressing Cftr apically in seawater- and hypersaline-acclimated gills. In the intestine, Nkcc-stained apical brush border was found in Nka-immunoreactive cells at greater levels under hypersaline conditions. These findings provided new insights into the responsiveness of these genes and tissues under hypersalinity challenge, specifically the posterior intestine being vital for salt absorption and iono-osmoregulation in the Mozambique tilapia; its ability to survive in hypersalinity may be in part related to its ability to up-regulate key ion transporters in the posterior intestine. The findings pave the way for future iono-regulatory studies on the Mozambique tilapia esophageal-gastrointestinal tract.

The invasion of land required amphibious fishes to evolve new strategies to avoid toxic ammonia accumulation in the absence of water flow over the gills. We investigated amphibious behaviour and nitrogen excretion strategies in six phylogenetically diverse Aplocheiloid killifishes ( Anablepsoides hartii, Cynodonichthys hildebrandi, Rivulus cylindraceus , Kryptolebias marmoratus, Fundulopanchax gardneri , and Aplocheilus lineatus ) in order to determine if a common strategy evolved . All species voluntarily emersed (left water) over several days, and also in response to environmental stressors (low O 2 , high temperature). All species were ammoniotelic in water and released gaseous ammonia (NH 3 volatilization) during air exposure as the primary route for nitrogen excretion. Metabolic depression, urea synthesis, and/or ammonia accumulation during air exposure were not common strategies used by these species. Immunostaining revealed the presence of ammonia-transporting Rhesus proteins (Rhcg1 and Rhcg2) in the skin of all six species, indicating a shared mechanism for ammonia volatilization. We also found Rhcg in the skin of several other fully aquatic fishes, implying that cutaneous ammonia excretion is not exclusive to amphibious fishes. Overall, our results demonstrate that similar nitrogen excretion strategies while out of water were used by all killifish species tested; possibly the result of shared ancestral amphibious traits, phenotypic convergence, or a combination of both.

The stomach, which is characterized by acid peptic digestion in vertebrates, has been lost secondarily multiple times in the evolution of the teleost fishes. The Cypriniformes are largely seen as an agastric order; however, within the superfamily Cobitoidea, the closely related sister groups Nemacheilidae and Balitoridae have been identified as gastric families. The presence of these most recently diverged gastric families in an otherwise agastric clade indicates that either multiple (>2-3) loss events occurred with the Cyprinidae, Catostomidae and Cobitidae, or that gastric reinvention arose in a recent ancestor of the Nemacheilidae/Balitoridae sister clade. In the present study, the foregut regions of Cobitidae, Nemacheilidae/Balitoridae and the ancestral Botiidae family members were examined for the presence of gastric glands and gastric proton pump (Atp4a) α subunit expression by histology and immunohistochemistry respectively. Atp4a gene expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Gastric glands expressing apical H+/K+-ATPase α subunit and isolated partial sequences of atp4a, identified using degenerate primers showing clear orthology to other vertebrate atp4a sequences, were detected in representative species from Nemacheilidae/ Balitoridae and Botiidae, but not Cobitidae (Misgurnus anguillicaudatus). In summary, we provide evidence for an uninterrupted gastric evolutionary lineage in the Cobitoidea, making it highly improbable that the stomach was reinvented in the Nemacheilidae/Balitoridae clade consistent with Dollo's principle. These results also indicate that the gastric trait may be present elsewhere in the Cobitoidea.

The origin of extracellular digestion in metazoans was accompanied by structural and physiological alterations of the gut. These adaptations culminated in the differentiation of a novel digestive structure in jawed vertebrates, the stomach. Specific endoderm/mesenchyme signalling is required for stomach differentiation, involving the growth and transcription factors: 1) Shh and Bmp4, required for stomach outgrowth; 2) Barx1, Sfrps and Sox2, required for gastric epithelium development and 3) Cdx1 and Cdx2, involved in intestinal versus gastric identity. Thus, modulation of endoderm/mesenchyme signalling emerges as a plausible mechanism linked to the origin of the stomach. In order to gain insight into the ancient mechanisms capable of generating this structure in jawed vertebrates, we characterised the development of the gut in the catshark Scyliorhinus canicula . As chondrichthyans, these animals retained plesiomorphic features of jawed vertebrates, including a well-differentiated stomach. We identified a clear molecular regionalization of their embryonic gut, characterised by the expression of barx1 and sox2 in the prospective stomach region and expression of cdx1 and cdx2 in the prospective intestine. Furthermore, we show that gastric gland development occurs close to hatching, accompanied by the onset of gastric proton pump activity. Our findings favour a scenario in which the developmental mechanisms involved in the origin of the stomach were present in the common ancestor of chondrichthyans and osteichthyans.

Our previous study identified 8 risk and 9 protective plasma miRNAs associated with progression to end-stage kidney disease (ESKD) in diabetes. This study aimed to elucidate preanalytical factors that influence the quantification of circulating miRNAs. Using the EdgeSeq platform, which quantifies 2,002 miRNAs in plasma, including ESKD-associated miRNAs, we compared miRNA profiles in whole plasma versus miRNA profiles in RNA extracted from the same plasma specimens. Less than half of the miRNAs were detected in standard RNA extraction from plasma. Detection of individual and concentrations of miRNAs were much lower when RNA extracted from plasma was quantified by RNA sequencing (RNA-Seq) or quantitative reverse transcription PCR (qRT-PCR) platforms compared with EdgeSeq. Plasma profiles of miRNAs determined by the EdgeSeq platform had excellent reproducibility in assessment and had no variation with age, sex, hemoglobin A1c, BMI, and cryostorage time. The risk ESKD-associated miRNAs were detected and measured accurately only in whole plasma and using the EdgeSeq platform. Protective ESKD-associated miRNAs were detected by all platforms except qRT-PCR; however, correlations among concentrations obtained with different platforms were weak or nonexistent. In conclusion, preanalytical factors have a profound effect on detection and quantification of circulating miRNAs in ESKD in diabetes. Quantification of miRNAs in whole plasma and using the EdgeSeq platform may be the preferable method to study profiles of circulating cell-free miRNAs associated with ESKD and possibly other diseases.

Freshwater fishes generally increase ammonia excretion in acidic waters. The new model of ammonia transport in freshwater fish involves an association between the Rhesus (Rh) protein Rhcg-b, the Na+/H+ exchanger (NHE), and a suite of other membrane transporters. We tested the hypothesis that Rhcg-b and NHE3 together play a critical role in branchial ammonia excretion in common carp (Cyprinus carpio) chronically exposed to a low-pH environment. Carp were exposed to three sequential environmental treatments—control pH 7.6 water (24 h), pH 4.0 water (72 h), and recovery pH 7.6 water (24 h)—or in a separate series were simply exposed to either control (72 h) or pH 4.0 (72 h) water. Branchial ammonia excretion was increased by ∼2.5-fold in the acid compared with the control period, despite the absence of an increase in the plasma-to-water partial pressure NH3 gradient. Alanine aminotransferase activity was higher in the gills of fish exposed to pH 4 versus control water, suggesting that ammonia may be generated in gill tissue. Gill Rhcg-b and NHE3b messenger RNA levels were significantly elevated in acid-treated relative to control fish, but at the protein level Rhcg-b decreased (30%) and NHE3b increased (2-fold) in response to water of pH 4.0. Using immunofluorescence microscopy, NHE3b and Rhcg-b were found to be colocalized to ionocytes along the interlamellar space of the filament of control fish. After 72 h of acid exposure, Rhcg-b staining almost disappeared from this region, and NHE3b was more prominent along the lamellae. We propose that ammoniagenesis within the gill tissue itself is responsible for the higher rates of branchial ammonia excretion during chronic metabolic acidosis. Unexpectedly, gill Rhcg-b does not appear to be important in gill ammonia transport in low-pH water, but the strong induction of NHE3b suggests that some NH4 + may be eliminated directly in exchange for Na+. These findings contrast with previous studies in larval zebrafish (Danio rerio) and medaka (Oryzias latipes), underlining the importance of species comparisons.