Potential urinary biomarkers of acute kidney injury in domestic animals

Acute kidney injury (AKI) is a rapid loss of kidney function, which can be a consequence of ischemic, toxic or obstructive insult to the tubules, a reduction of the filtering capacity of the glomeruli or tubulointerstitial inflammation. The diagnosis and prognosis of AKI are problematic due to the lack of sufficiently specific and sensitive markers. The aims of these studies were to assess how kidney impairment impacts on the urinary proteome and to reveal the pathophysiological processes in kidney tissue caused by toxic insult. Changes in the urine proteome after toxic insult to the kidneys were studied by measuring urine enzyme activities, total protein and creatinine concentrations, and using proteomic methods. The usefulness of urinary enzyme activities in kidney impairment diagnosis was studied with in dogs bitten by Vipera berus berus (common European adder) and in sheep with ketoprofen-induced AKI. In both cases, the urinary enzyme activities were demonstrated to be promising markers of kidney impairment. Two-dimensional gel electrophoresis (2D-GE) and two-dimensional differential gel electrophoresis (2D-DIGE) were used to detect potential new urinary protein markers in diagnosing AKI resulting from intoxication in sheep and dogs. The detected differentially expressed proteins were identified using a peptide mass fingerprinting method with either a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF) or a liquid chromatograph hybrid quadrupole mass spectrometer (LC-MS/MS). As a result, several potential urinary markers of kidney impairment were identified: retinol binding protein 4, calbindin D28k, CD1d, complement C3 and complement C4 in our sheep model of AKI, and alpha-1-antirypsin, β-2-microglobulin, fetuin-B and superoxide dismutase (Cu-Zn) in dogs bitten by the common European adder. The pathophysiological process in kidney tissue in renal impairment was examined using immunohistochemical methods. The possible involvement of calbindin D28k, CD1d and complement (C) components in the pathophysiology of AKI in our sheep model was also investigated. All antigens were localized in kidney tubule epithelial cells and the tubular lumina of the exposed sheep, confirming acute tubular injury detected by histological stains. In summary, the measurement of urine enzyme activities proved to be an efficient method to evaluate kidney function in two domestic animal species. Several potential urinary markers were found, and warrant further investigation in clinical veterinary patients suffering from kidney impairment. The complement system was activated in kidney tissue in the ketoprofen-induced sheep model of AKI.Akuutti munuaisvaurio tarkoittaa munuaisten toimintakyvyn äkillistä heikkenemistä iskeemisen tai toksisen altistuksen, tukoksen aiheuttaman munuaistiehyiden vaurion, munuaiskeräsen alentuneen suodatuskyvyn tai tubulointerstitiaalisen tulehduksen vuoksi. Akuutin munuaisvaurion diagnosoiminen on haastavaa niin ihmis- kuin eläinlääketieteessä spesifisten ja sensitiivisten merkkiaineiden puutteen vuoksi. Tämän väitöskirjan tarkoituksena oli tutkia minkälaisia proteiineja suodattui virtsaan munuaisten vaurioituessa munuaistoksisen altistuksen jälkeen. Altistuksina toimivat kokeellinen ketoprofeenin yliannos lampailla sekä tahattomat käärmeenpuremat kotikoirilla. Tarkoituksena oli myös selvittää toksiineille altistuneen munuaiskudoksen patofysiologisia prosesseja. Altistuksen jälkeen virtsan sisältämien proteiinien muutoksia tutkittiin mittaamalla virtsan entsyymiaktiivisuuksia sekä proteiini- ja kreatiniinipitoisuuksia. Kaksisuuntaista geelielektroforeesia (2D-GE ja 2D-DIGE) käyttäen löydettiin useita mahdollisia virtsan merkkiaineita toksisen altistuksen aiheuttaman akuutin munuaisvaurion diagnosoimiseen. Löydetyt mielenkiintoiset proteiinit tunnistettiin massaspektrometrian avulla (LC-MALDI-TOF tai LC-MS/MS). Munuaiskudoksen patofysiologisia prosesseja munuaistoksisen altistuksen jälkeen arvioitiin immunohistokemiallisin menetelmin. Virtsan entsyymiaktiivisuuksien mittaamisen todettiin olevan hyvä keino munuaisvaurion havaitsemiseen molemmilla eläinlajeilla. Tunnistetuista uusista akuutin munuaisvaurion merkkiaineista retinolia sitova proteiini 4, kalbindiini D28k, CD1d, C3 ja C4 liittyivät ketoprofeenin yliannostukseen ja alfa-1-antitrypsiini, beeta-2-mikroglobuliini, fetuiini-B ja SOD1 kyyn puremaan. Ketoprofeiinin yliannostuksen seurauksena havaitsimme calbindin D28k ja CD1d -proteiinien aktivoitumisen munuaistiehyiden epiteelisoluissa. Lisäksi havaitsimme komplementtijärjestelmän aktivaatioproteiinien C1q, C3, C4, C5, C9 sekä tekijä H:n kerääntymistä munuaistiehyeiden epiteelisoluihin komplementtijärjestelmän komponenttien C1q, C3, C4, C5, C9 sekä niiden inhibiittorin faktori H:n sekä kalbindiini D28k ja CD1d -proteiinien aktivoitumisen munuaistiehyiden epiteelisoluissa ketoprofeenin yliannoksen seurauksena

Språkpolicy vid svenskspråkiga daghem i Finland: tvåspråkiga barns handlingar och agens

The aim of this article is to examine communicative actions of nine Swedish-Finnish bilingual children enrolled in Swedish-medium early childhood education and care in Finland. We discuss the interplay between declared monolingual language policy, and the ideology of supporting the language background of the bilingual child. As a result we found a dual practiced language policy. With the agency and actions of bilingual children we could identify both monolingual policy being practiced, but also a bilingual policy being constructed at the same time. Neither of the practiced dual policies seemed to be explicitly resisted either by the children or the pedagogical practitioners. The communicative actions of the nine children showed that they are individuals and they all constructed their bilingual policies in varied ways, some of the children even, quite clearly, preferring the monolingual policy in their communicative actions.peerReviewe

Activation of complement system in kidney after ketoprofen-induced kidney injury in sheep

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Temperature sensitivity patterns of carbon and nitrogen processes in decomposition of boreal organic soils – Quantification in different compounds and molecule sizes based on a multifactorial experiment

Climate warming and organic matter decomposition are connected in a recursive manner; this recursion can be described by temperature sensitivity. We conducted a multifactorial laboratory experiment to quantify the temperature sensitivity of organic carbon (C) and nitrogen (N) decomposition processes of common boreal organic soils. We incubated 36 mor and 36 slightly decomposed Carex-Sphagnum peat samples in a constant moisture and ambient temperature for 6 months. The experiment included three temperature and two moisture levels and two food web manipulations (samples with and without fungivore enchytraeid worms). We determined the release of carbon dioxide (CO2) and dissolved organic carbon (DOC) in seven molecular size classes together with ammonium N and dissolved organic N in low molecular weight and high molecular weight fractions. The temperature sensitivity function Q10 was fit to the data. The C and N release rate was almost an order of magnitude higher in mor than in peat. Soil fauna increased the temperature sensitivity of C release. Soil fauna played a key role in N release; when fauna was absent in peat, the N release was ceased. The wide range of the studied C and N compounds and treatments (68 Q10 datasets) allowed us to recognize five different temperature sensitivity patterns. The most common pattern (37 out of 68) was a positive upwards temperature response, which was observed for CO2 and DOC release. A negative downward pattern was observed for extractable organic nitrogen and microbial C. Sixteen temperature sensitivity patterns represented a mixed type, where the Q10function was not applicable, as this does not allow changing the sign storage change rate with increasing or decreasing temperature. The mixed pattern was typically connected to intermediate decomposition products, where input and output fluxes with different temperature sensitivities may simultaneously change the storage. Mixed type was typical for N processes. Our results provide useful parameterization for ecosystem models that describe the feedback loop between climate warming, organic matter decomposition, and productivity of N-limited vegetation.Peer reviewe

Effects of intrauterine devices on proteins in the uterine lavage fluid of mares

Intrauterine devices block luteolysis in cyclic mares, but the underlying mechanism is unknown. To clarify the mechanisms, the protein profile of the endometrial secretome was analyzed using two-dimensional difference gel electrophoresis (2D-DIGE). Twenty-seven mares were classified according to whether they were inseminated (AI) or had an intrauterine device (IUD), a water-filled plastic sphere, inserted into the uterus on Day 3 after ovulation. Uterine lavage fluids were collected on Day 15 from pregnant inseminated mares (AI-P; n = 8), non-pregnant inseminated mares (AI-N; n = 4), and mares with IUD (n = 15). The IUD group was further divided into prolonged (IUD-P; n = 7) and normal luteal phase (IUD-N; n = 8) groups on the basis of ultrasound examinations, serum levels of progesterone and PGFM on Days 14 and 15, and COX-2 results on Day 15. Four mares from each group were selected for the 2D-DIGE analyses. Ten proteins had significantly different abundance among the groups, nine of the proteins were identified. Malate dehydrogenase 1, increased sodium tolerance 1, aldehyde dehydrogenase 1A1, prostaglandin reductase 1, albumin and hemoglobin were highest in pregnant mares; T-complex protein 1 was highest in non-pregnant mares; and annexin A1 and 6-phosphogluconolactonase were highest in IUD mares. The results suggest that the mechanism behind the intrauterine devices is likely related to inflammation.Peer reviewe

Matrix metalloproteinase (MMP)-2, MMP-9, semen quality and sperm longevity in fractionated stallion semen

Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of the pro-form of MMP-2, active MMP-2 and total MMP-9 were evaluated using gelatin zymography, and all were present in all fractions of the stallion's ejaculate, with higher relative activity of the latent than active forms and the highest relative activity in the HIGH fraction. The relative activities of MMP-2 and MMP-9 were positively correlated to sperm concentration and total sperm count, but only in the HIGH fraction and not in LOW or WE. The relative activities of MMPs were not related to differences in sperm longevity during cooled storage, measured as sperm motility and DFI. There was a harmful effect of SP on DFI during storage, but this effect was not associated with differences in the relative activities of MMPs. In conclusion, the relative activities of MMPs are not useful as markers for semen quality (other than sperm concentration), or sperm survival during storage in horses. (C) 2021 Elsevier Inc. All rights reserved.Peer reviewe

Circadian variation in ghrelin and certain stress hormones in crib-biting horses

Crib-biting is classified as an oral stereotypy, which may be initiated by stress susceptibility, management factors, genetic factors and gastrointestinal irritation. Ghrelin has been identified in the gastric mucosa and is involved in the control of food intake and reward, but its relationship to crib-biting is not yet known. The aim of this study was to examine the concentration and circadian variation of plasma ghrelin, cortisol, adrenocorticotropic hormone (ACTH) and β-endorphin in crib-biting horses and non-crib-biting controls. Plasma samples were collected every second hour for 24 h in the daily environment of eight horses with stereotypic crib-biting and eight non-crib-biting controls. The crib-biting horses had significantly higher mean plasma ghrelin concentrations than the control horses. The circadian rhythm of cortisol was evident, indicating that the sampling protocol did not inhibit the circadian regulation in these horses. Crib-biting had no statistically significant effect on cortisol, ACTH or β-endorphin concentrations. The inter-individual variations in β-endorphin and ACTH were higher than the intra-individual differences, which made inter-individual comparisons difficult and complicated the interpretation of results. Further research is therefore needed to determine the relationship between crib-biting and ghrelin concentration.Peer reviewe

Metabolomics Applied to the Study of Extracellular Vesicles

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities—particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies

Extracellular vesicles derived from hypoxic colorectal cancer cells confer metastatic phenotype to non-metastatic cancer cells

Background/Aim: Tumor-secreted extracellular vesicles (EVs) play an important role as mediators of intercellular communication. Hypoxia is a common feature of solid tumors frequently associated with an aggressive clinical behavior. This study aimed to gain a deeper understanding into the functions of EVs in intercellular communication between primary and metastatic cancer cells under hypoxic conditions. Materials and Methods: EVs were isolated from two isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured under normoxic and hypoxic conditions. Their uptake and effects in SW480 and SW620 cells were studied using EV uptake, proliferation, spheroid-formation, wound healing and invasion assays. Results: Our data showed that hypoxia enhanced the release of EVs from CRC cells in a Hypoxia Induced Factor (HIF)-1-dependent manner. Hypoxic EVs were taken up by CRC cells more efficiently than normoxic EVs. Hypoxic EVs stimulated motility, invasiveness and sternness of primary tumour-derived SW480 cells, whereas they had a little effect on metastasis-derived SW620 cells. Conclusion: Hypoxic colorectal cancer-derived EVs confer aggressiveness and invasiveness to hypoxia-naive cancer cells.Peer reviewe

Syöpä muuttaa solunulkoisten vesikkelien metabolista sormenjälkeä

Cancer alters cell metabolism. How these changes are manifested in the metabolite cargo of cancer-derived extracellular vesicles (EVs) remains poorly understood. To explore these changes, EVs from prostate, cutaneous T-cell lymphoma (CTCL), colon cancer cell lines, and control EVs from their noncancerous counterparts were isolated by differential ultracentrifugation and analyzed by nanoparticle tracking analysis (NTA), electron microscopy (EM), Western blotting, and liquid chromatography-mass spectrometry (LC-MS). Although minor differences between the cancerous and non-cancerous cell-derived EVs were observed by NTA and Western blotting, the largest differences were detected in their metabolite cargo. Compared to EVs from noncancerous cells, cancer EVs contained elevated levels of soluble metabolites, e.g., amino acids and B vitamins. Two metabolites, proline and succinate, were elevated in the EV samples of all three cancer types. In addition, folate and creatinine were elevated in the EVs from prostate and CTCL cancer cell lines. In conclusion, we present the first evidence in vitro that the altered metabolism of different cancer cells is reflected in common metabolite changes in their EVs. These results warrant further studies on the significance and usability of this metabolic fingerprint in cancer.Peer reviewe