Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

Background and purpose:We have established an in vitro model of acute tubular necrosis in rat kidney tubular cells, using combined oxygen-glucose deprivation (COGD) and screened a library of 1280 pharmacologically active compounds for cytoprotective effects. Experimental approach: We used in vitro cell-based, high throughput, screening, with cells subjected to COGD using hypoxia chambers, followed by re-oxygenation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the Alamar Blue assay measured mitochondrial respiration and the lactate dehydrogenase assay was used to indicate cell death. ATP levels were measured using a luminometric assay. Key results: Adenosine markedly reduced cellular injury, with maximal cytoprotective effect at 100 mM and an EC50 value of 14 mM. Inosine was also found to be cytoprotective. The selective A3 adenosine receptor antagonist MRS 1523 attenuated the protective effects of adenosine and inosine, while an A3 adenosine receptor agonist provided a partial protective effect. Adenosine deaminase inhibition attenuated the cytoprotective effect of adenosine but not of inosine during COGD. Inhibition of adenosine kinase reduced the protective effects of both adenosine and inosine during COGD. Pretreatment of the cells with adenosine or inosine markedly protected against the fall in cellular ATP content in the cells subjected to COGD. Conclusions and implications:The cytoprotection elicited by adenosine and inosine in a model of renal ischaemia involved both interactions with cell surface adenosine receptors on renal tubular epithelial cells and intracellular metabolism and conversion of adenosine to ATP.

BackgroundDeveloping chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only a few parental cell lines.MethodsParallel cell populations were initiated for two breast cancer cell lines (MDA-MB-231 and MCF-7) and these were treated independently for 18 months with doxorubicin or paclitaxel. IC50 values against 4 chemotherapy agents were determined to measure cross-resistance. Chromosomal instability and karyotypic changes were determined by cytogenetics. TaqMan RT-PCR measurements were performed for resistance-candidate genes. Pgp activity was measured by FACS.ResultsAll together 16 doxorubicin- and 13 paclitaxel-treated cell lines were developed showing 2–46 fold and 3–28 fold increase in resistance, respectively. The RT-PCR and FACS analyses confirmed changes in tubulin isofom composition, TOP2A and MVP expression and activity of transport pumps (ABCB1, ABCG2). Cytogenetics showed less chromosomes but more structural aberrations in the resistant cells.ConclusionWe surpassed previous studies by parallel developing a massive number of cell lines to investigate chemoresistance. While the heterogeneity caused evolution of multiple resistant clones with different resistance characteristics, the activation of only a few mechanisms were sufficient in one cell line to achieve resistance.

Thyroid hormone (TH) is present in the systemic circulation and thus should affect all cells similarly in the body. However, tissues have a complex machinery that allows tissue-specific optimization of local TH action that calls for the assessment of TH action in a tissue-specific manner. Here, we report the creation of a TH action indicator (THAI) mouse model to study tissue-specific TH action. The model uses a firefly luciferase reporter readout in the context of an intact transcriptional apparatus and all elements of TH metabolism and transport and signaling. The THAI mouse allows the assessment of the changes of TH signaling in tissue samples or in live animals using bioluminescence, both in hypothyroidism and hyperthyroidism. Beyond pharmacologically manipulated TH levels, the THAI mouse is sufficiently sensitive to detect deiodinase-mediated changes of TH action in the interscapular brown adipose tissue (BAT) that preserves thermal homeostasis during cold stress. The model revealed that in contrast to the cold-induced changes of TH action in the BAT, the TH action in this tissue, at room temperature, is independent of noradrenergic signaling. Our data demonstrate that the THAI mouse can also be used to test TH receptor isoform-specific TH action. Thus, THAI mouse constitutes a unique model to study tissue-specific TH action within a physiological/pathophysiological context and test the performance of thyromimetics. In conclusion, THAI mouse provides an in vivo model to assess a high degree of tissue specificity of TH signaling, allowing alteration of tissue function in health and disease, independently of changes in circulating levels of TH.

The aim of this study was to examine the prevalence of major and minor anomalies according to the increase of NT thickness. Methods: This is a long-term retrospective study in which singleton gestations of euploid fetuses with increased NT were analyzed. NT measurement was performed in the first trimester examination according to the criteria of fetal medicine foundation (FMF) when the fetal crown-rump length (CRL) was 45 to 84 mm. The cases were followed up from 1 to 5 years postpartum to assess the presence of CHD and to point out other anomalies that could be associated with increased NT. Results: The outcome of 133 cases could be analysed out of 198 pregnancies of which in 55 cases some congenital anomalies (minor or major) were revealed up to the 5 years of life (prevalence of 41.4%). The prevalence of CHDs, including the defects of the great vessels, stood out among the others. In the group with NT between 95th and 99th centiles four cases with minor heart problems were identified (11.1%, 4/36). The rate of major cardiac defects proved to be 13.3% (6/45) in the group with NT between 3.5-4.4 mm, and 17.3% (9/52) in the group with NT > 4.5 mm. Among the 35 healthy children with various minor health problems not related to the presence of increased nuchal translucency there were 7 cases with hydrocele. In 3 of them it was associated with unilateral inguinal hernia but in 3 it was isolated and one was part of a complex malformation (The rate of other organ-specific anomalies did not prove to be significant). In the whole study population only thirteen cases (9.8%) ended up in intrauterine death, or arteficial abortion. Conclusion: The prevalence of major cardiac defects as well as other major anomalies increases with fetal nuchal thickness. Since the prevalence of CHD is 100 times higher in the population of fetuses with NT above 4.5 mm, specialist fetal echocardiography should be offered in the second trimester together with other follow-up investigations. Among the children without any major abnormalities, a high number of minor anomalies were revealed during the long-term follow-up. These anomalies do not have significant disadvantage to the quality of life, but some of them necessitates short or long-term medical treatment and this should also be leveled with the future parents. Despite the numerous investigations the exact etiology of increased NT remains unknown. The relatively high prevalence of hydrocele in the newborns in our material raises the question wheather it is related to the presence of NT in the fetal period because of abnormal lymphatic development or alterations in the extracellular matrix. Further long-term follow-up studies could probably contribute to find explanation on the etiology of increased NT in the first trimester. These data can be used when counseling parents of euploid fetuses with increased fetal NT.