Angela Skalweit

Induction of translationally controlled tumor protein (TCTP) by transcriptional and post-transcriptional mechanisms

Expression of the human TPT1 gene coding for translationally controlled tumor protein (TCTP) was investigated in Calu‐6 and Cos‐7 cells under the influence of 4β‐phorbol 12‐myristate 13‐acetate (PMA), forskolin, dioxin and the heavy metals copper, nickel and cobalt. Transcriptional and post‐transcriptional aspects of the mechanism were analyzed by TCTP mRNA/protein quantification, luciferase reporter gene assays depending on TPT1 promoter sequences or TCTP mRNA 5′/3′‐UTRs and investigation of the interaction of RNA‐binding proteins with UTRs by UV‐crosslinking. PMA, forskolin, dioxin, cobalt and nickel induced TCTP expression in 24 h in both cell lines about 2.2–3.2‐fold at the mRNA level and 1.6–2.2‐fold at the protein level. The highest induction rate, 4.5–5.0‐fold at the mRNA level and 3.5–4.0‐fold at the protein level, was observed with copper. TPT1 promoter assays showed transcriptional activation by PMA, forskolin and dioxin (2.0–3.1‐fold) and a 7.0–8.0‐fold increase by copper, whereas cobalt and nickel had no effect. Deletion analysis revealed that copper‐dependent transcriptional control was transmitted by a metal‐responsive element residing in the TPT1 promoter. Post‐transcriptional activation of TCTP expression was associated with the action of dioxin, nickel, cobalt (1.8–2.3‐fold) and copper (2.5–3.0‐fold), whereas stimulation of TCTP synthesis by copper was mediated by the TCTP mRNA 3′‐UTR (3.2‐fold) but not by the 5′‐UTR (0.5‐fold). mRNA stabilization was found to mediate these effects of cobalt and nickel. Post‐transcriptional regulation was associated with qualitative and quantitative changes in the binding of specific RNA‐binding proteins to UTRs.

Aldosterone and vasopressin affect α- and γ-ENaC mRNA translation

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of α-, β- and γ-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of γ-ENaC synthesis were studied. γ-ENaC–mRNA 3′-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with γ-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase γ-ENaC 3′-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA–protein interaction for the up-regulation of γ-ENaC synthesis. We document that aldosterone and the V2 receptor agonist dDAVP act on synthesis of α- and γ-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of γ-ENaC–mRNA/HuR complexes document the significance of γ-ENaC–mRNA–3′-UTR/HuR interaction for hormonal control of ENaC synthesis.

Detailing renal hemodynamics and oxygenation in rats by a combined near-infrared spectroscopy and invasive probe approach

We hypothesize that combining quantitative near-infrared spectroscopy (NIRS) with established invasive techniques will enable advanced insights into renal hemodynamics and oxygenation in small animal models. We developed a NIRS technique to monitor absolute values of oxygenated and deoxygenated hemoglobin and of oxygen saturation of hemoglobin within the renal cortex of rats. This NIRS technique was combined with invasive methods to simultaneously record renal tissue oxygen tension and perfusion. The results of test procedures including occlusions of the aorta or the renal vein, hyperoxia, hypoxia, and hypercapnia demonstrated that the combined approach, by providing different but complementary information, enables a more comprehensive characterization of renal hemodynamics and oxygenation.

Aldosterone and vasopressin affect {alpha}- and {gamma}-ENaC mRNA translation.

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of α-, β- and γ-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of γ-ENaC synthesis were studied. γ-ENaC–mRNA 3′-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with γ-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase γ-ENaC 3′-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA–protein interaction for the up-regulation of γ-ENaC synthesis. We document that aldosterone and the V2 receptor agonist dDAVP act on synthesis of α- and γ-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of γ-ENaC–mRNA/HuR complexes document the significance of γ-ENaC–mRNA–3′-UTR/HuR interaction for hormonal control of ENaC synthesis.

Aldosterone and vasopressin affect - and -ENaC mRNA translation

Vasopressin and aldosterone play key roles in the fine adjustment of sodium and water re-absorption in the nephron. The molecular target of this regulation is the epithelial sodium channel (ENaC) consisting of α-, β- and γ-subunits. We investigated mRNA-specific post-transcriptional mechanisms in hormone-dependent expression of ENaC subunits in mouse kidney cortical collecting duct cells. Transcription experiments and polysome gradient analysis demonstrate that both hormones act on transcription and translation. RNA-binding proteins (RBPs) and mRNA sequence motifs involved in translational control of γ-ENaC synthesis were studied. γ-ENaC–mRNA 3′-UTR contains an AU-rich element (ARE), which was shown by RNA affinity chromatography to interact with AU-rich element binding proteins (ARE-BP) like HuR, AUF1 and TTP. Some RBPs co-localized with γ-ENaC mRNA in polysomes in a hormone-dependent manner. Reporter gene co-expression experiments with luciferase γ-ENaC 3′-UTR constructs and ARE-BP expression plasmids demonstrate the importance of RNA–protein interaction for the up-regulation of γ-ENaC synthesis. We document that aldosterone and the V2 receptor agonist dDAVP act on synthesis of α- and γ-ENaC subunits mediated by RBPs as effectors of translation but not by mRNA stabilization. Immunoprecipitation and UV-crosslinking analysis of γ-ENaC–mRNA/HuR complexes document the significance of γ-ENaC–mRNA–3′-UTR/HuR interaction for hormonal control of ENaC synthesis.

Monitoring hemodynamics and oxygenation of the kidney in rats by a combined near-infrared spectroscopy and invasive probe approach

We have developed a hybrid approach to investigate the dynamics of perfusion and oxygenation in the kidney of rats under pathophysiologically relevant conditions. Our approach combines near-infrared spectroscopy to quantify hemoglobin concentration and oxygen saturation in the renal cortex, and an invasive probe method for measuring total renal blood flow by an ultrasonic probe, perfusion by laser-Doppler fluxmetry, and tissue oxygen tension via fluorescence quenching. Hemoglobin concentration and oxygen saturation were determined from experimental data by a Monte Carlo model. The hybrid approach was applied to investigate and compare temporal changes during several types of interventions such as arterial and venous occlusions, as well as hyperoxia, hypoxia and hypercapnia induced by different mixtures of the inspired gas. The approach was also applied to study the effects of the x-ray contrast medium iodixanol on the kidney.