Concerted EP2 and EP4 receptor signaling stimulates autocrine prostaglandin E(2) activation in human podocytes

Glomerular hyperfiltration is an important mechanism in the development of albuminuria. During hyperfiltration, podocytes are exposed to increased fluid flow shear stress (FFSS) in Bowman's space. Elevated Prostaglandin E2 (PGE(2)) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE2 autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE(2), 15-keto-PGE(2), and 13,14-dihydro-15-keto-PGE(2) levels. hPC were treated with PGE(2) with or without separate or combined blockade of prostaglandin E receptors (EP), EP2, and EP4. Furthermore, the effect of FFSS on COX2, PTGER2, and PTGER4 expression in hPC was quantified. In hPC, stimulation with PGE(2) led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE(2). PTGER4 was downregulated after PGE(2) stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE(2) and 15-keto-PGE(2) levels were observed in isolated glomeruli obtained from a well-established rat model with glomerular hyperfiltration, the Munich Wistar Frömter rat. COX2 and PTGER2 were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE(2) pathway in hPC linked to concerted EP2 and EP4 signaling

Characterization of the c-specific promoter of the gene encoding human endothelin-converting enzyme-1 (ECE-1)

AbstractHuman ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform. A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained. Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (−142/−240 and −240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5′-RACE revealed multiple transcriptional start sites located at about −110, −140 and −350 bp. Site-directed mutagenesis demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity. Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities

Key Learning Outcomes for Clinical Pharmacology and Therapeutics Education in Europe: A Modified Delphi Study.

Harmonizing clinical pharmacology and therapeutics (CPT) education in Europe is necessary to ensure that the prescribing competency of future doctors is of a uniform high standard. As there are currently no uniform requirements, our aim was to achieve consensus on key learning outcomes for undergraduate CPT education in Europe. We used a modified Delphi method consisting of three questionnaire rounds and a panel meeting. A total of 129 experts from 27 European countries were asked to rate 307 learning outcomes. In all, 92 experts (71%) completed all three questionnaire rounds, and 33 experts (26%) attended the meeting. 232 learning outcomes from the original list, 15 newly suggested and 5 rephrased outcomes were included. These 252 learning outcomes should be included in undergraduate CPT curricula to ensure that European graduates are able to prescribe safely and effectively. We provide a blueprint of a European core curriculum describing when and how the learning outcomes might be acquired

Polymorphisms in the MAPT gene and risk of Parkinson disease in a Greek population: A case-control study.

The extended tau haplotype (H1) that covers the entire human microtubule-associated protein tau (MAPT) gene has been implicated in Parkinson’s disease (PD). We studied the frequency of H1 haplotype and H1 subhaplotypes of the tau gene in PD in Greek population. As two previous conflicting results were reported, we addressed this question again. We studied 122 PD patients and 123 age matched control subjects. We found significantly higher frequency of H1H1 genotype compared with H1H2 and H2H2 among PD patients (OR for H1H1 vs. H1H2 and H2H2: 1.566; 95% CI, 1.137- 2.157; p = 0.006). Whereas when adjusted for sex, the strong association with H1H1 genotype remained only for males. We further analyzed two single nucleotide polymorphisms frequencies in patients and subjects carrying H1H1 genotype which demonstrated no significant association with PD. In conclusion, these results show that Greek population is consistent with the hypothesis that MAPT alters risk in PD. However, the previously supported association of H1 sub-haplotypes with PD could not be confirmed in our study

Role of the H1 haplotype of microtubule-associated protein tau (MAPT) gene in Greek patients with Parkinson's disease

The extended tau haplotype (H1) that covers the entire human microtubule-associated protein tau (MAPT) gene has been implicated in Parkinson's disease (PD). Nevertheless, controversial results, such as two studies in Greek populations with opposite effects, have been reported. Therefore, we set out to determine whether the H1 haplotype and additional single nucleotide polymorphisms (SNPs) included in H1 are associated with PD in a sample of Greek patients. METHODS: We analysed MAPT haplotypes in cohorts of 122 patients and 123 controls of Greek origin, respectively. SNP genotyping was performed with Taqman assays and genotyping results were confirmed by sequencing. RESULTS: The presence of the H1 haplotype was significantly associated with PD (odds ratio for H1H1 vs. H1H2 and H2H2: 1.566; 95% CI: 1.137-2.157; P = 0.006) and remained so after adjustment for sex. Further analysis of H1 sub-haplotypes with three single nucleotide polymorphisms (rs242562, rs2435207 and rs3785883) demonstrated no significant association with PD. CONCLUSION: Our data support the overall genetic role of MAPT and the H1 haplotype for PD susceptibility in Greek patients. However, the previously supported association of H1 sub-haplotypes with PD could not be confirmed in our study