Eelco D. Keuning

Translational Control of the Xenopus laevisConnexin-41 5′-Untranslated Region by Three Upstream Open Reading Frames

The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5′-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5′-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stageXenopus embryos. The translational efficiency of the wild-type Cx41 5′-UTR was only 2% compared with that of the β-globin 5′-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5′-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5′-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency inXenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

Novel Role for Mast Cells in Omental Tissue Remodeling and Cell Recruitment in Experimental Peritoneal Dialysis

Because of its dynamic structure, the omentum plays a key role in the immunity of the peritoneal cavity by orchestrating peritoneal cell recruitment. Because mast cells accumulate in the omentum upon experimental peritoneal dialysis (PD) and may produce angiogenic/profibrotic factors, it was hypothesized that mast cells mediate omental tissue remodeling during PD. Daily treatment with conventional PD fluid (PDF) for 5 wk resulted in a strong omental remodeling response, characterized by an approximately 10-fold increase in mast cell density (P < 0.01), an approximately 20-fold increase in vessel density (P < 0.02), an approximately 20-fold increase in the number of milky spots (P < 0.01), and a four-fold increase in submesothelial matrix thickness (P < 0.0003) in wild-type rats. In contrast, all PDF-induced omental changes were significantly reduced in mast cell-deficient Ws/Ws rats or in wild-type rats that were treated orally with a mast cell stabilizer cromoglycate. A time-course experiment showed mast cell accumulation immediately before the formation of blood vessels and milky spots. Functionally, PDF evoked a peritoneal cell influx, which was significantly reduced in Ws/Ws rats (P < 0.04) and in wild-type rats that were treated with cromoglycate (P < 0.03). Cromoglycate treatment also completely prevented PDF-induced omental adhesions to the catheter tip (P = 0.0002). Mesothelial damage, angiogenesis, and fibrosis of mesentery and parietal peritoneum as well as glucose absorption rate and ultrafiltration capacity proved to be mast cell independent. Data strongly support the hypothesis that mast cells mediate PDF-induced omental tissue remodeling and, subsequently, peritoneal cell influx and adhesion formation, providing therapeutic possibilities of modulating omental function.

Translational control of the Xenopus laevis Connexin41 5′UTR by three upstream open reading frames

The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5′-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5′-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stageXenopus embryos. The translational efficiency of the wild-type Cx41 5′-UTR was only 2% compared with that of the β-globin 5′-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5′-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5′-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency inXenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.

A Novel Rat Model of Vitamin D Deficiency: Safe and Rapid Induction of Vitamin D and Calcitriol Deficiency without Hyperparathyroidism

Vitamin D deficiency is associated with a range of clinical disorders. To study the mechanisms involved and improve treatments, animal models are tremendously useful. Current vitamin D deficient rat models have important practical limitations, including time requirements when using, exclusively, a vitamin D deficient diet. More importantly, induction of hypovitaminosis D causes significant fluctuations in parathyroid hormone (PTH) and mineral levels, complicating the interpretation of study results. To overcome these shortcomings, we report the successful induction of vitamin D deficiency within three weeks, with stable serum PTH and minerals levels, in Wistar rats. We incorporated two additional manoeuvres compared to a conventional diet. Firstly, the vitamin D depleted diet is calcium (Ca) enriched, to attenuate the development of secondary hyperparathyroidism. Secondly, six intraperitoneal injections of paricalcitol during the first two weeks are given to induce the rapid degradation of circulating vitamin D metabolites. After three weeks, serum 25-hydroxyvitamin D3 (25D) and 1,25-dihydroxyvitamin D3 (1,25D) levels had dropped below detection limits, with unchanged serum PTH, Ca, and phosphate (P) levels. Therefore, this model provides a useful tool to examine the sole effect of hypovitaminosis D, in a wide range of research settings, without confounding changes in PTH, Ca, and P.

Protective Effects of Paricalcitol on Peritoneal Remodeling during Peritoneal Dialysis

Peritoneal dialysis (PD) is associated with structural and functional alterations of the peritoneal membrane, consisting of fibrosis, angiogenesis, and loss of ultrafiltration capacity. Vitamin D receptor activation (VDRA) plays an important role in mineral metabolism and inflammation, but also antiangiogenic and antifibrotic properties have been reported. Therefore, the effects of active vitamin D treatment on peritoneal function and remodeling were investigated. Rats were either kept naïve to PDF exposure or daily exposed to 10 mL PDF and were treated for five or seven weeks with oral paricalcitol or vehicle control. Non-PDF-exposed rats showed no peritoneal changes upon paricalcitol treatment. Paricalcitol reduced endogenous calcitriol but did not affect mineral homeostasis. However, upon PDF exposure, loss of ultrafiltration capacity ensued which was fully rescued by paricalcitol treatment. Furthermore, PD-induced ECM thickening was significantly reduced and omental PD-induced angiogenesis was less pronounced upon paricalcitol treatment. No effect of paricalcitol treatment on total amount of peritoneal cells, peritoneal leukocyte composition, and epithelial to mesenchymal transition (EMT) was observed. Our data indicates that oral VDRA reduces tissue remodeling during chronic experimental PD and prevents loss of ultrafiltration capacity. Therefore, VDRA is potentially relevant in the prevention of treatment technique failure in PD patients.