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Frauscher, B.
Establishment of a new murine CKD-MBD model and the role of autophagy in uremic vascular media calcification
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2018. pp. [OPEN ACCESS]
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Authors Med Uni Graz:
Frauscher Bianca
Advisor:
Eller Kathrin
Eller Philipp
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Abstract:
Part A Introduction: Chronic kidney disease is characterized by long-term morbidity and mortality. We developed a murine model of CKD and evaluated the kidney-phenotype along with the systemic changes in endocrine and metabolic parameters. Methods: DBA/2NCrl mice are susceptible to ectopic renal calcification when exposed to increased oral phosphate loads. In order to cause renal damage, these mice were fed standard chow diet or high phosphate diet for four days or 7 days with subsequent return to SCD for 84 days. Serum and urine samples,and samples for histology and qPCR were taken from kidney and bone. Additionally, transthoracic echocardiography was performed. Results: CKD mice developed uremic kidney disease with marked calcification of the kidneys and a marked reduction in measured glomerular filtration rate as determined by FITC-inulin clearance. Echocardiographic evaluations revealed a trend of an increase of left ventricle mass, whereas left-ventricular function was not affected. The abdominal aorta in HPD fed mice showed signs of media calcification. Histomorphometrical analysis revealed reduced bone volume/tissue volume as well as a decreased mineral apposition rate in HPD mice when compared with SCD mice. Additionally, serum Pth levels were increased in HPD mice. The described phenotype was more pronounced in mice from the HPD7 group than in mice from HPD4 group. Conclusion: We here present a model of chronic kidney disease-mineral and bone disorder which reflects important features of the human equivalent. Our model is characterized by media calcification, secondary hyperparathyroidism and low-turnover bone disease. This animal model may serve a useful tool for the investigation of pathogenic features of CKD-MBD without the need for surgical methods. Part B Introduction: Chronic kidney disease and diabetes mellitus are associated with extensive media calcification. The calcification of the tunica media is not only driven by systemic factors such as hyperphosphatemia and hyperglycemia, but is also dependent on vascular smooth muscle cells. The aim of our study was to investigate the role of autophagy in the pathogenesis of uremic media calcification. Methods: DBA/2NCrl mice were fed with high phosphate diet (HPD) for 5 or 12 days. DBA2/N mice on standard chow diet (SCD) were used as controls. To influence autophagy mice were treated intraperitoneally with 0.5 mg rapamycin per kg body weight starting on day -3. For our in vitro experiments, mouse muscular smooth muscle cells were used. The medium was supplemented with ascorbin acid and beta-glycerolphosphate to induce calcification. To influence autophagy, cells were treated with rapamycin or 3-methyladenin or bafilomycin. Aorta samples as well as cells were analyzed using qPCR, Western Blot, histology. Results: DBA2/NCrl mice on HPD developed a severe vascular calcification and also the cells showed calcification in presence of calcifying medium. qPCR of mouse aortas revealed a significantly higher gene expression of autophagy related genes such as igfbp3 or trp53in on day 5 and 12 on HPD. The protein levels of the autophagy markers LC3-II/I were also significantly increased in aortas of mice on HPD. In line protein levels of p62 were downregulated. By using histological staining, we showed that autophagy is located in the tunica media of the aortas and that it increases with the time on HPD. Rapamycin treatment further increased autophagy in vivo and in vitro. Rapamycin treatment resulted in a significant decrease of vascular calcification in vivo and in vitro. Furthermore, Rapamycin-treated mice survived longer compared to vehicle-treated controls. Conclusion: These findings indicate that autophagy plays an important role in the development of uremic media calcification. Furthermore, induction of autophagy by Rapamycin protects cells and mice from uremic media calcification.

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