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In vivo Analyse zellulärer Dynamik der renalen Tubulogenese in der Entwicklung und zystischer Nierenerkrankung

Antragsteller Dr. Soeren Lienkamp
Fachliche Zuordnung Nephrologie
Förderung Förderung von 2014 bis 2020
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 249641671
 
Erstellungsjahr 2020

Zusammenfassung der Projektergebnisse

The main objective of this project was to gain insight into the molecular mechanisms of renal tubule morphogenesis and inherited renal disease, such as cystic kidney disease and nephronophthisis, using two model systems: Xenopus and mice. An initial expression screen for acto-myosin-associated proteins enriched in the kidney uncovered genes previously linked to kidney disease and renal specific extracellular matrix components, but did not find obvious modulators of myosin mediated tubule morphogenesis specific to the developing nephron. We invested a substantial amount of effort into adopting state of the art transgenesis and genome editing technology and established CRISPR based genome editing routinely in Xenopus tropicalis. We focused our attention on a protein (Nme3) that strongly interacts with previously investigated nephronophthisis proteins (Nek8, Anks6). Depletion of Nme3 leads to morphogenetic defects of tubule extension. Nme3 was shown to supply dinucleotides in conditions of DNA-replication stress. Because we find that Nme3 localizes to the base of the cilium and its depletion elicits ciliopathy typical phenotypes in Xenopus, our data supports the notion that DNA-damage response is involved in the pathogenesis of nephronophthisis by implicating novel key protein players. The routine employment of genome editing techniques enabled us to investigate molecular mechanisms of related renal anomalies and ciliopathies and facilitated new and fruitful collaboration with human geneticists. Using ex vivo cultures of embryonic mouse kidneys, we found that genetic labeling of the nephron progenitor population can be used to observe all stages of embryonic nephron development, including pretubular aggregate, comma- and s-shape body formation and extension of the early tubules. We optimized live cell imaging conditions over extended periods using 3D printed custom sample holders and automated image acquisition. Efforts to apply these elaborate imaging techniques to a model of genetic renal disease did not result in conclusive findings. In addition, we investigated transcriptional regulatory mechanisms of renal cell type specification. We found a number of transcription factors that would ectopically induce renal tubules in ectodermal explants of Xenopus and identified four factors that in combination can reprogram human and mouse fibroblasts to renal tubule-like cells without undergoing dedifferentiation into a pluripotent or stem-cell like state. This work resulted in a publication in Nature Cell Biology and a number of follow up articles investigating the use of reprogrammed cells for disease modelling, nephrotoxicity testing, and metabolic profiling. We used reprogrammed cells to determine transcriptional differences of patient specific mutations in HNF4A that cause renal Fanconi syndrome. In summary, we gained substantial insights into the molecular mechanisms of tubulogenesis and ciliary related renal disease, propelled our research into new technological fields, including genome editing, direct reprogramming and machine learning.

Projektbezogene Publikationen (Auswahl)

 
 

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