Publikation: Silk fibroin wafers as a potential substrate for culture of RPTEC/TERT1 cells under atmospheric and physiological oxygen tension
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Kidney damage induced by the ingestion of aristolochic acid affects many individuals worldwide. As with all toxins transport and metabolism plays an important role in the mechanism of toxicity. In this work, the transport and metabolism of AAI with respect to the formation of AAI-DNA adducts. Current kidney research pertaining to toxicity of substances is performed in 2D cell culture. However, in the past 15 years, organ-on-a-chip technologies have seen a boom in development. These organ-on-a-chip are connected to microfluidic systems which allow for the manipulation of flow small volumes. Taken together, these two mimic the in vivo setting with respect to organ cell composition and fluid flow rates. Recently the increased trend development of hypoxia champers allowed for the use of physiological oxygen tension to mimic the actual physiological oxygen environment of the organ examined. These three factors, organ-on-a-chip, microfluidic flow and physiological oxygen tension contribute to in vitro experiments which are as close as possible to the in vivo setting. The current lack of organ-on-a-chip models for the determination of nephrotoxicity is disconcerting. The first part of this thesis is focused on the roles of OAT4 and NQO1 in their contribution to AAI toxicity and AAI-DNA adduct formation are demonstrated. Here it is shown that while OAT4 does in fact transport AAI into the cell, it prefers sulfated molecules such as estrone sulfate and that inhibition by probenecid is fairly low compared to OAT1 and OAT3, where probenecid is a more potent inhibitor. This suggests that sulfate conjugates of AAI are more likely to be transported by OAT4. Furthermore, the role NQO1 plays in AAI toxicity was deemed minor as knockdown experiments performed here showed no impact on DNA-adduct formation in RPTEC/TERT1 cells, while a minimal yet significant increase in adduct formation was seen in OAT1-NQO1 over expressing cells. In the second part of this thesis, silk fibroin is shown to be an excellent biomaterial for the production of wafers in cell culture. Furthermore, immortalized cell lines of two key sections of the kidney, the proximal tubule and glomerulus, are shown to attach to the silk fibroin wafer developed. The examined RPTEC/TERT1 and PODO/TERT256 cells show the proper morphology compared to the in vivo setting. SEM analysis of RPTEC/TERT1 cells show a brush border and solitary cilia. Likewise, TEM analysis showed the formation of tight junctions which play an important physiological role observed in the proximal tubule. Gene analyses showed that RPTEC/TERT1 cells express the appropriate transporters for their physiological role. While Western Blot analysis confirmed the presence of all three components of the tight junction: tight junction proteins 1 and 3 and claudin2, a brush border enzyme GGT1 and two important transporters SGLT2 and the Na+/K+ ATPase. Confocal microscopy showed localization of tight junction protein 1 and claudin 2 on the cell membrane. In the case of PODO/TERT256 cells, interdigitation can be observed via SEM. The aim of this thesis was to firstly find a suitable material for wafer production to be used with the microfluidic organ-on-a-chip model in development and characterize the cells to be used. To that end silk fibroin showed all the necessary properties. Secondly, further steps in the unraveling of the mechanism of toxicity of aristolochic acid have been taken. It is our hope that once the microfluidic organ-on-a-chip is functional that this can be taken a step further, combining organ-on-a-chip models of different organs can not only show the organ specific happenings but the influence of other organs on each other.
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MUCIC, Goran, 2024. Silk fibroin wafers as a potential substrate for culture of RPTEC/TERT1 cells under atmospheric and physiological oxygen tension [Dissertation]. Konstanz: Universität KonstanzBibTex
@phdthesis{Mucic2024fibro-72117, title={Silk fibroin wafers as a potential substrate for culture of RPTEC/TERT1 cells under atmospheric and physiological oxygen tension}, year={2024}, author={Mucic, Goran}, address={Konstanz}, school={Universität Konstanz} }
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The first part of this thesis is focused on the roles of OAT4 and NQO1 in their contribution to AAI toxicity and AAI-DNA adduct formation are demonstrated. Here it is shown that while OAT4 does in fact transport AAI into the cell, it prefers sulfated molecules such as estrone sulfate and that inhibition by probenecid is fairly low compared to OAT1 and OAT3, where probenecid is a more potent inhibitor. This suggests that sulfate conjugates of AAI are more likely to be transported by OAT4. Furthermore, the role NQO1 plays in AAI toxicity was deemed minor as knockdown experiments performed here showed no impact on DNA-adduct formation in RPTEC/TERT1 cells, while a minimal yet significant increase in adduct formation was seen in OAT1-NQO1 over expressing cells. In the second part of this thesis, silk fibroin is shown to be an excellent biomaterial for the production of wafers in cell culture. Furthermore, immortalized cell lines of two key sections of the kidney, the proximal tubule and glomerulus, are shown to attach to the silk fibroin wafer developed. The examined RPTEC/TERT1 and PODO/TERT256 cells show the proper morphology compared to the in vivo setting. SEM analysis of RPTEC/TERT1 cells show a brush border and solitary cilia. Likewise, TEM analysis showed the formation of tight junctions which play an important physiological role observed in the proximal tubule. Gene analyses showed that RPTEC/TERT1 cells express the appropriate transporters for their physiological role. While Western Blot analysis confirmed the presence of all three components of the tight junction: tight junction proteins 1 and 3 and claudin2, a brush border enzyme GGT1 and two important transporters SGLT2 and the Na+/K+ ATPase. Confocal microscopy showed localization of tight junction protein 1 and claudin 2 on the cell membrane. In the case of PODO/TERT256 cells, interdigitation can be observed via SEM. The aim of this thesis was to firstly find a suitable material for wafer production to be used with the microfluidic organ-on-a-chip model in development and characterize the cells to be used. To that end silk fibroin showed all the necessary properties. Secondly, further steps in the unraveling of the mechanism of toxicity of aristolochic acid have been taken. 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