Investigating flow-related effects of Chronic Kidney Disease on renal drug toxicity in a human-derived proximal tubule microphysiological system

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Examensarbete för masterexamen
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Publicerad
2020
Författare
Magnusson, Otto
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Sammanfattning
The kidney proximal tubule is responsible for the active expulsion of drugs from the blood to the urine and is, therefore, of great importance when evaluating drug safety. Chronic kidney disease (CKD) is a major cause of reduced renal filtration function, reducing the fluid forces experienced by human renal proximal tubule eptihelial cells (HRPTEC). Developing a physiologically representative in vitro model for drug toxicity studies in both healthy and diseased HRPTEC would therefore be highly valuable. As CKD is a major cause of reduced renal function and may affect the function of proximal tubule cells, this thesis focused on the investigation of how flow-related aspects of chronic kidney disease affects drug transport and toxicity in HRPTEC cultured in 3D in the Nortis microphysiological system. qPCR was used to evaluate differential gene expression of drug transporters, proximal tubule and stress markers in response to flow exposure and nutrient deprivation in 3D and 2D cultured HRPTEC, alongside LDH in the supernatant and Live/Dead stain to evaluate cell viability. Phenotype by gene expression remained unchanged in the 3D proximal tubule model when exposed to a 5 week 2-fold increase to fluid shear stress (FSS) from 0.9 to 1.7 dyne/cm2, as well as in 2D cultures exposed to orbital flow (1.9 or 5.3 dyne/cm2) for 1 week compared to static cultures. 2D cultured cells also displayed no change to transport (P-gp) function in response to orbital flow when evaluated with Calcein-AM. Interestingly, gene expression of several drug transporters was increased in 3D HRPTEC cultures (1, 5 weeks) compared to 2D, including regained expression of OAT1 and OAT3 and upregulation of OCT2 (SLC22A2, 3.6 ± 1.1 fold [5 weeks]), MATE1 (SLC47A1, 41.3 ± 3.4 fold, 33.2 ± 13.2 fold), MATE2-K (SLC47A2, 71.9 ± 8.9 fold , 99.6 ± 61.7 fold) and the endocytosis receptor Megalin (LRP2, 46.7 ± 13.1 fold, 67.5 ± 34.1 fold). Moreover, this phenotype remained stable from 1 to 5 weeks in culture. The antibiotic polymyxin B (50 μM, 48 h) showing reduced viability to 83 ± 7.0% as evaluated by Live/Dead stain, demonstrating sensitivity to know nephrotoxicants in 3D cultured HRPTEC. Phenotype by gene expression was unaffected by nutrient and flow deprivation for 3 days. Although the aim of replicating flow related aspects of chronic kidney disease in the 3D proximal tubule model used in this study was not achieved, it was established that the 3D model displayed stable phenotype for up to five weeks with regained and significantly increased expression of drug transporter and endocytosis receptor genes when compared to 2D cultured cells. This suggests that the 3D proximal tubule model used in this study is a more physiologically relevant model for future long-term drug toxicity studies.
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chronic kidney disease, 3D in vitro model, drug safety, drug toxicity and transport, primary human proximal tubule cells, microfluidics, microphysiological system
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