Investigating flow-related effects of Chronic Kidney Disease on renal drug toxicity in a human-derived proximal tubule microphysiological system
Typ
Examensarbete för masterexamen
Program
Biotechnology (MPBIO), MSc
Publicerad
2020
Författare
Magnusson, Otto
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
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.
Beskrivning
Ämne/nyckelord
chronic kidney disease , 3D in vitro model , drug safety , drug toxicity and transport , primary human proximal tubule cells , microfluidics , microphysiological system