Optimization of cell culture protocols of HK‐2 and RPTE cells as cellular models for renal proximal tubule
Thesis (Dissertationen, Habilitationen)
 
ID 4627971
Author Boo, Michelle
Author at UniBasel Meyer zu Schwabedissen, Henriette
Year 2014
Title Optimization of cell culture protocols of HK‐2 and RPTE cells as cellular models for renal proximal tubule
Type of Thesis Masterarbeit
Start of thesis 13.01.2014
End of thesis 06.06.2014
Name of University University of Basel
Name of Faculty Philosophisch-Naturwissenschaftliche Fakultät
Supervisor(s) / Fachvertreter/in Meyer zu Schwabedissen, Henriette
Abstract

The kidneys play a pivotal role in the body’s clearance of water‐soluble substances. The proximal tubule
plays a central role as its main function is the reabsorption of the majority of the primary filtrate and
secretion of harmful substances. These processes are mediated by transmembrane transport proteins.
Transporters identified in the renal proximal tubule are amongst others organic anion transporters
(OAT1, SLC22A6; OAT2, SLC22A7; OAT3, SLC22A8; OAT4, SLC22A11; and OAT10, SLC22A13), organic
cation transporters (OCT1, SLC22A1; OCT2, SLC22A2; and OCT3, SLC22A3), organic cation transporter,
novel, type (OCTN1, SLC22A4, OCTN2, SLC22A5), urate anion exchanger 1 (URAT1, SLC22A12), multidrug
and extrusion protein (MATE1, SLC47A1), P‐glycoprotein (P‐gp, MDR1, ABCB1), and multidrug resistance‐
associated protein (MRP2, ABCC2; MRP4, ABCC4).
The aim of this master thesis was to establish an optimized protocol for renal proximal epithelial tubule
cells (RPTEC) and human kidney 2 (HK‐2) cells as a cellular model for the human renal proximal tubule
with focus on drug transport. The idea originates in the well‐established cell model intestine Caco‐2 for
human intestine. We wanted to determine the quality of RPTE and HK‐2 cells as a good cellular model for
transport assays for the proximal tubule. Additionally we compared HK‐2 cells and RPTEC to Caki‐1, Caki‐
2, RCCEW, and HEK‐293 partial used as renal models. We conducted quantitative Real‐time PCR, Western
blot, and immunofluorescence to compare the expression. In addition, Standard Operation Protocols
(SOPs) were composed for TEER value measurements and transport assays and experiments were carried
out accordingly.
TaqMan® analysis showed no significant differences in expression of transporter mRNA between RPTEC
cultivated on PET‐membrane (Transwell® inserts) and tissue culture treated plastic surface. We found no
or marginal expressions of organic anion transporters and OCT2 in all investigated samples. OCT3, OCTN1
and 2, P‐gp, and MRP2 showed moderate expression in RPTEC on both surfaces. OCT1 and MATE1
expression was rather low compared to the kidney and we measured a high expression of MRP4 especially
in RPTEC. Comparing the expression in cells cultivated on transwell or plastic surface showed that
especially the expression of OCT1 and OCT3 was significantly higher in HK‐2 cells grown on normal cell
culture material. Western Blot analysis and immunofluorescence assay verified results gained from
TaqMan® analysis. Determination of cell layer density as preliminary experiments for transport assays
were determined by microscopy and TEER value. We observed that both RPTEC and HK‐2 cells reached a
monolayer on day 7 followed by formation of leaky multilayers. Therefore, transport assays were
conducted on day 7 showing no active transport of the model substrates E1S, MPP+ and PAH through
RPTEC and HK‐2 cells. We assumed that flux is mediated through the cell monolayer as a result of
diffusion through the cell layer or the involvement of transport proteins that were not investigated in this
study.
To conclude we can state that results of our investigations indicated that human kidney 2 cells are not an
ideal cell line used as a model for the proximal tubule. We further hypothesize that the most eligible
candidate for further studies are primary renal proximal tubule epithelial cells further research is
necessary with respect to increase confluence and to avoid the loss of transporter expression during
cultivation.

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