Bioengineered kidney tubules efficiently excrete uremic toxins

The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.


Chemicals and cell culture materials
The uremic toxin p-cresylsulfate (pCS) was synthesized via a modified literature procedure [1]. NaOH was used instead of KOH to afford pCS as its sodium salt.

Cell culture of ciPTEC-OAT1 and -OAT3
In standard cell culture approaches, cells were seeded in well plates or Transwell inserts using a density of 63,000 cells/cm 2 or 110,000 cells/cm 2 for ciPTEC-OAT1, respectively, and 82,000 cells/cm 2 for ciPTEC-OAT3 and were cultured for 24 h at 33°C, 5 % (v/v) CO 2 , to proliferate and subsequently transferred to 37°C, 5 % (v/v) CO 2 for 7 days to mature.
After uptake arrest, intracellular fluorescence was detected at excitation wavelength 485 nm and emission wavelength 535 nm, using a Victor TM X3 multilabel platereader (Perkin-Elmer, Groningen, The Netherlands).

Cell viability assay
Matured ciPTEC-OAT1 were exposed to a concentration range (mM -µM) of indoxyl sulfate or kynurenic acid in serum-free culture medium in the presence or absence of the BCRP and MRP4 efflux pump inhibitors, KO143 (10 µM) and MK571 (5 µM), for 24h at 37°C, 5 % (v/v) CO 2 . After 4 hours of incubation, the intracellular accumulated precipitate was detected by measuring samples at a wavelength of 570 nm from which the background was subtracted, using a Benchmark Plus plate reader (Bio-rad Laboratories, Veenendaal, The Netherlands).

Monolayer polarization and transepithelial barrier function
To investigate the barrier function of matured ciPTEC-OAT1 cultured on HFM, the fibers were mounted on a separated inlet and outlet glass cannula (DMT Trading, Aarhus, Denmark) stabilized by a frame glued to a petri-
Semi-quantification of real-time data was performed using Image J software (version 1.40g). From each single replicate 4 different cellular regions in focus were analyzed.

Methods to figure S1
Transepithelial barrier function of ciPTEC-OAT1 monolayers in 2D