Three-dimensional cell models for renal replacement therapy and drug screening

Abstract

The kidneys are organs with a central role in homeostasis. Their principal function is the excretion of the organic and inorganic wastes of metabolism and the reabsorption of valuable nutrients. These functions are mediated by the proximal tubule epithelial cells (PTEC) containing segment, via the concerted action of different transporters (e.g. organic anion transporter 1, OAT1). Aging, diabetes and hypertension lead to kidney malfunction. This can progress to chronic kidney disease (CKD), in which the kidneys no longer function well enough to meet the daily needs and, as a consequence, uremic toxins (UTs) accumulate in the patients’ blood. The first aim of this thesis was to develop and characterize the functionality of an in vitro bioartificial kidney device (BAK) for the removal of uremic toxins. We developed the bioartificial kidney tubule as an in vitro platform that was validated by studying the protein-bound UTs, indoxyl sulfate (IS) and kynurenic acid (KA), removal. This novel tool, named bioengineered kidney tubules, is the combination of polyethersulfone hollow fiber membranes (PES-HFM) with conditionally immortalized proximal tubules epithelial cells (ciPTEC) overexpressing OAT1. Additionally, we characterized the effect of HSA on IS transepithelial transport using the bioengineered tubules, with attention given to the modified form of HSA as present in CKD patients. Also, we characterized the transport kinetics of IS in free and bound fractions to either healthy- or CKD-HSA. CKD-HSA, with its low binding and high affinity for IS, promoted a lower turnover of the free IS fraction, accompanied with less IS secretion. Furthermore, bioengineered kidney tubules demonstrated active secretion of IS from plasma of healthy donors and of CKD patients, demonstrating that, despite unfavorable binding kinetics, plasma can still be cleared from UTs in patients. We describe the first BAK upscaling attempt. In this respect, a three-PES-HFM bioreactor with a total surface area of 4 cm2 was built and characterized. Subsequently, ciPTEC were cultured in the upscaled devices and organic cation transporter 2 (OCT2) function was demonstrated. The second aim of this thesis was to use natural scaffolds instead of synthetic ones for the development of a new 3D kidney model. Decellularized organs constitute a promising field within regenerative medicine. These newly generated scaffolds can provide an alternative to synthetic ones, as the native extracellular matrix expected to guide tissue development in a physiologically relevant manner. Recellularizing these native scaffolds with human cell sources can better recapitulate the in vivo situation. The novel platform was applied in nephrotoxicity testing by culturing ciPTEC-OAT1 on decellularized native kidney scaffolds from rats and compared to standard 2D ciPTEC-OAT1 cultures. To validate the platform, three different nephrotoxicants were studied: cisplatin, tenofovir and cyclosporin A. The 3D model demonstrated increased sensitivity to cisplatin and tenofovir toxicity, related to improved physiology of the platform. Altogether, this work shows two ways of developing 3D renal cell models to study kidney physiology, pharmacology and pathology, and providing novel solutions within the regenerative nephrology field