Human proximal tubule cells form functional microtissues

Abstract

The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.

Impaired autophagy bridges lysosomal storage disease and epithelial dysfunction in the kidney

“…As the intracellular positioning of lysosomes coordinates autophagy in response to nutrient availability 20 , we tested whether the perinuclear clustering of lysosomes reflects changes in autophagy. We used mPTCs, because this primary culture system provides a particularly suited model to decipher the molecular mechanisms underpinning the endolysosome disorders within epithelial cells 21 , 22 . Autophagy was assessed by quantifying the conversion of the non-lipidated form of LC3-I to the lipidated, autophagosome-associated form LC3-II, and the numbers of LC3 vesicles 23 in mPTCs cultured in nutrient-rich media (hereafter referred to as “fed”) or in nutrient-deprived conditions (hereafter referred to as “starved”).…”

Overcoming Endocytosis Deficiency by Cubosome Nanocarriers

“…Subsequently, the absorbance at 450nm was determined using an Infinite M200 Pro TECAN spectrophotometer (Tecan, Männedorf, Switzerland). Equal numbers of untreated cells were used as controls 20 .…”

“…Investigations using the wellestablished Clcn5 knock-out mouse model of Dent disease have demonstrated that the disease is caused by a generalized trafficking defect, with loss of apical megalin and cubilin receptors, leading to the massive urinary loss of albumin and LMW solutes 14,16 . The uptake of albumin and LMW tracers is routinely investigated in various cellular systems modelizing PT cells, characterized by variable levels of differentiation and endocytic uptake capacity 20 .…”

Co‐cultures of renal progenitors and endothelial cells on kidney decellularized matrices replicate the renal tubular environment in vitro

“…Indeed, changes on the composition and thickness of the BM are linked with pathological conditions, such as inflammatory processes, renal fibrosis and epithelial-to-mesenchymal transitions. 5 Major efforts have been accomplished in this area, namely, the creation of functional kidney microtissues, 6 microphysiological fluidic kidney systems 7,8 or even strategies to mimic the organic cation transport. 9 Although these systems were developed with the main aim of preclinical screening, they are far from replicating the in vivo contact with the BM and the cellular crosstalk between endothelial and epithelial cells, both crucial factors on the filtration process of the kidney.…”