Functionality of a co-culture kidney model: testing the renal barrier with [18F]fluorodeoxyglucose

Introduction:

When administering radiopharmaceuticals, the kidney is one of the dose-limiting organs [1]. Micro-physiological systems (MPS) for the in vitro evaluation of new potential radiopharmaceutical candidates should therefore be equipped with a functional kidney equivalent [2]. Hence the proximal tubule is responsible for most toxic interactions we generated a model of this part using a co-culture of human renal proximal tubule epithelial cells (RPTEC) and human umbilical vein endothelial cells (HUVEC) [3]. The functionality of the renal barrier was characterized by measuring transepithelial/transendothelial electrical resistance (TEER) and permeation of [18F]fluorodeoxyglucose (FDG).
Methods:
Investigations were performed in 24-well plates equipped with transwell inserts with a permeable membrane (pore size 0.4 µm). Viability and density of the cellular bilayer were measured using calcein staining. Apical and basolateral sites of the membrane were populated with RPTEC and HUVEC, respectively (each site 8x104 cells). Ionic conductance in the cell layers was measured using TEER. Basolateral-apical passage of [18F]FDG (produced in-house; 4 MBq/mL, equivalent to about 5 nmol/mL) through the cellular bilayer was measured using a gamma counter and expressed as percent passage.
Results:
In the proximal renal tubule model, calcein staining showed that the cellular bilayer reached confluence after 3 to 4 days. At this time point, the barrier showed a higher electrical resistance of 533 ± 47 Ω*cm2 across the cellular bilayer compared to 306 ± 26 Ω*cm2 across transwell blanks. Consistent with these results, [18F]FDG showed only 4 ± 1 % basolateral-apical passage through the intact cellular bilayer compared to 13.5 ± 1.5 % across transwell blanks. Morphological, electrical, and functional readouts therefore confirmed the integrity of the renal barrier in this model.
Conclusions:
This study demonstrates that measurement of [18F]FDG passage provides a functional test of the renal barrier in a proximal renal tubule model of the kidney. While the TEER value reflects the ionic conductance of the paracellular pathway in the epithelial layer, the flux of nonelectrolyte tracers such as [18F]FDG indicates the paracellular water flux as well as the pore size of the tight junctions [4]. In the next steps, apical-basolateral [18F]FDG transport will be investigated to functionally characterize reabsorption via sodium/glucose cotransporter 2 (SGLT2), which is mainly found in the proximal part of the renal tubule [5]. After characterization of the proximal renal tubule model in the static system, this kidney equivalent will be integrated into the fluidics system of a multicompartment MPS chip that also contains a liver equivalent and a tumor spheroid for use in radiopharmacological assays. With kidney and liver organoids first evidence for uptake, metabolism and excretion of a radioligand will be investigated.
 
Acknowledgements:
The authors like to express great appreciation for the financial support from the German Federal Ministry of Education and Research (BMBF) (Funding number 161L0275A/B).
References:
[1] Klaus et al., Radiat Oncol 2021, 16, 43. [2] Sihver et al., Curr Dir Biomed Eng 2022, 8, 532. [3] Schmieder et al., Curr Dir Biomed Eng 2019, 5, 1. [4] Srinivasan et al., J Lab Autom 2015, 20, 107. [5] Weber et al., Kidney Int 2016, 90, 627.