Proximal Tubule Transport Function Screening Assay Used to Optimize Function of Kidney Organoids

Abstract

BackgroundThe mechanisms that mediate solute filtration, reabsorption, secretion and excretion in the kidney are well established. However, there are a paucity of data regarding the functional properties of tubular epithelia cells within organoids. The goal of our current work is to define specific function properties of tubular epithelia cells within kidney organoids.Study objectivesTo develop quantitative assays adapted to human kidney organoids to measure proximal tubule function and test whether they can be used to optimize function in developing kidney organoids.MethodsOrganoids were generated from induced pluripotent stem cells (hiPS) derived from human fibroblasts. 6‐carboxyfluorescein (6CF) was used as a fluorescent prototype of organic anion transport (OAT), and was loaded into live organoids (1 mM in OptimemTM, with orbital rotation for 45 min), to measure basolateral uptake using confocal microscopy with tile scanning to test organoid populations and allow quantitation of transport function. To mimic continuous fluid shear stress which occurs in the PT, one population of organoids were placed on an orbital shaker (at 146 or 74 rpm for at least 30 hrs) prior to loading with 6CF. Organoids from the same culture batch at d 14 of differentiation were divided into two treatment groups, static and shaken, and loaded with 6CF, and imaged by a blinded investigator. Quantitation of average maximum intensity of basolateral uptake was done on organoid stacks using LASX software.ResultsOrganoids derived from the LRP2:mtagBFP2 reporter line (available through the Rebuilding a Kidney consortium) confirmed the PT segments within organoids and showed colocalization with 6CF. Concurrent treatment with probenecid (an OAT inhibitor) and 6CF inhibited uptake of 6CF into PT segments. Blinded quantitation of several organoid populations showed increased intensity of 6CF in PT segments subjected to chronic orbital shear. Assays demonstrated basolateral uptake and/or luminal secretion.ConclusionsKidney organoids subject to continuous fluid shear stress have increased basolateral anion transport capacity relative to static cultured controls. A screening assay for PT basolateral uptake, assumed to be OAT mediated, can be used to test and optimize PT function within developing human kidney organoids.Support or Funding InformationSupport from NIDDK: U01 DK107350RBK, 2 P30 DK079307‐07, R01 DK18726‐01.