Fig. 7: Assessment of the filtration capacity of RPTEC tissue showing the effects of HUVECs and flow induced shear stress.

a The generic protocol for quantification of renal reabsorption and excretion using the PToC. Measurements commence on day 14 irrespective of the assay in hand and the tissue layer configuration. The media in both epithelial and vasculature microchannels are circulated to improve diffusion of large-molecule substrates (e.g. BSA-AF488). Transfer rates (reabsorption of albumin/glucose or apparent permeability to Rh123) were estimated by conducting linear regression on time course data. b Relative expression levels (vs. ACTB) of structural genes, CDH6 (K-Cadherin) and EpCAM, and functional genes, namely, ABCB1 (MDR1), AQP1, SLC22A2 (OCT2), SLC5A2 (SGLT2), vs. the level of ACTB gene in the RPTEC-only and RPTEC/HUVEC systems. Four groups are presented, namely, (i), RPTECs on D14 in the culture dishes; (ii), RPTECs of the bilayer under static culture condition; (iii), RPTECs of the bilayer under perfused culture conditions; (iv) Single layer RPTECs under perfused media. Proximity with HUVECs under media perfusion has augmented the expression levels of AQP1, EpCAM, MDR1, OCT2, and SGLT2 (compare groups iii and iv). Independently, perfusion has increased the expression levels of the same genes (with the possible exception of SGLT2) in the co-culture (compare groups ii and iii). n = 5 PCR replicates were analyzed for each gene/sample obtained from one set of experiment. c Transfer rates of the glucose probe, 2NBDG measured in static and perfused culture conditions. Both reabsorption (a → b) and reverse transfer rates (b → a) were quantified. The inhibitory effect of phlorizin (P.) on glucose transport was also examined; n = 3 independent chips. d Apparent permeabilities to Rh123 in both excretion (b → a) and reverse (a → b) directions. Verapamil (V.), a contender for Rh123 extrusion, was applied to attenuate Rh123 efflux and confirm the function of Pgp; N = 3 independent chips. e Fluorescent confocal z-stacked images of the RPTEC tissue layer in bilayer and single layer configurations. A considerably higher amount of BSA was precipitated in the basolateral milieu of RPTECs in the bilayer system indicating a higher intake of the substrate. Some BSA is precipitated in the single layer RPTEC on D17 (white arrowhead). Scale bar is 50 μm. f Transport rates of AF488-conjugated bovine serum albumin (BSA-AF488) in both directions. In this study, we examined the effect of reducing the incubation temperature (to 4 °C) on selective transport of BSA-AF488; minimum N = 6 independent chips. In (b–d, f) two-sample t-test was run between pair of data sets as indicated. Error bars represent standard deviation; N.D., not detected; N/A, not available; N.S., not significant; *, **, ***, ****, for p ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. a → b, apical to basal; b → a, basal to apical.