Bioengineered kidney tubules efficiently excrete uremic toxins

Abstract

The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.

Figure 1. Concentration-dependent inhibition of OAT1- and OAT3-mediated fluorescein uptake by anionic uremic toxins.

(a–h) A concentration range of eight uremic toxins were exposed to matured ciPTEC-OAT1 (•) and -OAT3 (o) in the presence of 1 μM fluorescein, a known OAT model substrate. The intracellular fluorescent signal was detected by measuring samples at excitation wavelength 485 nm and emission wavelength 535 nm. Blank data were subtracted and data were normalized to control (absence of uremic toxin). Nonlinear regression analysis was performed using Graphpad Prism 5.02. Data are shown as mean ± S.E.M. of three independent experiments performed, at least, in duplicate.

Figure 2. Handling of indoxyl sulfate and kynurenic acid by OAT1, BCRP and MRP4 in flat monolayers.

In matured ciPTEC-OAT1, a concentration-dependent uptake of (a) IS and (b) KA (white) was shown using LC-MS/MS analysis of the uremic toxins. In the presence of probenecid (P, grey), the uptake of both toxins was attenuated. Moreover, the IS uptake was inhibited in the presence of KA (100 μM; black), and vice versa. The role of BCRP and MRP4 in the efflux of IS and KA was shown using an MTT assay. The experiment was performed in the absence (•) or presence (◦) of efflux pump inhibitors (KO143 (10 μM) and MK571 (5 μM)). Nonlinear regression analysis was performed using Graphpad Prism 5.02. Data are shown as mean ± S.E.M. of three independent experiments performed in triplicate. *p < 0.05, ***p < 0.001 using one-way ANOVA analysis followed by Dunnett’s multiple comparison test.

Figure 3. Bioengineered renal tubules show transepithelial barrier function and polarized characteristics.

FITC-inulin leakage was measured in matured ciPTEC-OAT1 seeded on coated HFM. (a) Schematic presentation of the experimental set-up in the absence (upper panel) or presence (lower panel) of cells. (b) Representative eal-time images of the two different conditions are shown; unseeded (square) and seeded (circle) HFM. In the absence of cells, a bright green fluorescent signal was detected at the apical membrane, whereas in the presence of cells the signal was clearly attenuated. Representative real-time images of the two different conditions were shown. (c) Semi-quantification of FITC-inulin diffusion in the absence (square) and the presence of ciPTEC-OAT1 (circle) on HFM. Seeded HFM showed significantly less FITC-inulin leakage demonstrating monolayer tightness. From each single replicate 4 different regions in focus were analyzed. (d) The expression of ZO-1 (green) was demonstrated in a homogenous cell monolayer cultured on HFM. Scale bar: 10 μm. (e–g) The OAT1, BCRP and MRP4 mRNA expression levels were investigated in matured ciPTEC-OAT1 cultured in well plates and on HFM. Gene expression levels were normalized to expression levels of the reference gene GADPH (h) and were expressed as fold increase when cultured on HFM (black) compared to matured cells in well plates (white). Data are shown as mean ± S.E.M. of three independent experiments performed, at least, in duplicate. ***p < 0.001, using an unpaired t test.

Figure 4. OAT1-mediated fluorescein uptake in bioengineered renal tubules.

Fluorescein was used as a model substrate in an advanced fiber perfusion system. (a) Schematic presentation of the experimental set-up: I. Fluorescein perfusion, II. Fluorescein perfusion in the presence of efflux pump inhibitors (EP; (5 μM)), III and IV. Fluorescein perfusion in the presence of efflux pump inhibitors and indoxyl sulfate (III; 100 μM) or kynurenic acid (IV; 30 μM), V. Fluorescein perfusion in the presence of probenecid (P; 100 μM). (b) Representative real-time images of the five different conditions. Scale bare: 10 μm. (c) Semi-quantification of fluorescein uptake data in the absence or presence of the efflux pumps inhibitors solely, in combination with IS and KA, and in the presence of probenecid. From each single replicate 4 different regions in focus were analyzed. Data were fitted according to one-site total binding saturation curve using non-linear regression analysis. (d) Vmax values from all conditions were calculated from the corrected AFU data according to michaelis-menten kinetics using non-linear regression analysis. For statistical analysis, the Vmax values obtained from condition EP and P where compared to only fluorescein uptake (F). Conditions IS + EP and KA + EP were compared to EP. Data are shown as mean ± S.E.M. of three independent experiments performed in duplicate. ***p < 0.001, using one-way ANOVA followed by Dunnett’s multiple comparison test.

Figure 5. Transepithelial clearance of indoxyl sulfate and kynurenic acid in the presence of human serum albumin, and albumin-FITC handling in bioengineered renal tubules.

(a–d) Transepithelial clearance of indoxyl sulfate and kynurenic acid. Schematic presentation of the experimental set up of IS and KA transepithelial transport in the absence (a) or presence (b) of HSA is reported. Quantification of IS (c) and KA (d) clearance in the absence (−) or presence (+) of HSA. The protein bound fraction of IS was 73 ± 5% and 63 ± 8% of KA was bound to albumin. Data are shown as mean ± S.E.M. of three independent experiments performed in duplicate. *p < 0.05, **p < 0.01 compared to IS in the absence of HSA, ^###p < 0.001 compared to IS in the presence of HSA. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. (e–g) Endocytosis-mediated albumin uptake in bioengineered renal tubules. Cellular BSA-FITC uptake (green) in renal tubules (nuclei: blue) after 30 min exposure from the (e) basolateral compartment at 37 °C (f ) apical compartment at 37 °C and (g) apical compartment at 4 °C. Active uptake was detected solely when BSA-FITC uptake was performed at 37 °C, as indicated by the red arrows. (h) Collagen IV expression (green) in renal tubules showed a highly similar pattern as BSA-FITC uptake as shown in b. Scale bare: 10 μm.