Functional Evaluation and Nephrotoxicity Assessment of Human Renal Proximal Tubule Cells on a Chip

Abstract

An in vitro human renal proximal tubule model that represents the proper transporter expression and pronounced epithelial polarization is necessary for the accurate prediction of nephrotoxicity. Here, we constructed a high-throughput human renal proximal tubule model based on an integrated biomimetic array chip (iBAC). Primary human renal proximal tubule epithelial cells (hRPTECs) cultured on this microfluidic platform were able to form a tighter barrier, better transporter function and more sensitive nephrotoxicity prediction than those on the static Transwell. Compared with the human immortalized HK2 model, the hRPTECs model on the chip gained improved apical-basolateral polarization, barrier function and transporter expression. Polymyxin B could induce nephrotoxicity not only from the apical of the hRPTECs, but also from the basolateral side on the iBAC. However, other chemotherapeutic agents, such as doxorubicin and sunitinib, only induced nephrotoxicity from the apical surface of the hRPTECs on the iBAC. In summary, our renal proximal tubule model on the chip exhibits improved epithelial polarization and membrane transporter activity, and can be implemented as an effective nephrotoxicity-screening toolkit.

Keywords: epithelial polarization; human renal proximal tubule cells; integrated biomimetic array chip; nephrotoxicity; transporter function.

Figure 1

hRPTECs model on the integrated biomimetic array chip (iBAC). (A) Picture of an integrated biomimetic array chip containing 24 units. (B) Schematic exploded view of a unit on the iBAC. (C) Diagram showing the hRPTECs cultured on the iBAC. (D,E) Immunofluorescence micrographs of the hRPTECs and HK2 cells cultured on the iBAC for 4 days labeled with acetylated tubulin (apical), villin (apical) and Na/K-ATPase (basolateral), (bar, 100 µm). (F) The coverage rate of the acetylated tubulin, villin and Na/K-ATPase on the hRPTECs and HK2 cells cultured on the iBAC on day 4. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

The barrier function of hRPTECs model on the iBAC. (A) TEER values of the hRPTECs cultured on the iBAC and Transwell during 9 days culture. (B) Cells viability of the hRPTECs cultured on the iBAC and Transwell on day 4. (C) TEER values of the hRPTECs and HK2 models cultured on the iBAC for 9 days. (D) The P_app values of FITC-labeled dextran (4 kDa, 40 kDa) through the hRPTECs and HK2 models cultured on the iBAC for 4 days. (E) Immunofluorescence micrographs of hRPTECs and HK2 cells cultured on the iBAC for 4 days labeled with ZO−1 (Green) and nuclear labeled with Dapi (Blue), (bar, 100 µm). * p < 0.05, *** p < 0.001.

Figure 3

Active absorption function of hRPTECs model on the iBAC. (A) The P_app value of FITC-labeled albumin, ASP+, or 5−CF through hRPTECs cultured on the iBAC and Transwell on day 4. (B) The P_app value of FITC-labeled albumin, ASP+, or 5−CF through the hRPTECs and HK2 cells cultured on the iBAC on day 4. The data were obtained from two independent experiments with three replicates. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Efflux protein function of hRPTECs model on the iBAC. (A) Photographs of P−gp substrate of rhodamine 123 and BCRP substrate of DiOC2 accumulated in the hRPTECs and HK2 cells cultured on the iBAC for 4 days (bar, 200 µm). (B) The P_app values of the rhodamine 123 through the hRPTECs and HK2 cells cultured on the iBAC for 4 days. (C) The P_app values of the DiOC2 through the hRPTECs and HK2 cells cultured on the iBAC for 4 days. ** p < 0.01.

Figure 5

Evaluation of cisplatin nephrotoxicity on the iBAC. (A) Viability of the hRPTECs treated with cisplatin for 48 h. (B) Relative release of LDH from the hRPTECs treated with cisplatin for 48 h. (C) Relative viability of the hRPTECs and HK2 cells treated with the cisplatin in the presence and absence of the cimetidine on the iBAC for 24 h. (D) Relative release of LDH from the hRPTECs and HK2 cells treated with cisplatin in the presence and absence of the cimetidine on the iBAC for 24 h. (E) Photographs of the hRPTECs and HK2 cells treated with cisplatin in the presence and absence of the cimetidine on the iBAC, green fluorescence represents living cells and red fluorescence represents dead cells (bar, 100 µm). ** p < 0.01.

Figure 6

Nephrotoxicity evaluation of model drugs on the iBAC. Cell viability of the hRPTECs (A) and the HK2 cells (B) on the iBAC adminstrated with doxorubicin, sunitinib and polymyxin B for 48 h.