Establishment of renal proximal tubule cell lines derived from the kidney of p53 knockout mice

Abstract

The human cell line HK-2 is most commonly used as a model of renal proximal tubular epithelial cells (PTECs) for various studies despite the absence or low expression of transporters characteristic of parental PTECs. In an effort to develop reliable PTEC models, several human cell lines have been newly established over the last decade. In contrast, reliable mouse PTEC models are still unavailable. In this study, we established immortalized renal cortex tubule cell lines derived from p53 knockout mice and evaluated various PTEC characteristics toward the development of reliable mouse PTEC models. Here, we focus on MuRTE61, one of 13 newly established clonal cell lines. Albumin uptake in MuRTE61 cells was verified by incubation with fluorescent dye-labeled albumin. RT-PCR confirmed the expression of efflux transporter genes characteristic of PTECs in the MuRTE61 cells. MuRTE61 cells exhibited high sensitivity to treatment with cisplatin, a nephrotoxic agent, accompanied by upregulated expression of the uptake transporter Slc22a2 gene. Furthermore, MuRTE61 cells consistently formed spheroids with a lumen and apicobasal polarity in three-dimensional Matrigel cultures. Apical brush border microvilli were also observed in the spheroids by transmission electron microscopy. These data validate that MuRTE61 can be characterized as a reliable mouse PTEC line. In future, detailed analysis of reliable mouse and human PTEC lines will provide an accurate extrapolation of results of experiments using mice and humans, and may help resolve apparent inconsistencies between mouse and human nephrotoxicity.

Keywords: 3D cell culture; Cell line; Cell spheroids; Matrigel; Renal proximal tubular epithelial cells.

Fig. 1

Renal proximal tubular epithelial cell type identification of MuRTE61 cells. a Relief phase contrast images of MuRTE cell lines at low density on culture plastic. Cells exhibited a rounded and slightly elongated morphology. b RT-PCR for the renal proximal tubular epithelial marker gene, Ggt1, in MuRTE cell lines. c Relief phase contrast image of the dome-like structure formed in the confluent MuRTE61 culture. d Immunofluorescent staining for epithelial cell adhesion molecules and renal tubular epithelial markers in MuRTE61 cells. e Immunofluorescent staining for megalin, uromodulin and Pax2 in MuRTE15 cells, primary cultured PTECs and kidney. Immunofluorescent staining for megalin showed specific staining of renal proximal tubules in kidney and punctate cytoplasmic staining in MuRTE61 cells and primary cultured PTECs. The thick ascending limb of loop of Henle marker, uromodulin, was not detected in MuRTE61 cells. A transcription factor Pax2 was not detected in renal cortex (data not shown) but nuclear staining of Pax2 was seen in MuRTE61 cells and primary cultured PTECs. f Relief phase contrast images of MuRTE61 before and after stimulation with 20 µg/mL BSA or 5 ng/mL TGF-β1. TGF-β1-stimulated MuRTE61s exhibited a spindle-shaped morphology. g Western blot analysis of α-SMA in cell lysates from MuRTE61 cells before and after stimulation with 20 µg/mL BSA or 5 ng/mL TGF-β1. h RT-PCR for Ggt1 in MuRTE61 cells before and after stimulation with 20 µg/mL BSA or 5 ng/mL TGF-β1. Ggt1 expression was clearly decreased in the BSA-stimulated MuRTE61 cells, or lost in TGF-β1-stimulated MuRTE61 cells. Scale bars, 100 µm (a, e) or 50 µm (c–e)

Fig. 2

MuRTE61 cells show gene expression and functional properties related to renal proximal tubular epithelial cells. a Fluorescence images of MuRTE61 cells after incubation with fluorescent dye-labeled albumin for 0, 15, or 60 min. b RT-PCR for renal proximal tubular transporter genes in the MuRTE cell lines. Expression data for Gapdh used as an internal control is identical to that in Fig. 2b. c, d Relative expression level of Slc47a1, Abcc2, Abcc4 and Abcb1a in the MuRTE cell lines (MuRTE61 and MuRTE15) and the primary cultured PTECs. Asterisk, p < 0.001. NS, not significant. e Graphical representation of the viability of MuRTE61 cells after incubation with cisplatin (0, 5, or 10 µM) for 24 h. In the plots, the upper left quadrant represents live cells and the upper right quadrant represents dead cells. f RT-PCR for Slc22a2 in MuRTE61 cells after incubation with cisplatin (0, 1, 5, or 10 µM) for 24 h. Scale bars, 50 µm (a)

Fig. 3

Three-dimensional cultured MuRTE61 cells. a Relief phase contrast and immunofluorescent images of MuRTE61 and HK-2 cells grown in Matrigel. b Percentage of spheroids with a lumen per cell grown in Matrigel; n = 5 wells. MuRTE15 and HK-2 cells did not form spheroids. Error bar represents standard deviation. ND, not detectable. c Confocal immunofluorescent images of MuRTE61-derived spheroids grown in Matrigel. d Confocal immunofluorescent images of reconstructed z-stack 3D structures of MuRTE61-derived spheroids grown in Matrigel. e Transmission electron microscopy images of MuRTE61-derived spheroids grown in Matrigel. Nu, nucleus; Mv, microvilli; TJ, tight junction; M, mitochondria; P, peroxisome; arrows, endocytic structures. Scale bars, 20 µm (a, c) or 2 µm (e)