Potential and Limits of Kidney Cells for Evaluation of Renal Excretion

Abstract

A large number of therapeutic drugs, herbal components and their metabolites are excreted by the kidneys. Therefore, generally applied models for estimating renal excretion, including freshly isolated rat proximal tubule cells, cultured tubule cells and immortalized kidney cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, were investigated regarding their predictive potential for active renal transport. Cultured proximal tubule cells showed an epithelial cell-like morphology and formed tight monolayers. However, mRNA expression analyses and immunohistochemical studies revealed patterns of tight junction proteins that were notably different from freshly isolated cells and distinct from those in vivo. High levels of mannitol permeation were found in NRK-52E, IHKE-1 and Caki-1 cells, suggesting that they are not suitable for bidirectional transport studies. Cultured cells and freshly isolated cells also differed in proximal tubule markers and transport proteins, indicating that cultured primary cells were in a state of dedifferentiation. Cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1 did not accurately reflect the characteristics of proximal tubules. The expression patterns of marker and transport proteins differed from freshly isolated primary cells. In summary, each of these models has profound disadvantages to consider when adopting them reliable models for the in vivo situation. Thus, they should not be used alone but only in combination.

Keywords: kidney cell lines; marker enzymes; renal excretion; transport proteins.

Figure 1

Proteins relevant for renal drug transport in humans and rats. OAT/Oat: Organic anion transporter; URAT: Urate transporter; OATP/Oatp: Organic anion transporting polypeptide; OC: Organic cation; OCTN/Octn: Organic cation transporter, novel; MATE/Mate: Multidrug and toxin extrusion protein; ABC: ATP-binding cassette; MDR/Mdr: Multidrug resistance; MRP/Mrp: Multidrug resistance-related protein; BCRP/Bcrp: Breast cancer resistance protein.

Figure 2

Phase contrast images of the renal cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, and of cultured PRPTCs. All cells were cultured on chambered glass slides for 5 days under standard conditions. Under these conditions, MDCKII, NRK-52E, IHKE-1 and PRPTC cells formed a confluent monolayer. In contrast, Caki-1 cells did not form a confluent monolayer and cell-free gaps (white triangles) remained even after extended culture periods. Scale bars = 200 µm.

Figure 3

Relative mRNA expression of tubule marker proteins in the renal cell lines NRK-52E (a), IHKE-1 (b) and Caki-1 (c), and in freshly isolated PRPTCs (d). The mRNA expression of each gene was normalized to GAPDH/Gapdh expression. Each bar represents the mean ± SD (n = 3).

Figure 4

Relative mRNA expression of the tubule marker proteins Vil1 (a), Ggt1 (b), Anpep (c), Alpl (d) and Hk1 (e) in freshly isolated and cultured PRPTCs. The mRNA expression of each gene was normalized to Gapdh expression and is shown in reference to the expression level of each gene in freshly isolated PRPTCs, which was set to 1. Each bar represents the mean ± SD (n = 3), ** p < 0.01.

Figure 5

Relative mRNA expression of tight junction proteins in the renal cell lines NRK-52E (a), IHKE-1 (b) and Caki-1 (c), and in freshly isolated PRPTCs (d). The mRNA expression of each gene was normalized to GAPDH/Gapdh expression. Each bar represents the mean ± SD (n = 3).

Figure 6

Relative mRNA expression of the tight junction proteins Tjp1 (a), Ocln (b), Cldn2 (c), Cldn7 (d) and Cldn10 (e) in freshly isolated and cultured PRPTCs. The mRNA expression of each gene was normalized to Gapdh expression and is shown in reference to the expression level of each gene in freshly isolated PRPTCs, which was set to 1. Each bar represents the mean ± SD (n = 3), * p < 0.05, *** p < 0.001, ns = not statistically different (p > 0.05).

Figure 7

Expression of the tight junction proteins tight junction protein 1, occludin, claudin-2 and claudin-7 in the renal cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, and in cultured PRPTCs. All cells were cultured on chambered glass slides for 5 days under standard conditions. For immunofluorescent staining, cells were incubated with primary and Cy3-conjugated secondary antibodies (red) and DAPI (blue). Negative controls incubated only with secondary antibodies showed no staining (not shown). Scale bars = 100 µm.

Figure 8

TEER values (a) and apparent permeability coefficients (P_app) of [¹⁴C]-mannitol (b) in the renal cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1 and in cultured PRPTCs. All cells were cultured on filter inserts for 5 days under standard conditions. For determination of P_app values, cell monolayers were incubated with 600 µM [¹⁴C]-mannitol (12.5 KBq/mL) for 120 min. Each bar represents the mean ± SD of 3 different sets of experiments. A correlation plot of TEER and P_app values is shown in (c).

Figure 9

Relative mRNA expression of transport proteins in the renal cell lines NRK-52E (a), IHKE-1 (b) and Caki-1 (c), and in freshly isolated PRPTCs (d). The mRNA expression of each gene was normalized to GAPDH/Gapdh expression. Each bar represents the mean ± SD (n = 3).

Figure 10

Change in relative mRNA expression of transport proteins in PRPTCs after 5 days in culture. The mRNA expression of each gene was normalized to Gapdh expression and is shown in reference to the expression level of each gene in freshly isolated PRPTCs, which was set to 1. Each bar represents the mean ± SD (n = 3).

Figure 11

mRNA expression levels of transporters in cell lines normalized to GAPDH/Gapdh expression (n = 3). < DL = below detection level, red= low expression intensity, green = high expression intensity.