Dual Effects of Bilirubin on the Proliferation of Rat Renal NRK52E Cells and ITS Association with Gap Junctions

Abstract

Objective: The effect of bilirubin on renal pathophysiology is controversial. This study aimed to observe the effects of bilirubin on the proliferation of normal rat renal tubular epithelial cell line (NRK52E) and its potential interplay with gap junction function.

Methods: Cultured NRK52E cells, seeded respectively at high- or low- densities, were treated with varying concentrations of bilirubin for 24 hours. Cell injury was assessed by measuring cell viability and proliferation, and gap junction function was assessed by Parachute dye-coupling assay. Connexin 43 protein was assessed by Western blotting.

Results: At doses from 17.1 to 513μmol/L, bilirubin dose-dependently enhanced cell viability and colony-formation rates when cells were seeded at either high- or low- densities (all p<0.05 vs. solvent group) accompanied with enhanced intercellular fluorescence transmission and increased Cx43 protein expression in high-density cells. However, the above effects of BR were gradually reversed when its concentration increased from 684 to 1026μmol/L. In high-density cells, gap junction inhibitor 12-O-tetradecanoylphorbol 13-acetate attenuated bilirubin-induced enhancement of colony-formation and fluorescence transmission. However, in the presence of high concentration bilirubin (1026μmol/L), activation of gap junction with retinoid acid decreased colony-formation rates.

Conclusion: Bilirubin can confer biphasic effects on renal NRK52E cell proliferation potentially by differentially affecting gap junction functions.

Keywords: Bilirubin; NRK52E cells; cell proliferation; connexin; gap junction; kidney injury.

FIGURE 1

The survival rates of NRK52E cells after challenge with BR at varying concentrations for 24 h. Data are presented as mean ± SD of three independent experiments. • p < 0.05 vs. solvent group, ▪ p < 0.05 vs. BR 513μmol/L.

FIGURE 2

BR-induced a dose-dependently biphasic effects on colony-formation rates of (A) low- and (B) high-density NRK52E cells. (C) Comparison between low- and high- density groups. Data are presented as mean ± SD of three independent experiments. • p < 0.05 vs. solvent group, ▪ p < 0.05 vs. BR 513μmol/L, ▴ p < 0.05 compare high-density cells to low-density cells.

FIGURE 3

BR influenced fluorescence transmission of high-density cells through gap junctions. Fig 3A–3I were the representative images from the parachute dye-coupling assay in which cells were exposed to BR at 0, 17.1, 85.5, 171, 342, 513, 684, 855, and 1026μmol/L respectively (obtained by fluorescence microscope, magnification ×200). Dye spread through GJ was assessed by the average number of communicating cells (number of receiver cells containing calcein from each donor cell). Bar graph 3J was quantified for the communicating cells after BR exposure at different concentrations mentioned above. Data are presented as means ± SD of three independent experiments. • p < 0.05 vs. solvent group, ▪ p < 0.05 vs. BR 513μmol/L.

FIGURE 4

Effects of GJ inhibition or activation on BR-induced alteration of colony-formation rates. (A and B) Cells at low- and high-density were pretreated with the GJ inhibitor TPA before BR 513μmol/L. (C and D) Cells at low- and high-density were pretreated with the GJ agonist RA before BR 1026μmol/L. ▪ p < 0.05 vs. BR 513μmol/L. Δ p < 0.05 vs. BR 1026μmol/L. The data are presented as mean ± SD of three independent experiments.

FIGURE 5

GJ function altered in response to varying BR concentrations following GJ inhibitor or GJ agonist for high-density cells. Fig 3A–3G were the representative images from the parachute dye-coupling assay in which cells were exposed to Solvent group, TPA group, BR 513μmol/L group, (TPA+BR 513μmol/L) group, RA group, BR 1026μmol/L group, and (RA+BR1026μmol/L) group respectively (obtained by fluorescence microscope, magnification ×200). Dye spread through GJ was assessed by the average number of communicating cells (number of receiver cells containing calcein from each donor cell). Bar graph 3H was quantified for the communicating cells treated with mentioned above. Data are presented as means ± SD of three independent experiments. • p < 0.05 vs. Solvent group, ▪ p < 0.05 vs. BR 513μmol/L, Δ p < 0.05 vs. BR 1026μmol/L.

FIGURE 6

BR affected the expression of Connexin43 in a dose-dependent biphasic manner for NRK52E cells. Cells were either solvent group or treated with BR at 171μmol/L, 513μmol/L, 684μmol/L, or 1026μmol/L and Cx43 protein expression were detected by Western blot. •< 0.05 vs. Solvent group.