Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

Sung Ju Lee; Chae Hwa Kwon; Yong Keun Kim

doi:10.4196/kjpp.2009.13.1.15

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Lee, Kwon, and Kim: Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

Original Article

Korean J Physiol Pharmacol 2009;13(1):15-22.

Published online: 17 February 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.1.15

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

Sung Ju Lee, Chae Hwa Kwon, Yong Keun Kim

Department of Physiology, MRC for Ischemic Tissue Regeneration, College of Medicine, Pusan National University, Busan 602-739, Korea

Corresponding to: Yong Keun Kim, Department of Physiology, College of Medicine, Pusan National University, Ami-dong, Suh-gu, Busan 602-739, Korea. (Tel) 82-51-240-7733, (FAX) 82-51-246-6001, (E-mail) kim430@pusan.ac.kr

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na⁺-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na⁺-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na⁺-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na⁺ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na⁺-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A₂ (cPLA₂). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA₂ specific inhibitor AACOCF₃. ATP depletion-induced alterations in Na⁺-dependent phosphate uptake and cell viability were prevented by AACOCF₃. Inhibition of Na⁺-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA₂ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

Keywords: ATP depletion, Membrane transport, cPLA₂, Serine/cysteine proteases

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Fig. 1.

Effect of ATP depletion on Na⁺-dependent phosphate uptake (A) and cell viability (B) in primary cultured renal proximal tubular cells. Cells were exposed to medium with 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) or without (Control) for 0~3 hr at 37°C. Na⁺-dependent phosphate uptake and cell viability were measured as described in ‘Materials and Methods'. Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with control. (C) Effect of ATP depletion on Na⁺-dependent phosphate uptake in primary cultured renal proximal tubular cells which were treated with ouabain. Cells were exposed to medium with 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) or without (Control) in the presence or absence of 2 mM ouabain for 120 min at 37°C. Na⁺-dependent phosphate uptake was measured as described in ‘Materials and Methods'. Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with control; ^#p<0.05 compared with ouabain alone.

Fig. 2.

(A) Time course of reactive oxygen species (ROS) generation in primary cultured renal proximal tubular cells during exposure to ATP depletion. Cells were exposed to 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) for various times and ROS generation was measured. Shown is the net increase of DCF fluorescence (arbitrary units) calculated by subtracting the values for the control cells from the corresponding values for KCN/IAA-treated cells. Data are mean±SEM of three independent experiments performed in duplicate. ∗p<0.05 compared with zero (0) time. (B-C) Effect of radical scavengers on inhibition of phosphate uptake (B) and cell death (C) induced by ATP depletion. Cells were exposed to KCN/IAA in the presence or absence of 500 units/ml catalase (Cat), 30 mM dimethylthiourea (DMTU), or 30 mM thiourea (TU) for 3 hr at 37°C. Na⁺-dependent phosphate uptake and cell viability were measured as described in ‘Materials and Methods'. Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with control; ^#p<0.05 compared with KCN/IAA alone.

Fig. 3.

Effects of amino acids on inhibition of phosphate uptake (A) and cell death induced by ATP depletion, (B) in primary cultured renal proximal tubular cells. Cells were exposed to 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) in the presence or absence of 5 mM glycine (Gly) or 5 mM alanine (Ala) for 3 hr at 37°C. Na⁺-dependent phosphate uptake and cell viability were measured as described in ‘Materials and Methods'. Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with control; ^#p<0.05 compared with KCN/IAA alone.

Fig. 4.

Effects of ATP depletion on arachidonic acid release in primary cultured renal proximal tubular cells. Cells were prelabeled with [³H]arachidonic acid for 20 hr, washed, and exposed to 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) for various times (A) or for 3 hr in the presence or absence of 20 μM AACOCF₃ (B). Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with zero time (A) and control (B); ^#p<0.05 compared with KCN/IAA alone. (C) RT-PCR analysis of cPLA₂ mRNA in cells subjected to ATP depletion. Cells were exposed to KCN/IAA for 0.2, 0.5, and 1 hr, and mRNA levels of were measured as described in ‘Materials and Methods'.

Fig. 5.

Effects of phospholipase A2 inhibitor on inhibition of Na⁺-dependent phosphate uptake (A) and cell death (B), induced by ATP depletion, in primary cultured renal proximal tubular cells. Cells were exposed to 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) in the presence or absence of 20 μM AACOCF₃ for 3 hr at 37°C. Na⁺-dependent phosphate uptake and cell viability were measured as described in ‘Materials and Methods'. Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with control; ^#p<0.05 compared with KCN/IAA alone.

Fig. 6.

Effects of serine/cysteine protease inhibitors on inhibition of phosphate uptake (A) and cell death (B), induced by ATP depletion, in primary cultured renal proximal tubular cells. Cells were exposed to 2.5 mM potassium cyanide/0.1 mM iodoacetic acid (KCN/IAA) in the presence or absence of 50 μM antipain (ANT) or 100 μM leupetin (LEU) for 3 hr at 37°C. Na -dependent phosphate uptake and cell viability were measured as described in ‘Materials and Methods'. Data are mean±SEM of four independent experiments performed in duplicate. ∗p<0.05 compared with control; ^#p<0.05 compared with KCN/IAA alone.

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