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

• Lee SJ
• Kwon CH
• Kim YK

• KMID: 2285366
• DOI: http://doi.org/10.4196/kjpp.2009.13.1.15

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.

Keyword

ATP depletion; Membrane transport; cPLA2; Serine/cysteine proteases

MeSH Terms

Adenosine Triphosphate
Amino Acids
Antipain
Arachidonic Acid
Arachidonic Acids
Catalase
Cell Death
Cell Survival
Cytosol
Diminazene
Hydrogen Peroxide
Hydroxyl Radical
Iodoacetic Acid
Membranes
Ouabain
Peptide Hydrolases
Phospholipases A2
Potassium Cyanide
Protease Inhibitors
Reactive Oxygen Species
RNA, Messenger
Trypan Blue
Adenosine Triphosphate
Amino Acids
Antipain
Arachidonic Acid
Arachidonic Acids
Catalase
Diminazene
Hydrogen Peroxide
Hydroxyl Radical
Iodoacetic Acid
Ouabain
Peptide Hydrolases
Phospholipases A2
Potassium Cyanide
Protease Inhibitors
RNA, Messenger
Reactive Oxygen Species
Trypan Blue

Figure

• 

  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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