Decreased iPLA₂γ expression induces lipid peroxidation and cell death and sensitizes cells to oxidant-induced apoptosis

Abstract

Our previous studies showed that renal proximal tubular cells (RPTC) express Ca²⁺-independent phospholipase A₂γ (iPLA₂γ) in endoplasmic reticulum (ER) and mitochondria and that iPLA₂γ prevents and/or repairs lipid peroxidation induced by oxidative stress. Our present studies determined the importance of iPLA₂γ in mitochondrial and cell function using an iPLA₂γ-specific small hairpin ribonucleic acid (shRNA) adenovirus. iPLA₂γ expression and activity were decreased in the ER by 24 h and in the mitochondria by 48 h compared with scrambled shRNA adenovirus-treated cells. Lipid peroxidation was elevated by 2-fold at 24 h and remained elevated through 72 h in cells with decreased iPLA₂γ. Using electrospray ionization-mass spectrometry, primarily phosphatidylcholines and phosphatidylethanolamines were increased in iPLA₂γ-shRNA-treated cells. At 48 h after exposure to the iPLA₂γ shRNA, uncoupled oxygen consumption was inhibited by 25% and apoptosis was observed at 72 and 96 h. RPTC with decreased iPLA₂γ expression underwent apoptosis when exposed to a nonlethal concentration of the oxidant tert-butyl hydroperoxide (TBHP). Exposure of control cells to a nonlethal concentration of TBHP induced iPLA₂γ expression in RPTC. These results suggest that iPLA₂γ is required for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial function and viability, providing further evidence for a cytoprotective role for iPLA₂γ from oxidative stress.

Phospholipase A₂ (PLA₂) hydrolyzes the sn-2 ester bond of glycerophospholipids. By this mechanism, PLA₂ generates free fatty acids and lysophospholipids, both of which have biological activity. The family of PLA₂ is large, with >20 isoforms that were separated historically into classes based on Ca²⁺ requirement or localization [cytosolic, secretory, or Ca²⁺-independent PLA₂ (iPLA₂)] and recently into 15 groups based on amino acids utilized for catalysis and other structural and functional similarities (groups I–XV) (1). The iPLA₂ family consists of at least seven members, with different isoforms being localized to different subcellular compartments, each displaying PLA₂ activity in the absence of Ca²⁺ (1).

PLA₂ is reported to perform diverse functions, including generation of signaling molecules [e.g., arachidonic acid (2), membrane remodeling (3), and protection or repair of membranes from oxidative damage (4–6)]. In support of a protective role for PLA₂, PLA₂ has been hypothesized to participate in the removal of oxidized fatty acids from biological membranes to maintain membrane integrity (7). Fatty acids at the sn-2 position of glycerophospholipids are preferred targets of reactive oxygen species due to their relatively high degree of unsaturation. van Kuijk et al. (7) proposed that when lipid peroxidation occurs, the oxidized sn-2 fatty acid of phospholipids becomes less hydrophobic and moves closer to the hydrophilic head of the phospholipid. The movement of this fatty acid increases the space between the polar head groups and exposes the sn-2 ester bond to PLA₂. This phenomenon would result in an apparent preference of certain PLA₂s for oxidized phospholipids. The cleavage of oxidized fatty acids releases a lysophospholipid, which can be reacylated with a nonoxidized fatty acid by an acyltransferase and reinserted into the membrane, preserving membrane integrity (8). The oxidized fatty acids can be detoxified through classical glutathione peroxidases, but only after they are released by the action of PLA₂ (9, 10). Secretory PLA₂ and cytosolic PLA₂ have been used in vitro to test the hypothesis that PLA₂ can act as repair enzymes in synthetic membranes or isolated microsomes (11, 12), but the native PLA₂ enzyme(s) responsible for the repair of oxidized phospholipids in intact cellular systems has not been identified.

Two members of the iPLA₂ family, iPLA₂β and iPLA₂γ (groups VIA and VIB, respectively), are reported to protect cells or organelles during oxidative stress (4–6). Overexpression of iPLA₂β in insulinoma cells and CHO cells results in mitochondrial localization of iPLA₂β and protects against oxidant-induced apoptosis (6). In primary cultures of rabbit renal proximal tubule cells (RPTCs), inhibition of iPLA₂γ with racemic bromoenol lactone (BEL) potentiated oxidant-induced lipid peroxidation and necrotic cell death (4). Furthermore, in isolated rabbit renal cortex mitochondria (RCM), pretreatment with the selective iPLA₂γ inhibitor R-BEL (the R-enantiomer of BEL) accelerated Fe²⁺-induced lipid peroxidation and mitochondrial swelling (5). In summary, several studies have reported that iPLA₂ enzymes are important mediators of cell and organelle protection from oxidative stress and lipid peroxidation.

The goal of these experiments was to determine the importance of iPLA₂γ in basal mitochondrial and cellular function. Since previous results from our laboratory suggested that the iPLA₂γ isoform protects renal cells and mitochondria from oxidative damage (4, 5), we hypothesized that decreasing iPLA₂γ expression in RPTC would increase lipid peroxidation, impair mitochondrial function, and decrease viability. To test this hypothesis, RPTCs were infected with adenovirus expressing small hairpin ribonucleic acid (shRNA) to decrease the expression of iPLA₂γ. We also examined the sensitivity of these cells to the model oxidant tert-butyl hydroperoxide (TBHP).

EXPERIMENTAL PROCEDURES

Materials

Annexin V-FITC was purchased from Biovision (Mountain View, CA). All other reagents were from Sigma (St. Louis, MO) or reported previously (13, 14).

Development of the iPLA₂γ shRNA adenovirus

An shRNA insert corresponding to the iPLA₂γ small interfering RNA (sense strand, 5′-gcaagcaacuguauuucuutt-3′; Ambion, Austin, TX) was designed using the Insert Design Tool for pSilencer Vectors on the Ambion website (www.ambion.com). This oligonucleotide was used to generate an adenoviral iPLA₂γ shRNA vector using the pSilencer Adeno-CMV system (Ambion) according to the manufacturer's protocol. The crude viral lysate was then transferred to the Viral Vector Core Facility at the Medical University of South Carolina for amplification, purification, and titer determination. The negative control adenovirus, Ad-CMV-RNAi, was obtained from Vector Biolabs (Philadelphia, PA).

Isolation of rabbit RPTCs, culture conditions, and adenovirus treatment

Rabbit RPTCs were isolated using the iron oxide perfusion method and grown in 35 mm tissue culture dishes under improved conditions as described previously (15). Confluent monolayers were exposed to either the iPLA₂γ shRNA adenovirus or a negative control scramble shRNA adenovirus at a concentration of 1 × 10⁷ plaque-forming units per 35 mm dish for 24 h. After 24 h, the culture medium was changed to normal culture medium and the cells were cultured routinely and processed as described below.

Immunoblot analysis

Immunoblot analysis of the expression of 88 kDa iPLA₂γ in RPTC mitochondrial and endoplasmic reticulum (ER) fractions was performed using an anti-iPLA₂γ antibody as described previously by our laboratory (5). Heat shock protein 60 was used as a mitochondrial marker and loading control for mitochondrial samples.

Measurement of iPLA₂ activity

PLA₂ activity was determined under linear reaction conditions in RPTC mitochondrial and ER fractions as described previously (5). Activity was measured using synthetic (16:0, [³H]20:4) plasmenylcholine (100 μM) in the presence of 4 mM EGTA.

Measurement of apoptosis

Annexin V and propidium iodide (PI) staining were determined using flow cytometry as described previously by our laboratory (16). RPTCs were washed, fixed, and stained with 4′,6-diamidino-2-phenylindole (DAPI) as described previously (16). At least 200 nuclei were counted for each treatment group in each experiment.

Measurement of oxygen consumption

RPTC monolayers were washed and gently detached from the dishes with a rubber policeman, suspended in 37°C culture medium, and transferred to the oxygen consumption (QO₂) measurement chamber. QO₂ was measured polarographically using a Clark-type electrode as described previously (14, 17). For measurement of uncoupled QO₂, 1 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) was used.

Measurement of cellular ATP levels

Intracellular ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche) as described previously (18).

Measurement of lipid peroxidation

Lipid peroxidation in RPTCs was monitored as the production of malondialdehyde using thiobarbituric acid-reactive substances (TBARS) as described previously (4).

Measurement of cellular phospholipids by electrospray ionization-mass spectrometry

At the end of the experimental period, culture media were removed from the dishes, the monolayers were rinsed with 1 ml of phosphate-buffered saline, and the cells were scraped into 1 ml of methanol-water (2.5:1). RPTCs from four dishes were pooled for each experiment. Whole cells were used to identify lipid changes because of the likelihood of lipid mixing during mitochondrial isolation. The cell suspension was then transferred to a glass vial, bubbled extensively with nitrogen, and stored at −80°C until extraction.

Lipids were isolated using a modified method of Bligh and Dyer (19) as described by Zhang, Peterson, and Cummings (20). Following extraction, the samples were evaporated under a stream of argon gas to near dryness. The lipids were then reconstituted in 95 μl of chloroform-methanol (2:1) and 5 μl of 1 mg/ml deuterated palmitate (m/z 259.5) in chloroform-methanol (2:1), as an internal standard for ESI-MS. Total lipid phosphorus was measured in each sample using malachite green (21), and the samples were diluted to 5 nmol/ml total lipid phosphorus in chloroform-methanol (2:1). ESI-MS was performed as described previously (20) using a Trap XCT ion-trap mass spectrometer (Agilent Technologies, Santa Clara, CA) with a nitrogen-drying gas flow rate of 3 l/min at 300°C and a nebulizer pressure of 10 p.s.i. The scanning range was from 100 to 2,200 m/z on 5 μl of sample scanned for 2.5 min with a mobile phase of acetonitrile-methanol-water (2:3:1) in 0.1% ammonium formate. Phospholipids were analyzed in both positive and negative ion detection modes.

Statistical analysis

Each experiment was performed using RPTCs from a single rabbit and was repeated at least four times. The appropriate ANOVA was performed for each data set using SigmaStat statistical software. Individual means were compared using Fisher's protected least significant difference test with P < 0.05 considered indicative of a statistically significant difference among mean values.

To analyze the mass spectrometry data, the m/z peaks were sorted by m/z and aligned to a master list compiled from the Lipid Maps Consortium (www.lipidmaps.org). Values from Lipid Maps were increased by one for positive mode and decreased by one in negative mode. The negative ion mode spectra were first normalized to the deuterated palmitate peak and then, using the central limit theorem, transformed as described previously (22, 23). Data from the positive mode were first normalized to the mean values of all of the samples and then transformed using the central limit theorem transformation method. After data normalization, the data were analyzed using ANOVA, and when significant effects were found (P < 0.05), individual means were compared using Duncan's multiple range test (α = 0.05). The only comparisons that were considered meaningful were when iPLA₂γ shRNA (24 or 48 h) treatments were significantly different from uninfected control and scrambled shRNA (24 or 48 h).

RESULTS

Adenoviral shRNA-mediated knock-down of RPTC iPLA₂γ

Infection of RPTCs with the iPLA₂γ shRNA adenovirus resulted in ∼80% of cells expressing the transgene, as determined by β-galactosidase staining (data not shown). Mitochondrial iPLA₂γ protein expression and activity were reduced by ∼40% at 48 h after iPLA₂γ shRNA adenovirus treatment compared with RPTCs exposed to an equivalent amount of scramble shRNA adenovirus (Fig. 1A, C). Mitochondrial iPLA₂γ protein and activity levels continued to decrease to ∼40% of scramble shRNA-treated cell values at 72 and 96 h. No significant changes in expression or activity of iPLA₂γ in noninfected control cells and scramble shRNA adenovirus-treated cells were observed (data not shown). In contrast, protein levels of iPLA₂γ in the ER fraction of RPTC decreased after 24 h (∼65% of scramble shRNA) and decreased to ∼40% of scramble shRNA at 48, 72, and 96 h after exposure to iPLA₂γ shRNA (Fig. 1B). ER-iPLA₂γ activity was also decreased (∼25%) at 24 h after exposure to the iPLA₂γ shRNA adenovirus, and activity decreased to ∼35% of scramble shRNA at 72 and 96 h (Fig. 1D). In summary, adenovirus-mediated knock-down of iPLA₂γ protein levels and activity occurred in the ER fraction prior to occurring in the mitochondria, but it resulted in similar decreases (60–70%) in both organelles by 72 h.

Fig. 1.

Small hairpin ribonucleic acid (shRNA)-mediated decreases in mitochondrial and endoplasmic reticulum (ER) Ca²⁺-independent phospholipase A₂ (iPLA₂γ) in rabbit renal proximal tubular cells (RPTCs). Confluent RPTCs were exposed to either adenoviral iPLA₂γ or scramble shRNA and cultured normally for 4 days. iPLA₂γ expression was measured by immunoblot analysis (A, B) and iPLA₂ activity was measured as described in Experimental Procedures (C, D) in mitochondrial (A, C) and ER (B, D) fractions at 24, 48, 72, and 96 h after exposure. The amount of iPLA₂γ expression and activity in cells exposed to iPLA₂γ shRNA is expressed as means + SEM, percentage of iPLA₂γ expression and activity in cells exposed to scramble shRNA for the same amount of time. Insets show representative immunoblots. Means with different superscripts are significantly different from each other (P < 0.05, n = 4–5).

Knock-down of RPTC iPLA₂γ causes increases in RPTC phospholipids

We determined the effect of iPLA₂γ knock-down on RPTC phospholipids. A total of 267 m/z values were examined for the phospholipids in each ion mode. Of these, one positive and nine negative m/z species were putatively identified as being specifically increased by decreasing the expression of iPLA₂γ (Table 1). The single positive phospholipid that increased was m/z 865 [42:5 (sn-1 + sn-2) phosphatidylcholine (PC), 36:1 phosphatidylinositol (PI), or 43:6 phosphatidylglycerol (PG)] after 24 h, which returned to control levels by 48 h. For the negative phospholipids, four m/z species were increased by 24 h {578 [24:0 phosphatidylethanolamine (PE)], 730 [32:2 phosphatidylserine (PS)], 795 [31:1 PI or 38:5 PG], and 985 [40:8 PI phosphate]}, while five m/z species were elevated by 48 h [693 (30:0 PG), 725 (26:0 PI or 33:5 PG), 844 (40:1 PS, 40:0 PC, or 44:7 PE), 860 (41:0 PS, 42:7 PS, or 42:6 PC), and 873 (37:3 PI, 43:1 PG, 44:8 PG)]. Specific identification of these phospholipid species will require tandem MSⁿ to identify exactly which they are. In summary, ablation of 60% of cellular iPLA₂γ resulted in the increase of several specific cellular phospholipids of the phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol classes.

TABLE 1.

Phospholipids increased by knock-down of iPLA₂γ in RPTCs

Positive mode phospholipids	[M+H]
	865
Control	−0.52 ± 0.41a
shRNA, 24 h	1.26 ± 0.38b
shRNA, 48 h	−0.17 ± 0.52a
Scrambled, 24 h	−0.17 ± 0.24a
Scrambled, 48 h	−0.55 ± 0.13a
Negative mode phospholipids	[M-H]
	578	693	725	730	795	844	860	873	985
Control	−0.27 ± 0.13a	−0.37 ± 0.07a	−0.23 ± 0.13a	−0.31 ± 0.07a	−0.36 ± 0.07a	−0.23 ± 0.14a	−0.29 ± 0.19a	−0.38 ± 0.03a	−0.21 ± 0.06a
shRNA, 24 h	1.31 ± 1.01b	−0.35 ± 0.13a	−0.25 ± 0.13a	1.18 ± 0.92b	1.16 ± 0.94b	−0.30 ± 0.16a	−0.49 ± 0.32a	0.09 ± 0.29ab	1.32 ± 1.09b
shRNA, 48 h	−0.33 ± 0.07a	1.27 ± 0.75b	1.18 ± 0.81b	−0.27 ± 0.15a	−0.38 ± 0.12a	1.15 ± 0.80b	1.21 ± 0.61b	1.05 ± 0.84b	−0.32 ± 0.00a
Scrambled, 24 h	−0.40 ± 0.00a	−0.31 ± 0.20a	−0.39 ± 0.12a	−0.46 ± 0.08a	−0.46 ± 0.11a	−0.24 ± 0.19a	−0.02 ± 0.23a	−0.39 ± 0.07a	−0.20 ± 0.11a
Scrambled, 48 h	−0.05 ± 0.30a	−0.36 ± 0.07a	−0.53 ± 0.03a	−0.45 ± 0.13a	−0.50 ± 0.35a	−0.35 ± 0.10a	−0.32 ± 0.00a

iPLA₂, Ca²⁺-independent phospholipase A₂; [M+H], gain of a proton; [M−H], loss of a proton; small hairpin ribonucleic acid, shRNA; RPTC, renal proximal tubular cell. iPLA₂γ was knocked down in RPTCs and whole cell lipids were isolated using a modified Bligh and Dyer (19) method. Phospholipids were identified using ESI-MS in both positive and negative ion modes. Data were normalized using the central limit theorem as described in Experimental Procedures and are presented as means ± SEM from four or five different rabbits. Within lipid species, means with the same letters were not significantly different as determined by Duncan's multiple range test. Where SEM is equal to zero, the particular lipid species was not identified by ESI-MS in any of the samples from that treatment. Boldface values are statistically significant within that particular phospholipid.

Knock-down of iPLA₂γ increases RPTC lipid peroxidation

Previous studies from our laboratory demonstrated that inhibition of iPLA₂γ led to increased oxidant-induced lipid peroxidation in RPTCs (4) and in isolated renal mitochondria (5). Accordingly, RPTC lipid peroxidation was measured in RPTCs after exposure to the scramble or iPLA₂γ shRNA adenovirus or diluent (control) using the TBARS assay (Fig. 2). Lipid peroxidation was elevated by ∼2-fold at 24 h after exposure to the iPLA₂γ shRNA adenovirus compared with control or scramble shRNA adenovirus (Fig. 2). TBARS remained elevated by 2-fold above control through 72 h. These results indicate that knocking down iPLA₂γ in RPTCs increases lipid peroxidation prior to the onset of cell death.

Fig. 2.

Effects of decreased iPLA₂γ expression on RPTC lipid peroxidation. The thiobarbituric acid-reactive substances (TBARS) assay was performed in noninfected (control), scramble shRNA, and iPLA₂γ shRNA adenovirus-treated RPTCs at 24, 48, and 72 h after virus exposure as a measure of cellular lipid peroxidation. Data are presented as means + SEM, percentage of control TBARS formation. Means with different superscripts are significantly different from each other (P < 0.05, n = 4).

Knock-down of RPTC iPLA₂γ causes changes in cellular and nuclear morphology and apoptosis

Light microscopy revealed that knock-down of iPLA₂γ resulted in RPTC cell death at 72 and 96 h (Fig. 3). No toxicity was observed through 96 h in control cells (data not shown) or RPTCs exposed to the scramble shRNA adenovirus (Fig. 3). The cell death observed consisted of RPTC shrinkage and detachment of cells from the plate. Examination of control (data not shown) and scramble shRNA-treated RPTC nuclear morphology, using the nuclear stain DAPI, revealed diffuse nuclear staining consistent with healthy RPTC. In contrast, 72 and 96 h after iPLA₂γ shRNA exposure, RPTC nuclei were condensed (15–20%) or fragmented (4–6%) (Figs. 3, 4).

Fig. 3.

Effects of decreased iPLA₂γ expression on cell and nuclear morphology in RPTCs. Confluent RPTCs were exposed to either adenoviral iPLA₂γ or scramble shRNA and cultured normally for 96 h. Representative photomicrographs are shown for cell (A, B) and nuclear (C, D) morphology in RPTCs at 96 h after exposure to scramble shRNA adenovirus (A, C) and iPLA₂γ shRNA adenovirus (B, D). Cells were fixed and stained with the nuclear dye, 4′,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology. Photomicrographs are representative of four separate RPTC preparations.

Fig. 4.

Characterization of cell death induced by decreasing iPLA₂γ expression. The percentage of cells with condensed or fragmented nuclei and cells with externalized phosphatidylserine was estimated by counting 10 high-power fields (per treatment group and per experiment) of DAPI-stained nuclei (A, B) and by flow cytometry of cells stained with annexin V-FITC and propidium iodide (PI) as described in Experimental Procedures (C) in noninfected (control), scramble shRNA, and iPLA₂γ shRNA adenovirus-treated RPTCs at 24, 48, 72, and 96 h after virus exposure. Data are presented as means + SEM, percentage of total nuclei that displayed condensed or fragmented morphology (A, B) or annexin V staining in the absence of PI staining (C). Means with different superscripts are significantly different from each other (P < 0.05, n = 3–4).

Annexin V-FITC and PI staining coupled with flow cytometry was used as an additional marker of apoptosis after knock-down of iPLA₂γ in RPTC. Annexin V-FITC binds to externalized phosphatidyserine on apoptotic cells, while PI stains the DNA of necrotic cells. Beginning at 72 h after iPLA₂γ shRNA adenovirus exposure and persisting at 96 h, annexin V staining increased compared with control or scramble shRNA-treated RPTCs (Fig. 4C). No increases in PI staining were detected in any treatment group at any time point (data not shown). In summary, the data reveal that knock-down of iPLA₂γ in RPTCs results in apoptotic cell death.

Knock-down of RPTC iPLA₂γ inhibits RPTC mitochondrial function

To determine the effect of decreasing iPLA₂γ expression on mitochondrial function in RPTCs, basal and uncoupled QO₂ rates and ATP levels were determined. Uncoupled QO₂ is a measure of the maximum rate that electrons can be shuttled through the electron transport chain. A 25% decrease in basal QO₂ was observed at 96 h after iPLA₂γ shRNA adenovirus exposure compared with scramble shRNA treatment (Fig. 5A). Uncoupled QO₂ was inhibited by 25% after 48 h and remained inhibited through 96 h in RPTCs with decreased iPLA₂γ expression (Fig. 5B). No significant changes in basal or uncoupled QO₂ were observed in control or scramble shRNA-treated RPTCs over 96 h (data not shown). Similar to basal QO₂, cellular ATP levels were reduced at 96 h after iPLA₂γ shRNA exposure (Fig. 5C).

Fig. 5.

Effects of decreased iPLA₂γ expression on RPTC mitochondrial function. Basal (A) and uncoupled (B) oxygen consumption (QO₂) and cellular ATP levels (C) were measured in scramble shRNA and iPLA₂γ shRNA adenovirus-treated RPTCs at 24, 48, 72, and 96 h after virus exposure. The rate of QO₂ or ATP level in cells exposed to iPLA₂γ shRNA is expressed as means + SEM, percentage of cells exposed to scramble shRNA for the same amount of time. No differences in QO₂ or ATP levels between control RPTCs and scramble shRNA-treated RPTCs were detected at any time point. Means with different superscripts are significantly different from each other (P < 0.05, n = 4–5).

ER and mitochondrial iPLA₂ activity measurements were correlated with basal mitochondrial QO₂ (Fig. 6A, E). Uncoupled QO₂ was well correlated over time only with mitochondrial iPLA₂ activity but not with ER iPLA₂ activity (r² = 0.920 vs. 0.564, respectively; Fig. 6B, F). The high correlation between uncoupled QO₂ and mitochondrial PLA₂ activity is consistent with the strong correlation between ATP levels and mitochondrial iPLA₂γ activity (Fig. 6C, G). Both ER and mitochondrial iPLA₂ activity measurements were correlated with the percentage of apoptotic cells after exposure to the iPLA₂γ shRNA adenovirus (Fig. 6D, H), suggesting that RPTC iPLA₂γ activity is required for cell viability.

Fig. 6.

Correlation of RPTC iPLA₂ activity with mitochondrial function and cell viability. Shown are iPLA₂ activity in ER (A–D) and mitochondria (E–H) isolated from RPTCs and basal (A, E) and uncoupled (B, F) QO₂, cellular ATP levels (C, G), and percentage of cells staining positive for annexin V-FITC and negative for PI (D, H) at the indicated time points after iPLA₂γ shRNA adenovirus exposure. Data are reported as means ± SEM, percentage of scramble shRNA-treated control RPTC ER or mitochondrial iPLA₂ activity, QO₂, ATP levels, and annexin V-positive cells from the same time point.

Apoptosis is induced by a nonlethal concentration of TBHP in RPTCs with decreased iPLA₂γ expression

To determine the effect of iPLA₂γ knock-down on sublethal oxidant stress, RPTCs with decreased iPLA₂γ, either in the ER fraction only (at 24 h after infection) or in the mitochondrial and ER fractions (at 48 h after infection), were treated with TBHP (75 μM) or diluent for 3 h. While this concentration of TBHP had no effect on annexin V staining in any treatment group at 24 h after infection (Fig. 7A) in RPTCs exposed to diluent (control) or scramble shRNA for 48 h, it caused a 2-fold increase in annexin V staining in RPTCs with decreased ER and mitochondrial iPLA₂γ expression (Fig. 7B). No increases in PI staining were detected in any treatment group at any time point (data not shown). In summary, RPTCs with decreased iPLA₂γ undergo apoptosis in the presence of a low concentration of the oxidant TBHP, whereas RPTCs with control levels of iPLA₂γ or a limited decrease in iPLA₂γ protein do not.

Fig. 7.

Effects of decreased iPLA₂γ expression on oxidant-induced apoptosis in RPTCs. After 24 h (A) and 48 h (B) of exposure to diluent (control), scramble shRNA, or iPLA₂γ shRNA adenovirus, RPTCs were treated with tert-butyl hydroperoxide (TBHP; 75 μM) or diluent for 3 h and cell death was measured by flow cytometry after annexin V-FITC and PI staining. No significant changes in PI staining were detected in any group at any time point. Data are presented as means ± SEM; percentage of total cells analyzed that displayed annexin V staining in the absence of PI staining. Means with different superscripts are significantly different from each other (P < 0.05, n = 3).

Nonlethal oxidative stress induces iPLA₂γ expression in RPTCs

Previous studies from our laboratory and the results of the current study demonstrate that iPLA₂γ is an important protective enzyme in RPTCs during oxidative stress. Accordingly, we investigated whether iPLA₂γ expression in RPTC ER and mitochondria is induced in the presence of nonlethal oxidant exposure. RPTCs were exposed to 50 μM TBHP or diluent for 24 h, and the expression of iPLA₂γ was measured by immunoblot analysis (Fig. 8A). These treatment conditions did not result in RPTC cell death, as confirmed by annexin V and PI staining (data not shown). Twenty-four hours after exposure to 50 μM TBHP, expression of iPLA₂γ was significantly induced (∼30%) in both the ER and mitochondria of RPTCs (Fig. 8B).

Fig. 8.

Effects of nonlethal oxidative stress on iPLA₂γ expression in RPTCs. Control RPTCs were exposed to TBHP (50 μM) or diluent for 24 h and cells were harvested and fractionated into ER and mitochondrial fractions. iPLA₂γ expression was determined by immunoblot analysis (A) as described in Experimental Procedures. Data are expressed as means + SEM, percentage of control iPLA₂γ expression (determined by densitometry) (B). Means with different superscripts are significantly different from each other (P < 0.05, n = 4).

DISCUSSION

The development and use of an iPLA₂γ shRNA adenoviral vector in primary cultures of rabbit RPTCs enabled us to examine the role of this enzyme in cell physiology and in the setting of increased oxidative stress. Decreased expression of this enzyme in RPTCs caused an immediate and sustained increase in cellular lipid peroxidation. The increase in peroxidized lipids was followed by a decrease in mitochondrial function and eventually apoptotic cell death. Furthermore, knock-down of iPLA₂γ sensitized RPTCs to nonlethal concentrations of an oxidant. These results support the hypothesis that iPLA₂γ participates in the prevention of oxidation or repair of oxidized phospholipids and highlights the importance of this enzyme in mitochondrial and ER function.

After exposure of RPTCs to iPLA₂γ shRNA, expression of ER-iPLA₂γ decreased by 40% at 24 h. The finding that the 24 h level of mitochondrial iPLA₂γ expression was not different from the control level suggests that the rate of turnover of ER-iPLA₂γ is faster than mitochondrial iPLA₂γ in RPTCs. Furthermore, because cellular lipid peroxidation was significantly elevated at 24 h, we suggest that ER-iPLA₂γ is a vital component of the cellular lipid peroxidation repair pathway in RPTCs. At 48 and 72 h after iPLA₂γ shRNA exposure (when mitochondrial iPLA₂γ expression and activity were decreased by 30–60%), lipid peroxidation remained elevated but did not increase above the 24 h level. Thus, we hypothesize that cells with lipid peroxidation greater than the level observed in the 24 h iPLA₂γ knock-down group underwent cell death and detached from the culture dish.

In this model, we promoted a shift from the physiological condition to a pathological condition by decreasing iPLA₂γ activity through the introduction of shRNA for iPLA₂γ. During the first 48 h, phospholipid changes occurred in the ER and mitochondria, resulting in mitochondrial dysfunction. By 72 h, the cells with decreased iPLA₂γ could not withstand these phospholipid changes (oxidation, phospholipid substrate buildup, etc.) and began to die. Phospholipid changes were not measured once cell death began because of the difficulty in determining whether the phospholipid changes were the result of the loss of iPLA₂γ or were due to the cell death process.

With ESI-MS analysis of the phospholipids, we identified the most classes to be affected. Increased levels of these phospholipids suggested that the loss of iPLA₂γ led to changes in the cellular milieu that affected cellular activity. The transience of these changes likely reflected time-dependent differences in the loss of iPLA₂γ between the mitochondria and ER or compensatory changes that occurred over time when other PLA₂ activities possibly became affected.

The loss of iPLA₂γ also resulted in an increase in substrate accumulation: saturated and unsaturated fatty acid-containing phospholipids. The accumulated unsaturated cellular phospholipids are potential targets of oxidative attack, leading to further damage. This hypothesis is supported by the TBARS data that showed that loss of iPLA₂γ led to greater lipid peroxidation in RPTCs. In addition, changes in saturated fatty acid-containing phospholipids may affect membrane fluidity, affecting organelle functionality.

The effect of iPLA₂γ shRNA on mitochondrial function was most likely the result of decreased mitochondrial iPLA₂γ protein expression, as the loss of mitochondrial function was not observed until 48 h after infection when mitochondrial iPLA₂γ expression and activity were decreased. The initial defect in mitochondrial function was identified at 48 h by determining the maximum capacity of the electron transport chain using the uncoupler FCCP. This stress test revealed a 25% decrease in electron transport capacity without a decrease in basal QO₂ or ATP levels. At 96 h, mitochondrial function was further decreased, as reflected by decreased basal QO₂ and decreased ATP. Because mitochondrial iPLA₂γ is localized to the inner membrane (5), it is likely that the loss of mitochondrial iPLA₂γ resulted in insufficient maintenance of the phospholipid composition or accumulation of oxidized phospholipids in the inner membrane, which, in turn disrupted electron transport. iPLA₂γ may selectively cleave oxidized phospholipids, which are generated by reactive oxygen species during normal oxidative metabolism, in the inner mitochondrial membrane to preserve membrane integrity and prevent the impairment of complex III function (24). Alternatively, or additionally, iPLA₂γ may be responsible for generating lysocardiolipin, which is reacylated during inner mitochondrial membrane remodeling, a critical process for the optimal function of complexes I, III, and IV of the electron transport chain (25, 26). In summary, the localization of iPLA₂γ to the inner mitochondrial membrane, the importance of inner mitochondrial membrane phospholipids to the electron transport chain, and our observations that mitochondrial function decreased with decreased iPLA₂γ suggest that iPLA₂γ activity is required to maintain mitochondrial function.

The induced decrease in iPLA₂γ expression resulted in elevated apoptosis, as measured by increased numbers of cells with condensed and fragmented nuclei and annexin V staining at 72 and 96 h. Apoptosis observed under these conditions may be the result of several mechanisms. Lipid peroxidation in the ER may have increased ER Ca²⁺ permeability, resulting in an elevated cytosolic free Ca²⁺ concentration that is known to induce apoptosis in this and other models (16, 27). Ca²⁺ accumulation by mitochondria may have initiated the mitochondrial permeability transition (MPT), a well-known signal for apoptosis (28). In addition, mitochondrial lipid peroxidation possibly initiated the MPT and caused the release of apoptotic mediators, including cytochrome c (29), or it may have led to an increase in Ca²⁺-dependent PLA₂ activity. Finally, a lipid peroxidation end product, 4-hydroxynonenal, which reportedly induces apoptosis by modulating the activities of Jun and p38 kinases (30, 31), may have accumulated in RPTCs with decreased iPLA₂γ. In summary, several mechanisms may mediate apoptosis induced by decreased iPLA₂γ.

Upregulation of other lipid peroxidation repair enzymes and/or cellular antioxidants may compensate for the gradual decrease in iPLA₂γ expression. One alternative pathway involves the L- and S-isoforms of phospholipid hydroperoxide glutathione peroxidase (32). Although phospholipid hydroperoxide glutathione peroxidase can directly reduce peroxidized phospholipids in membranes (33), its activity has been reported to be up to 500-fold less than that of cytosolic glutathione peroxidase (34). As mentioned above, the rate-limiting step in cytosolic glutathione peroxidase activity is PLA₂-mediated hydrolysis of the peroxidized fatty acid. A second potential lipid peroxidation repair enzyme, peroxiredoxin VI, is highly expressed in the lung (35). It is unknown at this time whether one or both of these pathways are functional in rabbit RPTCs. Upregulation of catalase, superoxide dismutase, or glutathione may also be consequences of iPLA₂γ knock-down. In summary, upregulation of other antioxidant enzymes and molecules may have occurred to compensate for the gradual and prolonged decrease in iPLA₂γ.

In apparent contrast to a previous study in RPTCs demonstrating that pharmacologic inhibition of iPLA₂γ deceases cisplatin-induced apoptosis (36), knock-down of iPLA₂γ in this study resulted in RPTC apoptosis after oxidant exposure. In the cisplatin-induced RPTC apoptosis model, cell death is not mediated by oxidative stress but by DNA damage and iPLA₂γ-mediated caspase activation (36, 37). The mechanism by which iPLA₂γ knock-down sensitizes RPTCs to oxidant-induced apoptosis has not been examined, but it may be the result of increased cytosolic Ca²⁺ due to ER lipid peroxidation and the effect of Ca²⁺ and lipid peroxidation on mitochondria (inducing MPT), as addressed above. Evidence for opposing roles of iPLA₂γ in RPTCs after oxidant versus nonoxidant insults has accumulated in recent years. For example, in RPTCs and isolated RCM, iPLA₂γ inhibition accelerates oxidant-induced lipid peroxidation, mitochondrial dysfunction, and cell death (4, 5). In contrast, iPLA₂γ mediates cisplatin-induced RPTC apoptosis (36) and Ca²⁺-induced MPT in RCM (38), demonstrating that RPTC iPLA₂γ can be protective or detrimental in oxidant and nonoxidant stress, respectively.

Because all available data regarding the role of iPLA₂γ in oxidant-induced lipid peroxidation, mitochondrial damage, and cell death suggest that iPLA₂γ is part of the RPTC defense against oxidative stress, we hypothesized that its expression would be upregulated in the presence of nonlethal oxidative stress. We confirmed this hypothesis. Additional studies are required to determine the mechanism by which iPLA₂γ expression is increased by oxidative stress and whether iPLA₂γ expression is induced by other sources of oxidative stress.

In conclusion, we demonstrate that selectively decreasing iPLA₂γ expression and activity resulted in increased lipid peroxidation, impaired mitochondrial function, and ultimately apoptotic cell death in RPTCs. Exposure of RPTCs, with decreased expression of iPLA₂γ, to TBHP caused a 2-fold increase in apoptotic cell death, whereas no toxicity was observed in controls. These results are consistent with previous experiments in RPTCs and in isolated RCM showing that pharmacological inhibition of iPLA₂γ increased lipid peroxidation, mitochondrial damage, and cell death induced by oxidants. Furthermore, the expression of iPLA₂γ was increased in both ER and mitochondria as part of the response of RPTCs to oxidative stress. Collectively, these data suggest that iPLA₂γ is a protective enzyme in the kidney under basal conditions and during oxidative stress.

Abbreviations

• BEL, bromoenol lactone

• DAPI, 4′,6-diamidino-2-phenylindole

• ER, endoplasmic reticulum

• FCCP, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone

• iPLA₂, Ca²⁺-independent phospholipase A₂

• MPT, mitochondrial permeability transition

• PI, propidium iodide

• PLA₂, phospholipase A₂

• QO₂, oxygen consumption

• RCM, renal cortex mitochondria

• RPTC, renal proximal tubular cell

• TBARS, thiobarbituric acid-reactive substances

• TBHP, tert-butyl hydroperoxide