Plasmalogen Phospholipid Hydrolysis During Hypoxic Injury of Rabbit Proximal Tubules

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Kidney Intemationa4 VoL 47 (1995), pp. 1087—1094

Plasmalogen phospholipid hydrolysis during hypoxic injury of rabbit proximal tubules

DIDIER PORTILLA and MICHAEL H. CREER

Division of Nephrology, Department of Medicine and Department of Pathology, University of Arkansas for Medical Sciences, and John L. McClellan Memorial Veterans Administration Medical Centet Little Rock, Arkansas, USA

Plasmalogen phospholipid hydrolysis during hypoxic injury of rabbit unsaturated fatty acids. These specific molecular structural proximal tubules. We have identified and quantified the major species of features would contribute to altered molecular dynamics of arachidonate-containing phospholipids in proximal tubules by high performance liquid chromatographic and gas chromatographic analyses. plasmalogens as compared to diacyl phospholipids which could Araehidonate was found to comprise 53% of the total mass of fatty acids have a profound effect on membrane protein-phospholipid esterified at the sn-2 position of ethanolaminc phospholipids, and 51% of interactions and integral membrane protein function [8]. The that amount resides in three plasmenylethanolamine species containing physiological role of plasmalogen phospholipids in the kidney the vinyl ethers of palmitaldehyde, oleylaldehyde or stearylaldehyde at the sn-i position. Choline phospholipids contained 21% arachidonylated is currently unknown. species and 33% of that amount resides in a single plasmenylcholine In many tissues the plasmalogen phospholipid subclass appears species containing the vinyl ether of palmitaldehyde at the sn-i position. to be selectively enriched in esterified arachidonic acid (AA) [12, Ten minutes of hypoxia did not cause a significant change in the total phospholipid mass of ethanolamine or choline phospholipids; however, 131. Thus, accelerated turnover of plasmalogen phospholipids phosphate analysis of the individual phospholipid molecular species would contribute importantly to the generation of unesterified containing esterified arachidonic acid in isolated proximal tubules dem- AA during ischemia. In the kidney, marked accumulation of AA onstrated a 24% reduction in the mass of the plasmenylethanolamine is associated with little change in the total phospholipid mass early molecular species with the vinyl ether of olcylaldehyde at the sn-i position and a 35% reduction in the mass of plasmenyicholine species within the course of ischemia/hypoxie cell injury [i, 3]. By the time net paimitaldehyde at the sn-i position. These studies underscore the patho- reduction of phospholipid mass could be demonstrated in these physiological importance of plasmalogen phospholipid hydrolysis and studies irreversible cell damage was present [3]. These observa- suggest that activation of PLA2s, which utilize endogenous proximal tions suggest that unesterifled AA may be derived by the early tubule plasmalogen substrates, may play an important role in the early generation of araehidonic acid and accompanying phospholipid catabo- hydrolysis of selective phospholipid pools highly enriched in AA lism during hypoxic cell injury. before irreversible cell injury occurs. Our recent studies in freshly isolated rabbit proximal tubules suggest that the activation of an intracellular calcium-independent plasmalogen-selective proximal Catabolism of phospholipids containing arachidonic acidtubule PLA2 activity occurs before the development of irrevers- esterified at the sn-2 position plays a central role in theible hypoxic cell injury and temporally parallels the generation of pathogenesis of renal ischemic cell injury [1—4] and mayAA [14, iS]. Thus, activation of this enzyme(s) may account for produce membrane-localized second messengers that influencethe early production of AA. critical ion channels [5—7]. In mammalian cell phospholipids, The present study was undertaken to: (1) characterize the arachidonate is found esterified to diaeyl, plasmalogen andphospholipid composition of rabbit proximal tubules; (2) deter- alkyl ether phospholipids [8]. Although previous studies havemine if hydrolysis of plasmalogen phospholipids in proximal described the existence of plasmalogen phospholipids in kidneytubules represents an important source of unesterified AA during tissue [9—11], their nephron segment localization as well ashypoxia; and (3) to define whether hypoxia results in selective their possible functional role in hypoxic cell injury have nothydrolysis of arachidonylated phospholipid pools with particular been previously investigated. emphasis on defining the relative contribution of plasmalogen Plasmalogens are distinguished from diacylphospholipids byversus diacylglycerophospholipid turnover. We demonstrate here the presence of 1-O-alk-1'-enyl ether (vinyl ether) linkage atfor the first time that a significant fraction (51%) of the AA in the sn-i position as compared to the 1-0-acyl ester linkageproximal tubule ethanolamine phospholipids is esterified to plas- present in diacylglyeerophospholipids. Furthermore, the sn-2malogen molecular species. Our studies also show the rapid and hydroxyl group of plasmalogens is usually esterified to highlyselective catabolism of arachidonate-containing choline and ethanolamine plasmalogen phospholipids within the first 10 minutes of hypoxic injury. These results underscore the functional impor- Received for publication September 9, 1994 tance of the high content of plasmalogens in proximal tubules and and in revised form November 3, 1994 suggest that the rapid activation of plasmalogen-selective intra- Accepted for publication November 7, 1994 cellular phospholipase(s) A2 during hypoxic cell injury may ac- © 1995 by the International Society of Nephrology count for the early generation of AA.

1087 1088 Portillaand Creer: Proximal tubule plasmalogens

Methods Rapport [20] as described by Kates [21]. The alkyl ether content Proximal tubule isolation of CGP and EGP was determined by quantification of lipid phosphorus in the lysophospholipid fraction remaining after Rabbit proximal tubules were isolated as previously describedsequential, exhaustive base and acid-catalyzed hydrolysis of the [15] and resuspended in DMEM/F12 medium at a protein con- diradylphospholipids [12]. The lysophosphatidylcholine and lyso- centration of 2 mg/mI. Prior to the hypoxic experiments, isolatedphosphatidylethanolamine fractions were isolated by HPLC as proximal tubules were transferred from ice to a 37°C shaking previously described [22]. water bath and pre-equilibrated for 20 minutes in a 95% 02-5% CO2atmosphere. Hypoxia was achieved by subjecting the tubulesGas chromatographic characterization of the fatty acid and alkenyl to a 95% N2-5% CO2 atmosphere that was maintained for ether composition of rabbit proximal tubules variable time periods at 37°C. Samples were obtained before hypoxia and at different hypoxic intervals for measurements of The fatty acid/fatty aldehyde composition of the major proximal cytosolic LDH release. For the measurement of lactic dehydro-tubule phospholipids classes separated by normal phase HPLC genase (LDH) release, 1 ml of tubule suspension was centrifugedwas determined by gas liquid chromatography (GLC) analysis of through 0.4 ml of a mixture of dibutyl phthalate and dioctylthe fatty acid methyl ester (FAME) and dimethylacetal (DMA) phthalate (2:1) to separate the tubules from the extracellularderivatives produced after acid catalyzed methanolysis [12, 231. medium. LDH activity was measured spectrophotometrically onThese products were injected onto a 30 m (length) by 0.25 mm Triton-permeabilized 0.5 ml aliquots of tubule suspensions and(i.d.) fused silica capillary GC column coated with SP 2330 corresponding supernatants as previously described [14, 15]. utilizing a split ratio of 5 to 10:1 and eluted isothermally at 190°C utilizing an HP model 5980A chromatographic system with he- Extraction, separation and analysis of phospholipid classes lium as a carrier gas at a flow rate of 0.5 mltmin. Components To characterize the phospholipid composition of proximaleluting from the column were detected and quantified with a tubules, a suspension of isolated rabbit proximal tubules contain-flame-ionization detector. Identification of the individual FAME ing approximately 40mg of total cellular protein was washed twicespecies was established by comparison of their GLC retention with phosphate buffered saline (PBS) and resuspended in a finaltimes with commercially available standards. Individual DMA volume of 2 ml. Three 50 d aliquots were removed for assay ofspecies were identified by comparison of their GLC retention total protein. Cellular phospholipids were extracted at 4°C withtimes with the DMA derivatives produced after acid-catalyzed chloroform and methanol by the method of Bligh and Dyer [16].methanolysis of lysoplasmenylcholine derived from bovine heart The extract was evaporated to dryness under N2 and the lipidcholine phospholipids as previously described [24]. GLC quanti- residue resuspended in 750 tl of chloroform-methanol 2:1 (volttation of individual FAME and DMA species was accomplished vol). Three 5 tl aliquots were removed for measurement of totalby comparison of the integrated flame-ionization detector re- lipid phosphorus, and 100 l aliquots were injected onto an HPLCsponse for each FAME and DMA species with the integrated response of the FAME species derived from arachidic acid which column for phospholipid class separation (see below). was added as an internal standard prior to derivatization. The Normal phase HPLC separation of major proximal tubule observed integrated areas were corrected for differences in the phospholipid classes detector response factor for individual FAME and DMA species which has been previously shown to be proportional to their To separate proximal tubule phospholipids, we utilized a Wa- ters HPLC system comprised of two Model 501 HPLC pumps, amolecular weights [23]. Quantification of phospholipid classes by GLC was accomplished as described by Kates [25]. Waters pump controlled module, Millenium software automated gradient controller, U6K injector, and a Model 486 tunable Separation and quantification of individual choline and absorbance detector (wavelength set at 203 nm) interfaced to an NEC PC 386t33i computer. Phospholipids were separated into ethanolamine glycerophospholipid molecular species classes by normal phase HPLC using an Ultrasphere-Si column Individual CGP and EGP molecular species were resolved by (4.5 X 250 mm; 5 jtm Beckman) as the stationary phase with anreversed phase HPLC utilizing an octadecyl silica stationary phase initial linear gradient over 12 minutes from a mobile phase of(Ultrasphere-ODS) (4.6 x 250 mm; 5 .tm particles) with isocratic hexane/isopropanoitwater (48.5:48.5:3, vol/vol) to hexanetisopro-elution at 2 ml/min and a mobile phase comprised of methanolt panoltwater (47.75:47.75:4.5, vol/vol). The composition of theacetonitriletwater 90.5/2.5/7 vol/vol containing 20 m choline latter mobile phase was held constant for 5 minutes followed bychloride as previously described [26]. Column eluents were mon- several step changes to hexane/isopropanoltwater (46.5:46.5:7,itored by UV detection at 203 nm. The identification of individual vol/vol) over a 45 minute period [17, 181. Column eluents corre-CGP and EGP molecular species was established by the following sponding to the following phospholipid classes (listed in order ofcriteria: (1) GLC characterization of the FAME and DMA elution) were: ethanolamine glycerophospholipids, phosphatidyl-derivatives produced after acid catalyzed methanolysis of the inositol, phosphatidylserine, choline glycerophospholipids andphospholipid species recovered in column effluents after extrac- sphingomyelin were collected, evaporated with N2 and subse-tion from the mobile phase with chloroform [24]; (2) comparison quently utilized for analysis. Phospholipid phosphorus was deter-of retention time and order of elution of individual species with mined by a microphosphate assay as previously described [19].authentic standards commercially available and; (3) identification The plasmalogen content of the choline glycerophospholipidof CGP and EGP plasmalogen species was also substantiated by (CGP) and ethanolamine glycerophospholipid (EGP) fractionsdemonstrating the loss of peaks corresponding to diradyl plas- was determined by quantification of the vinyl ether content basedmalogen molecular species following acid catalyzed hydrolysis of on a modification of the '2additionmethod of Gottfried andCGP and EGP. This was accomplished by comparison of the Portilla and Creer: Proximal tubule plasmalogens 1089

40 in rabbit proximal tubules were CGP (36%) and ethanolamine glycerophospholipids (31%), accounting for 67% of total extract- (I, C') able lipid phosphorus present in this preparation. Proximal tu- E bules also contained significant amounts of phosphatidylinositol D I 30 (6%), sphingomyelin (12%) and phosphatidylserine (11%). Less 0 than 4% of the total proximal tubule phospholipids corresponded to cardiolipin, lysophospholipids and phosphatidic acid. 00 Plasmalogen content determined by measurement of '2 addi- 20 tion to activated olefins and expressed as a percentage of the total phosphorus content of the corresponding phospholipid class demonstrated that ethanolamine glycerophospholipids contained 10 the majority of plasmalogens present in proximal tubules when compared to choline glycerophospholipids (35 3% and 10 2% by '2 addition, respectively). Together, choline and ethanolamine F glycerophospholipids quantitatively accounted for the total plas- 0 11 nnn malogen content of the isolated tubules. Alkaline methanolysis of CGP EGP P1 Ps Sph Other choline and ethanolamine glycerophospholipids and subsequent acid-catalyzed methanolysis, lipid extraction, separation of lyso- Phospholipid class phospholipids by 1-IPLC and quantitation by phosphate analysis Fig.1. Phosphoilpid composition of rabbit proximal tubules. Proximal demonstrated that proximal tubule CGP contained only minor tubule phospholipids were extracted and separated by normal phase I-TPLC and phospholipid classes and subclasses quantified as described amounts of alkyl ether phospholipids (1.20.5). This phospho- Methods. The percent distribution of phospholipid phosphonis in the lipid subclass was not detected in proximal tubule ethanolamine major membrane phospholipid classes and subclasses is indicated by the glycerophospholipids; however, we cannot rule out the presence height of the vertical bars. Abbreviations are: CGP, choline glycerophos- of small amounts of alkylether EGP species. pholipids; EGP, ethanolamine glycerophospholipids; P1, phosphatidylinositol; PS, phosphatidylserine; Sph, sphingomyelin; Other, refers collcc- tively to cardiolipin, phosphatidic acid and lysophospholipids. Data shown represent the means SEM of 5 separate measurements. Symbols are: (U) Characterization of the fatty acid and alkenyl ether composition of diacyl; ()plasmalogen;(•)alkylether. rabbit proximal tubule phospholipids

The presence of dimethylacetal derivatives in both choline and reverse phase HPLC chromatograms from CGP and EGP beforeethanolamine glycerophospholipids after acid-catalyzed meth- and after acid hydrolysis which confirmed that the peaks thatanolysis further corroborated the presence of plasmalogens phos- disappeared after acid treatment corresponded by GLC analysispholipids in proximal tubules. Identification of the peaks corre- to the plasmalogen species [23]. Quantification of individualsponding to the dimethylacetals of palmitic, stearic or oleic phospholipid molecular species was achieved by measurement ofaldehydes was accomplished by comparison of retention times the integrated UV detector response and by determination ofwith standards obtained from acid-catalyzed methanolysis of lipid phosphorus in reverse phase column effluents as previouslylysophasmenylcholine. The gas chromatographic coelution of described [23]. these peaks supported these assignments. Proximal tubule ethanolamine and choline glycerophospholipid methanolysates con- Arachidonic acid release tained the dimethylacetal of palmitic, stearic and oleic aldehydes AA was measured following a modified method previously(Table 1). The saturated fatty acid methyl ester (FAME) deriva- described [14, 15]. Briefly, AA was extracted from a 2 ml aliquottives of choline glycerophospholipids consisted predominantly of of the 2 mg/mi tubule suspensions. After addition of 20 nano-species with 16 carbon atoms, whereas the saturated FAME grams of octadeuterated (d8) AA as internal standard, AA wasderivatives of ethanolamine glycerophospholipids consisted of isolated on C18 columns and converted to the pentafiuorobenzoylalmost equal amounts of species with 16 and 18 carbon atoms. ester. Samples were then evaporated to dryness and dissolved inThe saturated DMA derivatives of choline glycerophospholipids acetonitrile for mass spectrometric analysis. The quantity of AAwere significantly smaller in quantity when compared to the was determined by high pressure liquid chromatography/particleethanolamine glycerophosholipids and were comprised predomi- beam/mass spectrometry (HPLC/PB/MS) in the electron capturenantly of 16 and 18 carbon species. The unsaturated FAME negative ion chemical ionization mode by monitoring m/z 303 forderivatives of choline glycerophospholipids contained predomi- AA and m/z 311 for octadeuterated AA as previously describednantly 18 carbon atoms with one or two double bonds while [14, 15]. ethanolamine glycerophospholipids yielded the FAME derivative Results of arachidonic acid (20:4) as the predominant unsaturated species. Composition of the major phospholipid classes in isolated Phosphatidylinositol and phosphatidylserine acid methanoly- proximal tubules sates contained the FAME derivatives of stearic, linoleic and The distribution of phospholipid phosphorus mass among thearachidonic acid as the principal species. There were no dimethy- various proximal tubule phospholipid classes and subclasses islacetal derivatives identified in the phosphatidylinositol or phos- shown in Figure 1. The predominant phospholipid classes presentphatidylserine fractions. 1090 Portillaand Creer: Proximal tubule plasmalogens

Table 1. Composition of fatty acid methyl ester (FAME) and dimethylacetal (DMA) derivatives prepared from rabbit proximal tubule phospholipids Derivative FAME/DMA derivative (%of Phospholipid total) class 16:0(D) 16:0 18:0(D) 18:1(D) 18:0 18:1 18:2 20:4 CGP 2.3 0.2 27.2 1.9 1.8 0.1 0.7 0.1 13.20.6 11.8 0.7 32.4 1.1 12.4 0.8 EGP 9.21.6 4.60.5 7.5 1.4 6.1 1.2 11.00.8 15.6 1.3 16.9 1.7 28.5 1.6 P1 ND 19.9 1.8 ND ND 42.75.0 10.02.0 12.54.8 14.8 2.5 PS ND 7.70.8 ND ND 40.3 0.9 11.5 0.9 34.72.0 5.6 0.5 Proximal tubule glycerophospholipids were extracted, the major classes separated by normal phase HPLC, and the isolated fractions corresponding to choline glycerophospholipids (CGP), ethanolamine glycerophospholipids (EGP), phosphatidylinositol PT, and phosphatidylserine (PS) were subjected to acid catalyzed methanolysis and the volatile derivatives were characterized by GLC as described under Methods. The mole % composition of each derivative was determined by comparing the integrated flame ionization detector response with that of the arachidic acid (20:0) internal standard after correction for differences in detector response factors for each derivative. The composition of FAME and DMA derivatives is given as chain length: number of double bonds. D refers to dimethylacetal derivatives. Values shown represent the mean SEM of 5 separate measurements. Abbreviation is ND, none detected.

A Reverse phase separation and quantification of individual phospholipid molecular species f Since phospholipids containing choline and ethanolamine bases 1.10 m accounted for the majority of plasmalogen proximal tubule phospholipids, the composition of individual molecular species in these phospholipid classes was further characterized. The elution pro- files for choline and ethanolamine glycerophospholipid molecular 1.00 species after reverse phase HPLC are shown in Figure 2 and the corresponding composition and relative mass contribution of each species are given in Table 2. The molecular identity of individual species was established by three independent criteria previously described under Methods. In Table 2 diacyl glycerophospholipids 0.90 q are referred to by the abbreviation "Ptd" and plasmalogen glycerophospholipids by "Plas." Choline glycerophospholipids are 0 20 40 60 80 100 represented by "Cho" and ethanolamine glycerophospholipids by "Eth." Time,minutes Theelution profile of choline glycerophospholipids from prox- B g imal tubules (Fig 2)demonstratedpredominant peaks at 30(d), 40(g),59(m), and 61(n) minutes, representing diacyl molecular species (Table 2). The predominant peaks at 50(h), 52(i), and 76(o) minutes in the elution profile of ethanolamine glycerophos- 1.10 pholipids represented plasmalogen molecular species containing esterified arachidonic acid. It is important to note that it is not possible to estimate the relative mass of each molecular species by 1.00 direct inspection of the UV detector response due to the high molar absorptivity of the alkenyl ether functional group relative to that of other olefin groups [23]. r 0.90 Pq Compositionof proximal tubule choline and ethanolamine 0 20 40 60 80 100 glycerophospholtpid molecular species

Time, minutes PtdCho was predominantly comprised of species with 16 carbon atoms at the sn-i position as opposed to PlasCho which was Fig.2. Reverse-phase HPLC analyses of individual molecular species of ethanolamine (A) and choline (B) glycerophospholipids from isolated prox- comprised of both 16:0 and 18:0 species. PlasEth was comprised imal tubules. Proximal tubule phospholipids were extracted and separated of almost equal amounts of 16:0 and 18:0 species at the sn-i into classes by normal phase HPLC. The individual molecular species ofposition, while PtdEth was mostly comprised of 18:0 carbon atoms EGP and CGP were separated by reverse phase HPLC and detected by monitoring UV absorption at 203 nm as described under Methods. The in the sn-i position. These results shown in Table 2, are in letters above each peak correspond to the lettering scheme used toagreement with the results obtained by GLC analysis (Table 1). In identify individual phospholipid molecular species given in Table 2. CGP 21% of the total phospholipid mass is comprised of species Portilla and Creer: Proximal tubule plasmalogens 1091

Table 2. Composition of proximal tubule choline and ethanolamine glycerophospholipid molecular species determined following reverse phase HPLC isolation

Peak % Total mass U, 20 - Composition C,, Choline glycerophospholipids E a (18:2, 18:3)-PtdCho 1.80.1 -o b (16:1, 18:2)-PtdCho 1.3 0.1 0.... c (18:2, 20:4)- 1.80.2 PlasCho d (18:2, 18:2) PtdCho 7.80.6 .oE ...-. e (16:1, 20:4) PtdCho 1.80.4 10- f (16:0, 20:4) PtdCho 5.10.5 g (16:0, 18:2) PtdCho 22.2 1.3 CUO h (18:1, 18:2) PtdCho 9.40.7 i (16:0, 20:4) PlasCho 4.80.4 'O j (16:0, 18:2) PlasCho 1.1 0.2 -c k (16:0, 16:0) PtdCho 1.40.3 0 1 (16:0, 18:1) PtdCho 4.80.5 m (18:0, 20:4) PtdCho 8.1 0.4 rj (18:0, 18:2) PtdCho 4.50.9 Diacyl Plasmalogen Diacyl Plasmalogen o (16:0, 18:0) PtdCho 17.4 1.2 p (18:0, 18:1) PtdCho 3.00.3 Ethanolamine Choline q (16:0, 20:0) PtdCho 2.00.7 glycerophospholipids glycerophospholipids Ethanolamine glycerophospholipids a (16:1, 20:4) PtdEth 1.80.6 Fig. 3. Arachidonic acid content in ethanolamine (EGP) and choline b (18:2, 20:4) PtdEth 2.1 0.3 (CGP) glycerophospholipid molecular species in rabbit proximal tubules. A c (18:2, 18:2) PtdEth 3.30.2 total of 4 different proximal tubule preparations were used and in each d (unidentified, acid 2.9 0.4 preparation the phospholipid mass was measured 3 times. Values repre- labile, 20:4) sent the mean and SEM of these 4 independent proximal tubule PlasEth preparations. e (16:0, 20:4) PtdEth 3.60.1 f (18:1, 20:4) PtdEth 8.5 0.7 Table 3. Effects of hypoxia on arachidonic acid (AA) and lactic g (18:1, 18:2) PtdEth 12.1 1.4 dehydrogenase (LDH) release in proximal tubules h (16:0, 20:4) PlasEth 8.60.6 i (18:1, 20:4) PlasEth 8.5 0.5 AA release LDH release j (18:1, 18:2) PlasEth 3.3 0.4 Experimental condition pmol/mg prot % k (16:0, 18:1) PtdEth 2.20.4 1 (18:1, 18:1) PtdEth 2.90.3 Normoxia 9.9 3 6 2 m (18:0, 20:4) PtdEth 10.91.2 10 minutes hypoxia 46.2 9 12 3 n (18:0, 18:2) PtdEth 12.3 0.9 20 minutes_hypoxia 92.4 i3 31 5' o (16:0, 18:1) PlasEth 3.20.2 Values are means SE; N =4experiments for each condition. p (18:0, 20:4) PlasEth 7.60.9 Abbreviations are: AA, arachidonic acid; LDH, lactate dehydrogenase. q (18:0, 18:2) PlasEth 2.3 0.3 Significantly different from normoxic value (P < 0.05) Purified choline and ethanolamine glycerophospholipids were analyzed by reverse phase high performance liquid chromatography as described in Figure 2. The mass of each individual phospholipid molecular species was quantified by a microphosphate assay as described under Methods. The composition of the sn-i and sn-2 groups of individual phospholipid Documentation of proximal tubule hypoxic injuly molecular species is abbreviated as: (a:b, c:d) where a:b represents chain Hypoxia caused a time-dependent release of lactic dehydroge- length: number of double bonds for the sn-01 radyl group and c:dnase (LDH) from 6 2% (normoxia) to 31 5% (20 mm represents the corresponding designations for the sn-2 radyl group. Ptd hypoxia, N = and this was accompanied by a time-dependent refers to diacyl phospholipids, and Plas to plasmalogens species. Eth 4), represents ethanolamine glycerophospholipids and Cho represents cho- release of arachidonic acid (AA) mass from 9.93 (normoxia) to line glycerophospholipids. Values are means SEM from 5 separate 92,4 13 pmol/mg protein (20 mm hypoxia, N 4) (Table 3). experiments. Effect of hypoxia on the total phosphate mass of proximal tubule choline and ethanolamine glycerophospholipids and the mass of containing esterified arachidonate with PlasCho species account- individual molecular species ing for 33% of the total arachidonylated CGP mass. In EGP 53% In order to investigate the functional role of plasmalogen of the total EGP mass is comprised of arachidonylated speciesphospholipids in proximal tubules we measured the mass of with 51% of the total esterified arachidonate in PlasEth species.proximal tubule ethanolamine and choline glycerophospholipids The relative distribution of esterified AA between diacyl andunder control conditions (normoxic) and after 10 and 20 minutes plasmalogen molecular species for CGP and EGP is summarizedof hypoxic injury. As shown in Figure 4 there were no significant in Figure 3. In addition to arachidonate we also found thatchanges in the total mass of both ethanolamine and choline linoleate in both CUP and EUP was esterified to the sn-2 positionglycerophospholipids after 20 minutes of hypoxic injury. We also in both diacyl and plasmalogen species (Table 2), which wasmeasured the mass of all the individual molecular species in similar to what has been previously described in isolated proximalproximal tubule ethanolamine and choline glycerophospholipids tubules. under normoxia (control) and after 10 and 20 minutes of hypoxic 1092 Portillaand Creer: Proximal tubule plasmalogens

100 A 7000

U) 6000 0 U) 2 5000 o 60 0.--- 2 ° 4000 0-. o0) 40 •.cE 3000 U) oE -c0 20 2000 o -c a- 1000 0 0 Control 10 mm hypoxia 20 mm hypoxia Time of hypoxia, minutes

Fig.4.Effectof hypoxia on total mass of choline (1 CGP) and ethanol- Time amine (, EGP) glycerophospholipids from proximal tubules. A total of 4 B different proximal tubule preparations were used and in each preparation the phospholipid mass was measured 3 times. Values represent the mean and SEM of these 4 independent proximal tubule preparations. 4000 U) 0 -a.--3000 injury. There was no significant reduction in the mass of CGP or U)'— EGP molecular species containing oleate (18:1), linoleate (18:2) or saturated fatty acids at the sn-2 position. For species containing 2000 esterified arachidonate (20:4) there was no significant alteration in the mass of arachidonylated diacyl CGP or EGP species. U) However, there were arachidonylated plasmalogen species which 2 1000 were found to exhibit accelerated hypoxia-induced catabolism a- (decreased mass). As shown in Figure 5, the 16:0, 20:4 plasmenylcholine species did show a persistent decrease (35%) during 0— hypoxia. We also observed a 24% decrease in the mass of 18:1, Control 10 mm hypoxia 20 mm hypoxia 20:4 plasmenylethanolamine species. There appeared to be a small increase in the mass of 18:0, 18:2 diacyl EGP within the first Time 10 minutes of hypoxic injury (data not shown). Fig.5. EJfrct of hypoxia on the mass of arachidonylated molecular species from ethanolamine (EGP) and choline (CGP) glycerophospholipids. A Discussion represents (18:1, 20:4) plasmenylethanolamine and B represents (16:0, 20:4) plasmenylcholine. A total of 4 different proximal tubule preparations In the present study we demonstrated by various independentwere used and in each preparation the phospholipid mass was measured 3 analytical techniques the presence of significant amounts oftimes. Values represent the mean and SEM of these 4 independent plasmalogen in proximal tubule choline (10%) and ethanolamineproximal tubule preparations. *Significantlydifferent from control values (35%) glycerophospholipids. The total plasmalogen content ofP < 0.05. rabbit glycerophospholipids in proximal tubules is significantly greater than that previously reported in isolated membrane fractions of rat kidney cortex [11]. This suggests that the total plasmalogen content in the kidney may vary significantly amongrenal proximal tubules suggests that they may be involved in the different animal species. In particular, the total plasmalogenregulation of transmembrane ion movements in the kidney. This content of rat kidney appears to be significantly lower than thatcould occur by virtue of the ability of plasmalogen phospholipids previously reported in rabbit [36] and human kidney [27]. The lowto modulate the activity of integral membrane proteins such as content of plasmalogens in rat tissues has also been found in manysurface membrane ion pumps and channels [30]. other organ systems [27]. The species and organ-related differ- Our studies show that linoleate and AA are the predominant ences in plasmalogen content are greatest for choline as com-fatty acids esterified to the sn-2 position of CGP and EGP. The pared to ethanolamine glycerophospholipids. In addition, thereesterified linoleate was found predominantly in diacyl phospho- may be profound differences in the distribution of plasmalogenslipid species in both CGP and EGP. Our study demonstrates that and diacylglycerophospholipids in different subcellular membranearachidonate, however, is found esterified to both choline and compartments. The high vinyl ether content of membrane phos-ethanolamine diacyl and plasmalogen molecular species. It is very pholipids seems to be a ubiquitous characteristic of electricallysignificant that plasmalogens accounted for 33% of the total active tissues such as heart and brain [9, 12]. Their presence inarachidonylated CGP mass and 51% of the total arachidonylated Portilla and Creer: Proximal tubule plasmalogens 1093

EGP mass, despite the fact that plasmalogens represent approx-suggest that accumulation of lysoplasmalogens may also represent imately 10% and 36% of the total CGP and EGP mass, respec-an early event in hypoxic cell injury in the proximal tubule. tively. Thus, plasmalogens are highly enriched in esterified AA as Several studies implicate changes in calcium homeostasis as an compared to diacyl phospholipids. The relative enrichment of AAimportant contributor to the pathogenesis of cell injury during in proximal tubule plasmalogen phospholipids suggests that thishypoxia in the proximal tubule [28, 291. This presumably reflects phospholipid pool may be an important source of AA for thethe effects of calcium on enzymes which catalyze the hydrolysis of generation of biologically active eicosanoids. membrane phospholipids. Recent studies in isolated rabbit Several studies, using in vivo and in vitro models of ischemic cellproximal tubules subjected to ATP depletion in high calcium injury in the kidney have investigated the effects of ischemia/medium have shown gradual increases in intracellular calcium hypoxia on various end points of phospholipid turnover. Theassociated with substantial polyphosphoinositide hydrolysis specific end points previously examined include measurement ofand total disintegration of cell structure. Decreases in the mass total phospholipid mass, lysophospholipid production and freeof phospholipids other than polyphosphoinositides were mod- fatty acid accumulation. In these studies, lysophospholipid pro-est; however, palmitic, linoleic, stearic and arachidonic acid duction and free fatty acid accumulation are relatively earlywere increased significantly suggesting that hydrolysis of di- events, occurring within 15 to 20 minutes after the onset ofverse phospholipids was indeed occurring to some degree. This hypoxia/isehemia [3, 31, 321. Depletion of cellular phospholipids,observation further corroborates the notion that measurements however, did not occur until the ischemic/hypoxic interval hadof the total mass of individual phospholipid classes does not been extended for greater than 60 minutes [32]. Since theprovide a sensitive index of phospholipid hydrolysis as opposed lysophospholipid production and fatty acid accumulation reflectto fatty acid release or measurements of the mass of individual accelerated phospholipid catabolism, the inability to detect aphospholipid molecular species. In the study of Garza-Ouin- measurable decrease in total phospholipid mass may relate to thetero et al [291, lowering medium calcium to 100 n preserved relative insensitivity of depletion of total phospholipid mass as athe integrity of mitochondria and brush border microvilli but measure of phospholipid turnover, Indeed, Humes et al [321, didwas still associated with profound changes in the fragmentation not find phospholipid depletion even when the hypoxic intervalof endoplasmic reticulum, Golgi apparatus, peroxisomes and was extended to 60 minutes in rabbit proximal tubules, despitelysosomes. In addition, lowering calcium did not prevent cell marked increases in fatty acid production which were evident atdeath. Our present results demonstrating the selective turnover earlier time points. Our results confirm the observations ofof plasmalogens that is temporally correlated with the activa- Humes et al [32], showing no change in the total phospholipidtion of calcium-independent plasmalogen selective PLA2 [14] mass in either CGP or EGP after 10 and 20 minutes of hypoxia.suggests that plasmalogens may contribute to those changes However, when we analyzed the turnover of individual phospho-[29] which did not require increased intracellular calcium and lipid molecular species, we noted a statistically significant turn-which were also accompanied by lethal cell injury. We propose over of those molecular species containing esterified arachidonatethat accelerated hydrolysis of plasmalogens contributes to the in both ethanolamine and choline glycerophospholipids. Thechanges seen under conditions which did not require increased hydrolysis of CGP and EGP during hypoxia was highly selectiveintracellular calcium, Proof of this hypothesis will require for plasmalogen molecular species containing esterified AA (thatfurther studies to characterize the subeellular distribution of is, 16:0, 20:4 PlasCho and 18:1, 20:4 PlasEth) and was detectedplasmalogen phospholipids in proximal tubular cells and to within the first 10 minutes of hypoxic injury. The amount ofdetermine whether increased hydrolysis of plasmalogen phos- phospholipid hydrolyzed from these two molecular species con-pholipids contributes directly to morphological and functional taining arachidonate (approximately 1300 pmol/mg protein) ischanges in those membrane compartments enriched in plas- more than sufficient to account for the observed release of AAmalogen molecular species. (approximately 92 pmol/mg protein) from isolated tubules in the first 10 minutes of hypoxia (Table 3). Acknowledgments The diacyl phospholipids containing esterified AA did not This work was supported by a grant from the National Institutes of change significantly. Accordingly, our results demonstrate that inHealth, R29 DK46914. The authors would like to thank Aditi Duffield and the proximal tubule there is a selective turnover of arachidony-Janet Jones for their technical assistance and Ellen Satter for her lated plasmalogen phospholipid molecular species which occurssecretarial assistance. early in the course of hypoxic cell injury. Recently, we have found that a significant fraction of phospho- Reprint requests to Didier Portilla, MD., University of Arkansas for Medical Sciences, Department of Medicine, Division of Nephrology, 4301 W. lipase A2 activity in the proximal tubules is calcium-independentMarkham, Slot 501, Little Rock Arkansas 72205-7199, USA. and selective for plasmalogen substrates [14]. 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