Renal cortical ceramide patterns during ischemic and toxic injury: assessments by HPLC-mass spectrometry

Thomas Kalhorn and Richard A. Zager

Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington 98109


    ABSTRACT
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INTRODUCTION
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RESULTS
DISCUSSION
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Ceramides are a class of signaling molecules that can acutely accumulate in tissues as part of a "stress response." They are classically measured by the diacylglycerol kinase assay, which, in general, measures total ceramide rather than individual moieties within the diverse ceramide family. The present study was undertaken to 1) adapt current HPLC-mass spectrometry technology for measuring individual renal ceramides, and 2) use this technique to more fully characterize the nature of the renal ceramide "stress" reaction. Renal cortical tissues were obtained from CD-1 mice under control conditions and 2 or 18 h after renal injury (ischemia-reperfusion and glycerol-mediated myohemoglobinuria). C24, C22, and C16 ceramides were identified in normal renal cortex, constituting 70, 10, and 20% of the total ceramide pool, respectively. Within each of these families, heterogeneity was apparent because of differing degrees of unsaturation (0-3 double bonds) in the constituent fatty acid of ceramide. Renal injury dramatically changed ceramide profiles: 1) total ceramide increased by ~300%; 2) although all ceramides participated in this reaction, they did so to differing degrees; 3) this caused pronounced changes in ceramide distribution patterns; 4) injury induced a striking shift toward unsaturated (vs. saturated) fatty acids within the C22 and C24 (but not the C16) ceramide pools; and 5) the extent of these qualitative changes differed according to the etiology of the initiating renal damage. Thus we conclude that ceramide stress response involves major qualitative (and not simply quantitative) changes in ceramide expression that are partially disease dependent. These findings underscore the fact that simply measuring total renal ceramide content (e.g., by diacylglycerol kinase assay) substantially oversimplifies the nature and, hence, the potential implications of the ceramide stress reaction.

acute renal failure; stress response; myoglobinuria; glycerol; ischemia; sphingomyelin; high-performance liquid chromatography


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE PAST 10 years, ceramide has come under increasing scrutiny as an important signaling molecule, potentially initiating apoptosis, growth inhibition, and a prodifferentiated state (8, 9, 16, 18, 19, 27, 37). Structurally, ceramide is composed of a long-chain aliphatic amine, sphingosine, coupled to a single fatty acid via amide linkage. Because variable-length fatty acids, with differing degrees of unsaturation, can be linked to sphingosine, the term "ceramide" refers to a class of molecules rather than one specific entity. In addition to their important modulating effects on cell homeostasis, ceramides are also pivotal substrates for the synthesis of sphingomyelin and hundreds of cell surface glycosphingolipids. Via the action of ceramidases, they are also sources for sphingosine and sphingosine 1-phosphate, additional signaling molecules (11, 20, 24, 25). In composite, these facts support the concept that alterations in ceramide expression could have important protean effects on cell homeostasis.

Helping to illustrate the above principle are observations that diverse forms of physiological stress, imposed on highly diverse cell types, can evoke rapid ceramide accumulation, potentially impacting cell survival (13, 29, 33). Consequently, ceramide is now widely regarded as a "stress response" molecule (4, 7, 10, 12, 23), in a sense analogous to the heat shock proteins. Recent observations from our laboratory indicating that diverse forms of renal "stress" (ischemia-reperfusion, myohemoglobinuria, acute glomerulonephritis, and fluorinated anesthetic exposure; see Refs. 15, 34, 35) evoke rapid renal cortical ceramide accumulation underscore the relevance of this response to the in vivo kidney. Parallel observations have been made in cultured porcine (LLC-PK1) and human (HK-2) proximal tubular cells (29, 33). This indicates that in vivo renal ceramide accumulation is an intrinsic cellular response rather than one stemming from systemic cytokine stimulation (e.g., by tumor necrosis factor-alpha or interleukin-1beta ; e.g., see Ref. 21). The exact biochemical pathways leading to ceramide accumulation have not been completely defined. However, sphingomyelinase-mediated sphingomyelin hydrolysis (14, 15), de novo ceramide synthesis (3, 29), and decreased ceramide catabolism (33) have each been implicated.

In virtually all studies of this area, ceramide has been quantified by the diacylglycerol (DG) kinase assay (22, 31). This method takes advantage of the fact that when cell extracts are incubated with bacterial DG kinase plus [32P]ATP, phosphorylation of ceramide, and not just DG, occurs. In a sense then, this assay is made possible by the relative nonspecificity of the bacterial DG kinase used in the reaction. Once formed, 32P-labeled ceramide and 32P-labeled DG are separated by TLC, allowing for their individual quantitation. Although this assay permits an approximation of ceramide content, at least two significant caveats exist: first, high tissue DG levels (e.g., as could occur with cell injury; see Ref. 17) could partially mask the magnitude of ceramide accumulation because of DG-vs.-ceramide competition in the DG kinase reaction (31); and second, it is virtually impossible to separate the various ceramides from one another by TLC (e.g., see Fig. 2 in Ref. 15). Hence, total ceramide, rather than individual ceramides, is typically reported. Because it is likely that different constituent fatty acids impact the biological effects of ceramide, it could be important to characterize and quantitate which particular ceramides participate in this stress reaction.

Given the burgeoning interest in the biological effects of ceramide, and in light of the limitations inherent to the current DG kinase assay, we have applied recent advances in mass spectrometry (MS) for the purpose of quantifying both total and individual ceramides within renal cortex. Having established this methodology, we have used it to 1) define which particular ceramides compose the renal cortical ceramide pool (an issue that, to our knowledge, has never been addressed), and 2) ascertain the magnitude and the pattern of renal cortical ceramide expression in response to ischemia-reperfusion and nephrotoxic renal injury. As a result of these studies, the potential utility and the limitations of the currently used DG kinase assay can be better assessed.


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General Procedures: Tissue Procurement and Extraction

The animal protocols, which are described in detail in Animal Protocols, were conducted on male CD-1 mice (25-50 g; Charles River Laboratories, Wilmington, MA) housed under routine vivarium conditions. They were maintained under pentobarbital anesthesia (~2 mg, administered ip) and then subjected to a midline abdominal incision for the purpose of renal and at times hepatic tissue extraction. Immediately after being harvested, the tissues were iced and ~50 mg were homogenized for ~30 s in four parts of methanol. The homogenates were then subjected to chloroform-methanol extraction according to the method of Bligh and Dyer (2), as previously employed in this laboratory (36). The chloroform phase was dried under nitrogen and stored at -20°C until the time of ceramide analysis. One part of each extract was saved for inorganic phosphate analysis according to the method of Van Veldhoven and Mannaerts (30).

HPLC-MS

The residue obtained from the above extraction procedure (representing 50 mg of tissue) was reconstituted in 450 µl of MeOH and 50 µl of an internal standard solution containing 1 nmol N-acetylsphingosine (C2:0 ceramide, no. 110145; Calbiochem, San Diego, CA) in MeOH. The resulting suspension was sonicated at room temperature for 3 min to complete dissolution/extraction. Fifty microliters of this solution were transferred to a 13 × 100-mm culture tube and evaporated to dryness. The nonvolatile fraction was dissolved in 0.5 ml of methylene chloride and applied to a 200-mg silica solid-phase extraction (SPE) column (no. 304159; Alltech, Deerfield, IL) that had been previously conditioned with 2.0 ml of methylene chloride. The initial sample was pulled through the column under light vacuum. The original culture tube was rinsed with an additional 0.5 ml of methylene chloride, which was also applied to the SPE column. Nonpolar cellular lipids (e.g., triacylglycerides and fatty acid esters) were eluted to waste with an additional 1.0 ml of methylene chloride. The ceramide fraction was then eluted with two 2.0-ml aliquots of 30% isopropanol in methylene chloride. The eluant was collected and evaporated to dryness under nitrogen at room temperature.

Samples were analyzed by HPLC-MS on the basis of modifications of the method of Couch et al. (5). In brief, samples were reconstituted in 200 µl of MeOH, vortexed thoroughly, and transferred to injection vials. Ceramide separation was achieved by applying 6-µl samples to a Zorbax 2.1 × 150-mm C-8 5 µ dp column (Hewlett Packard, Palo Alto, CA) on a Hewlett Packard series 1100 liquid chromatography (LC)-MS system. This used an isocratic mobile phase of 97% MeOH-3% 30 mM aqueous acetic acid at a flow rate of 0.2 ml/min. All solvents used were of analytic (Optima) grade and were obtained from Fisher Scientific (Pittsburgh, PA). Ceramide and sphingomyelin standards were obtained from Sigma (St. Louis, MO) and Biomol (Plymouth Meeting, PA), respectively.

Ceramides were detected with the use of an atmospheric pressure chemical ionization interface. Positive ions were acquired in either the scanning (100-1000 atomic mass units) or single-ion monitoring (SIM) mode. The MS was set up with the use of nitrogen as both the drying (3.0 l/min, 350°C) and the nebulizing (60 psi) gas. The vaporizer was maintained at 400°C and the capillary at 2.5 kV. The corona current was set at 4.0 µA. The fragmentor voltage was varied from 60 to 180 V depending on the type of experiment being run. For quantitative SIM work, it was maintained at 80 V. These evaporation/ionization conditions were adjusted to maximize the signal resulting from the loss of one molecule of water from the parent plus H+ ion. During quantitative analysis, ion abundances at mass-to-charge ratios (m/z) of 324.3, 518.5, 520.5, 602.6, 604.6, 626.6, 628.6, 630.6, and 632.6 were recorded with the use of high SIM resolution and a dwell time of 196 µs.

To standardize for possible interassay differences in recovery, 3 samples/day were run with and without the addition of exogenous C16 ceramide (100 pmol, in 50 µl MeOH). This standard was added to the tissue extract/internal standard solution before evaporation and SPE. The response factor was taken as the mean of the C16:0 signal in samples plus the standard minus the C16:0 signal in the sample alone. All other signals were then normalized with the use of this C16:0 response factor.

Assessments of Assay Performance

First, limit of quantification was taken as the amount of injected C16:0 that resulted in a signal of >5× background with a coefficient of variation (CV) of <10% (n = 5). Linearity was determined to 5 pmol of C16:0 applied to the column. Second, recovery studies were conducted by comparing the response of C16:0 ceramide (100 pmol) before and after the sample work up. All runs were done in quadruplicate. Third, reproducibility was confirmed by running three random samples through the entire extraction/analytic processes five times each. Whereas the recovery studies focused only on the C16:0 standard, the reproducibility studies included analysis of all ceramide peaks.

Qualitative Ceramide Analysis

Standards for individual physiological saturated and unsaturated ceramides are not available. Hence, identification of individual ceramides was based on their chromatographic behavior and mass spectral characteristics. The rationale for this approach was as follows.

Chromatographic behavior. First, retention times of derivatized fatty acids during reverse-phase HPLC increase with the number of carbons and decrease with the degree of unsaturation (1, 28). This was consistent with the chromatographic behavior of the putative ceramides in the kidney cortex samples, as the retention times decreased from C24:0 to C16:0 ceramide and from C24:0 to C24:3 ceramide. Second, an employed C18-C24 ceramide mixture, obtained from bovine brain (Sigma no. C-2137), also contained small amounts of the same ceramides detected in mouse kidney test samples.

Mass spectral analyses. The observed peaks were indicative of ceramide structures, as follows. 1) The even parent and base m/z signal indicated an odd number of nitrogens in the compound. 2) The molecular weights were 0.5-0.6 atomic mass units greater than the molecular weight calculated by using integral atomic weights, consistent with 60-90 hydrogens/molecule. 3) The relative intensity of the m+1 peaks were consistent with 30-60 carbons. 4) Changing fragmentor voltage resulted in ions that were consistent with previously characterized ceramide fragmentation patterns (6) (Fig. 1). Decreasing voltage resulted in a parent peak; conversely, increasing fragmentation generated a peak consistent with dehydrated sphingosine (m/z 264; and no sphinganine). 5) A stock solution of sphingomyelin, extracted from bovine brain (Sigma no. C2137), showed essentially no ceramide response.


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Fig. 1.   Full-scanning mass spectra of N-palmitoylsphingosine (C16 ceramide) at fragmentor settings of 80 (A), 140 (B), and 180 V (C). All conditions were as presented in METHODS. m/z, mass-to-charge ratio.

Animal Protocols

Two-hour ischemia-reperfusion injury protocol. Four mice were subjected to a midline laparotomy and right nephrectomy. The left renal pedicle was isolated and then occluded with a microvascular clamp for 45 min. The vascular clamp was then removed, permitting a 75-min vascular reperfusion period. Throughout these procedures, body temperature was carefully monitored and maintained at 36-37°C. On completion of the reflow period, the left kidney was removed and the cortex was isolated by dissection on an iced plate with a razor blade. It was then subjected to lipid extraction, as noted above. Kidneys extracted from mice not subjected to ischemia-reperfusion (I/R) injury provided control tissue samples.

Eighteen-hour I/R injury protocol. Four mice were subjected to unilateral nephrectomy and left renal ischemia, as noted above. After vascular clamp removal, the mice were sutured and allowed to recover from anesthesia while body temperature was maintained at 36-37°C. Approximately 18 h later, the mice were reanesthetized with pentobarbital, a 0.5-ml blood sample was obtained from the abdominal vena cava [for blood urea nitrogen (BUN) determination as an index of renal failure], and then the postischemic kidneys were removed. The cortices were separated and subjected to lipid extraction. Kidneys resected from normal mice and right nephrectomized kidneys obtained at the time of the initial surgical procedures were used to establish normal ceramide concentrations.

Glycerol-induced myohemoglobinuria: 2-h injury phase. Four mice maintained under normal vivarium conditions were anesthetized with pentobarbital and subjected to intramuscular glycerol injection (10 ml/kg body wt; administered in 2 equally divided doses into the upper hindlimbs), inducing myohemoglobinuria (32). They were then maintained at a 36-37°C body temperature for 2 h post-glycerol injection. Finally, they were given an additional dose of pentobarbital, the abdomen was opened through a midline abdominal incision, and one kidney from each mouse was removed and processed for ceramide analysis. Four kidneys obtained simultaneously from normal mice were used to establish normal ceramide concentrations.

Glycerol-induced myohemoglobinuria: 18-h injury phase. Four mice were injected with glycerol, as noted above, and then allowed to recover from anesthesia. Free food and water access was provided. Approximately 18 h post-glycerol injection, the mice were reanesthetized, a 0.5-ml blood sample was obtained for BUN analysis, and then the left kidneys were removed for ceramide assay. Four kidneys obtained from normal mice were used to determine normal ceramide concentrations.

Comparison of ceramide profiles in kidney vs. liver. The following experiment was undertaken to ascertain whether the pattern of epithelial cell ceramide expression is, in part, organ specific. To this end, three mice were anesthetized, and then the kidneys and livers were removed. Renal cortical and hepatic samples were then processed and analyzed simultaneously for ceramide content.

Ceramide Levels in Isolated Proximal Tubules Treated With Sphingomyelinase

Because mouse renal ceramides are not commercially available, the following experiment was undertaken to substantiate that the ceramide peaks reported from whole renal cortex by MS were, in fact, ceramides rather than some unidentified substance with similar LC-MS characteristics. Toward this end, three normal mice were anesthetized, and cortical proximal tubule segments were isolated, as routinely performed by this laboratory (36). Each of the three tubule preparations were divided into two aliquots and incubated for 30 min under control oxygenated conditions with or without exogenous bacterial sphingomyelinase (0.2 units/ml, SE-108; Biomol). At the completion of the incubations, the tubules were subjected to lipid extraction (36) and then analyzed for ceramides, as noted above.

Calculations and Statistics

The ceramide levels were calculated as picomoles per nanomoles of phospholipid phosphate. All values are given as means ± 1 SE. Statistics were performed by unpaired Student's t-test. The Bonferroni correction was applied if multiple comparisons were made.


    RESULTS
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HPLC-MS Characteristics

A representative total ion chromatogram showing the separation of the different ceramides is presented in Fig. 2. Single ion scans of renal cortical extract are shown in Fig. 3. The ions correspond to the following ceramides detected in renal cortex (values in parentheses are m/z): C16:1 (518.5), C16:0 (520.5), C22:1 (602.6), C22:0 (604.6), C24:3 (626.6), C24:2 (628.6), C24:1 (630.6), and C24:0 (632.6). Extensive work was conducted searching for other ceramides ranging in molecular weight from C14:2 to C26:0 with the use of appropriate SIM programs designed to detect the p-18 signal characteristic of ceramide MS. No other signals consistent with ceramides could be found within the kidney cortex samples (see Fig. 3 for representative mass spectral analyses). Fragmentation of each ceramide peak generated sphingosine (but not sphinganine) derivatives, supporting the identity of a ceramide as the base compound.


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Fig. 2.   A total ion chromatogram showing separation of different ceramides. Components are as follows: 3.14 min, C2:0 (internal standard); 3.78 min, C16:1; 4.21 min, C16:0; 5.52 min, C24:3; 6.26 min, C22:1 and C24:2; 7.17 min, C22:0 and C24:1; 7.6 min, C24:1; and 8.8 min, C24:0.



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Fig. 3.   Single-ion monitoring chromatograms of ceramides from mouse renal cortex processed 2 h post-glycerol injection. From top to bottom: m/z 324 = C2:0 (internal standard), 518.5 = C16:1, 520.5 = C16:0, 602.6 = C22:1, 604.6 = C22:1, 626.6 = C24:3, 628.6 = C24:2, 630.6 = C24:1 (2 species), and 632.6 = C24:0.

Standard curves showed excellent linearity to 30 fmol applied to the column. The response was linear to 5 pmol. This was ~5× that required for individual ceramide detection; hence, no effort was made to work beyond this concentration.

The recovery of exogenously added C16:0 ceramide from kidney samples was 101.5 ± 3%. The analysis showed excellent reproducibility, with the average CV for all ceramides being 3.98 ± 4%.

Ceramide Levels in Normal Renal Cortex

Normal renal cortex had an overall (total) ceramide concentration of ~4.5 pmol/nmol phosphate (Fig. 4). Three major ceramide pools, as denoted by fatty acid length, were observed: C16, C22, and C24 ceramides, composing ~20, 10, and 70% of the total ceramide content, respectively (Fig. 5). The amounts of individual ceramides within each of these families are given in Table 1. Two distinct C24:1 ceramides were detected, the difference presumably being due to the position of the single double bond.


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Fig. 4.   Absolute amounts of C16, C22, and C24 ceramides (cer) in normal mouse cortex and in mouse cortex 2 h after glycerol (glyc)-induced myohemoglobinuria or ischemia-reperfusion (I/R) injury (45 and 75 min, respectively). Ceramide values are given as pmol/nmol phospholipid phosphate. Totals of either C16, C22, or C24 ceramides and overall total of all of ceramides (total cer) are presented. Individual ceramide concentrations are given in Table 1. Both forms of injury caused a significant increase of each ceramide class compared with normal control (c) values. Comparisons between 2 injury models are presented as P values above bars; NS, not significant. * P < 0.01 compared with control tissues.



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Fig. 5.   Percent contribution of either C16, C22, or C24 ceramide to total ceramide pool after 2 h of injury. Both forms of injury caused a relative increase in C16 ceramide expression and a relative decrease in C24 expression compared with ceramide distribution in normal kidney. These changes were significantly exaggerated in I/R- vs. glycerol-mediated injury. * P < 0.01 vs. normal control tissue.


                              
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Table 1.   Individual ceramide concentrations in renal cortex after I/R- or glycerol-induced injury

Ceramide Levels 2 h Post-Renal Injury

Total ceramide levels. As shown in Fig. 4, total renal cortical ceramide concentrations were dramatically increased by 2 h post-glycerol injection or postinduction of I/R injury. There was no significant difference in the degree of total ceramide elevations induced by the two injury models (both with ~3- to 4-fold increments). Each of the major ceramide pools (C16, C22, and C24) and individual ceramides within each pool significantly contributed to the overall ceramide increments (Table 1).

Relative changes in ceramide expression. As noted above, C16, C22, and C24 ceramides composed ~20, 10, and 70% of the total ceramide content of normal kidneys, respectively. These percent distributions were profoundly altered by both glycerol- and I/R-mediated renal injury (Fig. 5). With both forms of injury, the most prominent change was a relative increase in C16 ceramide, with a corresponding relative decrease in C24 ceramide. The %C22 ceramide remained largely unaffected. The two injury models significantly differed in the magnitude of these percent changes, being more exaggerated with I/R vs. the glycerol-mediated injury (i.e., greater ratio of %C16 ceramide increments to %C24 decrements with I/R vs. glycerol treatment).

Relative changes in unsaturated vs. saturated ceramides in individual ceramide pools. Despite the striking injury-induced increments in total C16 ceramide, the relative proportion of unsaturated vs. saturated moieties within the C16 ceramide pool (i.e., the unsaturated-to-saturated fatty acid ratios) were not dramatically affected (Fig. 6). In contrast, both the C22 and C24 ceramide pools became highly enriched in unsaturated fatty acid moieties with both glycerol and I/R injury (Fig. 6; increase in unsaturated-to-saturated ceramide fatty acid ratios).


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Fig. 6.   Ratio of unsaturated to saturated fatty acid (unsat'd/sat'd) moieties within each of major ceramide pools as observed 2 h after tissue injury. Both forms of injury caused a significant shift from saturated to unsaturated C22 and C24 ceramides: * P < 0.01 compared with normal control tissues. Conversely, in the C16 ceramide family, this shift was not expressed: tau  P < 0.05 vs. controls.

Ceramide Levels 18 h Post-glycerol and -I/R Injury

Total ceramide levels. Total ceramide levels remained dramatically elevated 18 h after both glycerol and I/R injury (Fig. 7). Unlike the 2-h results described in Ceramide Levels 2 h Post-Renal Injury, there was a significant difference in total ceramide elevations between the two groups by 18 h postinjury (33% greater with I/R- vs. glycerol-mediated injury; P < 0.03). This difference predominantly reflected an increase in C16 ceramide, although a generalized increase in all of the ceramide pools was observed (Table 1). The higher total ceramide levels in the I/R vs. postglycerol group could not be linked to differences in the severity of renal injury, since comparable degrees of azotemia were observed in the two groups (BUN: I/R, 151 ± 8 mg/dl; glycerol, 158 ± 37; not significant).


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Fig. 7.   Absolute amounts of C16, C22, and C24 ceramides in normal mouse cortex and in mouse cortex 18 h after glycerol-induced myohemoglobinuria or I/R injury. See Fig. 4 legend for details. Both injury models caused significant increases in each of ceramides compared with control tissues: * P < 0.01. Differences between 2 injury models appear above bars.

Relative changes in ceramide expression. The same pattern of altered relative ceramide expression observed at 2 h post-renal injury was also apparent at the 18-h time point (increase and decrease in the %C16 and %C24 ceramides, respectively; Fig. 8). Unlike the 2-h findings, at 18 h the relative contribution of C22 ceramide was also perturbed, increasing from 10 to 14% with glycerol treatment.


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Fig. 8.   Percent contribution of either C16, C22, or C24 ceramide to total ceramide pool 18 h after renal injury. Both forms of injury caused a relative increase in C16 ceramide expression and a relative decrease in C24 expression compared with ceramide distribution in normal kidney. These changes were essentially identical to those observed at 2-h time point, as depicted in Fig. 5. * P < 0.01 vs. normal control tissue.

Relative changes in unsaturated vs. saturated ceramides in individual ceramide pools. The percent contribution of unsaturated vs. saturated fatty acid ceramides remained altered at the 18-h postinjury time point, with a persistent enrichment of unsaturated fatty acids in the C22 and C24 ceramide pools (Fig. 9). However, unlike the 2-h time point, at 18 h there were significant decrements in unsaturated ceramides within the C16 ceramide pool. This change was significantly greater with I/R- vs. glycerol-mediated injury.


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Fig. 9.   Ratio of unsaturated to saturated fatty acid moieties within each of major ceramide pools 18 h after tissue injury. Both forms of injury caused a significant shift from saturated to unsaturated C22 and C24 ceramides. Conversely, C16 ceramide showed an opposite shift, i.e., toward greater saturated fatty acid ceramide content. The latter change was significantly exaggerated in I/R vs. glycerol group. * P < 0.05 compared with normal ("c", control) tissue.

Renal vs. Hepatic Ceramide Composition

Total hepatic and renal cortical ceramide levels did not significantly differ (4.0 ± 0.3 vs. 4.66 ± 0.25 pmol/nmol phosphate, respectively). However, the relative ceramide distribution patterns were dramatically different (Fig. 10). The kidney was relatively enriched in %C16 ceramide, whereas the liver had a strikingly greater percentage of C22 ceramide (Fig. 10, left). Kidney also showed a relative predominance of unsaturated vs. saturated fatty acids compared with liver (Fig. 10, right).


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Fig. 10.   Comparison of renal vs. hepatic ceramide expression. As shown at left, liver contained a significantly lower percentage of C16 ceramide and a significantly higher percentage of C22 ceramide vs. kidney. As shown at right, liver had dramatically lesser amounts of unsaturated vs. saturated fatty acid ceramides compared with kidney. This was observed in each of ceramide pools.

Ceramide Levels in Isolated Tubules Exposed to Exogenous Sphingomyelinase

Each of the ceramides identified in renal cortex was also observed in the isolated tubule preparations (Table 2). Treatment with exogenous sphingomyelinase caused marked and statistically significant elevations of each of these ceramides (thereby providing confirmation of their nature/origin).

                              
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Table 2.   Ceramide concentrations in isolated proximal tubules with and without treatment with bacterial sphingomyelinase


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Because ceramides cannot be adequately separated or identified by either TLC or HPLC, quantitation of individual species within either normal or injured renal cortex has not been previously reported. The present study has taken advantage of recent advances in MS, coupled with HPLC, to provide insights into this issue. Using adaptations of a previously described HPLC-MS method (5), we have established a rapid and relatively simple approach for quantifying individual renal ceramides. This technique yields highly reproducible results (CV, ~4%), it does not require sample derivatization (e.g., benzoylation) (5, 26), and it eliminates the need for 32P, high amounts being required for the DG kinase assay (22, 31). In addition, MS eliminates the potential problem of DG-induced competitive inhibition during the DG kinase assay. This can be a particular problem under conditions in which accelerated DG generation or decreased DG metabolism are expected (17).

By using HPLC-MS, we have identified in renal cortex nine different ceramides on the basis of their chromatographic and spectral characteristics. These can be grouped into three families on the basis of their individual constituent fatty acid length. The C24 family clearly predominates, constituting ~70% of the total ceramide pool. C16 and C22 ceramides make up the remainder, contributing 20 and 10%, respectively. Although we cannot definitively exclude the possibility that other ceramides might also be present (e.g., C18 ceramide), the extreme sensitivity of MS detection should ensure that if others do exist in renal cortex, they presumably do so in only trace amounts. Within each of these three major ceramide pools, considerable heterogeneity exists, based on differing degrees of fatty acid unsaturation. For example, total C16, C22, and C24 ceramides contained 24, 40, and 66% unsaturated (vs. saturated) fatty acids, respectively. In addition, the pattern of ceramide expression appears to be highly organ specific. For example, C22 ceramide and saturated C24 ceramide were observed to be far more pronounced in liver compared with renal cortex.

It has previously been suggested that when cells are subjected to diverse forms of stress, ceramide accumulation can result. The relevance of this ceramide stress response to the in vivo kidney was first documented by observations from this laboratory that showed dramatic ceramide accumulation after ischemic, toxic, and immunologic renal injury (15, 34, 35). Given the apparent ubiquitous nature of this cellular stress response, we now have attempted to better characterize it in the in vivo kidney by HPLC-MS. Four noteworthy observations have emerged from these investigations.

1) The magnitude of total ceramide accumulation appears to be far more dramatic than we previously reported with the use of the DG kinase assay (34, 35). In those studies, we observed ~50-100% ceramide increments after I/R- and glycerol-mediated renal injury. However, in the present study, ~300% increases were consistently noted. Because all of these studies have employed the same experimental protocols, it appears that these quantitative differences likely stem from DG-induced competitive inhibition of ceramide detection during the DG kinase assay (31). No such interference exists by MS. Hence, the present studies suggest that HPLC-MS detection is likely to provide more accurate quantitation of total ceramide content.

2) Ceramide elevations after renal injury do not simply result from an increase in a single ceramide pool; rather, they stem from a broad-based reaction to which each individual ceramide contributes (see Table 1). However, the extent to which each contributes differs substantially. This produces marked perturbations in relative ceramide profiles. For example, as noted in the RESULTS, C16 and C24 ceramides compose ~20 and ~70% of the normal renal cortical ceramide pool, respectively. However, 2 h after I/R injury, this difference is largely obliterated, with C16 and C24 ceramide now contributing almost equally (~45%). In contrast, the relative contribution of C22 ceramide to total ceramide remains relatively constant (~10%) despite an overall increase in its absolute concentration. These shifting profiles indicate that simply measuring total renal cortical ceramide levels grossly oversimplifies the ceramide changes that occur.

3) Different forms of renal injury may affect the pattern of ceramide expression, independent of total ceramide accumulation. For example, after 2 h of I/R- or glycerol-mediated injury, essentially identical total ceramide elevations were observed. However, far greater %C16 increments (and %C24 decrements) were seen with I/R- compared with glycerol-mediated injury. These same findings were also apparent 18 h after injury, suggesting that they do not simply represent a transient biological event. It is noteworthy that I/R and glycerol injection induced essentially identical degrees of azotemia. This implies that these different ceramide profiles likely stemmed from differences in the type of injury rather than the severity of injury per se.

4) The present study documents for the first time that a component of the acute ceramide stress response can be a shift from saturated to unsaturated ceramide fatty acid content. This change was observed with both injury models and at both time points, suggesting a generalized and durable response. Surprisingly, C22 and C24 ceramides, but not C16 ceramides, were involved. This suggests that this shift is a regulated phenomenon rather than a nonspecific injury response.

A key question emerging from this study is whether the consequences of ceramide accumulation after cell injury are determined, at least in part, by total ceramide, specific ceramide(s), or relative changes within the total ceramide pool. Unfortunately, these are exceedingly difficult, if not impossible, issues to resolve at this time because of an inability to pharmacologically manipulate individual ceramide levels. For example, one cannot simply add differing mixtures of ceramides to cells in culture to produce a specific ceramide profile, since long-chain (i.e., physiological) ceramides are relatively membrane impermeant, largely precluding cell uptake. Furthermore, although inhibitors of ceramide synthesis exist (e.g., fumonisin B1; see Refs. 14, 31), they presumably cause a generalized decrease in ceramide expression rather than a specific tailoring of the ceramide pool. Hence, although the present study clearly advances our understanding of ceramide expression in response to tissue injury, it also renders future exploration and interpretation of these changes far more complex.

In conclusion, the present study describes a reliable HPLC-MS method for quantifying ceramide profiles in renal cortex. Nine different ceramides have been identified, falling within one of three ceramide (C16, C22, C24) pools. Differing degrees of fatty acid unsaturation exist within each pool, producing further degrees of heterogeneity. Acute ischemic and toxic renal injury each induce striking increases in total cortical ceramide content, far more dramatically than previously appreciated in studies employing the standard DG kinase assay method. In addition to marked increases in absolute ceramide levels, tissue injury causes dramatic shifts in relative ceramide profiles within the total ceramide pool. The most notable of these changes are 1) a relative decrease in C24 ceramide, 2) a relative increase in C16 ceramide, and 3) a shift toward unsaturated (vs. saturated) fatty acids within the C22 and C24 ceramide pools. Documentation of these changes underscores the complexity of the ceramide stress response. This will undoubtedly further complicate efforts aimed at unraveling the biological influences of these enigmatic lipids.


    ACKNOWLEDGEMENTS

We thank Ali Johnson, Kristin Burkhart, and Ben Sacks for expert technical assistance with this project.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants RO1-DK-54200 and RO1-DK-38432.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. A. Zager, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Rm. D2-190, Seattle, WA 98109 (E-mail: dzager{at}fhcrc.org).

Received 16 March 1999; accepted in final form 18 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Physiol 277(5):F723-F733
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