Nitric oxide (NO) and oxygen-derived radicals such as superoxide (O2-) have been implicated in a wide range of physiological and pathophysiological processes as intracellular signaling molecules or toxic mediators, respectively1,2,3. It is well established that the balance between NO and O2- at sites of injury determines the net outcome and may direct cell responses toward resolution and repair of injury, or toward chronic disease4,5,6. Glomerular mesangial cells are specialized cells sharing a variety of smooth muscle cell properties, and under inflammatory conditions they also depict features of macrophages7. In contrast to other vascular endothelial cells, the microcapillary endothelial cells form a fenestrated monolayer that lines the glomerular capillaries and can easily be passed by circulating macromolecules as well as by immune cells. As elsewhere, the glomerular endothelial cells are directly exposed to all substances circulating in the blood stream, suggesting a key regulatory role of glomerular endothelial cells not only in normal glomerular physiology, but also in the pathophysiology of glomerular diseases. For example, in the course of developing glomerulosclerosis, endothelial cells die by massive apoptosis, which results in destruction of the glomerular capillary network and contributes to the onset of sclerosis8,9.
In cell culture glomerular endothelial cells also respond to inflammatory cytokines, like tumor necrosis factor-
, lipopolysaccharide and other stress factors, with increased rates of apoptosis10. Both glomerular endothelial and mesangial cells are able to produce reactive oxygen species (ROS)11,12,13, and it has been suggested that these autocoids can trigger cytotoxic as well as apoptotic responses thereby promoting the degradation of the basal membrane and accelerating the progression of glomerular diseases14,15.
Moreover, mesangial cells have also been reported to express an inducible NO synthase (iNOS) in response to interleukin-1
(IL-1)16,17 and cAMP stimulation17,18 with subsequent high output production of NO. It is noteworthy that coincubation of mesangial cells with NO and O2- in a balanced ratio produced cross-protection against the toxicity by each compound individually, suggesting that the simultaneous generation of NO relative to O2- reflects a protective principle by antagonizing the destructive capacity of individually acting radicals5,19.
The sphingolipid ceramide is another molecule that has gained a lot of interest as a possible regulator of apoptosis20,21,22,23. It is generated by the action of either an acid or neutral sphingomyelinase, which hydrolyzes the membrane constituent sphingomyelin and thereby releases ceramide and the headgroup phosphocholine. Recently, NO has been reported to trigger ceramide production in glomerular mesangial and endothelial cells24,25. Furthermore, we previously showed that superoxide is also a potent inducer of ceramide formation in glomerular endothelial cells26. Our current study demonstrates that costimulation of glomerular endothelial cells with NO and O2-, which generates peroxynitrite, leads to a potentiation of ceramide formation. In contrast, costimulation with NO and O2- in mesangial cells causes a decreased ceramide formation as compared to the NO or O2- stimulations alone. The levels of ceramide correlate well with the extent of apoptosis observed under the various conditions. Most importantly, the interaction of NO and O2- critically depends on the cell-type involved thus illustrating the role of not yet fully defined protective principles in the microenvironment of cells.
METHODS
Chemicals
(Z)-1-[2-(2-Aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (Deta-NO), 3-morpholinosydnonimine (SIN-1) and 2,3-dimethoxy-1-naphthoquinone (DMNQ) were from Alexis Corporation (Läufelfingen, Switzerland); glucose oxidase was from Calbiochem-Novabiochem (Schwalbach, Germany); [14C]serine (specific activity, 53 Ci/mol) was from Amersham Pharmacia Biotech Europe GmbH (Freiburg, Germany); the DNA fragmentation enzyme-linked immunosorbent assay (ELISA) was from Roche Diagnostics; fetal calf serum (FCS) was from Hyclone (Erembodegem-Aalst, Belgium). All other cell culture nutrients were from Life Technologies (Karlsruhe, Germany).
Cell culture
Bovine glomerular endothelial cells were cultivated as described27. Individual clones of endothelial cells were characterized by positive staining for Factor VIII-related antigen and uniform uptake of fluorescent acetylated low-density lipoproteins. Negative staining for smooth muscle actin and cytokeratin excluded mesangial cell and epithelial cell contaminations. Cells were utilized at passages 5 to 19. Rat mesangial cells were cultivated and characterized as described previously28. In a second step, single cells were cloned by limited dilution on 96-well plates. Clones with apparent mesangial cell morphology were characterized by positive staining for the intermediate filaments desmin and vimentin, which is considered to be specific for myogenic cells, positive staining for Thy 1.1 antigen, and negative staining for Factor VIII-related antigen and cytokeratin, excluding endothelial and epithelial contaminations, respectively. For the experiments passages 8 to 20 were used.
Lipid extraction and ceramide quantitation
Confluent cells in 30-mm diameter dishes were labeled for 24 hours with [14C]-serine (0.2 
Ci/mL) and stimulated as indicated. Lipids were extracted according to the method established by Bligh and Dyer29, and ceramide was resolved by sequential one-dimensional thin-layer chromatography (TLC) using chloroform/methanol/ammonia (65:35:7.5; vol/vol) followed by chloroform/methanol/acetic acid (9:1:1; vol/vol). Spots corresponding to ceramide were analyzed and quantitated using a Phosphoimager (Fuji, Tokyo, Japan).
Apoptosis assay
Confluent endothelial cells in 60 mm-diameter dishes were incubated with the indicated stimuli in DMEM containing 1% FCS for the indicated time periods. Thereafter, oligonucleosomal DNA fragmentation, a characteristic biochemical feature of apoptotic cell death, was measured using a nucleosome DNA enzyme-linked immunosorbent assay (ELISA; Roche Diagnostics, Mannheim, Germany), which quantitatively records histone-associated DNA fragments.
Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA). For multiple comparisons with the same control group, the limit of significance was divided by the number of comparisons according to Bonferroni using using GraphPad InStat version 3.00 for Windows NT (GraphPad Software, San Diego California USA).
RESULTS
In view of our previous finding that nitric oxide (NO) can induce ceramide production in mesangial and endothelial cells24,25, and the fact that NO reacts with superoxide (O2-) to form peroxynitrite, a substance potentially showing cytotoxic effects30, we studied the combination of one NO donor and an O2--generating substance. As shown in Figure 1a, costimulation of glomerular endothelial cells with the NO donor spermine-NO and the O2- producing redox cycler DMNQ caused a synergistic increase in ceramide generation when compared to NO donor or DMNQ stimulation alone, suggesting that the peroxynitrite formed is more active than either NO or O2- alone in triggering ceramide formation in endothelial cells. This finding is in contrast to the situation in glomerular mesangial cells, as in those cells DMNQ dose-dependently reduced NO-induced ceramide formation, although DMNQ alone evoked a marked ceramide generation Figure 1b.
Figure 1.
Effect of nitric oxide on DMNQ-induced ceramide formation in glomerular endothelial cells (A) and mesangial cells (B). (A) Confluent [14C]serine-labeled glomerular endothelial cells were stimulated for 24 hours with either vehicle (—) or spermine-NO (0.5 mmol/L) in the presence of the indicated concentrations of DMNQ (in mol/L). (B) Confluent [14C]serine-labeled glomerular mesangial cells were stimulated for 24 hours with either vehicle (—) or spermine-NO (1 mmol/L) in the presence of the indicated concentrations of DMNQ. Thereafter, lipids were extracted and [14C]ceramide was analyzed as described in the Methods section. Results are expressed as % of control values and are means 
SD (N = 2 to 4). *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference compared to the DMNQ- or spermine-NO–stimulated values, respectively.
Furthermore, 3-morpholinosydnonimine (SIN-1), a substance that simultaneously releases NO
and superoxide (O2-) and thus generates peroxynitrite31, also stimulated ceramide production in glomerular endothelial cells Figure 2a whereas it failed to do so in mesangial cells Figure 2b.
Figure 2.
Effect of SIN-1 on ceramide formation in endothelial cells (A) and mesangial cells (B). Confluent [14C]serine-labeled glomerular endothelial cells (A) or mesangial cells (B) were stimulated for 24 hours with either vehicle (Co) or the indicated concentrations of SIN-1. Thereafter, lipids were extracted and [14C]ceramide was analyzed as described in the Methods section. Results are expressed as % of control values and are means SD (N = 2–4). ***P < 0.001, statistically significant difference compared to the unstimulated control.
Since O2- can dismutate in aqueous solution to H2O2, we evaluated whether the effects seen with ROS were related to H2O2 formation. Addition of exogenous H2O2 as well as incubation of endothelial cells with glucose oxidase, an endogenous H2O2-generating system, led to a dose-dependent increase in ceramide levels in endothelial cells Figure 3a. Again, mesangial cells differ in this respect from endothelial cells in that H2O2 did not evoke a significant increase in ceramide production Figure 3b, whereas glucose oxidase triggered a moderate increase in ceramide levels, which was much less than the levels observed in endothelial cells Figure 3b.
Figure 3.
Effect of H2O2 and glucose oxidase on ceramide formation in endothelial cells (A) and mesangial cells (B). Confluent [14C]serine-labeled glomerular endothelial cells (A) or mesangial cells (B) were stimulated for 24 hours with either vehicle (Co) or the indicated concentrations of H2O2 and glucose oxidase, as indicated. Thereafter, lipids were extracted and [14C]ceramide was analyzed as described in the Methods section. Results are expressed as % of control values and are means SD (N = 2–4). *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference compared to the unstimulated control.
When oligonucleosomal DNA fragmentation, a typical feature of programmed cell death, was measured, SIN-1 caused a concentration-dependent increase in DNA fragmentation in glomerular endothelial cells, whereas mesangial cells were resistant to SIN-1 Figure 4a. In addition, costimulation of endothelial cells with Deta-NO and DMNQ caused a synergistic increase in apoptosis Figure 4b which again contrasts to the situation previously reported for mesangial cells, as in these cells DMNQ dose-dependently reduced NO-induced apoptosis5,19.
Figure 4.
Effect of simultaneous nitric oxide (NO) and superoxide (O2-) generation reactive oxygen species (ROS) on DNA fragmentation of glomerular endothelial cells and mesangial cells. (A) Confluent endothelial cells (
) or mesangial cells () were stimulated for 24 hours with either vehicle (CO) or SIN-1 (in mmol/L). (B) Confluent endothelial cells were stimulated with either vehicle (—) or Deta-NO (1 mmol/L) in the presence of the indicated concentrations of DMNQ. Thereafter, DNA fragmentation was measured as described in the Methods section. Results are expressed as % of control values and are means SD (N = 3 to 6). *P < 0.05, **P < 0.01, ***P < 0.001, statistically significant difference compared to the unstimulated control value (A) or to the Deta-NO–stimulated value (B).
DISCUSSION
Reactive oxygen species are commonly generated during normal metabolism when oxygen is reduced by leakage out of electrons from the electron transport chain during respiration in mitochondria. The metabolism of ROS in a cell is very complex and involves several O2- synthesizing and eliminating enzymes, which are not only found in mitochondria, but also in various other cell compartments. The enzymes participating in ROS metabolism comprise plasma membrane NADPH-oxidase, cytoplasmic xanthine oxidase, peroxisomal cytochrome P-450 oxidases and perinuclear cyclooxygenases, just to name a few. In addition, other enzymes like superoxide dismutase, catalase or myeloperoxidase produce O2--derived ROS, like hydrogen peroxide, hydroxyl radicals and hypohalous acid, that exert diverse biological effects11,12,32,33,34, including damage of DNA, RNA, proteins and lipids, which finally can lead to cell death. Especially in vascular endothelial cells, reactive oxygen and nitrogen species have been implicated in the pathogenesis of atherosclerosis and hypertension35,36,37,38.
Previously we showed that ceramide is an important mediator of NO- as well as superoxide-induced apoptosis in glomerular endothelial and mesangial cells24,25,26. Mechanistically, we showed that NO caused the activation of the ceramide generating enzymes, the neutral and acidic sphingomyelinases, which was paralleled by an inhibition of the ceramide-degrading enzymes, the neutral and acidic ceramidases, thus resulting in a drastic and long-lasting up-regulation of ceramide levels24.
Our current study reports that NO and superoxide act additively in endothelial cells to induce ceramide formation Figure 1a as well as apoptosis Figure 4. Coincubation of NO and superoxide will rapidly lead to the generation of peroxynitrite (ONOO-), and evidence has shown that peroxynitrite is cytotoxic and induces DNA damage and apoptosis30,39,40. By contrast, a balanced and simultaneous generation of NO and O2- is nondestructive for glomerular mesangial cells or macrophages, and it seems that signaling cascades initiated as a consequence of the NO/O2--interaction redirect apoptosis-inducing signals toward cell protection5,41,42,43. In line with this hypothesis, SIN-1, which simultaneously releases NO and superoxide and thus leads to a continuous generation of peroxynitrite, also causes a dose-dependent increase in ceramide Figure 2a and apoptosis Figure 4a in endothelial cells, but lacks effects on mesangial cells.
A similar differential action of peroxynitrite was reported by Pfeifer and Meyer, who found that only exogenous peroxynitrite caused tyrosine nitration, whereas simultaneous NO plus O2- generation did not44, suggesting that a low steady-state concentration of peroxynitrite is inefficient in tyrosine nitration45. Furthermore, peroxynitrite was shown to act in a cardioprotective manner in vivo43, whereas in an in vitro system, it exerted a rather deleterious effect46.
Mechanistically, the cytotoxic action of ONOO- is still poorly defined but may involve lipid peroxidation47, oxidation of sulfhydryls48 or inactivation of enzymes in the mitochondria electron transport chain49. In contrast to NO, ONOO- causes tyrosine nitration, which thereby inhibits enzyme activities50,51. Recently, it was shown that nitration of tyrosine inhibits the phosphorylation of the nitrated tyrosine residue52, thus disrupting the signaling cascades.
To counteract the damaging effect of oxidative stress, cells have developed two important defense mechanisms: (1) a thiol reducing buffer containing small proteins with a redox-active sulfhydryl group, such as glutathione and thioredoxin, and (2) enzymatic systems that decompose ROS, including superoxide dismutase, catalase and glutathione peroxidase. In this context it is worth noting that exposure of mesangial cells to NO leads to the activation of genes encoding a heterogenous panel of protective antioxidant defense enzymes including copper/zinc superoxide dismutase53, heme oxygenase-142 and the inhibitor of apoptosis protein family54, which certainly serve to protect mesangial cells against the imbalanced formation of cytotoxic mediators [reviewed in55. It is tempting to speculate that one or more of these enzymes are differentially regulated in mesangial cells and endothelial cells.
Whether or not the modification of certain functional residues of proteins in the sphingolipid signaling cascade by peroxynitrite contributes to its apoptotic effect is presently unclear, but certainly deserves further investigation.
Besides superoxide26 and peroxynitrite Figure 4, hydrogen peroxide also induces ceramide formation and apoptosis in endothelial cells Figure 3a. Again, mesangial cells were rather resistant to hydrogen peroxide treatment as compared to endothelial cells with regard to ceramide formation Figure 3b and apoptosis.
Whether the net effect of redox signals is either beneficial or deleterious will be determined by the amount and duration of mediator formation and the microenvironment of a specific cell type, particularly its antioxidant capacity. Understanding the cell type-specific stress response and defense mechanisms will help to delineate optimized therapeutic strategies for the treatment of inflammatory kidney diseases.
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Acknowledgments
This work was supported by grants of the Deutsche Forschungsgemeinschaft (HU-842/2-2, PF 361/1-1 and SFB 553), the "Stiftung VERUM für Verhalten und Umwelt," the August-Scheidel Stiftung and a "Nachwuchsstipendium" by the University Hospital Frankfurt am Main.
