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(The FASEB Journal. 1999;13:869-882.)
© 1999 FASEB

Research Communications

Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats

STEFAN FRANK*, KAI ZACHAROWSKI{dagger}, GILLIAN M. WRAY{dagger}, CHRISTOPH THIEMERMANN{dagger} and JOSEF PFEILSCHIFTER*1

* Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany; and
{dagger} William Harvey Research Institute, St. Bartholomew's and The Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1 6BQ, United Kingdom

1Correspondence: Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der JWG-Universität Frankfurt/M., Theodor-Stern-Kai 7, D-60590 Frankfurt/M., Germany. E-mail: Pfeilschifter{at}em.uni-frankfurt.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To define the mechanism of nitric oxide (NO) action in the glomerulus, we attempted to identify genes that are regulated by NO in rat glomerular mesangial cells. We identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced in these cells by treatment with S-nitroso-glutathione as a NO-donating agent. Bacterial lipopolysaccharide (LPS) acutely decreased Cu/Zn SOD mRNA levels. The LPS-mediated decrease in Cu/Zn SOD is reversed by endogenously produced NO, as LPS also induced a delayed strong iNOS expression in these cells in vitro, which is accompanied by increased Cu/Zn SOD expression. NO dependency of Cu/Zn SOD mRNA recovery could be demonstrated by inhibition of this process by L-NG-monomethylarginine, an inhibitor of NOS enzymatic activity. To demonstrate the in vivo relevance of our observations, we have chosen LPS-treated rats as a model for induction of a systemic inflammatory response. In these animals, we demonstrate a direct coupling of Cu/Zn SOD expression levels to the presence of NO, as Cu/Zn SOD mRNA levels declined during acute inflammation in the presence of a selective inhibitor of iNOS. We propose that the up-regulation of Cu/Zn SOD by endogenous NO may serve as an adaptive, protective mechanism to prevent the formation of toxic quantities of peroxynitrite in conditions associated with iNOS induction during endotoxic shock.—Frank, S., Zacharowski, K., Wray, G. M., Thiemermann, C., Pfeilschifter, J. Identification of copper/zinc superoxide dismutase as a novel nitric oxide-regulated gene in rat glomerular mesangial cells and kidneys of endotoxemic rats.


Key Words: gene expression regulation • glomerular mesangium • superoxide anion • endotoxemia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
REACTIVE OXYGEN SPECIES (ROS)2 have been implicated in the pathogenesis of several inflammatory renal diseases, including acute renal failure, renal graft rejection, and different types of immune-mediated glomerulonephritis (1, 2) . Central to the latter process is the mesangial cell, which is responsible for structural and functional integrity of the glomerulus under physiological conditions and functions as a source of secreted inflammatory mediators, including cytokines, and the autacoid nitric oxide (NO) and superoxide anions (O2.-) under pathological conditions (3, 4) . Mesangial cells do not only produce, but also respond, to ROS by decreasing the glomerular filtration rate in pathological situations (5) . Another key player during the process of acute and chronic inflammation is NO. The mode of action of NO is clearly dependent on the microenvironment surrounding the source of NO formation, as the toxic potential of NO is determined through its concentration. High amounts of NO are released from the inducible NO synthase (iNOS) isoform in response to inflammatory stimuli from a variety of cell types (6) . In addition to their ability to produce ROS, mesangial cells are capable of expressing the iNOS on stimulation with tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin-1ß (IL-1ß), and thus produce tremendous amounts of NO (7, 8) .

Cells have to stringently control intracellular O2.- pools to prevent generation of intermediates that are even more reactive than their precursors, especially hydroxyl radicals (OH.). The fastest diffusion-limited reaction of O2.- with a clear toxicological significance occurs with NO leading to the formation of highly reactive peroxynitrite (ONOO-) (9, 10) . ONOO- represents a potent oxidant that readily nitrates tyrosine residues in proteins (10 11 12 13) . For those reasons, eukaryotic cells dismutate any O2.- formed by superoxide dismutase (SOD), which are present at high concentrations throughout the cell (14) . As NO is the only target for O2.- that reacts fast enough to compete with SOD, cells avoid the formation of toxic ONOO- concentrations through high SOD levels and activity. Inflammatory conditions, however, can lead to markedly elevated NO and O2.- concentrations, which result in ONOO- formation rather than dismutation of O2.- by SOD (15) .

Mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of NO during TNF-{alpha}, IL-1ß, and LPS-triggered inflammatory conditions in the glomerulus (7, 8, 16 17 18 19) , but only little is known about the mechanism by which mesangial cells cope with the problem of potential ONOO- formation and toxicity. In this study we have analyzed the LPS-mediated regulation of manganese superoxide dismutase (Mn SOD) and copper/zinc superoxide dismutase (Cu/Zn SOD) in rat renal mesangial cells and whole kidney homogenates from LPS-treated rats, used as an in in vivo model for acute, systemic inflammation. Our data provide strong evidence for the dependence of ongoing Cu/Zn SOD expression on continuously high production rates of NO during endotoxic shock. This may constitute an important protective feedback mechanism to prevent deleterious formation of ONOO- and OH. radicals resulting from high output NO generation during inflammatory conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Surgical procedure
This study was carried out on male rats (Tuck, Rayleigh, Essex, U.K.) weighing 240–320 g and receiving a standard diet and water ad libitum. The investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London.

All animals were anesthetized with thiopentone sodium (Intraval Sodium, 120 mg/kg intraperitoneal) and anesthesia was maintained by supplementary injections of thiopentone sodium [~1–2 mg/kg/h intravenous (i.v.) as required]. The trachea was cannulated to facilitate respiration and rectal temperature was maintained at 37°C with a homeothermic blanket (BioScience, Sheerness, Kent, U.K.). The left carotid artery was cannulated and connected to a pressure transducer (Senso-Nor 840, Senso-Nor, Horten, Norway) to measure phasic and mean arterial blood pressure and heart rate, which were displayed on a data acquisition system (MacLab 8e, ADINtsruments, Hastings, U.K.) installed on an Apple Macintosh computer. The femoral vein was cannulated for the administration of drugs. The bladder was also cannulated to collect urine. On completion of the surgical procedure, cardiovascular parameters were allowed to stabilize for 15 min.

Experimental design
After recording baseline hemodynamic parameters, animals received Escherichia coli lipopolysaccharide (LPS, 10 mg/kg i.v., n=30) as a slow injection over 15 min. In addition, one group of rats, which had received vehicle rather than LPS (sham-operated animals, n=15), were also studied. Then, all animals received a continuous infusion of saline (6 ml.kg-1.h-1 i.v.). At 2 h after the injection of LPS, this infusion of saline was either maintained (control, n=15) or replaced by an infusion of the selective iNOS inhibitor L-N6-l (iminoethyl) lysine dihydrochloride (L-NIL) (3 mg/kg/h, n=15) (20) . These infusions of L-NIL were preceded by bolus injections of 3 mg/kg i.v. The dose of L-NIL used here is sufficient to abolish the rise in nitrite and nitrate caused by endotoxin (within 6 h) in the rat (21) . Three animals subjected to one of the above treatment regimes were killed by an overdose of anesthetic at 0.5 h, 1 h, 2 h, 4 h, or 6 h after injection of LPS or vehicle (n=3 per time point). Subsequently, the right kidney was removed, snap frozen in liquid nitrogen, and stored at -70°C until used for RNA isolation. All animals received a total fluid replacement of 4 ml/kg/h (as an i.v. infusion into the femoral vein throughout the experiment).

Sodium thiopentone (Intraval Sodium) was obtained from Rhone Merieux Ltd. (Harlox, Essex, U.K.) and L-NIL was obtained from Alexis Corporation (Nottingham, U.K.). Bacterial LPS (E. coli serotype 0.127:B8) was from Sigma (Poole, Dorset, U.K.).

Cell culture and treatment of mesangial cells
Rat glomerular mesangial cells were cultured and cloned as described previously (22) . Cells were grown in RPMI 1640 supplemented with 10% (v/v) fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml), and bovine insulin at 0.66 U/ml. Passages 10–18 were used. For the induction experiments, cells were grown to confluency without changing the medium and were rendered quiescent by a 24 h incubation in RPMI 1640 without serum. Cells were then incubated for varying periods in fresh RPMI 1640 containing 20% FCS, 10 ng/ml epidermal growth factor (EGF), 10 ng/ml transforming growth factor-ß2 (TGF-ß2), 10 ng/ml basic fibroblast growth factor (bFGF), 2 nM IL-1ß, 2 nM TNF-{alpha}, 250 µM S-nitroso-glutathione (GSNO), 500 µM GSNO, 50 µg/ml E. coli LPS, 2 mM NG-monomethyl-L-arginine.AcOH (L-NMMA), 1 mM N6,2'-O-dibutyryladenosine 3',5' cyclic monophosphate (dibutyryl-cAMP), 1 mM 8-bromoguanosine 3',5' cyclic monophosphate (8-bromo-cGMP), or 1 µM 1H- (1, 2, 4) oxodiazole (4,3-a) quionoxalin-1-one (ODQ), respectively. Cells were harvested at different time points after addition of these agents and used for RNA isolation. Each experiment was repeated at least twice. FCS and DMEM were purchased from Life Technologies, Inc. (Eggenstein, Germany), growth factors and cytokines were from Boehringer Mannheim Biochemicals (Mannheim, Germany), LPS and bovine insulin were from Sigma Biochemicals, and L-NMMA was from Alexis Corporation (Grünberg, Germany).

RNA isolation and RNase protection analysis
RNA isolation was performed as described (23) . Twenty micrograms of total RNA from the different experimental time points of the cell culture experiments were used for RNase protection assays. RNase protection assays were carried out as described (24) . All protection assays were performed with at least three different sets of RNA from independent experiments.

Probe DNAs
The rat Cu/Zn SOD cDNA probe was cloned by polymerase chain reaction, using 5'-GCT GAA GGG CGA CGG TCC GGT-3' as a 5'-primer and 5'-TCT TGT TTC TCG TGG ACC ACC-3' as a 3'-primer. The amplified cDNA fragment corresponds to nucleotides 20 - 370 of the published sequence (25) . Furthermore, we cloned a cDNA fragment of rat Mn SOD by polymerase chain reaction using 5'-GTC GCT TAC AGA TTG CCG CCT GC-3' as a 5'-primer and 5'-CTA CTA CAA AAC ACC CAC CAC GG-3'as a 3'-primer. The cDNA fragment corresponds to nucleotides 481–731 of the published sequence (26) .

Preparation of tissue lysates
Kidneys from control rats and LPS-treated rats were frozen in liquid nitrogen. Total kidney samples were homogenized in 2x homogenizing buffer (1x homogenizing buffer: 20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM phenylmethylsulfonly fluoride, 15 µg/ml leupeptin). The tissue extract was cleared by centrifugation and the supernatant was diluted 1:1 with water. Cytoplasmic and particulate fractions of total kidney lysates were prepared by a single centrifugation step at 100.000 g for 60 min, using an Optima TLX Ultracentrifuge (Beckman, München, Germany). The amount of protein in the cytoplasmic fractions of the lysates was determined using the Bio-Rad protein assay (Bradford method).

Isolation of glomeruli from rat kidneys
Whole glomeruli were isolated from the kidneys of male Wistar rats using a well-characterized three-stage sieving technique. Kidneys were removed from anesthetized rats and the cortex separated and chopped into 2–3 mm3 pieces. These were rinsed three times using ice-cold Hank's buffered salt solution (HBSS, Life Technologies, Paisley, U.K.) and then filtered through three metal sieves of decreasing pore size. The tissue pieces were first passed through a 300 µM sieve to separate glomeruli from larger fragments of renal tubules, vasculature, and interstitium, then through a 150 µM sieve to separate glomeruli from smaller tubular fragments and strip away the Bowman's capsules from the glomeruli. Finally, the mixture was passed through a 75 µM sieve on which glomeruli were retained on the surface. The sieve was then inverted over a plastic tissue culture plate and trapped glomeruli were washed off using HBSS. The glomerular suspension was then centrifuged (5 min, 1500 rpm, 200 g) and the pellet was resuspended in HBSS. The glomeruli were washed two more times using centrifugation and resuspended as described above. The final pellet was resuspended in 1 ml HBSS, snap frozen in liquid nitrogen, and stored at -70°C until used for RNA isolation.

Preparation of cell lysates
Glomerular mesangial cells were grown to confluency in RPMI 1640 with 10% FCS without changing the medium. Cells were rendered quiescent by a 24 h incubation in serum-free RPMI 1640. Subsequently, cells were treated for varying time points by 250 µM or 500 µM GSNO, respectively. Aliquots of cells were harvested before and at different time points after treatment. Cells were washed twice with 1x PBS (3 mM KCl, 1.5 mM KH2PO4, 0.15 mM NaCl, 6 mM Na2HPO4) and subsequently scraped into 1x homogenizing buffer. The cytoplasmic fraction of cell lysates was prepared as described above.

Western blot analysis
Protein (50 µg) from cytoplasmic fractions of control cells and GSNO-treated cells, or 50 µg of protein from cytoplasmic fractions of rat control kidney and kidneys after 30 min, 1 h, 2 h, 4 h, or 6 h after LPS treatment were separated using SDS-gel-electrophoresis. After transfer to a PVDF membrane, Cu/Zn SOD protein was detected by using a polyclonal antiserum directed against human Cu/Zn SOD (Upstate Biotechnology, Lake Placid, N.Y.), and iNOS protein was detected using a polyclonal antibody raised against mouse iNOS (27, 28) . A secondary antibody coupled to horseradish peroxidase and the ECL detection system were used to visualize Cu/Zn SOD protein. Phenylmethylsulfonyl fluoride and leupeptin were supplied from Sigma Biochemicals (Deisenhofen), the antibody coupled to horse radish peroxidase was from Biomol (Hamburg), and the ECL detection system was obtained from Amersham (Braunschweig).

Trypan blue exclusion test for cell viability
Rat renal mesangial cells from the late experimental time points were trypsinized from the cell culture plates (1x trypsin/EDTA, Life Technologies, Inc., Eggenstein, Germany), and diluted in fresh RPMI 1640. One part of the mesangial cell suspension was mixed with one part of 0.4% trypan blue (Life Technologies) and incubated for 3 min at room temperature. Subsequently, unstained (viable) and stained (nonviable) cells were counted by using a Neubauer chamber.

Nitrite determination
Nitrite, a stable NO oxidation product, was determined using the Griess reaction. Cell-free culture supernatants were collected (200 µl), adjusted to 4°C, and mixed with 20 µl sulfanilamide (dissolved in 1.2 M HCl) and 20 µl N-naphtylethylenediamine dihydrochloride. After 5 min at room temperature, the absorbance was measured at 540 nm, with a reference wavelength at 595 nm. Sulfanilamide and N-naphtylethylenediamine dihydrochloride were from Sigma Biochemicals, Inc. (Deisenhofen, Germany).

Statistical analysis
Data are shown as means ± SD The data are presented either as x-fold induction or as percent change compared with the control (100%). For each reagent in each set of conditions, data were analyzed by Student's t test by using the software SigmaPlot (Jandel Scientific, Erkrath, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of Cu/Zn SOD mRNA and protein expression by GSNO
We have previously demonstrated a substantial release of NO by rat glomerular mesangial cells after stimulation with TNF-{alpha} or IL-1ß (7, 8) . Due to the potency to generate large amounts of NO in vitro, NO release from mesangial cells has been suggested to contribute to the tissue injury seen in certain types of glomerulonephritis (16, 29) . Therefore, we were interested in NO-mediated gene expression in mesangial cells to determine potential players that might be involved in the progress of inflammatory processes in the kidney. Using cultured mesangial cells as an in vitro system, we found expression of the Cu/Zn SOD isoenzyme to be under stringent control of exogenously added NO. As shown in Fig. 1 A, basal levels of Cu/Zn SOD mRNA were detected already in quiescent mesangial cells. On addition of increasing amounts of the NO donor GSNO (250 µM, 500 µM), a strong induction of Cu/Zn SOD mRNA was observed. Maximal stimulation of Cu/Zn SOD expression was obtained at 250 µM of GSNO. Higher concentrations of the NO donor (500 µM) did induce Cu/Zn SOD mRNA levels, but the viability of cells was decreased due to induction of apoptosis by high concentrations of GSNO, as reported previously (30) . The effect of NO on Cu/Zn SOD expression was transient, and elevated Cu/Zn SOD mRNA levels returned to basal levels after 24 h. The same pattern of induction was seen after addition of spermine NONOate, a second NO-donating agent (data not shown). By contrast, Fig. 1C shows that expression of the second intracellular SOD isoenzyme, the mitochondrial Mn SOD, is not stimulated by exogenously added NO. These data clearly demonstrate that NO specifically induces Cu/Zn SOD.



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  		Figure 1. Induction of Cu/Zn SOD mRNA and protein expression by NO in cultured rat mesangial cells. A) RNase protection assay demonstrating the induction of Cu/Zn SOD mRNA expression by the NO-donating agent GSNO. Cells were rendered quiescent by serum starvation and stimulated with 250 µM or 500 µM GSNO for the indicated time periods. Samples of 20 µg of total cellular RNA were analyzed for Cu/Zn SOD mRNA expression. 1000 counts/min of the hybridization probe were used as a size marker. The concentration-dependent, GSNO-induced increase in Cu/Zn SOD mRNA as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gels is shown schematically in the right panel. Data are expressed as x-fold induction of unstimulated control. Mean percent change in Cu/Zn SOD mRNA levels ± SD are shown (n=3). *P<0.05; **P<0.01 compared with control. GSNO-stimulated expression of Cu/Zn SOD protein is shown in panel B. Serum-starved cells were harvested before and at different time points after treatment with GSNO as indicated. Cytoplasmic fractions of these cells were analyzed by immunoblotting for the presence of Cu/Zn SOD protein. Cu/Zn SOD protein of 15 kDa is indicated by an arrow. C) mesangial cells were rendered quiescent by serum starvation. They were stimulated with 250 µM or 500 µM GSNO for 1.5, 5, 8, or 24 h as indicated. 20 µg of total cellular RNA were analyzed by RNase protection analysis for Mn SOD mRNA expression. An ethidium bromide stain of 2 µg of analyzed RNA is shown below.

Cu/Zn SOD represents a homodimer consisting of subunits with a molecular weight of 15.7 kDa. To determine whether the observed induction of Cu/Zn SOD mRNA expression correlates with the induction of immunoreactive protein, we performed Western blot analysis of GSNO-treated cells (Fig. 1B ). In line with the kinetics of Cu/Zn SOD induction at the mRNA level, cells were harvested 6 h and 10 h after GSNO treatment. Fifty micrograms of total cytoplasmic protein was subsequently analyzed for the presence of Cu/Zn SOD protein by immunoblotting using an antiserum raised against human Cu/Zn SOD (see Materials and Methods). As shown in Fig. 1B , the antiserum detected only one band of Cu/Zn SOD protein with an estimated molecular mass of 15 kDa. Compared to basal Cu/Zn SOD protein expression, Cu/Zn SOD protein levels increased 3.2-fold and 4-fold after addition of 250 µM or 500 µM GSNO, respectively. Taken together, these data demonstrate that induction of Cu/Zn SOD mRNA by NO also correlates with induction Cu/Zn SOD protein.

Cu/Zn SOD mRNA levels are not regulated via growth factors and proinflammatory cytokines but are biphasically modulated by LPS
In a next step, we tested the potency of serum, purified serum growth factors, and proinflammatory cytokines to stimulate Cu/Zn SOD expression, as there were no data available concerning the regulation of Cu/Zn SOD expression in glomerular mesangial cells. As shown in Fig. 2 A, LPS was the only stimulus that resulted in an alteration of Cu/Zn SOD mRNA levels. Treatment of mesangial cells with LPS decreased Cu/Zn SOD mRNA levels by ~50–70% within 6 to 9 h. The decrease of Cu/Zn SOD mRNA levels was only transient, and Cu/Zn SOD mRNA levels started to increase again, reaching basal expression levels after 24 h. In addition to LPS, we tested serum, EGF, TGF-ß2, bFGF, IL-1ß, or TNF-{alpha} for their ability to alter Cu/Zn SOD expression in mesangial cells. In summary, none of these serum growth factors and proinflammatory cytokines modulated Cu/Zn SOD expression (data not shown). By contrast, Fig. 2B demonstrates a large induction of Mn SOD expression by LPS and by the proinflammatory cytokines IL-1ß and TNF-{alpha}. A 6- to 10-fold induction of Mn SOD mRNA was observed in mesangial cells stimulated with LPS or cytokines (Fig. 2B ), whereas serum and purified growth factors had only marginal effects on Mn SOD expression (data not shown). These data suggest that Cu/Zn SOD and Mn SOD expression is likely to be subjected to different regulatory mechanisms in glomerular mesangial cells.



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  		Figure 2. Regulation of Cu/Zn SOD and Mn SOD mRNA levels by LPS and inflammatory cytokines. A) Serum-starved mesangial cells were stimulated for different time periods with 50 µg/ml LPS. 20 µg of total RNA were analyzed by RNase protection analysis for the expression of Cu/Zn SOD. The changes in Cu/Zn SOD mRNA as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gels are shown schematically. Data are expressed as percent of unstimulated control (100%). Mean percent change in Cu/Zn SOD mRNA levels ± SD are shown (n=3). **P<0.01 compared with control. B) Serum-starved mesangial cells were stimulated for different time periods with 50 µg/ml LPS, 2 nM IL-1ß, or 2 nM TNF-{alpha} as indicated. 20 µg of total RNA were analyzed by RNase protection analysis for the expression of Mn SOD. The changes in Mn SOD mRNA as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gels are shown schematically below the autoradiograms. Data are expressed as x-fold induction of unstimulated control. Mean percent change in Mn SOD mRNA levels ± SD are shown (n=3). *P<0.05; **P<0.01 compared with control.

NO-mediated induction of Cu/Zn SOD expression is independent of activation of soluble guanylate cyclase
NO activates soluble guanylate cyclase (31, 32) , leading to an increase in the intracellular levels of cyclic GMP (cGMP), which is the second messenger for most of the physiological actions of NO. To investigate whether the NO-induced increase in Cu/Zn SOD expression is secondary to activation of soluble guanylate cyclase and hence cGMP, we treated cells with the membrane-permeable cGMP analog 8-bromo-cGMP (1 mM). We observed no alteration in Cu/Zn SOD mRNA expression levels by 8-bromo-GMP (Fig. 3, upper panel). Even a higher concentration of 8-bromo-cGMP (3 mM) did not result in increased Cu/Zn SOD mRNA levels (data not shown). Thus, the expression of Cu/Zn SOD caused by NO is independent on the formation of intracellular cGMP. Furthermore, 8-bromo-cGMP had an moderate effect on Mn SOD expression (Fig. 3 , upper panel), leading to a twofold induction after 24 h



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  		Figure 3. Induction of Cu/Zn SOD and Mn SOD mRNA expression by cyclic GMP, or cyclic AMP. Serum-starved mesangial cells were stimulated for 1.5, 5, 8, or 24 h with 1 mM cGMP or 1 mM cAMP, respectively. 20 µg of total cellular RNA were analyzed by RNase protection analysis for the expression of Cu/Zn SOD and Mn SOD mRNA. The degree of mRNA induction as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gels is shown schematically. Data are expressed as x-fold induction of unstimulated control. Mean percent change in Cu/Zn SOD and Mn SOD mRNA levels ± SD are shown (n=3). *P<0.05; **P<0.01 compared with control. Cu/Zn SOD or Mn SOD mRNA levels and the cyclic nucleotide used are indicated in the figure.

Mn SOD, but not Cu/Zn SOD expression is induced by cyclic AMP
In addition to the signaling pathways activated by growth factors and cytokines, activation of adenylate cyclase leading to an increase of intracellular cyclic AMP (cAMP) has been described as a second independent signaling pathway for iNOS and related enzyme induction in rat glomerular mesangial cells (33 34 35) . As shown in Fig. 3 , lower panel, Mn SOD mRNA levels are also strongly induced by 1 mM dibutyryl-cAMP, whereas Cu/Zn SOD expression is not influenced by this cAMP analog.

LPS-mediated decrease in Cu/Zn SOD expression levels is reversed by NO
Induction of iNOS and increased formation of NO by LPS has been demonstrated for mesangial cells (4, 34) . We speculated that the induction of iNOS and the subsequent production of endogenous NO modulates the levels of Cu/Zn SOD mRNA (Fig. 2A ), as exogenous NO induces Cu/Zn SOD expression in mesangial cells in vitro (Fig. 1) . To this end, we treated mesangial cells with LPS in the presence or absence of the specific NOS inhibitor L-NMMA. As shown in Fig. 4 , LPS treatment decreased Cu/Zn SOD mRNA levels by 70% within 9 h irrespective of the presence or absence of L-NMMA. Furthermore, Fig. 4A demonstrates that iNOS expression is strongly induced in mesangial cells by LPS, resulting in the release of large amounts of endogenously produced NO. This NO release could be measured as nitrite, a stable oxidation product of NO that accumulates in the cell culture supernatants after NOS action (Fig. 4E ). We hypothesized that if NO should trigger the recovery phase of Cu/Zn SOD mRNA, L-NMMA should prevent the reconstitution of basal Cu/Zn SOD mRNA levels after 24 to 30 h of LPS treatment. This was indeed the case as shown in Fig. 4 . The addition of L-NMMA weakened the initial increase in Cu/Zn SOD mRNA after 12 h and nearly completely inhibited up-regulation observed after 24 h, 27 h, and 30 h (Fig. 4B, C ). Cells remained fully viable (3.5% nonviable cells after 30 h, as measured using the trypan blue assay; see Materials and Methods) under these experimental conditions (data not shown). The inhibitory effect of L-NMMA on the recovery of Cu/Zn SOD mRNA levels is further strengthened by the fact that LPS-induced iNOS mRNA levels also decreased twofold in the presence of L-NMMA (data not shown), thus confirming our previous observation of an amplified NO-induced iNOS up-regulation in mesangial cells (36) . We then treated cells with LPS in the presence of 1 µM ODQ, a potent inhibitor of soluble guanylate cyclase. Recovery of Cu/Zn SOD mRNA levels after LPS treatment occurred equally in the presence or absence of 1 µM ODQ, indicating that NO signaling is not mediated via activation of soluble guanylate cyclase (Fig. 4A , panel 3). Furthermore, application of the specific iNOS inhibitor L-NIL in the presence of LPS also nearly completely inhibited the restoration of Cu/Zn SOD mRNA levels after 30 h (Fig. 4D ). In summary, these data provide strong evidence that expression of Cu/Zn SOD is controlled by NO in rat glomerular mesangial cells in a cGMP-independent manner.



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  		Figure 4. Transient decrease of Cu/Zn SOD mRNA expression in LPS-treated mesangial cells. A) Mesangial cells were rendered quiescent by serum starvation and subsequently treated with 50 µg/ml LPS. Total cellular RNA was harvested before and at different time points after LPS treatment in the presence or absence of 2 mM L-NMMA or after addition of 1 µM ODQ, as indicated. 20 µg of total RNA were analyzed by RNase protection assay for the presence of Cu/Zn SOD mRNA. The same set of total cellular RNA isolated from LPS-treated cells was used for analysis of iNOS mRNA levels (lower panel). Nitrite accumulation in the cell culture supernatants during the incubation period was measured as a readout of iNOS enzymatic activity (E). The changes in Cu/Zn SOD mRNA levels as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gels are shown schematically in panels B and C. Data are expressed as percent of unstimulated control (100%). Mean percent change in Cu/Zn SOD mRNA levels ± SD are shown (n=3). *P<0.05; **P<0.01 compared with control. #P<0.05; ##, P<0.01 compared with the conditions as indicated by the brackets. D) Serum-starved mesangial cells were treated with 50 µg/ml LPS in the presence or absence of the specific iNOS inhibitor L-NIL (2 mM) or ODQ (1 µM). Total cellular RNA was harvested for the indicated time points and analyzed by RNase protection assay for the presence of Cu/Zn SOD mRNA. One representative experiment is shown.

Rapid and transient decrease of Cu/Zn SOD expression in kidneys from endotoxemic rats
As a next step, we used a rat model of endotoxic shock to investigate whether NO also regulates the expression of Cu/Zn SOD in vivo. Figure 5 shows the kinetics of Cu/Zn SOD expression in total kidney homogenates of LPS-treated rats from three independent experiments. Treatment of rats with LPS led to a rapid decrease in Cu/Zn SOD mRNA (12.5-fold) and protein levels (5- to 10-fold) in whole kidney extracts (Fig. 5A ). Cu/Zn SOD mRNA levels declined rapidly to reach a minimum after 30 min, followed by reduced Cu/Zn SOD protein levels after 60 min, thus indicating a rapid turnover of this enzyme. The reduction in Cu/Zn SOD protein was only transient, as high levels of Cu/Zn SOD protein are restored after 2 h of LPS treatment. These data demonstrate that LPS treatment of rats transiently reduced Cu/Zn SOD levels in the kidney, confirming the results seen in mesangial cells in vitro. It is remarkable that the observed increases of Cu/Zn SOD mRNA and protein levels correlated with the induction of iNOS protein caused by LPS (Fig. 5A ). To confirm published observations and to complete our own data, we also measured Mn SOD mRNA levels in total kidney homogenates of LPS-treated rats. As shown in Fig. 5B , Mn SOD mRNA expression increased ~15-fold within 6 h after LPS, clearly indicating a differential regulation pattern for the O2.- dismutating enzymes in vivo, as already indicated in the cell culture experiments. As the necessary antibodies to Mn SOD are currently not commercially available, the kinetics of the expression of Mn SOD protein could not be determined.



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  		Figure 5. Effects of endotoxemia on the kinetics of expression of mRNA and protein of Cu/Zn SOD and mRNA of Mn SOD in the kidney (whole kidney extracts). A) 30 µg of total cellular RNA from kidneys obtained from rats treated with either vehicle (for LPS) or LPS at 30 min, 60 min, 2 h, 4 h, or 6 h after injection of LPS was analyzed by RNase protection analysis (upper panel). 50 µg of protein from isolated cytoplasmic fraction of total kidney homogenates obtained from rats treated with either vehicle (for LPS) or LPS at 30 min, 60 min, 2 h, 4 h, and 6 h after injection of LPS was analyzed by immunoblotting for the presence of Cu/Zn SOD protein. Cu/Zn SOD protein with an estimated mol wt of 15 kDa was detected and is indicated with an arrow. Three independent series of control rats and LPS-treated rats (n=3 for every data point) were used for analysis as indicated by #1, #2, and #3 with numbers. 100% refers to the expression level of Cu/Zn SOD in sham-operated animals that were not treated with LPS, as Cu/Zn SOD is constitutively expressed in the kidney. 50 µg of protein from the cytoplasmic fraction of total kidney homogenates was analyzed for the expression of iNOS protein by immunoblotting. The detected iNOS protein of ~130 kDa is indicated with an arrow (lower panel). B) 30 µg of total cellular RNA from kidneys obtained from rats treated with either vehicle (for LPS) or LPS at 30 min, 60 min, 2 h, 4 h, and 6 h after injection of LPS was analyzed for MnSOD mRNA levels by RNase protection analysis. An ethidium bromide stain of 2 µg of the same batch of RNA is shown in the lower panel. The kinetic alterations in the expression of mRNA and protein of Cu/Zn SOD (A) and in the expression of Mn SOD (B) were assessed by PhosphoImager (Fuji) and scanning densitometry, expressed as % of control (pre-LPS value), and shown in the right panels. For Mn SOD mRNA expression, data are expressed as x-fold induction of unstimulated control. Mean percent change in Mn SOD mRNA levels ± SD are shown (n=3). *P<0.05; **P<0.01 compared with control. Every data point depicted in this figure represents an individual experiment (animal). None of the results were pooled and the variability between experiments was relatively small.

Mn SOD expression is not modulated by NO in kidneys from endotoxemic rats
As shown in Fig. 6 A, the induction of Mn SOD mRNA expression strongly correlated with a large induction of iNOS mRNA in total kidney tissue. To analyze whether there is a functional link between NO formation and induction of Mn SOD expression in endotoxic shock, rats were treated simultaneously with LPS and L-NIL, a selective inhibitor of iNOS enzymatic activity (20, 21) . Figure 6B demonstrates that L-NIL did not alter the increase in Mn SOD mRNA levels observed in kidney after LPS treatment. Moreover, an early and strong induction of the inflammatory mediators IL-1ß and TNF-{alpha} occurred in parallel with the induction of Mn SOD mRNA levels (Fig. 6C ), confirming results obtained for mesangial cells in vitro.



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  		Figure 6. Induction of Mn SOD mRNA expression is not dependent on NO in LPS-treated rat kidney (whole kidney homogenates). Mn SOD and iNOS mRNA expression levels were analyzed by RNase protection analysis using total cellular RNA (30 µg) from kidneys obtained from rats treated with either vehicle (for LPS) or LPS in the presence (B, C) or absence (A) of L-NIL at 30 min, 60 min, 2 h, 4 h, and 6 h after injection of LPS. Every data point depicted in this figure is a representative example of three individual experiments (animals). C) The same batch of RNA used in panel A was analyzed for IL-1ß and TNF-{alpha} mRNA expression in LPS-treated rats and correlated to Mn SOD mRNA levels (upper panel).

Direct correlation between NO formation and Cu/Zn SOD expression levels in kidneys from endotoxemic rats
As a next step, we had to examine whether Cu/Zn SOD mRNA expression is controlled by NO formation in kidneys of rats with endotoxic shock. As already shown in Fig. 5A , there is a tight temporal correlation between elevated levels of Cu/Zn SOD and iNOS mRNA in rats treated in the absence of the selective iNOS inhibitor L-NIL (Fig. 7 A). Figures 7 B, C show two independent series of rats exposed to LPS in the presence of L-NIL (given 2 h after LPS). In both experiments, the recovery of Cu/Zn SOD mRNA levels after the initial LPS-mediated decrease is partially (Fig. 7C ) or fully prevented (Fig. 7B ) by the selective iNOS inhibitor L-NIL. There is considerable interindividual variability concerning the time course of iNOS induction in the rats with very early onsets of iNOS mRNA expression after 30–60 min (Fig. 7B ) or a more delayed type (2 to 4 h) of iNOS mRNA expression, as seen in Fig. 7C . This difference may explain why in Fig. 7B the initial LPS-induced decay in Cu/Zn SOD mRNA is only seen allusively and almost fully compensated for by the early increase in iNOS. By contrast, a clear down-regulation of Cu/Zn SOD mRNA is seen in Fig. 7C , where iNOS induction is delayed. Moreover, in both experiments, inhibition of iNOS by L-NIL (given as infusion starting at 2 h after LPS addition) abolished up-regulation of Cu/Zn SOD mRNA levels at later time points (4 h for Fig. 7B and 6 h for both experiments reported in Fig. 7B, C ).



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  		Figure 7. Cu/Zn SOD mRNA expression levels in LPS-treated rats correlate with induction of iNOS after LPS treatment. Cu/Zn SOD and iNOS mRNA expression levels were analyzed by RNase protection analysis using total cellular RNA (30 µg) from kidneys obtained from rats treated with either vehicle (for LPS) or LPS in the presence (B, C) or absence (panel A) of L-NIL at 30 min, 60 min, 2 h, 4 h, and 6 h after injection of LPS. Every data point depicted in this figure is a representative example of three individual experiments (animals).

Cu/Zn SOD expression by isolated glomeruli
Finally, we investigated Cu/Zn SOD expression patterns in isolated glomeruli obtained from LPS- or LPS/L-NIL-treated rats. The pattern of Cu/Zn SOD mRNA expression by glomeruli was determined to further strengthen the observed link between in vitro and in vivo data, since the mesangial cell represents a major cell type within the glomerulus. As shown in Fig. 8 , isolated glomeruli revealed a biphasic effect of LPS on Cu/Zn SOD expression. Notably, glomeruli isolated from LPS/L-NIL-treated rats were significantly lower in Cu/Zn SOD mRNA levels compared with glomeruli isolated from rats treated with LPS alone, thus supporting our hypothesis of a NO-dependent restoration of Cu/Zn SOD mRNA levels.



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  		Figure 8. Cu/Zn SOD mRNA expression by glomeruli isolated from LPS- or LPS/L-NIL-treated rats. Cu/Zn SOD mRNA expression levels were analyzed by RNase protection assay using total cellular RNA (10 µg) obtained from isolated glomeruli from rats treated with either vehicle (for LPS) (upper panel) or LPS in the presence of L-NIL (lower panels) at 30 min, 60 min, 2 h, and 5 h after injection of LPS. Every data point depicted in this figure represents an individual experiment (animal).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mesangium is a highly specialized pericapillary tissue that is involved in most pathological processes in the renal glomerulus. A very prominent proinflammatory feature of intrinsic mesangial cells is the secretion of a variety of mediators that is initiated by cross-communication with invading immune cells (4, 37) . Recently, we have demonstrated that the inflammatory cytokines TNF-{alpha} and IL-1ß induce the expression of iNOS in rat glomerular mesangial cells, resulting in the formation of large amounts of NO in vitro (7, 8) . A potential role for NO as a novel mediator serving the communication between cells by triggering cellular gene expression became more evident in the last few years (38 39 40) . Therefore, our interest focused on the identification of novel NO-regulated genes in mesangial cells. We have discovered that exogenous and endogenous NO (from iNOS) causes the expression of Cu/Zn SOD and, hence, that Cu/Zn SOD is a novel, NO-regulated gene in mesangial cells. It is remarkable that the induction of Cu/Zn SOD is clearly restricted to NO, as none of the many growth factors and inflammatory cytokines tested were able to induce Cu/Zn SOD expression. By contrast, Mn SOD expression is clearly induced by LPS, TNF-{alpha}, and IL-1ß in mesangial cells in vitro. Besides spatial separation of Cu/Zn SOD and Mn SOD, these findings clearly also implicate a functional separation of intracellular signaling pathways leading to induction of O2.- dismutating enzyme systems. Our data confirm and extend previously published data demonstrating an up-regulation of Mn SOD expression in rat glomerular mesangial and epithelial cells by inflammatory mediators such as heat-aggregated IgG, LPS, IL-1{alpha}, or IL-1ß (41, 42) . Another stimulus reported to increase Mn SOD expression in mesangial cells is exogenously added hydrogen peroxide (H2O2), indicating an important role for reactive oxygen species (ROS) in regulating SOD expression in mesangial cells (43) . It has been shown that the transcriptional induction of the Mn SOD gene caused by TNF-{alpha} is dependent on ROS-mediated activation of NF{kappa}B in lung-derived cell lines (44, 45) . In contrast to the expressional regulation of Mn SOD by inflammatory stimuli, Cu/Zn SOD is constitutively expressed in glomerular mesangial and epithelial cells and is not regulated by cytokines and growth factors (42, 43) .

We report here for the first time that LPS, which potently induces Mn SOD expression in mesangial cells, clearly decreases Cu/Zn SOD mRNA levels in the cells (Figs. 2 and 4) . This finding indicates a clearly separated and differential regulation of the cytoplasmic and mitochondrial component of the cellular O2.- dismutating enzyme system in mesangial cells. It is well established that inflammatory mediators like TNF-{alpha}, IL-1ß, and LPS trigger the formation of NO in mesangial cells by stimulation of iNOS expression (7, 34) . Furthermore, mesangial cells have been shown to express the functional LPS receptor CD14, and subsequent expression of iNOS is induced via activation of NF{kappa}B (46) . Our data clearly indicate that NO does not mediate the initial LPS-mediated reduction of Cu/Zn SOD mRNA levels; instead, NO compensates for the LPS-mediated decrease of Cu/Zn SOD mRNA levels, and exogenously as well as endogenously produced NO strongly up-regulates Cu/Zn SOD mRNA and protein (Figs. 1 and 4) . Most convincingly, the inhibitor of iNOS enzymatic activity (L-NMMA) almost completely blocked recovery of Cu/Zn SOD mRNA levels after 24–30 h of LPS treatment (Fig. 4) .

To evaluate the possible relevance of our in vitro data for an in vivo situation, we chose a rat model of septic shock induced by endotoxin application (47) . The finding that inhibitors of NOS activity attenuate the hypotension and vascular hyporeactivity to vasoconstrictor agents caused by endotoxin in animals (48 49 50 51) , together with the discovery that mice in which the iNOS gene has been inactivated by gene targeting (iNOS knockout mouse) exhibit only a minor fall in blood pressure when challenged with endotoxin (52, 53) , supports the hypothesis that enhanced NO production due to iNOS induction is largely responsible for the circulatory failure associated with septic shock. As we have identified Cu/Zn SOD to be regulated exclusively by NO in vitro, we wanted to gain insight into this novel regulatory mechanism in vivo. Remarkably, the main enzyme for dismutating O2.- in the cytoplasm, Cu/Zn SOD, is not regulated via inflammatory cytokines, but expression is likely to be triggered by NO after LPS treatment in vivo and counteracts LPS-induced acute down-regulation of Cu/Zn SOD (Fig. 7) . We have recently demonstrated that inhibition of iNOS activity with either L-NIL or 1400W attenuates the hypotension (circulatory failure), but not the renal, liver, or pancreatic injury caused by endotoxin in the rat (21) . As the enhanced formation of NO by iNOS is essential to trigger the expression of Cu/Zn SOD and selective inhibition of iNOS with L-NIL (20) prevents the recovery of Cu/Zn SOD in rats with endotoxemia (this study), we propose that inhibition of iNOS activity may impair the ability of tissues (such as the kidney) to dismutate superoxide anions. This may (in part) explain why inhibitors of iNOS activity improve blood pressure and organ perfusion pressure without preventing the development of tissue injury in endotoxemia. We believe that the NO-induced up-regulation of Cu/Zn SOD can be envisaged as a defense mechanism that serves to protect cells that simultaneously produce NO and O2.-, as mesangial cells do in an inflammatory environment. Increased levels of Cu/Zn SOD will dismutate the amplified production of O2.- and limit the cytotoxic activity of O2.- itself as well as the formation of peroxynitrite, a potentially harmful reaction product of O2.- and NO. We have recently shown that the simultaneous generation of NO relative to O2.- reflects a protective principle by antagonizing the destructive role of individually active radicals. However, a disturbance of the endogenous NO/O2.- balance (which does not need to be a 1:1 molar ratio) by an appropriate increase or decrease of only one radical induces mesangial cell apoptosis (28, 54) . NO-mediated Cu/Zn SOD expression might be a novel mechanism to protect cells and tissues against the imbalanced formation of cytotoxic mediators such as O2.- or ONOO- in conditions associated with local or systemic inflammation.


   ACKNOWLEDGMENTS
 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 553) and by grants of the Commission of the European Communities (Biomed 2, PL 90979) and the Paul and Ursula Klein-Stiftung. We thank C. Rordorf, Basel, for kindly providing recombinant IL-1ß and U. Messmer, Frankfurt, for providing freshly synthesized GSNO. C.T. is a Senior Fellow of the British Heart Foundation (FS/98015). We thank Nicole Kolb for her excellent technical assistance.


   FOOTNOTES
 
2 Abbreviations: bFGF, basic fibroblast growth factor; 8-bromo-cGMP, 8-bromoguanosine 3',5' cyclic monophosphate; Cu/Zn SOD, copper/zinc superoxide dismutase; dibutyryl cAMP, N6,2'-O-dibutyryladenosine 3',5' cyclic monophosphate; EGF, epidermal growth factor; FCS, fetal calf serum; GSNO, S-nitroso-glutathione; IL, interleukin; iNOS, inducible nitric oxide synthase; i.v., intravenous; L-NIL, L-N6-l (iminoethyl) lysine dihydrochloride; L-NMMA, NG-monomethyl-L-arginine.AcOH; LPS, lipopolysaccharide; Mn SOD, manganese superoxide dismutase; NO, nitric oxide; O2.-, superoxide anion; ODQ, 1H- (1, 2, 4) oxodiazole (4,3-a) quionoxalin-1-one; OH., hydroxy radical; ONOO-, peroxynitrite; ROS, reactive oxygen species; TNF, tumor necrosis factor.

Received for publication August 3, 1998. Revision received December 21, 1998.
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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