Increased nitric oxide formation in recurrent thrombotic microangiopathies: A possible mediator of microvascular injury

Increased Nitric Oxide Formation in Recurrent Thrombotic Microangiopathies: A Possible Mediator of Microvascular Injury Marina Noris, ChemPharmD, Piero Ruggenenti, MD, Marta Todeschini, Chemist, Marina Figliuzzi, BiolSciD, Daniela Macconi, BiolSciD, Carla Zoja, BiolSciD, Simona Paris, Chemist, Flavio Gaspari, ChemD, and Giuseppe Remuzzi, MD • The term thrombotic microangiopathy (TMA) has been used extensively to encompass hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, two syndromes of hemolytic anemia, and thrombocytopenia associated with renal or brain involvement or both. There is evidence that endothelial damage is a crucial feature in the sequence of events that precedes the development of microvascular lesions. More recent studies would suggest that endothelial dysfunction could be a consequence of neutrophil activation. Activated neutrophils generate superoxide anions (02-) that, combining with endothelial-derived nitric oxide (NO), form the highly cytotoxic hydroxyl radical. Seven patients with recurrent forms of TMA and seven healthy volunteers were studied. Plasma concentrations of the NO metabolites, nitrites/nitrates, were elevated in the acute phase of TMA, indicating an increased NO synthesis in vivo. In addition, elevated serum concentrations of tumor necrosis factor, a potent inducer of endothelial NO synthase, were found in acute TMA. Serum from patients with acute TMA induced NO synthesis in cultured endothelial cells more than normal serum. Enhanced stimulatory activity was no longer found in the recovery phase. Release of 02- by neutrophils ex vivo was higher than normal in patients with acute TMA, but decreased in the recovery phase. Exactly the same trend was observed for plasma malondialdehyde and conjugated dienes, indicating that excessive oxygen radical formation in acute TMA is associated with increased lipid peroxidation. Thus, in recurrent forms of TMA, NO formation was increased as compared with controls. This was associated with signs of lipid peroxidation, likely the consequence of the interaction of NO with neutrophilderived oxygen products. © 1996 by the National Kidney Foundation, Inc. INDEX WORDS: Nitric oxide; thrombotic microangiopathy; hemolytic uremic syndrome; thrombotic thrombocitopenic purpure; oxygen radicals; peroxynilbr~e; endothelial cells; neutrophils.

"EMOLYTIC uremic syndrome and throm,botic thrombocytopenic purpura are rare syndromes of microangiopathic hemolysis and thrombocytopenia with signs of renal and brain involvement secondary to widespread microthrombi and reactive endothelial proliferation.~ Recently the term thrombotic microangiopathy (TMA) has been revived to provide a natural link between the two syndromes and to encompass all of the above lesions. Classical and sporadic forms have a remarkably better prognosis than recurrent forms.~ The latter usually occur in families and have a very

H

From the Mario Negri Institute for Pharmacological Research, and the Division of Nephrology and Dialysis, Ospedali Riuniti di Bergamo, Bergamo, Rome, Italy. Received November 14, 1995; accepted in revised form January 29, 1996. Supported in part by a grant (93.00874.CT04)from the Consiglio Nazionale delle Ricerche, Rome, Italy. Presented in part at the 27th Annual Meeting of the American Society of Nephrology, Orlando, FL, October 26-29, 1994. Address reprint requests to Marina Noris, ChemPharmD, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. © 1996 by the National Kidney Foundation, Inc. 0272-6386/96/2706-000453.00/0

790

poor outcome, with end-stage renal failure or death in more than 50% of cases. ~ Because endothelial cell swelling and detachment are constant features of the syndrome, efforts in the past have concentrated on defining endothelial cell dysfunction in the hope to clarify the pathophysiology of microvascular lesions. ~ More recent studies focused on the possibility that endothelial damage could derive from neutrophil activation, with the consequent excessive release of neutrophil-derived enzymes. 2-4 Recent data that endothelial cells can be induced to form nitric oxide (NO) by bacterial and viral products 5'6 or by increased local levels of cytokines TM led us to consider NO as a potential mediator of vascular damage in this disease. NO has potent cytotoxic properties 5'7'9'~°per se that are further enhanced by its interaction with activated neutrophils. Specifically, NO interacts with neutrophil-derived superoxide anion (02-) to form peroxynitrite, a potent cytotoxin j ~.~2that is further transformed into a highly reactive hydroxyl radical ( H O - ) (Fig 1). j t.]2 We explored the possibility that in patients with chronic recurrent forms of TMA, an exaggerated formation of NO could increase in vivo lipid peroxidation, possibly as a consequence of NO interacting with neutrophil-derived products.

American Joumal of Kidney Diseases, Vol 27, No 6 (June), 1996: pp 790-796

NITRIC OXIDE IN THROMBOTIC MICROANGIOPATHY

~

'_~ ONOO-+H + peroxynltrite

-

~

ti with TMA, human umbilical vein endothelial cells ( '.(2) were exposed for 24 hours in vitro to serum (dill, 2 with phosphate-buffered saline) from patients or c( in the presence of 0.5 #Ci [3H]L-arginine (New Engh ,uclear, Boston, MA; 56.4 Ci/mmol). Incubation was stop~ ed by adding one volume of ice-cold 15% trichloroacetic acid ~nd NO synthesis was evaluated by measuring the conversi~m of [3H]L-arginine to [3H]L-citrulline. Results were expressed as pmoles/IOs cells by correcting data in counts per minute (CPM) for the specific activity of [~H]L-arginine, calculated on the basis of serum endogenous content of uarginine as measured by high-performance liquid chromatography (HPLC). 6 Because tumor necrosis factor a (TNFa) is a potent inducer of NO synthesis in endothelial cells,7 concentrations of TNFa were measured in the serum from patients and controls by using an enzyme-linked immunosorbent assay (Amersham, Buckinghamshire, UK; detection limit: 3 pg/mL). To evaluate whether in recurrent forms of TMA activated neutrophils release into the circulation highly reactive oxygen radicals, an additional aliquot of peripheral blood was drawn to assess ex vivo 02- production by polymorphonnclear cells. Plasma lipid peroxidation was assessed in patients with TMA and in controls by measurement of concentrations of malondialdehyde (MDA) using the thiobarbituric acid method.'5 Conjugated diene formation also was evaluated in plasma extracts (500 #L)) 6 The lipid residue was dissolved in 2.5 mL cyclohexane and the absorbance measured at 233 nm. Results were expressed as relative absorbance per milligram phospholipid.

~ )

,

OOH ~ H O ' + NO2 hydroxyl rodicol

791

/

Fig 1. Proposed mechanism for the formation of hydroxyl radical (HO.) through the interaction betwssn NO and superoxide anion (O=-).

PATIENTS AND METHODS

Study Design Seven consecutive adult patients (four men, three women, 18 to 47 years of age), admitted to our Hospital between January 1993 and April 1995 with acute forms of recurrent TMA, were included in this study. TMA was diagnosed on the basis of anemia of hemolytic type, severe thrombocytopenia, and renal or neurological symptoms. Only two of the seven patients manifested severe renal insufficiency during the acute phase of the disease. In one patient the relapsing episodes consistently followed, within 2 to 3 days, an upper respiratory tract infection. Neither signs of infection nor other obvious triggers could be recognized in the other patients. Clinical details of the patients are given in Table 1. Patients were treated with plasma exchange (n = 4) or infusion (n = 3) according to the previously described protocols./3 Patients were studied twice, first during the acute phase of the disease (before treatment), then later, at recovery (at least 24 hours after the end of the last plasma infusion). Seven sexand age-matched volunteers (four men, three women, 20 to 42 years of age) were studied simultaneously as controls. To have an in vivo index of NO synthesis in TMA, plasma concentrations of nitrites and nitrates (NO2-/NO3-), the stable NO metabolites, were evaluated in patients and controls. All subjects fasted overnight (at least 10 hours) before plasma collection to minimize exogenous nitrate intake attributable to the diet# To investigate whether substances that increase vascular NO synthesis were present in the circulation of pa-

Cell Isolation and Culture HUVEC were obtained from human umbilical veins as described, e The cells were first plated in 5 5 - c m 2 tissue culture plates (Falcon Labware Division, Becton Dickinson, Milan, Italy) precoated with 0.2% bovine gelatin. The growth medium consisted of Medium 199 (Gibco, Grand Island, NY), supplemented with 20% newborn calf serum (Gibco), 20 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, Merck, Darmstadt, Germany), 100 U/mL penicillin, 100 #g/mL streptomycin, 250 ng/mL fungizone, 2 mmol/L glutamine (Gibco), 15 U/mL heparin (Parke-Davis, Milan, Italy), and 50 #g/mL endothelial cell growth factor.

Table 1. Clinical and Laboratory Data During the Acute and Remission Phase of TMA PlateletCount (×1,000//zL) Patient No.

Sex (M/F)

Age (yr)

Acute

1 2 3 4 5 6 7

M F M M F F M

18 23 27 29 47 27 28

50 105 25 50 59 27 56

Hemoglobin (g/dL)

Remission A c u t e 256 206 274 218 241 230 226

Abbreviation: LDH, lactate dehydrogenase.

11.0 7.3 14.1 12.6 9.3 10.8 11.5

Remission 11.5 7.5 12.6 14.0 8.6 12.0 11.0

LDH (U/L) Acute 3,870 562 517 681 749 637 742

SerumCreatinine (mg/dL)

Remission 444 393 311 388 318 265 346

Acute 1.7 4.4 3.5 1.8 1.1 0.9 1.8

Remission 1.1 3.6 1.8 1.6 1.0 1.0 1.7

792 Cultures were grown at 37°C in 5% CO2-95% air. Confluent HUVEC were passaged with 0.025% trypsin-0.02% ethylenediaminetetraacetic acid (Gibco) and were used at the second to third passage. For the experiments, HUVEC were plated on six-well tissue culture plates (Falcon) and grown in complete medium until confluence was reached.

NORIS ET AL 120

E G) O

*O

90

E t-

Determination of the Conversion of [3H]L-Arginine to [3H]L-Citrulline TCA-treated samples were centrifuged at 10,000g to precipitate proteins. Thereafter, supernatant was extracted with ether, vacuum lyophilized, and resuspended in 2 mL HEPES, pH 5.5, and applied to 2 mL wet bed volumes of Dowex AG 50 WX-8 (100 to 200 mesh, Bio-Rad, Richmond, CA) (Li + form) followed by 2 mL water. [3H]L-citrulline was quantitated in the column effluent by liquid scintillation counting and identified as described. 6

'o'~ z

60

,o~ z ~

30

O.

Controls

Measurement of 02- Production by Polymorphonuclear Cells Polymorphonuclear cells were isolated from heparinized blood as described. ~s Superoxide generation was measured by superoxide dismutase-inhibitable reduction of ferricytochrome c. ~8 Polymorphonuclear cells (1 × 106) were incubated in triplicate samples in 1 mL Hank's balanced salt solution ('Iris, 27.4 mmol/L; Na~HPO4, 0.7 mmol/L; KH2PO4, 0.74 mmol/L; KCI, 5.4 mmol/L; NaCI, 136 mmol/ L; dextrose, 9.6 mmoi/L, supplemented with CaCI2 1.87 mmol/L and MgSO4 0.8 mmol/L in the presence of 80 #mol/ L ferricytochrome c (horse heart, grade III, Sigma) with or without 30 #g/mL superoxide dismutase (approximately 3,000 U/mg protein, Sigma). After 30 minutes of incubation at 37°C in a shaking bath, cells were removed by centrifugation (500g for 7 minutes) and the amount of reduced cytochrome c was determined from the absorbance at 550 nm by using the extinction coefficient of 21.1/cm/mmol/L. Data were expressed as nmoles of O2-/10 6 cells/30 minutes. Particular care was taken in standardizing the interval be-

Recovery I

i

Plasma NO2-/N03- Measurement NO2-/NO3- concentrations were measured by semiautomatic HPLC analysis (Beckman Instruments Inc., Berkeley, CA) according to the method of Green et al, '7 with some modifications. Briefly, plasma samples were treated with zincum sulfate (60 #mol/L, final concentration) and centrifuged to eliminate proteins. Supernatants were eluted onto a Dowex AG 50 WX-8 column followed by a cadmium column, which catalyzes the reduction of nitrate to nitrite (eluent: borate buffer, pH = 8.5). The postcolumn eluate reacted with Griess reagent (5% H3PO4, 1% sulphanilic acid, 0.1% n-[1naphthyl]-ethylenediamine, vol:vol:voi) (Sigma Chemical Co., St Louis, MO) to form a purple azo dye, and the color was detected by UV-VIS detector at h = 504 nm. The absorbance peak area was measured, and NO2-/NO 3- concentration in the sample was calculated by extrapolation from a curve obtained by injection of standard nitrate solutions. Values were corrected for recovery, which averaged 80% (range, 60% to 90%), as determined by addition of known amounts of standard nitrate to an additional aliquot of each plasma sample.

Acute

TMA Fig 2. Concentrations of the stable NO metabolites, nitbrite/nitrete (NO2-/NOs-) in plasma from patients with T M A (l~) (n = 7) and controls (E3) (n = 7). NO2-/NO3- plasma concentrations were measured by HPLC. T M A patients were studied both during the acute phase of the disease and at recovery. *P < 0.05 v controls; °P < 0.05 v T M A acute.

tween sample collection and neutrophil isolation, and between neutrophil isolation and superoxide anion measurement. For this assay, control samples were run in parallel with patient samples under identical in vitro incubation conditions.

Statistical Analysis Data are mean _+ SE. Data on plasma concentrations of NO2 /NO3-, HUVEC NO synthesis, L-arginine serum concentration, plasma MDA and conjugated dienes, and 02 production by polymorphonuclear cells were analyzed by MannWhitney U test or Wilcoxon rank sum test as appropriate. Data on serum TNFa were analyzed by Fisher's exact test (comparison between TMA patients and controls) or Wilcoxon rank sum test (comparison between TMA acute and TMA recovery). Linear regression analysis was performed to calculate correlation coefficients. The statistical level of significance was defined as P < 0.05.

RESULTS To establish whether NO synthesis was modified i n r e c u r r e n t f o r m s o f T M A , p l a s m a c o n c e n t r a t i o n s o f t h e s t a b l e N O m e t a b o l i t e s , NO2-~ NO3-, were measured in the acute and recovery phases of the disease. Results showed higher p l a s m a N O 2 - / N O 3 - in T M A p a t i e n t s s t u d i e d d u r i n g t h e a c u t e p h a s e o f t h e d i s e a s e as c o m p a r e d w i t h c o n t r o l s (94.1 ___ 15.9, n = 7, v 45.1 ___ 5 . 4 n m o l / m L , n = 7; P < 0 . 0 5 ; F i g 2). A t r e c o v e r y , N O 2 - / N O 3 - c o n c e n t r a t i o n s d e c r e a s e d i n all p a -

NITRIC OXIDE IN THROMBOTIC MICROANGIOPATHY

tients but remained significantly higher than control values (73.9 ± 9.5 nmoles/mL, n = 7; P < 0.05 v controls, P < 0.05 v TMA acute, Fig 2). HPLC measurement of serum L-arginine showed no difference between patients with TMA, either in the acute phase of the disease or at remission, and controls (controls: 59.6 ___ 2.9, n = 7; T M A acute: 68.9 ___ 8.7, n = 7; TMA recovery: 63.0 ___ 8.2 #mol/L, n = 7), indicating that higher NO synthesis in TMA was not the consequence of an increased bioavailability of the NO precursor. By contrast, serum concentrations of the potent NO synthase inducer T N F a were higher than in controls in the acute phase of recurrent T M A (controls: < 3 , n = 7; TMA acute: 11.92 ___5.02, n = 7, P < 0.01 v controls; T M A recovery: 4.09 ___ 0.52 pg/mL, n = 7, P < 0.05 v controls and T M A acute). Serum from patients with acute T M A induced a marked increase in NO synthesis by HUVEC compared with control serum (113 ± 14, n = 7, v 65 ___ 10 pmol/105 cells, n = 7, P < 0.05, Fig 3). In all patients but one, serum taken in the recovery phase had the same effect as control serum on NO synthesis by HUVEC (68 _ 18 ~k

135

O O

90 O

E v (U °_

45 ,o .--I

3E

Controls

Acute

Recovery

I

I

TMA Fig 3. Synthesis of NO by human umbilical vein endothelial celia (HUVEC) exposed to serum from TMA patients (Eq) (n = 7) and controls (I-]) (n = 7). HUVEC were exposed for 24 hours to serum from patients or conbx)ls in the presence of 0.5/~Ci [SH]L-arginine. IncubalJon was stopped by adding one volume TCA, and NO synthesis was evaluated by measuring the conversion of [affJL-arginine to [3H]L-citrulline. TMA patients were studied both during the acute phase of the disease and at recovery. *P < 0.05 v controls and TMA recovery.

793

pmoles/105 cells, n = 7, P = NS v control serum, P < 0.05 v acute TMA; Fig 3). No sign of cell detachment and death, as assessed by Trypan blue exclusion, was observed in HUVEC exposed either to T M A or control serum. Cell count after 24-hour incubation was comparable in HUVEC exposed to T M A and control serum (TMA acute: 501.4 ± 32.9; TMA recovery: 533.1 ___ 31.7; controls: 539.3 ± 30.9 × 103 cells/well), excluding the possibility that TMA serum exerts a direct cytotoxic effect on endothelial cells. In TMA patients, neither plasma NO2-]NO3(r = 0.39, P = 0.15) nor the effect of serum on NO synthesis by HUVEC (r = 0.40, P = 0.14) correlated with creatinine concentrations; this makes it unlikely that excessive NO synthesis in T M A were the consequence of renal function impairment. By contrast, a positive correlation was found between NO2-/NO3- plasma values and lactate dehydrogenase levels (r = 0.64, P < 0.05), the best available marker of disease activity. Ex vivo superoxide spontaneous release from polymorphonuclear cells was significantly higher in patients with acute T M A than in controls (controis: 3.8 ___ 0.2, n = 7; TMA acute: 9.6 ___ 0.8, n = 6; P < 0.001 v controls; T M A recovery: 5.3 + 0.5 nmoles/106 cells/30 min, n = 6, P < 0.05 v controls, P < .05 v TMA acute, Fig 4), indicating excessive oxygen radical formation in T M A patients. M D A concentrations were significantly higher in plasma from patients with acute T M A as compared with controls (0.52 ___ 0.13, n = 7, v 0.13 ___0.02 nmol/mL, n = 7, P < 0.005, Fig 5), indicating enhanced lipid peroxidation. At recovery, plasma M D A tended to decrease but still remained significantly higher than in controls (0.29 ___ 0.05 nmol/mL, n = 7, P < .01 v controls, P < .05 v acute TMA, Fig 5). The same trend was observed for plasma levels of conjugated dienes (controls: 0.76 __+ 0.06, n = 7; acute TMA: 1.36 ___ 0.24, n = 7; P < 0.02 v controls, T M A recovery: 1.11 ± 0.19 A233/mg phospholipid, n = 7, P = NS v controls and acute TMA). Interestingly, in patients with recurrent TMA, either 02 release from polymorphonuclear cells or M D A values positively correlated with plasma L D H levels (r = 0.69, P < 0.01, and r = 0.82, P < 0.01, respectively).

794

NORIS ET AL DISCUSSION

In the last lO years, data have accumulated to

credit endothelial injury as the central event in TMA. l More recently, evidence has been provided that activated neutrophils contribute to microvascular injury.2-4 This is consistent with findings that cytotoxic activity of bacterial endotoxin--one of the causative agents of some forms of TMA~9--mainly results from its ability to promote neutrophil adhesion to endothelium2° and stimulate oxygen radical formation. 2~ Neutrophil activation in all inflammatory processes may upregulate a calcium-independent isoform of NO synthase in endothelial cells, through the action of cytokines, s'7'8 Here we present the first in vivo evidence that NO is formed in excessive amounts in patients with recurrent TMA in the acute phase of the disease. Additional evidence that serum from the same patients potently induces NO synthesis by endothelial cells in culture would suggest that the high NO levels measured in vivo depend on NO synthesis induction, most probably in vascular endothelial cells. Data of significantly higher plasma TNFa concentrations in TMA, already reported in literature 22 and confirmed by the current study, contribute to clarify the mechanism of enhanced NO 12. A

E

10.

z

8

0 ¢q

OD

II

0

cE

b Controls

Acute I

Recovery I TMA

Fig 4. Spontaneous ex vivo release of 02- by polymorphonuclear cells (PMNs) from patients with TMA (1~) (n = 6) and controls (n) (n = 7). PMNs (1 x 10e cells) were incubated in HBSS for 30 minutes. O2- release was measured by SOD-inhibitable reduction of ferricytochrome c. TMA patients were studied both in the acute phase of the disease and at recovery. *P < 0.001 v controls; °P < .05 v controls; Dp < 0.05 v TMA acute.

0.8-"7" (]) O

E ,< o

:E

0.6 /t

0.4

E OD

0.2

~L

~.

O Controls

Acute TMA

Recoveryfrom

TMA

Fig 5. Plasma malondialdehyde concenlralJons in patients with TMA (ll) (n = 7) and controls ((3) (n = 7). Malondialdehyde was measured by the thiobarbituric acid method. TMA palients were studied both during the acute phase of the disease and at recovery. *P < 0.005 v conlrols; °P < .01 v controls; Dp < 0.05 V TMA acute.

synthesis in this disease, because TNFa is one of the most potent inducers of the inducible NOsynthase enzyme in various cell systems.5'7'23 Apart from TNFa, other substances that accumulate in the blood of patients with TMA, such as bacterial cytotoxins23 and other cytokines (eg, interleukin-1 fl23), could theoretically contribute to endothelial NO synthesis induction in recurrent TMA. Activated inflammatory cells are also possible sources of NO. 24 In humans, there is indirect evidence of NO formation by macrophages 25 and peripheral blood monocytes26; however, conclusive proof is still lacking. 5 NO is a potent mediator of vascular damage,27 •28 and evidence is available that administration of the arginine analog N-monomethyl-L-arginine, which blocks NO formation, protects experimental animals from immune-mediated vascular injury.29 Overproduction of NO is part of a defense mechanism against invading microorganisms, but it can exert undesired cytotoxicity on the cells that produce it and on neighboring cells by inhibiting several intracellular functions such as DNA synthesis, mitocondrial respiration, and the citric acid cycle.9 Thus, autocytotoxicity of NO formed from the inducible enzyme has been recently reported on endothelial cell stimulation with T N F o t . 7 Moreover, the production of large amounts of NO has

NITRIC OXIDE IN THROMBOTIC MICROANGIOPATHY

been implicated in the cell and organ damage associated with endotoxic shock. 3°'3] In tum, NO releases TNFa and interleukin132,33 from human leukocytes, thus promoting secondary activation that may amplify inflammatory damage. 34 Data that polymorphonuclear cell-derived 02- ex vivo are remarkably higher than normal in patients with TMA provide further evidence of leukocyte activation in this disease. This excess 02 could react with NO, yielding peroxynitrite, a strong oxidant that has been recently implicated in tissue injury 35'36 because of its capability to form HO. and initiate lipid peroxidation, tLj2 The latter possibility is supported by the finding of higher MDA and conjugated dienes concentrations in the circulation of patients with acute forms of TMA as compared with healthy volunteers. Our results suggest the possibility of a novel pathway of injury that could account for microvascular damage in TMA. We speculate that the initial injury induces NO synthesis and release by endothelial cells that, by interacting with leukocyte-derived oxygen radicals, contributes to amplify the microvascular damage. Beside their possible relevance to the pathophysiology of tissue injury, the current findings may have interesting implications for the future management of this devastating disease. A preliminary report by Westberg et a137 has recently documented increased NO3- plasma levels in patients with active TMA, confirming our data. The authors suggest that increased NO synthesis in TMA may be an adaptive response to prevent platelet aggregation in this disease. Given the inhibitory properties of NO on platelet activation, 5 this possibility cannot theoretically be excluded. However, the very rapid interaction of NO with neutrophil-derived 02-1t likely prevents NO from exerting its beneficial effects in this syndrome. ACKNOWLEDGMENT The authors thank Dr Marina Morigi, Dr Sistiana AieUo, and Dr Miriam Galbusera for helpful discussion and assistance during these studies, and Daniela Coma, Carlo Stefano Cereda for excellent technical assistance.

REFERENCES 1. Remuzzi G, Ruggenenti P, Bertani T: Thrombotic microangiopathy, in Tisher CG, Brenner BM (eds): Renal Pathology With Clinical and Functional Correlations. Philadelphia, PA, Lippincott, 1994, pp 1154-1184

795 2. Henson PM, Johnston RB Jr: Tissue injury in inflammation: Oxidants, proteinases, and cationic proteins. J Clin Invest 79:669-674, 1987 3. Milford DV, Staten J, MacGreggor I, Dawes J, Taylor CM, Hill FG: Prognostic markers in diarrhoea-associated haemolytic-uraemic syndrome: Initial neutrophil count, human neutrophil elastase and von Willebrand factor antigen. Nephrol Dial Transplant 6:232-237, 1991 4. Forsyth KD, Simpson AC, Fitzpatrick MM, Barratt TM, Levinsky RJ: Neutrophil-mediated endothelial injury in haemolytic uraemic syndrome. Lancet 2:411-414, 1989 5. Moncada S, Higgs A: The L-arginine-nitric oxide pathway. N Engl J Med 329:2002-2011, 1993 6. Noris M, Benigni A, Boccardo P, Aie|lo S, Gaspari F, Todeschini M, Figliuzzi M, Remuzzi G: Enhanced nitric oxide synthesis in uremia: Implications for platelet dysfunctions and dialysis hypotension. Kidney Int 44:445-450, 1993 7. Estrada C, Gomez C, Martin C, Moncada S, Gonzalez C: Nitric oxide mediates tumor necrosis factor-a cytotoxicity in endothelial cells. Biochem Biophys Res Commun 186:475482, 1992 8. Lamas S, Michel T, Brenner BM, Marsden PA: Nitric oxide synthesis in endothelial cells: Evidence for a pathway inducible by TNFa. Am J Physiol 261:C634-C641, 1991 9. Drapier JC, Hibbs JB Jr: Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol 140:2829-2838, 1988 10. Volk T, Ioannidis 1, Hensel M, de Groot H, Kox WJ: Endothelial damage induced by nitric oxide: Synergism with reactive oxygen species. Biochem Biophys Res Commun 213:196-203, 1995 11. Pryor WA, Squadrito GL: The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am J Physiol 268:L699-L722, 1995 12. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA: Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87:1620-1624, 1990 13. Ruggenenti P, Galbusera M, Plata Cornejo R, Bellavita P, Remuzzi G: Thrombotic thrombocytopenic purpura: Evidence that infusion rather than removal of plasma induce remission of the disease. Am J Kidney Dis 21:314-318, 1993 14. Leone AM, Francis PL, Rhodes P, Moncada S: A rapid and simple method for the measurement of nitrite and nitrate in plasma by high performance capillary. Biochem Biophys Res Commun 200:951, 1994 15. Suryaprabha P, Das UN, Ramesha G, Kumar KV, Kumar GS: Reactive oxygen species, lipid peroxides and essential fatty acids in patients with rheumatoid arthritis and systemic lupus erythematosus. Prostaglandins Leukot Essent Fatty Acids 43:251-255, 1991 16. Buege JA, Aust SD: Microsomal lipid peroxidation, in Fleischer S, Packer L (eds): Methods in Enzymology, vol LII: Biomembranes. New York, NY, Academic Press, 1978, pp 302-310 17. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR: Analysis of nitrate, nitrite, and [~SN]nitrate in biological fluids. Anal Biochem 126:131 138, 1982

796

18. Macconi D, Zanoli A, Orisio S, Longaretti L, Magrini L, Rota S, Radice A, Pozzi C, Remuzzi G: Methylprednisolone normalizes superoxide anion production by polymorphonuclear cells from patients with ANCA-positive vasculitides. Kidney Int 44:215-220, 1993 19. Koster F, Levin J, Walker L, Tung KSK, Gilman RH, Rahaman MM, Majid MA, Islam S, Williams RC Jr: Hemolytic-uremic syndrome after shigellosis: Relation to endotoxemia and circulating immune complexes. N Engl J Med 298:927-933, 1978 20. Thomas PD, Hampson FW, Casale JM, Hunninghake GW: Neutrophil adherence to endothelial cells. J Lab Clin Med 111:286-292, 1988 21. Taylor CM, Powell HR: Oxygen-derived free radicals in the pathogenesis of the hemolytic uremic syndrome, in Kaplan BS, Trompeter RS, Moake JL (eds): Hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. New York, NY, Marcel Dekker, 1992, pp 355-372 22. Wada H, Kaneko T, Ohiwa M, Tanikawa S, Minami N, Takahashi H, Deguchi K, Nakano T, Shirakawa S: Plasma cytokine levels in thrombotic thrombocytopenic purpura. Am J Hematol 40:167-170, 1992 23. Kilbourn RG, Belloni P: Endothelial cell production of nitrogen oxides in response to interferon y in combination with tumor necrosis factor, interleukin-1, or endotoxin. J Natl Cancer Inst 82:772-776, 1990 24. Marietta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS: Macrophage oxidation of L-arginine to nitrite and nitrate: Nitric oxide is an intermediate. Biochemistry 27:8706-871 l, 1988 25. Denis M: Tumor necrosis factor and granuiocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: Killing effector mechanism depends on the generation of reactive nitrogen intermediates. J Leukoc Biol 48:380-387, 1991 26. Hunt NCA, Goldin RD: Nitric oxide production by monocytes in alcoholic liver disease. J Hepatol 14:146-150, 1992

NORIS ET AL

27. Mulligan MS, Moncada S, Ward PA: Protective effects of inhibitors of nitric oxide synthase in immune-complexinduced vasculitis. Br J Pharmacol 107:1159-1162, 1992 28. Seekamp A, Mulligan MS, Till GO, Ward P: Requirements of neutrophil products and L-arginine in ischemiareperfusion injury. Am J Pathol 142:1217-1226, 1993 29. Mulligan MS, Hevel JM, Marietta MA, Ward PA: Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc Natl Acad Sci USA 88:63386342, 1991 30. Stoclet JC, Fleming I, Gray G, Julou-Schaeffer G, Schneider F, Schott C, Schott C, Parratt JR: Nitric oxide and endotoxemia. Circulation 87:V77-V80, 1993 (suppl V) 31. Wizemann TM, Gardner CR, Laskin JD, Quinones S, Durham SK, Goller NL, Ohnishi ST, Laskin DL: Production of nitric oxide and perox,. ,fitrite in the lung during acute endotoxemia. J Leukoc Biol 56:759-768, 1994 32. Lander HM, Sehajpal P, Levine DM, Novogrodsky A: Activation of human peripheral mononuclear cells by nitric oxide-generating compounds. J Immunol 150:1509-1516, 1993 33. Magrinat G, Mason SN, Shami PJ, Weinberg JB: Nitric oxide modulation of human leukemia cell differentiation and gene expression. Blood 80:1880-1884, 1992 34. Larrick JW, Graham D, Toy K, Lin LS, Senyk G, Fendly BM: Recombinant tumor necrosis factor causes activation of human granulocytes. Blood 69:640-644, 1987 35. White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA: Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci USA 91:1044-1048, 1994 36. Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S: Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J Clin Invest 94:2407-2413, 1994 37. Westberg G, Herlitz H, Petersson A, Sigstrom L, Wennmalm A: The arginine-nitric oxide pathway is activated in thrombotic microangiopathy. J Am Soc Nephrol 6:458, 1995