Clin. exp. Immunol. (1992) 90, 326-329

Urinary excretion of nitrite and nitrate in experimental glomerulonephritis reflects systemic immune activation and not glomerular synthesis R. SEVER, T. COOK & V. CATTELL Department of Histopathology, St Mary's Hospital Medical

School, London, UK

(Accepted for publication I August 1992)

SUMMARY In immune-induced glomerulonephritis (gn), glomeruli (gl) synthesize nitric oxide (NO), and urinary nitrite (NO2 ) excretion is increased. In mammals

on a

low nitrate (NO3-) diet, urinary NO3- is

a

of endogenous NO3- synthesis. Excretion is increased after administration of macrophage activators, reflecting induction of NO production. To determine whether increased urinary NO2 gn is due to glomerular synthesis we studied urinary NO2-/NO3- in accelerated nephrotoxic nephritis induced by preimmunization with rabbit immunoglobulin G (IgG), followed by rabbit anti-rat nephrotoxic globulin, and in control rats similarly preimmunized with rabbit IgG, but followed by normal rabbit serum. Both urinary NO2- and NO3- were increased by i.p. preimmunization with rabbit IgG (peak 463+171 nmol NO2 /603+9-4 nmol NO3- /24 h, P < 0-001 for both NO2- and NO3- compared with preimmunization levels). Repeat immunization with i.v. rabbit anti-rat nephrotoxic globulin (nephritic rats) or normal rabbit globulin (control rats) again increased urinary

measure

NO2- and NO3-. There was no statistically significant difference in urinary NO2- and NO3- levels between nephritic rats where globulin had nephrotoxic activity and the control rats injected with normal rabbit globulin, despite increased NO2- synthesis in ex vivo nephritic glomeruli after nephrotoxic globulin (7 9 + 1 9 nmol/2000 gl/48 h; controls 3 2 + 1 0 nmol/2000 gl/48 h). Thus neither urinary NO2- nor NO3- levels reflect local activation of the NO pathway in glomeruli. As reported for other stimulants, we show here that systemic stimulation with foreign antigen increased NO

synthesis. Keywords nitric oxide nitrite nitrate glomerulonephritis

nephritic glomeruli [6]. This suggested to us that the source in urine was local NO production in the kidney. As the nephritis was induced by systemic administration of immunological stimuli, but the specific response was localized in the glomeruli (due to administration of basement membrane antibody which fixes in the glomerulus), we have now used this model to determine whether urinary NO2- is a reflection of glomerular NO production. Since NO may be metabolized to both NO2and NO3-, and NO2- may be oxidized in vivo to NO3- we have now measured both products.

INTRODUCTION Studies in man [1], mouse [2] and rat [3] have shown that urinary nitrate (NO3 ) is the net result of dietary intake, endogenous synthesis and metabolic loss. In animals fed a low NO3 diet, urinary NO3- excretion exceeds ingestion and is a measure of endogenous synthesis masked by ingestion of the levels of NO3in normal diets. Endogenous NO3- synthesis is increased by administration of stimulants of the mononuclear phagocytic system [2,4,5]; this increase is inhibited by L-monomethyl arginine (L-NMMA) [2], an inhibitor of nitric oxide synthase which suggests that the source of NO3- is induction of nitric oxide (NO) synthesis in macrophages and possibly other cells such as hepacytocytes and endothelium. During studies on activation of NO synthesis in glomeruli in immune-induced glomerulonephritis (accelerated nephrotoxic nephritis) in rats, we found increased levels of nitrite (NO2-) in urine, coincident with increased ex vivo NO2- production by

MATERIALS AND METHODS Animals Male Lewis rats bred at St Mary's Hospital Medical School were used; weight 200-250 g. To ensure minimal NO2-/NO3- contamination rats were housed individually in steel metabolic cages with recessed water spouts. Cages were cleaned daily and rinsed in NO2- /NO3- free distilled water.

Correspondence: Dr V. Cattell, Department of Histopathology, St Mary's Hospital Medical School, London W2, UK.

326

327

Urine NO2- and NO3- in glomerulonephritis In addition, urine was collected in a mixture of penicillin G 104 U, amikacin 10 mg and chloramphenicol 10 mg/ml to prevent bacterial growth [2]. Urine specimens were centrifuged and stored at 200C prior to assay. -

Diet The amino acid rodent diet [2] was prepared by Lillico Ltd (Surrey, UK), as a powder; this was mixed to a paste in NO2/ NO3-free distilled water. Rats received 30 g/day of freshly prepared mixture. The diet was first tested for effects on urinary N02-/NO3- levels in 26 normal rats. Rats used in immunization and lipopolysaccharide (LPS) experiments (see below) commenced a NO3 -free diet 3 days before induction of experiments.

Urinary NO2- and NO3Urinary NO2- was assayed by the Griess reaction as previously described [6]. Urinary NO3- was measured by reducing NO3- to NO2- with bacterial nitrate reductase, using Pseudomonas oleovorans (National Collection of Type Cultures, Colindale) [2].

Glomerular NO2Glomeruli were isolated from saline perfused kidneys and incubated for 48 h in phenol red-free Dulbecco's Modified Eagles medium, as previously described [6]. NO2- production was assayed by the Griess reaction and expressed as nmol NO2per 2000 gl/48 h.

Urinary protein Urinary protein method.

was

measured by the sulphosalicylic acid

Urinary creatinine Urinary creatinine was measured by the Jaffe reaction using Olympus A115021 analyser and Merck (UK) reagents. Induction of glomerulonephritis Accelerated nephrotoxic nephritis was induced as previously described [7]. Rats (n = 8) were preimmunized on day -7 with i.p. rabbit IgG (Sigma, UK) and Freund's complete adjuvant. On day 0, this group (accelerated nephrotoxic nephritis) received a single i.v. injection of rabbit anti-rat nephrotoxic globulin (NTG), 3 mg/100 g. Rats were killed on days 3 or 4. Control group immunization protocol Rats (n = 8) were preimmunized on day -7 with i.p. rabbit IgG, as in the accelerated nephrotoxic nephritis group. On day 0 they received a single i.v. injection of normal rabbit globulin (RG) at 3 mg/l 00 g. Both nephrotoxic and normal rabbit globulins were prepared by ammonium sulphate precipitation of serum under identical conditions. Rats were killed on days 3 or 4.

Lipopolysaccaride (LPS) The effects of LPS, known to produce an acute rise in urinary NO3- [4,5] were studied for comparison with immunological stimuli. Six rats on a NO2-/N03- free diet received either 1 mg/kg i.p. LPS (E. coli 055:B5, Sigma) (n 3) or i.p. saline injection (n = 3). Urine was collected for 24 h and glomeruli isolated for NO2- production. =

Drugs Lipopolysaccharide (E. coli 0.55: B5, Sigma) was used. RESULTS All rats on the NO2-/NO3- free diet survived the experimental

protocols. Accelerated nephrotoxic nephritis The characteristics of this model have been previously described [7]. Renal histology shows a proliferative glomerulonephritis with linear deposition of rabbit and rat IgG and rat C3 on glomerular capillary walls. Characterization of leucocyte infiltration by cell isolation and MoAb shows neutrophil infiltration at 4 h, and at subsequent time points a substantial macrophage infiltration. The mean proteinuria in the current experiments was 1 15 + 15 8 mg/24 h (RG-immunized controls 1 3 + 0 4) on day 3, and 24-8 + 10-8 mg/24 h (RG-immunized controls 1 65 + 1 1) on day 4.

Glomerular NO2- production Basal NO2- production in glomeruli isolated at sacrifice was 7.4+1.9 nmol/48 h in the nephritic group (RG-immunized controls 3-2 + 1-0) and was increased to 17 3 + 3-6 with 1 ug/ml LPS and 61-8+ 10 7 with 100 ,g/ml LPS in nephritic glomeruli (RG-immunized controls 3 2 + 1 0 and 4-8 + 0-5 respectively).

Urinary NO2- and NO3Twenty-six normal rats maintained on the NO2-/NO3- free diet showed a mean urinary output of 59-4+10-8 nmol NO2-/24 h (pre-diet 816+12 2) and 3 5+0 3 ,mol NO3-/24 h (pre-diet 40 1 + 6-0). These levels were achieved from 24 h of commencing the diet.

700 600 2 500E

400-

300

5 200100 C

-10

-8

-6

-4

-2

0

2

4

Day

Fig. 1. Urinary nitrite levels. Rats commenced a low nitrite/nitrate diet on day -9, and were immunized with i.p. rabbit IgG and Freund's complete adjuvant on day -7. On day 0 rats received either NTG (nephritic group, closed symbols) or RG (immunized controls, open

symbols).

328

R. Sever, T. Cook & V. Cattell 80 _

60

Fg 40-10 C:

S0 a~~ ~ ~~~~~a

D20 -JO

-8

-6

-2

-4

0

2

4

Day

Fig. 2. Urinary nitrate levels in rats following the same regime as in Fig. 1

The results for nephritic rats and RG immunized controls are shown in Figs 1 and 2. Following i.p. immunization with rabbit IgG and Freund's adjuvant there was a rise in NO2- and NO3- which peaked after 4-6 days (P< 0-001 for both N02and NO3-). The rise in NO3- appeared earlier than for NO2-. and peaked later. This increased production fell towards normal on the day of i.v. injection of NTG or RG. The subsequent rises in NO2- and NO3- were no different in nephritic rats injected with NTG from non-nephritic controls injected with RG; as with the i.p. injection the peak NO3- was later than NO2-. When NO2-/NO3- levels were expressed as a ratio of urine creatinine there was again no difference between the NTG and RG-immunized rats, and indeed there was no difference between the urine creatinines in the two groups (NTG 2 67+0 2, RG 2 71 +0-2 mmol/l). LPS In the first 24 h after LPS injection urinary NO3- rose from preinjection levels of 7 3 + 0 79 to 23 7 + 12 4 kmol/24 h. However this rise occurred in two out of the three rats tested; in one increased NO3- was not detected. NO2- was reduced in the LPS-treated group (42-0+45, saline controls 14555+134 nmol/24 h). In neither group was there NO2- synthesis by isolated glomeruli.

DISCUSSION Animals immunized intraperitoneally with a foreign antigen (rabbit IgG) showed a significant increase in urinary NO2- and NO3-. When this immunization was followed by intravenous injection of rabbit globulin there was a second rise in urinary N02- and NO3-; there was no difference between the rats which had received rabbit globulin with antiglomerular basement membrane activity (glomerulonephritic rats) and those which had received rabbit globulin without specific antiglomerular activity (immunized controls), despite evidence of glomerular N02- production in the glomerulonephritic group, as we have

previously described [6]. Therefore in the context of a systemic immunization, increased levels of NO2- and NO3- detected in urine are the result of systemic liberation of NO2- and NO3into the circulation which overwhelms any local production by nephritic glomeruli. This is the first demonstration of increase in urinary NO3due to antigenic activation. The time and degree of induction is very similar to previous studies administering intraperitoneal BCG [2] and mycobacteria [5] to the mouse, and fits other parameters of immunological stimulation. Further evidence linking this to immune activation is the temporal difference between our experiments with rabbit globulin and those with LPS. As has been previously reported [4,5], intraperitoneal administration of LPS results in a rapid (within 24 h) rise in urinary NO3-. It is of some interest to speculate whether the source of NO3-is the same in both cases. In previous studies on stimulated urinary NO3- production there is no information on levels of urinary NO2-, which was the parameter which first attracted our attention in relation to glomerulonephritis. We found levels of NO2- were a thousand-fold less than NO3-, which, as both are produced in similar amounts on activation of NO synthase, must reflect conversion of NO2- to NO3- within the circulation, as there was no difference in the pattern of elevation in glomerulonephritis compared with control immunized rats. Kelm et al. [8] have shown that in human plasma NO is converted to NO2- and NO3- with a ratio of approximately 14:1 but that in blood NO2- is rapidly converted to N03within erythrocytes. Indeed, our studies show that NO3-is a more reliable indicator of NO generation as the levels are higher and the variation less. We also however noted wide variations in NO3-, even on the low NO3- diet, and this has been previously reported [2,4] but remains unexplained. The major source of the increased urinary NO2-/NO3 with immune activation is most probably the macrophage [5]. Although there is no direct in vivo evidence for this, macrophages in vitro produce large amounts of NO2-/NO3- when exposed to IFN-y and other substances stimulating macrophage activation [9], and an inducible NO synthase has recently been identified in activated macrophages [10]. Other possible sources are endothelium or hepatocytes, and speculatively, the route of immune stimulation may have significant effects on the source of N02-/NO3-. In conclusion, our experiments show that urinary NO2- and NO3- are elevated in response to systemic immunization with foreign antigen. In glomerulonephritis, where a specifically directed immunological disease is induced with local generation of NO2- in the kidney, no additional NO2-/NO3- is detectable in the urine, and therefore urinary levels cannot be used to measure NO activity in renal disease.

ACKNOWLEDGMENTS Dr Ivor Brown, Department of Microbiology, St Mary's Hospital Medical School is thanked for culturing the P. oleovorans used in the NO3- reduction assay. We also wish to thank Anne Goldsmith, our laboratory assistant for the excellent care of the metabolic cages without which this study would not have been possible. The work was supported by a grant from The Wellcome Trust, UK.

REFERENCES 1 Green LC, Ruiz de Luzuriaga K, Wagner DA, Rand W, Istfan N, Young VR, Tannenburg SR. Nitrate biosynthesis in man. Proc Natl Acad Sci USA 1981; 78:7764-8.

Urine NO2- and NOy- in glomerulonephritis 2 Granger DL, Hibbs JR, Broadnax LB. Urinary nitrate excretion in relation to murine macrophage activation. J Immunol 1991; 146:1294-302. 3 Green LC, Goldman P. Nitrate synthesis in the germfree and conventional rat. Science 1981; 212:56-8. 4 Wagner DA, Young VR, Tannenbaum SR. Mammalian nitrate biosynthesis: incorporation of 15NH3 into nitrate is enhanced by endotoxin treatment. Proc Natl Acad Sci USA 1983; 80:4518-21. 5 Stuehr DS, Marletta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci USA 1985; 82:7738-42. 6 Cattell V, Cook HT, Moncada S. Glomeruli synthesize nitrite in experimental nephrotoxic nephritis. Kid Int 1990; 38:1056-60. 7 Cattell V, Smith J, Cook HT. Prostaglandin El suppresses macro-

329

phage infiltration and ameliorates injury in an experimental model of macrophage-dependent glomerulonephritis. Clin Exp Immunol 1990; 79:260-5. 8 Kelm M, Feelisch M, Grube R, Mott W, Strauer BE. Metabolism of endothelium-derived nitric oxide in human blood. Abstract; Biology of Nitric Oxide, 2nd International Meeting, London, 1991. 9 Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 1988; 141:2407-12. 10 Yui Y, Hatton R, Kosuga K, Eizawa H, Hiki K, kawai C. Purification of nitric oxide synthase from rat macrophages. J Biol Chem 1991; 19:12544-7.