Indian Pediatrics 2001; 38: 1025-1029  

Serum Nitrite and Urinary Nitrite Excretion in Nephrotic Syndrome

From the Pediatric Nephrology Division and Department of Biochemistry, Department of Pediatrics, Kalawati Saran Children’s Hospital, New Delhi 110 001, India and Institute of Pathology, Safdarjung Hospital, New Delhi 110 029, India.

Correspondence to: Dr. N.K. Dubey, A-11/25, Vasant Vihar, New Delhi 110 057, India.
E-mail: [email protected]

Manuscript received: April 10, 2000;Initial review completed: July 7, 2000;Revision accepted: March 1, 2001

The pathogenesis of nephrotic syndrome is unclear. Shalhoub postulated the role of lymphokines in increasing the permeability of the glomerular basement membranes(1). Later on, vascular permeability factor, glomerular permeability factor, and interieukin-2 were implicated in its pathogenesis(1-4). Although organic nitrates were known to be potent vasodilators for a century, the role of nitric oxide in the pathogenesis of nephrotic syn-drome has been postulated very recently(5).

Nitric oxide is synthesized in the body from L-arginine by enzyme nitric oxide synthase (NOS). It is a highly reactive free radical gas that easily decomposes into nitrite and nitrate in biological fluids. The concentration of these can be measured and used as markers of nitric oxide production(5). Studies have been pub-lished related to increased glomerular synthesis of nitric oxide in several well characterized models of immune glomerulonephritis in rats(6-8), however, not many studies have been conducted to investigate renal nitric oxide production in patients with nephrotic syn-drome. The present study aimed to estimate serum nitrite level and urinary nitrite excretion in children with nephrotic syndrome and their relation to steroid responsiveness.

Sixty five children (aged 1-12 years) with nephrotic syndrome attending the pediatric nephrology clinic were enrolled for the study. The controls were 35 children free of renal disease attending the pediatric nutrition and immunization clinics of Kalawati Saran Children’s Hospital, New Delhi. Patients were treated with steroids strictly in accordance with the therapeutic protocol of International Study of Kidney Disease in Children(9). Relapse was defined as proteinuria (2+ or more) for 3 consecutive days, while clinical remission was defined when urine was free of protein for 3 consecutive days.

Investigations included blood counts, urinalysis and culture, screening for tuber-culosis, and serum levels of protein, albumin, cholesterol, creatinine and nitrite in study and control groups. Initial urine and blood samples were taken at the time of presentation in newly diagnosed patients and subsequently when the child went into remission following steroid therapy. In previously diagnosed patients , samples were taken during relapse and remission of their disease. Children who did not respond to 8-weeks steroid therapy were subjected to renal biopsy for histopathological examination.

Three ml of blood and 2 ml of first morning urine were collected and frozen within two hours of collection. For blood, protein removal was done by protein precipitation technique (using zinc sulphate and sodium hydroxide). Serum and urinary nitrite were estimated by Griess reaction after reducing the nitrate to nitrite by granulated cadmium. One hundred µl of Griess reagent (1% sulfanilamide, 0.2% naphthylene diamine hydrochloride and 2.5% phosphoric acid) was added to 100 µl of treated specimen. Absorbance was read at 540 nm in an ELISA reader. Standard curves were prepared with known concentration of sodium nitrite and results expressed as millimoles per milligram of creatinine(10).

Statistical analysis was done using software Microstat and Statistix. Results were expressed as mean ± SD. Differences between various groups were compared by Student’s ‘t’ test. Small samples were compared by Mann Whitney test and p <0.05 was considered statistically significant.

The children in study and control groups were matched for age and sex (Table I). Eighty Per cent of patients were steroid responsive; the rest were steroid resistant. Comparison between patients and controls showed signi-ficant differences in total serum protein, albumin and cholesterol. No significant difference was found in the serum creatinine and nitrite levels between the two groups. However, the urinary nitrite excretion was significantly higher among the patients (p <0.001) (Table I).

The steroid responsive patients consisted of 24 (46%) newly diagnosed patients, 15 (28.8%) frequent relapsers and 13 (25%) infrequent relapsers. Urinary nitrite excretion in all these patients were significantly higher p <0.0001) as compared to controls. Of the 13 steroid resistant patients, 7 had focal segmental glomerulosclerosis and two each had IgA nephropathy, mesangial proliferative glome-rulonephritis and minimal change disease. All these patients had urinary nitrite levels similar to controls. Urinary nitrite levels were higher in patients with steroid responsive compared to patients with steroid resistant nephrotic syndrome (p <0.0005) (Table II).

All patients with steroid responsive nephrotic syndrome received corticosteroids for a mean period of 56.7 days (range 53-65 days). The mean period for achieving remis-sion in these patients was 21.4 days (range 16-30 days). Paired urinary nitrite samples (before steroid therapy and after achieving remission) were obtained in 24 patients during the initial episode. In the rest samples were obtained during the subsequent relapses or remission. There were no significant differences observed in urinary nitrite excretion in these patients during initial attack or relapse and remission (Table III). Thirty-four patients continued to receive steroids even after achieving remission while in 18 patients steroids were tapered off and stopped. The urinary nitrite excretion in patients receiving steroids compared to those off therapy was similar.

Table I - Comparison of baseline characteristics (Mean ± SD)

Table II  - Comparison of Urinary Nitrate (mmol/mg Creatinine)

p <0.005  

Table III - Comparison of Urinary Nitrate (mmol/mg Creatinine)

None of the differences between the groups was statistically significant.

High levels of nitrite in nephrotic glomeruli have been reported by many authors in experimental models(11, 12). The first in vivo evidence of its role in the pathogenesis of glomerulonephritis was reported by Weinberg et al.(13). Trachtman, et al.(5) reported increased level of urinary nitrite in children with nephrotic syndrome. In the present study also the urinary levels of nitrite were significantly elevated in children with steroid responsive nephrotic syndrome. Although nitric oxide is considered to have pre-dominantly pro-inflammatory effect(14-16), minimal change disease does not show any inflammatory cells. Further, persistence of elevated urinary nitrite during remission indicates that nitric oxide might not be directly involved in the pathogenesis of nephrotic syndrome. Since all patients were receiving Corticosteroids, it is probable that steroid therapy does not effect nitrite production.

That nitric oxide production is not an important and sole cause of the disease is further substantiated by near normal level of urinary nitrite found in children with steroid resistant nephrotic syndrome in the present study. Similar observations were made in earlier studies(16). Furusu, et al.(17) also found a decreased expression of endothelial nitric oxide synthase (eNOS) in patients with IgA nephropathy and lupus nephritis suggesting diminished physiological effect of eNOS (decreased generation of nitric oxide) in damaged glomeruli. This could be another explanation for near normal value found in case of IgA nephropathy and focal segmental glomerulosclerosis in the present study.

The persistently elevated nitrite excretion in relapse and remission suggested that renal nitric oxide synthesis does not modulate proteinuria. Further, the role of increased urinary nitrite excretion in mediating the manifestations of nephrotic syndrome requires study. The near normal levels of serum nitrite in children with nephrotic syndrome could be due to the fact that nitric oxide is produced within the kidney and has a very short life. The possible source for nitric oxide synthesis in patients with steroid responsive nephrotic syndrome may be lymphokines produced by activated suppressor T cells.

The findings of present study suggest that urinary nitrite excretion is increased in patients with steroid responsive nephrotic syndrome. Patients with steroid resistant nephrotic syndrome probably have different mechanisms for glomerular dysfunction.

Contributors: NKD conceptualized the study design was responsible for interpretation of results and drafting the manuscript. He will act as the guarantor for the study. PKD participated in collection of data and study material. SS reported on biopsy specimens. SB was responsible for measurement of different biochemical parameters. PK and AG participated in data collection and carrying out the study.

Funding: None.
Competing Interest: None stated.

1. Shalhoub RJ. Pathogenesis of lipoid nephrosis: A disorder of T cell function. Lancet 1974; 1: 556-559.

2. Lagrue G, Branellec A, Blanc C, Xheneumont S, Beaudoux F, Sohel A, et al. A vascular permeability factor in lymphocyte culture supernatants from patients with nephrofic syndrome: Pharmacological and physiological properties. Biomedicine 1975- 23: 73-75.

3. Koyama A, Fujisaki M, Kobayashi M, lgarashi M, Narita M. A glomerular permeability factor produced by human T cell hybridoma. Kidney lnt 1991; 40: 453-460.

4. Kawaguchi H, Yamaguchi Y, Nagata M, ltoh K. The effects of human recombinant interleukin-2 (IL-2) on the permeability of glomerular basement membrane in rats, Jpn J Nephrol 1987; 29: 1-11.

5. Trachtman H, Gauthier B, Frank R, Futterweil S, Goldstein A, Tomczak J. Increased urinary nitrite excretion in children with minimal change nephrotic syndrome. J Pediatr 1996; 128: 173-176.

6. Cattel V, Cook T, Moncada S. Glomeruli synthesize nitrite in experimental nephrotoxic nephritis. Kidney Int 1990; 38: 1056-1060.

7. Cook T, Sullivan RS. Glomerular nitrite synthesis in in situ immune complex glomerul-onephritis in rats. Am J Pathol 1991; 139: 1047-1052.

8. Cattel V, Largen P, de Heer E, Cook T. Glomeruli synthesize nitrite in active Hey-mann nephritis. The source in infiltrating macrophages. Kidney Int 1991; 40: 847- 851.

9. International Study of Kidney Disease in Children. Nephrotic syndrome in Children, A randomized trial comparing two prednisone regimens in steroid responsive patients who relapse early. J Pediatr 1979; 95: 239-243.

10. Stuchi DJ, Marietta MA. Synthesis of nitrite and nitrate in murine MO cell lines. Cancer Res 1987; 47: 5584-5590.

11. Jonsen A, Cook T, Michail T, Largen P, Rivenor MV, Moncada S, et al. Induction of nitric oxide synthesis in rat immune complex glomerulone-phritis. Kidney Int 1994; 46: 1215-1219.

12. Schultz PJ, Archer SL, Rosenberg ME. Inducible nitric oxide synthase MRNA activity in glomerular mesangial cells. Kidney Int 1994; 46: 683-689.

13. Weinberg JB, Granger DL, Pisetsky DS. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease. Increased nitric oxide production and nitric oxide synthase expression in MR-Lpr/Lpv mice and reduction of spontaneous glomerulonephritis and arthritis by administered NG-monomethyl-L-arginine. J Exp Med 1994; 179: 651-680.

14. Hibbs JB Jr, Tainlor RR, Vavrin Z, Rachlin EM. Nitric oxide: A cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 1988; 157: 87-94.

15. Hibbs JB Jr, Vavrin Z, Taintor RR. L-Arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J Immunol 1987; 138: 550-565.

16. Cattel V, Lianos E, Largen P. Glomerular NO synthase activity in mesangial cell immune injury. Exp Nephrol 1993; l: 36-40.

17. Furusu A, Miyazaki M, Abe K, Tru Kasuki S, Shioslita K, Sasekii O, et al. Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis. Kidney Int 1998; 53: 1760-1768.