In experimental immune complex glomerulonephritis nitric oxide (NO) is synthesized by nephritic glomeruli1,2,3,4. NO synthesis results from the induction of the high output, calcium independent isoform of nitric oxide synthase (iNOS or NOS2); NOS2 mRNA can be demonstrated in glomeruli by ribonuclease protection assay5 and reverse transcriptase (RT)-polymerase chain reaction (PCR)6,7 and NOS2 protein by immunohistochemistry8 in models of glomerulonephritis. The role of NOS2 in glomerulonephritis, however, remains controversial. NO has the potential to increase glomerular injury by its direct cytotoxic actions9, but may also have protective actions by improving glomerular hemodynamics10, inhibiting thrombosis11 and adhesion molecule expression12, and scavenging other free radicals such as superoxide13. Experiments to define a role for NOS2 have been hampered by the lack of completely selective inhibitors of NOS2; the major NOS inhibitors also inhibit endothelial NOS3 leading to hypertension, a factor known to exacerbate glomerulonephritis14. Some groups have therefore used indirect methods to manipulate NOS2 activity, such as alteration of substrate availability either by dietary supplementation with L-arginine15 or administration of arginase16 to deplete circulating arginine. Overall the results are inconclusive with some experiments pointing to a toxic role for NO17,18 and others suggesting that it is protective16. Therefore, in order to determine the role of NOS2 in glomerulonephritis, we have now examined the course of glomerulonephritis in mice that are homozygous for a disruption of the NOS2 gene and compared them with heterozygous littermates with functional NOS2.
METHODS
Mice
The derivation of mice with genetically disrupted NOS2 gene has been previously described19. The chimeras were bred into the inbred 129 strain (Harlan Olac, Bicester, UK). The mice were kept in an isolator at the University of Glasgow and maintained as homozygous or heterozygous littermates. DNA from each individual mouse was examined by Southern blot analysis to confirm the genotype for NOS2. Adult male mice (8 to 10 weeks of age were transferred to Imperial College School of Medicine where experiments were performed. Mice were negative for common murine viral pathogens by sera analyses.
Preliminary experiments establishing the model of accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis in the 129Sv mouse and techniques for mouse glomerular isolation were performed on adult male 129Sv mice from Harlan Olac.
Induction and monitoring of glomerulonephritis
Mice were immunised with a single intraperitoneal dose of 0.2 mg rabbit IgG (Sigma, Poole, UK) in Freund's complete adjuvant (Sigma). Rabbit anti-mouse glomerular basement membrane antibody (globulin concentration 15 mg/ml)20 was given intravenously seven days later. In preliminary experiments doses of between 0.2 to 3 mg anti-GBM globulin per mouse were used to establish a reproducible model of accelerated anti-GBM nephritis with glomerular NOS2 induction in the 129Sv mouse strain. Mice were killed at one, three or six days.
In the two principal experiments described here, NOS2 deficient and NOS2 heterozygous mice received either 0.6 mg anti-GBM globulin and were killed at 24 hours, or 0.3 mg anti-GBM globulin and were killed 24 hours or six days later. Kidneys were removed for glomerular isolation, histology and immunohistochemistry. Mice were housed in metabolic cages for urine collection.
Urinalysis
Urine was collected for 24 hour periods. Albuminuria was measured by rocket immunoelectrophoresis using sheep anti-rat albumin antibody21 (Biogenesis Ltd., Poole, UK). Creatinine was measured by the Jaffe reaction on an automatic analyzer.
Glomerular isolation
Magnetizable iron oxide was prepared as described22. A diethyl pyrocarbonate (DEPC) treated sterile suspension of iron oxide (1.25% wt/vol) in 0.9% saline solution was prepared and passed four times through a 25 gauge needle. Mice were killed by cervical dislocation and perfused through the left ventricle with 10 mls of iron oxide suspension. The kidneys were removed and sieved through a 75 
m sieve. The resulting suspension of glomeruli and tubules was washed three times with phosphate buffered saline (PBS) with centrifugation at 300 g and resuspended in 1 ml of DEPC treated PBS. Glomeruli were concentrated by washing three times with DEPC PBS under a magnetic field from a Dyna magnetic particle concentrator (MPC-E, Dynal, UK). Glomerular preparations obtained using this method routinely had a purity of > 95%.
Reverse transcription polymerase chain reaction
Glomerular pellets from individual animals were snap frozen and stored at -70°C before mRNA extraction which was carried out with RNAzol B (Biogenesis, Poole, UK) following the manufacturer's instructions. The entire RNA sample from each animal was reverse transcribed with Moloney murine leukemia virus enzyme (Gibco BRL, Paisley, UK) according to the manufacturer's instructions. The resulting cDNA was amplified by PCR using primers against NOS2 (735 bp product) and 
-actin (202 bp) as reported previously6 and for interleukin-1
(IL-1; sense, 5'-GCA-ACT-GTC-CCT-GAA-CTC-A-3'; antisense, 5'-GCC-CAA-GGC-CAC-AGG-GAT-T-3', 528 bp) and TNF
(sense, 5'-GGA-TCA-TCT-TCT-CAA-AAC-TCG-5'; antisense, 5'-TCA-CAG-AGC-AAT-GAC-TCC-AAA-3', 468 bp). PCR was carried out for 35 (NOS2, -actin) or 40 cycles with the following sequential steps: 72°C for one minute, 60°C for 30 seconds and 94°C for 30 seconds. The identity of the NOS2 PCR product was confirmed by restriction enzyme digestion with AvaI and with StuI, which produced products of the predicted sizes. PCR products were analyzed on 1% agarose gel and visualized with ethidium bromide staining and ultraviolet light transillumination.
Histology
Kidneys were fixed in 10% buffered formal saline, paraffin embedded and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). For neutrophil counts sections were stained with chloroacetate esterase and a minimum of twenty glomeruli per section counted; results are expressed as neutrophils per glomerular cross section.
Glomeruli thrombi were scored as 0 to 4 for each glomerulus depending on the number of quadrants involved and a mean obtained for at least twenty glomeruli as previously described16
Immunohistochemistry
Kidney was fixed in paraformaldehyde, lysine, periodate fixative23 for four hours at 4°C, washed in 7% sucrose overnight and frozen in isopentane cooled in liquid nitrogen. Tissue sections were cut and stained with monoclonal anti-mouse CD68 (FA/11; a kind gift from Dr. Andrew McKnight, Oxford, United Kingdom), to detect macrophages. The primary antibody was detected using biotinylated rabbit anti-rat antibody and streptavidin biotin peroxidase.
Statistics
Results are expressed as mean 
standard error and comparisons between groups are by Student's t-test.
RESULTS
Induction of glomerulonephritis
Immunization followed by anti-GBM antibody in the 129Sv wild type strain resulted in an accelerated nephrotoxic nephritis with persistent albuminuria up to 55 mg per 24 hours (normal 129Sv mice, albuminuria < 0.05 mg/24 hr). Histologically, at 24 hours there was neutrophil polymorphonuclear leukocyte infiltration and segmental glomerular thrombosis Figure 1. NOS2 mRNA was detected in glomeruli at 24 hours and nine days; NOS2 message was not present in normal glomeruli. From 6 to 9 days there was glomerular thrombosis and mild glomerular hypercellularity together with small cellular crescents in 10% of glomeruli Figure 2. There was focal tubular dilatation with eosinophilic casts and a focal, mild, interstitial mononuclear cell infiltrate. These changes increased in severity with increasing doses of anti-GBM antibody, and with 3 mg of antibody there was total glomerular thrombosis with non-perfusable kidneys.
Figure 1.
Glomerulus 24 hours after induction of glomerulonephritis. There is prominent thrombosis (arrows). An infiltrating neutrophil is present (arrowhead) (periodic acid-Schiff 
500).
Figure 2.
Glomerulus six days after induction of glomerulonephritis. The glomerulus is hypercellular with thrombosis (arrows) and a developing crescent (arrowheads) (periodic acid-Schiff 500).
The presence of neutrophils and a high level of albuminuria at 24 hours suggested an early neutrophil-dependent phase of disease, preceding autologous disease. In experiments to assess the effect of NOS2 deficiency in these two phases, we therefore examined early (24 hr) injury, using a dose of 0.6 mg anti-GBM antibody, and autologous phase injury at six days using a lower dose of 0.3 mg. The lower dose was chosen to ensure significant survival at six days.
Experiment 1. Early injury (24 hours)
Disease was induced in 10 NOS2 mutant and 10 NOS2 heterozygous mice by immunization followed by 0.6 mg anti-GBM antibody. Two heterozygous animals died within 24 hours. At 24 hours mice were killed and three mice from each group were perfused with iron oxide to obtain isolated glomeruli for mRNA preparation and RT-PCR. Kidneys were taken for histology from all mice.
There were no significant differences in the histological appearances or level of albuminuria between the mutant and the heterozygous mice Table 1 mice. NOS2 mRNA was induced in the glomeruli of heterozygous mice but was absent in NOS2 deficient mice Figure 3. IL-1 and TNF- mRNA was induced in both strains of mice Figure 4. Disease in the NOS2 heterozygous mice did not differ significantly from that in wild type 129Sv mice.
Figure 3.
Glomerular expression of NOS2 and 
-actin mRNA, demonstrated by RT-PCR, 24 hours after induction of anti-GBM glomerulonephritis in NOS2-deficient, heterozygous and wild-type 129Sv mice.
Figure 4.
Glomerular expression of interleukin 1
(IL-1) and tumor necrosis factor-
(TNF-) mRNA demonstrated by RT-PCR 24 hours after induction of glomerulonephritis. The left hand panel shows heterozygous mice (N = 2) and the right hand panel NOS2 deficient mice (N = 3). For each mouse, and for each cytokine, two dilutions (1:5 and 1:15) of cDNA were used for PCR.
Table 1 - TABLE 1. Anti-GBM glomerulonephritis in NOS2-deficient and heterozygous mice.
Full table (23K)Experiment 2. Autologous phase injury
Disease was induced in 12 NOS2 deficient and 12 NOS2 heterozygous mice by immunization followed by 0.3 mg anti-GBM antibody. One mutant mouse and 2 heterozygous mice died within 24 hours. Three mice from each group were killed at 24 hours for histological examination of kidneys. Two more mutant mice died on day five. The remaining mice were killed on day 6 and kidneys taken for histology and immunohistochemistry.
At 24 hours there was no difference in albuminuria or histological injury between the two groups Table 1 as found in experiment 1. The autologous phase results are shown in Table 1. There was no significant difference in survival, level of albuminuria, or histological appearances between the groups by ranking for histological severity, scoring thrombi, crescent formation or macrophage infiltration.
DISCUSSION
Using a well characterized rabbit anti-mouse glomerular basement membrane antibody previously shown to induce heterologous disease20,24 we developed a model of accelerated anti-GBM nephritis in the 129Sv mouse. Accelerated anti-GBM glomerulonephritis depends on an autologous response to heterologous immunoglobulin, with anti-GBM specificity, planted on glomerular capillary walls. The accelerated model was chosen as injury in this model in the rat is known to be macrophage dependent25. The disease produced was similar to that in the rat with NOS2 induction, heavy albuminuria and a diffuse acute glomerulonephritis. The level of glomerular thrombosis and crescent formation was slightly higher than normally seen in the Lewis rat in our laboratory and the degree of macrophage infiltration was less. There have been several recent reports of this model in other strains of mice, stimulated by the availability of transgenic animals. It has been reported that the mechanisms of injury vary with strain; in C57BL/6 mice, cell mediated responses appear more important than antigen/antibody complexes26, in contrast to BALB/c26 and CSF-1 deficient (op/op) mice27 where cell mediated mechanisms were not essential. As accelerated anti-GBM nephritis has not previously been reported in the 129Sv strain, which of these mechanisms of injury is most critical is unknown. The appearances in the glomeruli have features more in common with those described in the BALB/c strain with prominent thrombi and focal crescent formation but only a minor degree of macrophage infiltration.
In NOS2 deficient mice there was no induction of NOS2 message in glomeruli, contrasting with induction of NOS2 mRNA in the heterozygous and wild type mice during glomerulonephritis. Disruption of the NOS2 gene did not affect the level of injury in glomerulonephritis either at 24 hours or in the advanced autologous phase at six days. This lack of effect has also been found in a model of chronic glomerulonephritis: in a recent study on MRL-lpr/lpr mice, which develop a spontaneous lupus-like glomerulonephritis, targeted disruption of the NOS2 gene had no effect on glomerular disease although it protected against renal vasculitic lesions28.
The role of NOS2 in several other inflammatory reactions has now been studied using NOS2 deficient mice. A clear and major role in the control of the intracellular parasites Mycobacterium tuberculosis29, Leishmania major19 and Listeria monocytogenes30 has been demonstrated. However, the effects on other aspects of the inflammatory response are less clear. Acute dermal inflammation induced by carageenin was suppressed19 while acute, acetic acid-induced colitis was enhanced31 in mice lacking NOS2. In glomeruli we found no effect on neutrophil numbers. The macrophage influx in our model was also not affected by the lack of NOS2. This result is similar to that of MacMicking et al29, who found no effect on granuloma formation in NOS2-deficient mice despite greatly reduced survival and increased bacterial load, and no impairment in the recruitment of macrophages in response to intraperitoneal thioglycollate. As in this previous report29 there was no impairment of IL-1 and TNF- induction.
Lack of NOS2 did not affect the prominent glomerular thrombosis in this model. NO inhibition with L-NAME in the rat has previously been associated with enhanced glomerular thrombosis in endotoxin-induced injury11,32, and plasma arginine depletion using arginase increases glomerular thrombosis in acute nephrotoxic nephritis16. It has recently been reported that selective NOS2 inhibition with L-NIL in endotoxemia does not increase thrombi33, which suggests a critical role for NOS3 rather than NOS2. It is currently unclear how much NO production can result locally from stimulation of endothelial NOS. Nitrite production is not detectable by Griess reaction (a relatively insensitive measure of NO production) in normal glomeruli1, but it is known that this isoform is present in glomeruli34,35 and an increase in endothelial NOS mRNA has been reported in anti-Thy 1 glomerulonephritis5. It is important to emphasize that the role of NO in pathophysiology can only be clarified by distinguishing between the protective effects of constitutively generated NO and the potentially injurious effects of high levels of NO produced by NOS2
In conclusion, we have induced a model of autologous immune-mediated glomerulonephritis in mice, characterized by proteinuria and glomerular thrombosis with a minor degree of macrophage infiltration and focal crescent formation. There was glomerular induction of NOS2 mRNA. In NOS2-deficient mice there was no induction of NOS2 mRNA, but the disease did not differ from that in mice with an intact NOS2 gene. Specifically we found an equivalent degree of albuminuria, glomerular thrombosis, neutrophil and macrophage accumulation, and crescent formation. Although we have not yet examined compensatory changes in other NOS isoforms, (NOS1 or NOS3) we can conclude from our current experiments that NOS2 does not play an essential role in the initiation or progression of these events in this form of glomerulonephritis in the mouse.
References
| 1. | CATTELL V, COOK T & MONCADA S. Glomeruli synthesize nitrite in experimental nephrotoxic nephritis. Kidney Int 1990; 38: 1056−1060. | PubMed | ISI | ChemPort | |
| 2. | COOK HT & SULLIVAN RS. Glomerular nitrite synthesis in in situ immune complex glomerulonephritis in the rat. Am J Pathol 1991; 139: 1047−1052. | PubMed | |
| 3. | CATTELL V, LIANOS E, LARGEN P & COOK T. Glomerular NO synthase activity in mesangial cell immune injury. Exp Nephrol 1993; 1: 36−40. | PubMed | ISI | ChemPort | |
| 4. | CATTELL V, LARGEN P, DE HEER E & COOK T. Glomeruli synthesize nitrite in active Heymann nephritis; the source is infiltrating macrophages. Kidney Int 1991; 40: 847−851. | PubMed | ISI | ChemPort | |
| 5. | GOTO S, YAMAMOTO T, FENG L, YAOITA E, HIROSE S, FUJINAKA H, KAWASAKI K, HATTORI R, YUI Y, WILSON CB, ARAKAWA M & KIHARA I. Expression and localization of inducible nitric oxide synthase in anti-thy-1 glomerulonephritis. Am J Pathol 1995; 147: 1133−1141. | PubMed | ISI | ChemPort | |
| 6. | COOK HT, EBRAHIM H, JANSEN AS, FOSTER GR, LARGEN P & CATTELL V. Expression of the gene for inducible nitric oxide synthase in experimental glomerulonephritis in the rat. Clin Exp Immunol 1994; 97: 315−320. | PubMed | ISI | ChemPort | |
| 7. | SAKURAI H, HISADA Y, UENO M, SUGIURA M, KAWASHIMA M & SUGITA T. Activation of transcription factor NF-kappaB in experimental glomerulonephritis in rats. Biochim Biophys Acta Mol Basis Dis 1996; 1316: 132−138 10.1016/0925-4439(96)00022-1. |
| 8. | JANSEN A, COOK T, TAYLOR GM, LARGEN P, RIVEROS-MORENO V, MONCADA S & CATTELL V. Induction of nitric oxide synthase in rat immune complex glomerulonephritis. Kidney Int 1994; 45: 1215−1219. | PubMed | ISI | ChemPort | |
| 9. | STUEHR DJ & NATHAN CF. Nitric oxide: A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989; 169: 1543−1545 10.1084/jem.169.5.1543. | Article | PubMed | ChemPort | |
| 10. | BAYLIS C & QIU CB. Importance of nitric oxide in the control of renal hemodynamics. Kidney Int 1996; 49: 1727−1731. | PubMed | ISI | ChemPort | |
| 11. | SHULTZ PJ & RAIJ L. Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis. J Clin Invest 1992; 90: 1718−1725. | PubMed | ISI | ChemPort | |
| 12. | GABOURY J, WOODMAN RC, GRANGER DN, REINHARDT P & KUBES P. Nitric oxide prevents leukocyte adherence: Role of superoxide. Am J Physiol Heart Circ Physiol 1993; 265: H862−H867. |
| 13. | RUBBO H, RADI R, TRUJILLO M, TELLERI R, KALYANARAMAN B, BARNES S, KIRK M & FREEMAN BA. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen containing oxidized lipid derivatives. J Biol Chem 1994; 269: 26066−26075. | PubMed | ISI | ChemPort | |
| 14. | NEUGARTEN J, FEINER H, SCHACT RG, GALLO GR & BALDWIN DS. Aggravation of experimental glomerulonephritis by superimposed clip hypertension. Kidney Int 1982; 22: 257−263. | PubMed | |
| 15. | NARITA I, BORDER WA, KETTELER M, RUOSLAHTI E & NOBLE NA. L-arginine may mediate the therapeutic effects of low protein diets. Proc Natl Acad Sci USA 1995; 92: 4552−4556. | PubMed | ChemPort | |
| 16. | WADDINGTON S, COOK HT, REAVELEY D, JANSEN A & CATTELL V. L-arginine depletion inhibits glomerular nitric oxide synthesis and exacerbates rat nephrotoxic nephritis. Kidney Int 1996; 49: 1090−1096. | PubMed | ISI | ChemPort | |
| 17. | NARITA I, BORDER WA, KETTELER M & NOBLE NA. Nitric oxide mediates immunologic injury to kidney mesangium in experimental glomerulonephritis. Lab Invest 1995; 72: 17−24. | PubMed | ISI | ChemPort | |
| 18. | WEINBERG JB, GRANGER DL, PISETSKY DS, SELDIN MF, MISUKONIS MA, MASON SN, PIPPEN AM, RUIZ P, WOOD ER & GILKESON GS. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: Increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered. NG-monomethyl-L-arginine. J Exp Med 1994; 179: 651−660 10.1084/jem.179.2.651. | Article | PubMed | ISI | ChemPort | |
| 19. | WEI X, CHARLES IG, SMITH A, URE J, FENG G, HUANG F, XU D, MULLER W, MONCADA S & LIEW FY. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 1995; 375: 408−411 10.1038/375408a0. | Article | PubMed | ISI | ChemPort | |
| 20. | ASSMANN KJM, TANGELDER MM, LANGE WPJ, SCHRIVJER G & KOENE RAP. Anti GBM nephritis in the mouse: Severe proteinuria in the heterologous phase. Virchows Arch [A] 1985; 406: 285−300. |
| 21. | TOMOSUGI NI, CASHMAN SJ, HAY H, PUSEY CD, EVANS DJ, SHAW A & REES AJ. Modulation of antibody-mediated glomerular injury in vivo by bacterial lipopolysaccharide, tumor necrosis factor, and IL-1. J Immunol 1989; 142: 3083−3090. | PubMed | ChemPort | |
| 22. | COOK WF & PICKERING GW. A rapid method for separating glomeruli from rabbit kidney. Nature 1958; 182: 1103−1104. | PubMed | |
| 23. | MCLEAN IW & NAKANE PK. Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J Histochem Cytochem 1974; 22: 1077−1083. | PubMed | ISI | ChemPort | |
| 24. | FEITH GW, ASSMANN KJM, BOGMAN MJJT, VAN GOMPEL APM, SCHALKWIJK J & KOENE RAP. Lack of albuminuria in the early heterologous phase of anti-GBM nephritis in beige mice. Kidney Int 1993; 43: 824−827. | PubMed | ISI | ChemPort | |
| 25. | SCHREINER GF, COTRAN RS, PARDO V & UNANUE ER. A mononuclear cell component in experimental immunological glomerulonephritis. J Exp Med 1978; 147: 369−384. | Article | PubMed | ISI | ChemPort | |
| 26. | HUANG X-R, TIPPING PG, SHUO L & HOLDSWORTH SR. Th1 responsivenes to nephritogenic antigens determines susceptibility to crescentic glomerulonephritis in mice. Kidney Int 1997; 51: 94−103. | PubMed | ISI | ChemPort | |
| 27. | NEUGARTEN J, FEITH GW, ASSMANN KJM, SHAN Z, STANLEY ER & SCHLONDORFF D. Role of macrophages and colony stimulating factor-1 in antiglomerular basement membrane glomerulonephritis. J Am Soc Nephrol 1995; 5: 1903−1909. | PubMed | ISI | ChemPort | |
| 28. | GILKESON GS, MUDGETT JS, SELDIN MF, RUIZ P, ALEXANDER AA, MISUKONIS MA, PISETSKY DS & WEINBERG JB. Clinical and serologic manifestations of autoimmune disease in MRL-lpr/lpr mice lacking nitric oxide synthase type 2. J Exp Med 1997; 186: 365−373. | Article | PubMed | ISI | ChemPort | |
| 29. | MACMICKING JD, NORTH RJ, LACOURSE R, MUDGETT JS, SHAH SK & NATHAN CF. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci USA 1997; 94: 5243−5248. | Article | PubMed | ChemPort | |
| 30. | MACMICKING JD, NATHAN C, HOM G, CHARTRAIN N, FLETCHER DS, TRUMBAUER M, STEVENS K, XIE Q, SOKOL K, HUTCHINSON N, CHEN H & MUDGETT JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 1995; 81: 641−650. | Article | PubMed | ISI | ChemPort | |
| 31. | MCCAFFERTY D-M, MUDGETT JS, SWAIN MG & KUBES P. Inducible nitric oxide synthase plays a critical role in resolving intestinal inflammation. Gastroenterology 1997; 112: 1022−1027. | PubMed | ISI | ChemPort | |
| 32. | WESTBERG G, SHULTZ PJ & RAIJ L. Exogenous nitric oxide prevents endotoxin-induced glomerular thrombosis in rats. Kidney Int 1994; 46: 711−716. | PubMed | ISI | ChemPort | |
| 33. | SCHWARTZ D, MENDONEA M, SCHWARTZ I, ZIA Y, SATRIANO J, WILSON CB & BLANTZ RC. Inhibition of constitutive nitric oxide synthase (NOS) by nitric oxide generated by inducible NOS after lipopolysaccharide administration provokes renal dysfunction in rats. J Clin Invest 1997; 100: 439−448. | PubMed | ISI | ChemPort | |
| 34. | UJIIE K, YUEN J, HOGARTH L, DANZIGER R & STAR RA. Localization and regulation of endothelial NO synthase mRNA expression in rat kidney. Am J Physiol (Renal, Fluid Electrolyte Physiol) 1994; 267: F296−F302. |
| 35. | BACHMANN S, BOSSE HM & MUNDEL P. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. Am J Physiol 1995; 268: F885−F898. | PubMed | ISI | ChemPort | |
