Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis

Abstract

Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study the role of eNOS in renal inflammation, the development of accelerated anti-glomerular basement membrane (GBM) glomerulonephritis was examined in mice lacking a functional gene for eNOS and compared with wild-type (WT) C57BL/B6j mice. WT C57BL/6j mice (n = 12) and eNOS knockout (-/-) mice (n = 12) were immunized intraperitoneally with sheep IgG (0.2 mg in complete Freund's adjuvant). At day 6.5 after immunization, mice received a single i.v. injection of sheep anti-mouse GBM (1 mg in 200 microl PBS). Mice were sacrificed at day 1 and 10 after induction of the disease. All WT mice survived until day 10, whereas 1 eNOS-/- mouse died and 2 more became moribund, requiring sacrifice. At day 10, eNOS-/- mice had higher levels of blood urea nitrogen than WT mice (P < 0.02), although proteinuria was comparable. Immunofluorescence microscopy documented similar IgG deposition in both WT and eNOS-/- mice, but eNOS-/- mice had more extensive glomerular staining for fibrin at day 10 (P < 0.007). At day 10, light microscopy demonstrated that eNOS-/- mice had more severe glomerular thrombosis (P < 0.003) and influx of neutrophils (P < 0. 006), but similar degrees of overall glomerular endocapillary hypercellularity and crescent formation. In conclusion, accelerated anti-GBM glomerulonephritis is severely aggravated in eNOS-/- mice, especially with respect to glomerular capillary thrombosis and neutrophil infiltration. These results indicate that NO radicals generated by eNOS play a protective role during renal inflammation.

Figure 1.

Immunohistochemistry for eNOS in renal tissue from WT and eNOS−/− mice. A: No eNOS immunoreactivity was found in eNOS−/− mice (G indicates a glomerulus). B: In WT mice, strong immunoreactivity was found in arteries (inset), arterioles (arrows) and glomerular and peritubular capillaries. C−E: A focal and segmental decrease or loss (D, arrows) in glomerular eNOS expression was detected at days 1 and 10 after induction of accelerated anti-GBM glomerulonephritis. At these time points, strong expression of eNOS in cortical arteries could still be detected (C, inset; E, arrow). Original magnifications, ×250 (A−C), ×400 (D and insets), ×200 (E).

Figure 2.

Light microscopy of renal tissue from normal WT (A) and eNOS−/− (B) mice and at day 1 after administration of sheep anti-mouse GBM Ig. Focal and segmental glomerular thrombosis was observed in WT mice (C and E, PAS stain). More extensive glomerular thrombosis was found in eNOS−/− mice (D and F, PAS stain). Original magnifications, ×200 (A-D), ×400 (E and F).

Figure 3.

Light microscopy of renal tissue 10 days after administration of sheep anti-mouse GBM Ig in WT (A, B, PAS stain) and eNOS−/− mice (C-F, PAS stain). A: Overview of renal tissue in a WT mouse demonstrating mild glomerular hypercellularity and glomerular crescent formation. B: Glomerulus from a WT mouse showing crescent formation and mild periglomerular inflammatory cell accumulation. C: Overview of renal tissue from an eNOS−/− mouse that became moribund and was sacrificed at day 8 after sheep anti-mouse GBM Ig administration. Glomerular deposits of PAS positive material are prominent in all glomeruli. D: Glomerulus from the same mouse as in C showing extensive capillary thrombosis and necrosis. E: Overview of renal tissue from an eNOS−/− mouse at 10 days after administration of sheep anti-mouse GBM Ig. Glomeruli show extensive deposits of PAS-positive material. F: Glomerulus from the same mouse as in E showing deposits of PAS-positive material throughout the glomerulus, segmental karyorrhexis, and the development of a small crescent (top right). Original magnifications, ×200 (A, C, E) and ×400 (B, D, F).

Figure 4.

Semiquantitation of glomerular deposits of PAS-positive material as a measure of glomerular injury. Deposits of PAS-positive material in glomeruli was quantitated in WT and eNOS−/− mice at the indicated time points according to the following scoring system: 0, absent; 1, up to one-third of glomerulus involved; 2, one-third to two-thirds involved; 3, more than two-thirds involved. Fifty glomeruli per mouse were evaluated. Bars represent means ± SD.

Figure 5.

Immunofluorescence microscopy for mouse IgG and fibrinogen 10 days after administration of sheep anti-mouse GBM Ig in WT (A and C) and eNOS−/− (B and D) mice. A: Linear staining for mouse IgG along the glomerular capillary wall in a WT mouse. B: Linear staining for mouse IgG along the glomerular capillary wall in an eNOS−/− mouse. C: Glomerulus from a WT mouse showing segmental fibrin deposition. D: Glomerulus from an eNOS−/− mouse showing extensive global fibrin deposition. Original magnifications, ×400.

Figure 6.

Semiquantitation of glomerular fibrin deposition in WT and eNOS−/− mice. Fibrin deposition was evaluated in 30 glomeruli per mouse according to the following scoring system: 0, no fibrin deposits; 1, up to one-third of glomerulus involved; 2, one-third to two-thirds involved; 3, more than two-thirds involved. Bars represent mean ± SD.

Figure 7.

Quantitation of glomerular numbers of PMNs (A), CD11b-positive cells (B), FA/11-positive cells (C), and CD3-positive T cells (D) in WT and eNOS−/− mice during the course of accelerated anti-GBM glomerulonephritis. Glomerular infiltrates were evaluated by counting the number of positive cells within 30 glomeruli. Bars represent mean numbers per glomerular cross-section (gcs) ± SD

Figure 8.

Blood urea nitrogen (BUN) levels (A) and the development of albuminuria (B) after the induction of accelerated anti-GBM glomerulonephritis in WT and eNOS−/− mice. Bars represent mean ± SD.