Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice

Abstract

Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS(-/-) mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS(-/-) C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS(-/-) C57BLKS db/db exhibited decreases in GFR to levels <50% of that in eNOS(+/+) C57BLKS db/db, as confirmed by increased serum creatinine. In summary, eNOS(-/-) db/db mice provide the most robust model of type II DN that has been described to date and support a role for deficient eNOS-derived NO production in the pathogenesis of DN.

Figure 1

Metabolic and physiologic parameters. (A) Fasting blood glucose at 24 to 26 wk in db/db mice and age-matched controls ± eNOS^−/−. *P < 0.05 versus control and eNOS^−/−. Values are means ± SE of at least eight mice. (B) Systolic BP. *P < 0.05 eNOS^−/− or eNOS^−/− db/db versus control or db/db. Values are means ± SE of at least four mice.

Figure 2

Functional renal parameters. (A) Urinary albumin/creatinine ratio (ACR) at 24 to 26 wk of age. Values are means ± SE of at least eight mice. *P < 0.05 versus respective controls, db/db, and eNOS^−/−. (B) GFR in diabetic mice. *P < 0.05 versus control, db/db, and eNOS^−/−. Values are means ± SE of at least eight mice. (C) Correlation of serum creatinine with GFR.

Figure 3

Glomerular histopathology. (A) Representative glomerular lesions of diabetic mouse kidneys at 24 to 26 wk: control (a); db/db (b and e); eNOS^−/− (c); eNOS^−/−db/db (d and f). (f) Arteriolar hyalinosis (big arrow) and early nodular glomerulosclerosis (small arrow) in eNOS^−/− db/db mice (periodic acid-Schiff). (B) Glomerular fibronectin expression in control (a), db/db (b), eNOS^−/− (c), and eNOS^−/− db/db mice (d). (C) Glomerular injury scores in diabetic mice. Mesangial expansion and sclerosis were scored on a scale from 0 to 4⁺ as described in The Materials and Methods section. *P < 0.05, eNOS^−/− db/db (24 to 26 wk) mice versus age-matched controls, db/db, and eNOS^−/− mice. (D) Correlation of glomerular injury index with albuminuria. (E) Electron micrographs of glomeruli (16 wk). Opposed arrows indicate GBM. GBM thickening was notable in eNOS^−/−db/db mice (c) compared with control (a) and db/db mice (b). Magnifications: ×400 in A, a through d, and B; ×200 in A, e and f.