Nephrogenic diabetes insipidus in mice lacking all nitric oxide synthase isoforms

Abstract

Nitric oxide (NO) is produced in almost all tissues and organs, exerting a variety of biological actions under physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS), including neuronal, inducible, and endothelial NOSs. Because there are substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. Here, we have successfully developed mice in which all three NOS genes are completely deleted by crossbreeding singly NOS-/- mice. NOS expression and activities were totally absent in the triply NOS-/- mice before and after treatment with lipopolysaccharide. Although the triply NOS-/- mice were viable and appeared normal, their survival and fertility rates were markedly reduced as compared with the wild-type mice. Furthermore, these mice exhibited marked hypotonic polyuria, polydipsia, and renal unresponsiveness to an antidiuretic hormone, vasopressin, all of which are characteristics consistent with nephrogenic diabetes insipidus. In the kidney of the triply NOS-/- mice, vasopressin-induced cAMP production and membranous aquaporin-2 water channel expression were reduced associated with tubuloglomerular lesion formation. These results provide evidence that the NOS system plays a critical role in maintaining homeostasis, especially in the kidney.

Fig. 1.

NOS expression, NOS activity, and NOx level in triply NOS^-/- mice. (A) Appearance of NOS^-/- mice (KO, knockout). (Scale bars, 1 cm.) (B) PCR analysis for nNOS, iNOS, eNOS, and neomycin genes in genomic DNA. (C) Western blot analysis for nNOS, iNOS, and eNOS in the brain, the LPS-treated heart (20 mg/kg i.p.; 12 h after treatment), and the normal heart, respectively. (D) Total NOS activity in the brain, the heart, and the LPS-treated heart (n = 6 each). ^*, P < 0.05; #, P < 0.001; compared with wild-type C57BL/6 mice. (E) Plasma NOx concentrations and urinary NOx excretion (n = 6 each). ^*, P < 0.05; †, P < 0.01; #, P < 0.001; compared with C57BL/6 mice.

Fig. 2.

Survival, fertility, and hemodynamics in triply NOS^-/- mice. (A) Survival rate 10 months after birth (n = 13-40). ^*, P < 0.05 between each singly NOS^-/- and C57BL/6 mice; †, P < 0.01 between each doubly NOS^-/- and C57BL/6 mice; #, P < 0.001 between triply NOS^-/- and C57BL/6 mice. (B) Number of offspring produced from breeding pairs (n = 10-26). †, P < 0.01; #, P < 0.001; compared with C57BL/6 mice. (C) Systolic blood pressure (n = 9-16). ^*, P < 0.05 vs. C57BL/6 mice. (D) Heart rate (n = 9-16). ^*, P < 0.05 vs. C57BL/6 mice.

Fig. 3.

Body water balance, osmolality, and vasopressin kinetics in triply NOS^-/- mice. (A) Urinary volume (n = 6-13). ^*, P < 0.05; †, P < 0.01; compared with C57BL/6 mice. (B) Water intake (n = 6-13). ^*, P < 0.05; †, P < 0.01; compared with C57BL/6 mice. (C) Plasma osmolality (n = 6-13). ^*, P < 0.05; †, P < 0.01; compared with C57BL/6 mice. (D) Urinary osmolality (n = 6-13). ^*, P < 0.05; †, P < 0.01; compared with C57BL/6 mice. (E) Urinary vasopressin excretion (n = 6 each). (F) Changes of urinary volume in response to exogenously administered vasopressin (n = 6 each). Open circles, saline i.p.; closed circles, vasopressin i.p. (0.002 unit).

Fig. 4.

Histopathology in the kidney of triply NOS^-/- mice. (A) Renal tubular apoptosis (arrows) and regeneration (increased cell number) in triply NOS^-/- mice. H&E, hematoxylin and eosin staining. (Scale bar, 10 μm.) (B) Renal glomerulosclerosis (hyalinization, pink color) in triply NOS^-/- mice. PAS, periodic acid-Schiff staining. (Scale bar, 10 μm.) (C) Renal glomerular thrombi (arrows, dark blue) in triply NOS^-/- mice. PTAH, phosphotungstic acid-hematoxylin staining. (Scale bar, 10 μm.)

Fig. 5.

cAMP level and aquaporin-2 expression in renal collecting duct of triply NOS^-/- mice. (A) Basal and vasopressin-stimulated (1 μM; for 20 min) cAMP production in collecting duct (n = 6 each). NS, not significant. (B) Western blot analysis for aquaporin-2 (48 and 28 kDa) in kidney membrane fraction (n = 6 each). Actin served as an internal control. (C) Immunostaining for aquaporin-2 in renal collecting duct (arrows, brown staining). (Scale bars, 10 μm.)