The natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide system

Abstract

Natriuretic peptides (NPs), mainly produced in heart [atrial (ANP) and B-type (BNP)], brain (CNP), and kidney (urodilatin), decrease blood pressure and increase salt excretion. These functions are mediated by natriuretic peptide receptors A and B (NPRA and NPRB) having cytoplasmic guanylyl cyclase domains that are stimulated when the receptors bind ligand. A more abundantly expressed receptor (NPRC or C-type) has a short cytoplasmic domain without guanylyl cyclase activity. NPRC is thought to act as a clearance receptor, although it may have additional functions. To test how NPRC affects the cardiovascular and renal systems, we inactivated its gene (Npr3) in mice by homologous recombination. The half life of [125I]ANP in the circulation of homozygotes lacking NPRC is two-thirds longer than in the wild type, although plasma levels of ANP and BNP in heterozygotes and homozygotes are close to the wild type. Heterozygotes and homozygotes have a progressively reduced ability to concentrate urine, exhibit mild diuresis, and tend to be blood volume depleted. Blood pressure in the homozygotes is 8 mmHg (1 mmHg = 133 Pa) below normal. These results are consistent with the sole cardiovascular/renal function of NPRC being to clear natriuretic peptides, thereby modulating local effects of the natriuretic peptide system. Unexpectedly, Npr3 -/- homozygotes have skeletal deformities associated with a considerable increase in bone turnover. The phenotype is consistent with the bone function of NPRC being to clear locally synthesized CNP and modulate its effects. We conclude that NPRC modulates the availability of the natriuretic peptides at their target organs, thereby allowing the activity of the natriuretic peptide system to be tailored to specific local needs.

Figure 1

Targeted inactivation of the Npr3 gene. (a) The targeting strategy. (Top line) The region of the Npr3 gene that includes exon 1 (black bar). (Middle line) The targeting construct. (Bottom line) The targeted locus in which the gene is disrupted and from which 215 amino acids of the ligand-binding domain have been deleted. neo, neomycin resistance gene; tk, Herpes simplex thymidine kinase gene. Restriction sites are H, HindIII; N, NotI; S, SpeI; Xb, XbaI; Xh, XhoI. The positions of two probes, A and B, and of two primers, C and D, are indicated. (b) Southern blots of tail DNA from wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice digested with HindIII and hybridized to probe A. The sizes of the hybridizing bands in kb are shown. (c) An autoradiogram showing the presence or absence of the 70-kDa band corresponding to NPRC after SDS gel electrophoresis under reducing conditions of 4-azidobenzoyl [¹²⁵I]ANP photoaffinity-labeled lung plasma membranes from +/+ and −/− mice. The 135-kDa radio-labeled band corresponding to NPRA is indicated.

Figure 2

Clearance of ANP and water handling. (a) Decline in trichloroacetic acid precipitable radioactivity after injection of [¹²⁵I] rat ANP (–28) into wild-type (n = 5, filled circles) and homozygous mutant mice (n = 4, open circles). The plotted points (log cpm) are means ± SE of logarithms of trichloroacetic acid-precipitable counts normalized as described in Materials and Methods. Where not shown, error bars are smaller than the symbols. ∗, P versus wild type < 0.0001. (b) Ability to dilute and concentrate urine assessed by loading and depriving water; wild-type (n = 7, filled circles), heterozygous (n = 6, open triangles), and homozygous mutants (n = 4, open circles). Urinary osmolalities (Uosm) at time 0, and 1, and 2 hours after 4% body weight water loading by gavage, and after 12 hours of water deprivation, are shown as means ± SE in mOsm/kgH₂O. ^†, P versus wild type < 0.05; ^‡, P versus wild type < 0.02.

Figure 3

Skeletal phenotypes of Npr3 +/+ and −/− mice. (a) Wild-type (left) and homozygous mutant mice (right) at 2 months of age. (b–e) Soft x-ray analysis of phalanges [wild type (b) and homozygous mutant (c)] and lower bodies [wild type (d) and homozygous mutant (e)] of mice at 3 months of age. (f) Body and bone dimensions of homozygous mutants at 3 months of age as percent difference from the wild type. ∗, P versus wild type < 0.001.

Figure 4

(a and b) Histochemical localization of osteoclasts by tartrate-resistant acid phosphatase staining (dark brown) in developing femurs of 10-day-old Npr3 +/+ (a) and −/− mice (b); original magnification 40×. The arrow indicates a secondary ossification center. (c and d) Cartilagenous growth plates of femurs from 10-day-old +/+ (c) and −/− (d) mice; hematoxylin and eosin stain; original magnification 200×. (e and f) Picro-Sirus Red staining of bony trabeculae in femurs from 10-day-old Npr3 +/+ (e) and −/− mice (f); original magnification 400×. The amounts of unresorbed mineralized cartilage (MC) and newly deposited bone matrix (arrows) are both increased in the mutant. (g and h) In situ hybridization for mouse NPRC mRNA in trabecular bones of 10-day-old wild-type (g) and homozygous mutant mice (h); original magnification 400×. The signal is specifically localized to the osteoblastic cells lining the bony trabeculae (BT). (i) In vitro osteoclast formation assessed by osteoclast number and by the average number of nuclei per osteoclast. Open bars, wild type; filled bars, homozygous mutant. (j) In vitro osteoblast formation assessed by number of ALP-positive colonies and the average cell number per colony in bone marrow stromal cell cultures from wild-type (open bars) and homozygous mutant mice (filled bars). ∗, P versus +/+ < 0.05; ^†, P versus +/+ < 0.01; ^‡, P versus +/+ < 0.001.