Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death

Abstract

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

Exogenous C-type natriuretic peptide restores normal growth and prevents early growth plate closure in its deficient rats

“…For RT-PCR analysis, we used three-week-old male CNP-KO rats with homozygous Δ774 mutation, which caused massive deletion within the Nppc gene that included the translation initiation site. We previously confirmed that both Δ11 and Δ774 homozygous lines had CNP deficiency and exhibited almost identical impaired skeletal phenotype [ 14 ]. Genotyping of CNP-KO rats were performed by PCR analysis using genomic DNA from tail biopsy, as described in our previous report [ 14 ].…”

“…We previously confirmed that both Δ11 and Δ774 homozygous lines had CNP deficiency and exhibited almost identical impaired skeletal phenotype [ 14 ]. Genotyping of CNP-KO rats were performed by PCR analysis using genomic DNA from tail biopsy, as described in our previous report [ 14 ]. The animals were housed in a humidity and temperature-controlled environment with an automatic 12-hour light/dark cycle.…”

“…All animal care and experiments were conducted under the guidelines for animal experiments of Kyoto University and Asubio Pharma Co., Ltd., and approved by the Animal Research Committee of Kyoto University (Med Kyo 17218), and by the committee for ethics in animal experiments of Asubio Pharma Co., Ltd. CNP-KO rats were generated as previously reported [ 14 ]. The CNP-KO rats had a homozygous Δ11 deletion of nucleotides 192–202 (NM_053750.1), which generated a frame shift, and a premature stop codon at nucleotides 275–277 (NM_053750.1) in the natriuretic peptide precursor C ( Nppc ) gene.…”

“…The CNP-53 was dissolved in 0.03 M acetate buffer (pH 4.0), 1% benzyl alcohol, and 10% purified sucrose for use as dosing solutions. The concentrations of the dosing solutions were based on body weight data of CNP-KO and WT rats in our previous study [ 14 ], and calculated using the average body weight of CNP-KO and WT rats at four to six weeks of age, and six to eight weeks of age. The osmotic mini-pumps using the dosing solutions for four to six weeks of age were implanted in the rats at the start of the experiments.…”

“…We recently developed CNP-KO rats using zinc-finger nuclease technology [ 14 ]. The CNP-KO rats exhibit impaired skeletal growth, caused by a deficiency in endochondral ossification, which agrees with the findings in CNP-KO mice.…”