Targeted disruption of Adamts16 gene in a rat genetic model of hypertension

Abstract

A disintegrin-like metalloproteinase with thrombospondin motifs-16 (Adamts16) is an important candidate gene for hypertension. The goal of the present study was to further assess the candidacy of Adamts16 by targeted disruption of this gene in a rat genetic model of hypertension. A rat model was generated by manipulating the genome of the Dahl Salt-sensitive (S) rat using zinc-finger nucleases, wherein the mutant rat had a 17 bp deletion in the first exon of Adamts16, introducing a stop codon in the transcript. Systolic blood pressure (BP) of the homozygous Adamts16(mutant) rats was lower by 36 mmHg compared with the BP of the S rats. The Adamts16(mutant) rats exhibited significantly lower aortic pulse wave velocity and vascular media thickness compared with S rats. Scanning electron and fluorescence microscopic studies indicated that the mechanosensory cilia of vascular endothelial cells from the Adamts16(mutant) rats were longer than that of the S rats. Furthermore, Adamts16(mutant) rats showed splitting and thickening of glomerular capillaries and had a longer survival rate, compared with the S rats. Taken together, these physiological observations functionally link Adamts16 to BP regulation and suggest the vasculature as the potential site of action of Adamts16 to lower BP.

Fig. 1.

Screening animals for ZFN-targeted mutation at the Adamts16 locus. (A) Representative agarose gel image of PCR-amplified genomic DNA from an intercross of heterozygous Adamts16^mutant carriers. The normal allele of the Dahl S rat is 307 bp. Rats with alternate allele size of 290 bp are representative mutants. M, males; F, females; 95745–95769, rat tag numbers. (B) Representative sequencing results from the 307 bp and 290 bp bands shown in panel A confirmed the 17 bp deletion in the genomic DNA. The same 17 bp deletion was confirmed by amplification and sequencing of cDNA from the homozygous mutants. TGA in red depicts the stop codon in exon 1.

Fig. 2.

BP measurements of homozygous Adamts16^mutant (n = 6), heterozygous Adamts16^mutant (n = 5), and S (n = 7) rats. Levels of statistical significance for all data were analyzed by ANOVA followed by Tukey’s test. (A) Mean systolic BP effect ± SEM by the tail-cuff method. (B and C) BP measures of the same animals monitored after surgical implantation of radiotelemetry transmitters. Data plotted are the recordings obtained once every 5 min continuously for 24 h and averaged for 4 h intervals; **P < 0.01, ***P < 0.001.

Fig. 3.

Cardiac parameters of homozygous Adamts16^mutant (n = 6), heterozygous Adamts16^mutant (n = 5), and S (n = 7) rats. (A) Relative heart weight. (B) Left ventricular relative wall thickness. (C) Aortic pulse wave velocity. Left Ventricular (LV) RWT and aortic PWV were determined by echocardiography. Levels of statistical significance for all data were analyzed by ANOVA followed by Tukey’s test; *P < 0.05, ***P < 0.001.

Fig. 4.

Vascular features of homozygous Adamts16^mutant (n = 6) and S (n = 7) rats. (A) Quantitation of media area. (B) Images of vascular endothelial primary cilia. From left first and second panels, representative images of primary cilia from vascular endothelia observed by immunofluorescence microscopy. Arrows indicate green fluorescently labeled cilia. Cilia were stained with monoclonal acetylated α-tubulin (green), whereas the nuclei were stained with DAPI (blue). From left third panel, scanning electron micrographs of cilia in vascular endothelia; arrows point to cilia. (C) Quantitation of cilia length from scanning electron micrograph images. More than 200 cilia were counted in each strain; **P < 0.01, ***P < 0.001.

Fig. 5.

Renal parameters of experimental groups of animals. (A) Total 24 h urine protein was assessed in S (n = 15) and homozygous Adamts16^mutant rats (n = 12) as described in the Materials and Methods section. (B) Representative Jones silver-stained kidney sections. Yellow arrows in the top panel point to thickened arteries. White arrow in the bottom panel center image from the kidney of a homozygous Adamts16^mutant rat shows split glomerular capillary. (C) Quantitation of intrarenal artery thickness shown in the top panel of B; n = 3/group; ***P < 0.0001.

Fig. 6.

Kaplan–Meir plot. Data from rats (n = 10 S rats and n = 10 Adamts16^mutant rats) in the survival study were plotted, and median survival of each group was calculated using the Graphpad prism software. Median survival of the Adamts16^mutant rats was greater than the survival of the S rats; *P < 0.05.