The renin-angiotensin system plays an important role in the control of blood pressure (BP) and renal function. To illuminate the importance of renin in the context of a disease background in vivo, we used zinc-finger nucleases (ZFNs) designed to target the renin gene and create a renin knockout in the SS/JrHsdMcwi (SS) rat. ZFN against renin caused a 10-bp deletion in exon 5, resulting in a frameshift mutation. Plasma renin activity was undetectable in the Ren−/− rat, and renin protein was absent from the juxtaglomerular cells in the kidney. Body weight was lower in the Ren−/− rats (than in the Ren+/− or wild-type littermates), and conscious BP on low-salt diet (0.4% NaCl) was 58 ± 2 mm Hg in the Ren−/− male rats versus 117 mm Hg in the Ren+/− littermates, a reduction of almost 50 mm Hg. Blood urea nitrogen (BUN) and plasma creatinine levels were elevated in the Ren−/− strain (BUN 112 ± 7 versus 23 ± 2 mg/dL and creatinine 0.53 ± 0.02 versus 0.26 ± 0.02 mg/dL), and kidney morphology was abnormal with a rudimentary inner renal medulla, cortical interstitial fibrosis, thickening of arterial walls, and abnormally shaped glomeruli. The development of the first rat knockout in the renin-angiotensin system demonstrates the efficacy of the ZFN technology for creating knockout rats for cardiovascular disease on any genetic background and emphasizes the role of renin in BP regulation and kidney function even in the low-renin SS rat.
Recent studies in humans and animal models suggest that hypertension is determined by different genes in male and females. The Dahl Salt‐Sensitive hypertensive (SS) rat develops salt‐sensitive hypertension and substitution of Chr 13 from the normotensive Brown Norway (BN) rat onto the SS genetic background (SS.13BN consomic), attenuates the development of salt‐sensitive hypertension in both males and females, and restores renin levels and vasodilatory function. Introgression of a 4Mbp BN segment harboring the renin gene attenuated the development of salt‐sensitive hypertension in female but not in male SS rats, suggesting a sex‐specific effect of this gene on blood pressure. In order to narrow the region responsible for renin regulation and blood pressure we developed sequentially narrow overlapping subcongenic strains for this region. We measured blood pressure in these subcongenic strains, and we found two blood pressure loci located within 1 Mbp in females, with a 30 mmHg effect on blood pressure, 50 kb below the renin gene. We also found a locus for blood pressure in males 1 Mbp below renin, with a 20 mmHg effect on blood pressure. The data suggests that there are a different set of genes that regulate blood pressure in males and females in the renin region. By sequence and expression analysis of the candidate regions, we identified all sequence variants and prioritized the candidate genes for hypertension. HL‐82798
The catecholamine system plays an important role in the control of blood pressure and sodium excretion. One of the inactivation pathways of catecholamines is the enzymatic metabolism by catechol-O-methyltransferase (Comt). There are conflicting data regarding the role of Comt on the development of salt-sensitive hypertension, so the goal of our study was to evaluate the importance of Comt in the context of a susceptible background in vivo. Comt was mutated in the Dahl SS rat by zinc finger nucleases (ZFNs) injections targeting the sequence CTGTTCCAGGTCACCATCctcaatGGGGCATCCCAGGATCTT into SS/JrHsdMcwi (Dahl S) rat embryos. The resulting mutation was a 14-bp frameshift deletion in exon 4. Conscious blood pressure was measured by telemetry on male and female Comt knockout and wild type (WT) rats on low salt diet (0.4% NaCl) and during three weeks of high salt diet (8%NaCl). Disruption of Comt caused the protein not to express in the kidneys of the Dahl S rat. There were no differences in mean arterial pressure (MAP) between the Comt -/- and the Comt +/+ male rats at any time point during the day-night cycle at low or high salt diet. Body and organ weights, and protein and electrolyte excretion was also unchanged by the Comt mutation. On the other hand, female Comt -/- rats evidenced a higher MAP, which was only significantly higher at night during low salt diet (112±2 mmHg in Comt +/+ vs 125±2 mmHg in the Comt -/-, n>6) and both during day and night after 21 days of high-salt diet (∼ 30 mmHg difference between Comt +/+ and Comt -/- strains at both day and night). Systolic blood pressure differences were mostly responsible for the observed blood pressure diferences in females KO of the Comt gene, despite blood pressure effect, was not followed by a parallel difference in urine flow, electrolyte excretion or renal damage (protein and albumin excretion). In conclusion, we are the first ones to show that disruption of Comt enhances salt-sensitive hypertension in a gender-dependent manner.
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