Am J Physiol Regul Integr Comp Physiol. 2015 Sep;309(5):R467-74.
Viral transduction of renin rapidly establishes persistent hypertension in diverse murine strains.
Harlan SM, Ostroski RA, Coskun T, Yantis LD, Breyer MD, Heuer JG.
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana.
Mice provide a unique platform to dissect disease pathogenesis, with the availability of recombinant inbred strains and diverse genetically modified strains. Leveraging these reagents to elucidate the mechanisms of hypertensive tissue injury has been hindered by difficulty establishing persistent hypertension in these inbred lines. ANG II infusion provides relatively short-term activation of the renin-angiotensinogen system (RAS) with concomitant elevated arterial pressure. Longer-duration studies using renin transgenic mice are powerful models of chronic hypertension, yet are limited by the genetic background on which the transgene exists and the exposure throughout development. The present studies characterized hypertension produced by transduction with a renin-coding adeno-associated virus (ReninAAV). ReninAAV mice experienced elevated circulating renin with concurrent elevations in arterial pressure. Following a single injection of ReninAAV, arterial pressure increased on average +56 mmHg, an increase that persisted for at least 12 wk in three distinct and widely used strains of adult mice: 129/S6, C56BL/6, and DBA/2J. This was accomplished without surgical implantation of pumps or complex breeding and backcrossing. In addition, ReninAAV mice developed pathophysiological changes associated with chronic hypertension, including increased heart weight and albuminuria. Thus ReninAAV provides a unique tool to study the onset of and effects of persistent hypertension in diverse murine models. This model should facilitate our understanding of the pathogenesis of hypertensive injury.
KEYWORDS: adenovirus; hypertensive murine model; renin
Hypertension is a prevalent disease with diverse etiologies ranging from genetic, environmental, developmental and metabolic factors. Although treatment options exist for patients, a large population suffers from persistent elevations in arterial pressure as a result of either non-compliance to treatment regimens or development of resistant hypertension. It is well documented that persistent hypertension can cause not only detrimental effects to the cardiovascular system, but also end organ damage to diverse organ beds such as eye, kidney, vessels, and brain 1 (Figure 1). Understanding the contribution of hypertension to end organ damage relies on representative animal models. AngII infusion is a widely used approach to induce hypertension, however it is limiting for developing animal models of end organ damage caused by hypertension, as this approach only allows for weeks of elevated arterial pressure as opposed to more chronic hypertension needed for end organ damage. To induce persistent hypertension, transgenic mice and rats have been developed which overexpress renin, the rate limiting step in the renin angiotensinogen system (RAS) 2-4. These transgenic models have served as valuable tools to study end organ damage induced by persistent hypertension, but they are restrictive if one seeks to develop animal models of disease complicated by hypertension. It is well documented that many diseases are known to be complicated or accelerated by hypertension such as diabetic nephropathy, diabetic retinopathy, Alzheimer’s, and vascular diseases5, 6. A transferrable tool to induce persistent hypertension on the currently available models of these diseases would allow for development of more representative animal models. To generate such models using currently available renin overexpressing transgenic mice would require extensive breeding and backcrossing to the parental disease strain one wishes to induce hypertension on. Moreover, many of the transgenic models are found on the C57/BL6 background which is resistant to developing certain diseases such as kidney disease 7. Thus extensive breeding and backcrossing is required to obtain the transgene on desired genetic background. To circumvent the limitations of transgenic approach, we developed a novel adeno-associated virus (AAV) that overexpresses renin to allow for the rapid and transferrable approach of inducing persistent hypertension in any murine model and genetic background of choice.
Figure 1: Hypertension contributes to end organ damage
In this study, using radiotelemetry we demonstrated in wild type C57/BL6 mice that a single retro-orbital injection of ReninAAV induces rapid elevations in arterial pressure with effects detected within 24 hours post injection (Figure 2A). To demonstrate the elevations in arterial pressure were due to overactive RAS, mice were treated with the ACEi Lisinopril which led to reduction in arterial pressure. In a separate study the ability of ReninAAV to induce persistent elevations in arterial pressure were examined. Using the kidney disease prone 129/S6 background a dose response study was performed in which mice were followed for 12 weeks after injection with ReninAAV. The ReninAAV was shown to have a dose dependent effect on arterial pressure with no indication of decline after 12 week timepoint (Figure 2B). To determine if the elevated arterial pressure translated to renal disease the urine albumin was measured throughout the study. Within one week of ReninAAV injection elevations in ACR (urine albumin to creatinine ratio) were observed in the higher doses tested which persisted the entire 12 week duration of the study, supporting the contribution of hypertension to end organ damage to the kidney. To validate these physiological endpoints were due to overactive RAS, circulating RAS biomarkers were analyzed. There was a dose-dependent effect on plasma renin, renin activity and aldosterone. Importantly, at 12 week measurements there was no indication of a decline in plasma renin or plasma renin activity suggesting longer term studies could be performed as there was no indication of decline in AAV activity after 12 weeks.
Figure 2: ReninAAV rapidly induces persistent hypertension. Using Radiotelemetry, it was shown that a single retro-orbital injection of ReninAAV leads to elevations in mean arterial pressure (MAP) that can be detected within 24-hours post injection (A). Using tail cuff plethysmography, ReninAAV was demonstrated to elevate systolic arterial pressure (SAP) for up to 12 weeks post injection of ReninAAV (B).
To demonstrate the feasibility to rapidly induce persistent hypertension on diverse genetic backgrounds, the effect of ReninAAV was evaluated on three different genetic backgrounds: C57/BL6, 129/S6 and DBA/2J backgrounds. The data demonstrate that ReninAAV elevated arterial pressure in all three genetic backgrounds (Figure3). On the other hand, differential effects of ReninAAV on renal damage as accessed by elevations of ACR were detected. ReninAAV increased ACR in the 129/S6 and DBA/2J backgrounds with only modest elevations observed in C57/BL6 mice. This is consistent with published data on the C57/BL6 mice being kidney disease resistant and supports the development of a transferrable approach to induce persistent hypertension7.
In summary, the study characterizes a novel tool to rapidly induce persistent hypertension on any genetically modified murine model or background. We feel this is an invaluable tool to foster the development of novel animal models of diseases complicated by hypertension. In addition, it will serve as a tool permit the investigation of the role a gene of interest may have in development of disease developed by chronic hypertension. Most studies using genetically modified animals rely on AngII infusion for evaluating the role that a gene has in arterial pressure control or end organ damage induced by hypertension. However, these short term studies are not representative of the human condition in which we know chronic hypertension is needed for the development of many complications of the disease. Using AngII infusion the only means to develop models of persistent hypertension would require surgical reimplantation of pumps which is an added stress to the animals. In addition, there is a risk of decline in peptide effect at later timepoints8. The advantage of our approach is that a single injection of ReninAAV leads to persistent elevations of arterial pressure that is readily transferrable to any murine model of choice.
Figure 3: ReninAAV is a novel approach to induce persistent hypertension on diverse murine strains and backgrounds.
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