Abstract 525: Morphological and Functional Characterization of Targeted Renal Gene Silencing via Medullary Interstitial vs. Subcapsular siRNA Infusion in Mice
Advances in gene knockout technology have enhanced gene function studies, paving the way for the development of mouse lines for the functional analysis of genes, such as those that are germane to blood pressure regulation. Classic gene knockout requires gene disruption at the embryonic stem cell level, which may allow for compensatory mechanisms to develop. The development of conditional knockouts allows tissue-specific gene manipulation and reversible gene silencing. However, this technology involves a substantial investment of time and resources, and is fraught with low success rate. To address these limitations, we compared two novel approaches for targeting renal genes against the classic tail vein delivery technique. These techniques involved the use of osmotic minipumps to infuse gene-specific siRNA directly into either the renal medullary parenchyma or subcapsular space and required only a fraction of the tail vein siRNA concentration. The dopamine D5 receptor (D5R) gene Drd5 was selected because Drd5 knockout mice have the highest blood pressure among all the dopamine receptor null mice. The D5R is distributed in the PCT, DCT CCT PST, TAL and OMCD. In vivo xenograph imaging studies using AAV9-luciferase conjugate showed the infusate distribution was restricted to the target kidney. The lack of extra-renal spillage of the infusate was confirmed by the restricted distribution of exogenous siRNA-Alexa488 in the kidney, but not in the liver of the same mouse. Subcapsular and intramedullary infusion of Drd5-specific siRNA (vs. non-silencing “mock” siRNA) resulted in decreased expression of D5R (subcapsular: mock, 100±2 vs. Drd5-siRNA, 72±6; intramedullary: 100±6 vs. 78±6); However, there was no difference in D5R expression after tail vein infusion. The reduction in D5R expression was accompanied by a markedly increased systolic blood pressure in both subcapsular and intramedullary approaches (subcapsular: mock, 94±2 mm Hg vs. Drd5-siRNA, 116±3; intramedullary: 103±1.5 vs. 109±0.9), but not in the tail vein approach. There was no significant difference in body weight, heart rate and 24-hr urine Na+/K+. Our novel alternative techniques offer an economical, efficient, and kidney-restricted targeted gene silencing.
Author Disclosures: L.D. Asico: None. X. Wang: None. V.M. Villar: None. Y. Yang: None. P. Konkalmatt: None. P.A. Jose: None.
- © 2014 by American Heart Association, Inc.