Novel Approaches to Manipulate Gene Expression in the Mouse Kidney: siRNA Silencing and Gene Rescue
Authors: Asico Laureano D , Konkalmatt Prasad , Villar Van Anthony M , Wang Xiaoyan , Armando Ines , Lin Hai , Jose Pedro A 
Published: November 2023

Essential hypertension represents a multifactorial set of genetic dysfunctions that involve several organ systems, including the kidney. The mechanistic underpinnings of hypertension has been facilitated by the availability of knockout mouse models, which allow the functional analysis of specific genes involved in the regulation of blood pressure (BP) and ion transport. Recent innovations on knockout mouse models, e.g., conditional knockouts, allow the reversible silencing of genes in a limited number of tissues; however, this technology is tedious, expensive and not always successful. Moreover, the use of gene complementation techniques has been instrumental in confirming the function of genes of interest in vivo. Therefore, we evaluated the feasibility and efficiency of using two complementary techniques to study gene function in mice, namely, the (1) delivery of gene‐specific siRNA via an osmotic pump into the renal parenchyma, and (2) bolus retrograde injection of the gene of interest packaged in AAV9 viral vectors into the ureter of the kidney. Previously uninephrectomized and acclimated mice were surgically implanted with an osmotic pump equipped with an attached 28G‐tipped polyurethane tube to infuse siRNA dissolved in an in vivo transfection reagent into the subcapsular space of the kidney. A 28‐day siRNA infusion (3μg/day) which ensured optimum silencing of the targeted gene neither resulted in gross or histological changes in the renal morphology nor spillage into other organ systems, i.e. the heart and liver. We silenced the renal Drd2 gene expression by ~65% in four C57Bl/6J mice which resulted in increased systolic blood pressure (DSBP=21±4.5 mm Hg vs. 1.0±2.9 mm Hg, measured via the femoral artery, in non‐silencing “mock” siRNA‐treated mice), similar to the hypertension observed in Drd2 knockout mice; Drd2 encodes for the dopamine receptor type 2, D2R, whose absence results in hypertension. When a siRNA against a gene (e.g., USP48 which prevents the degradation of NHE3) whose absence should decrease BP was tested, systolic BP was effectively decreased (DSBP=−21±2 mm Hg; n=5), suggesting that the resultant phenotype depends upon the specific gene being silenced. We next compared the effect of unilateral infusion of Drd2‐specific siRNA in mice with both kidneys intact. The BP was unchanged after a 7‐day infusion, but increased after 28 days of infusion (DSBP=26±2 mm Hg; n=5), suggesting that the presence of the contralateral, non‐infused kidney could initially compensate for the loss of D2R but not in the long‐term. The unilateral renal infusion of siRNA did not change the D2R expression of the non‐infused kidney, regardless of the duration of infusion, demonstrating the feasibility to use one kidney as an internal control for the functional analysis of gene silencing in the targeted kidney. We next performed gene rescue experiments in C57Bl/6J mice that have been previously depleted of their renal D2R using a human Drd2 gene subcloned in a mammalian expression vector and packaged in an AAV9 viral vector. Fifteen days post‐silencing, a bolus of Drd2‐AAV9 was given via retrograde ureteral injection. The D2R expression increased 7.5‐fold (compared to control) and the blood pressure was normalized at 14 days post‐infusion; mock‐AAV9 had no effect n=3/group). Our novel strategies for gene silencing and gene rescue in mice may prove to be an easier and more versatile approach of genetic manipulation compared to traditional methods.Support or Funding InformationThe work was funded by grants from the US National Institutes of Health: P01HL074940, P01HL068686, R01HL092196, R37HL023081, R01DK039308, and DK090918.