In Vivo siRNA Delivery and Rebound of Renal LRP2 in Mice

Abstract

siRNA stabilized for in vivo applications is filtered and reabsorbed in the renal proximal tubule (PT), reducing mRNA expression transiently. Prior siRNA efforts have successfully prevented upregulation of mRNA in response to injury. We proposed reducing constitutive gene and protein expression of LRP2 (megalin) in order to understand its molecular regulation in mice. Using siRNA targeting mouse LRP2 (siLRP2), reduction of LRP2 mRNA expression was compared to scrambled siRNA (siSCR) in mouse PT cells. Mice received siLRP2 administration optimized for dose, administration site, carrier solution, administration frequency, and administration duration. Kidney cortex was collected upon sacrifice. Renal gene and protein expression were compared by qRT-PCR, immunoblot, and immunohistochemistry (IHC). Compared to siSCR, siLRP2 reduced mRNA expression in PT cells to 16.6% ± 0.6%. In mouse kidney cortex, siLRP2 reduced mRNA expression to 74.8 ± 6.3% 3 h and 70.1 ± 6.3% 6 h after administration. mRNA expression rebounded at 12 h (160.6 ± 11.2%). No megalin renal protein expression reduction was observed by immunoblot or IHC, even after serial twice daily dosing for 3.5 days. Megalin is a constitutively expressed protein. Although LRP2 renal mRNA expression reduction was achieved, siRNA remains a costly and inefficient intervention to reduce in vivo megalin protein expression.

TNF‐α/ENO1 signaling facilitates testicular phagocytosis by directly activating Elmo1 gene expression in mouse Sertoli cells

“…2). Noteworthy, the Ambion siRNA employed in the present study has been shown to be able to dramatically improve the stability, efficiency and specificity of siRNA‐mediated gene silencing in multiple pathophysiological systems, including kidney [26] and tumor xenografts [27], thus confirming its methodological benefits and irreplaceability to analyze biological functions of genes of interest. Nevertheless, in the present study, the effects of siRNA‐mediated ablation of ENO1 on murine spermatogenesis were not as effective as conventional gene knockout models in terms of thoroughness and durability of gene deletion.…”

Translation rescue by targeting Ppp1r15a upstream open reading frame in vivo

“…We next tested the efficacy of ASO-based induction of Ppp1r15a protein in vivo using our mouse model of endotoxemia. The tissue distribution of ASO is similar to in vivo siRNA 29,30 . Indeed, ASO enriches in the proximal tubules [31][32][33] where translation shutdown is most prominent in the kidney 12 .…”

Signaling Modulation by miRNA-221-3p During Tooth Morphogenesis in Mice

“…Therefore, it can be speculated that 1-week renal calcified teeth had comparatively increased expression patterns of Shh along the HERS in the inhibitor group compared to control and mimic ( Supplementary Figure 6 ). As the transient knockdown of genes can result in a regain of its expression at a certain time point ( Eadon et al, 2017 ), it can also be deduced that the inhibition of miRNA-221-3p may also be regained and upregulate the Shh expression in the HERS after 1-week.…”