Small hairpin inhibitory RNA delivery in the metanephric organ culture identifies long noncoding RNA as a modulator of cyst growth.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder characterized by the formation of kidney cysts that originate from the epithelial tubules of the nephron and primarily results from mutations in polycystin-1 () and polycystin-2 (). The metanephric organ culture (MOC) is an ex vivo system in which explanted embryonic kidneys undergo tubular differentiation and kidney development. MOC has been previously used to study polycystic kidney disease as treatment with 8-bromo-cAMP induces the formation of kidney cysts. However, the inefficiency of manipulating gene expression in MOC has limited its utility for identifying genes and pathways that are involved in cystogenesis. Here, we used a lentivirus and three serotypes of self-complementary adeno-associated viral (scAAV) plasmids that express green fluorescent protein and found that scAAV serotype D/J transduces the epithelial compartment of MOC at an efficiency of 68%. We used scAAV/DJ to deliver shRNA to knockdown , a long noncoding RNA, which was upregulated in kidneys from and mutant mice and humans with ADPKD. shRNA delivery by scAAV/DJ downregulated expression of by 45% and reduced the cyst index by 53% in wild-type MOCs and 32% in -null MOCs. Knockdown of decreased the level of c-MYC protein by 60% without affecting mRNA, indicating that regulation of c-MYC was posttranscriptional. These results identify as a long noncoding RNA that modulates cyst progression in MOC. This study identified scAAV/DJ as effective in transducing epithelial cells of the metanephric organ culture (MOC). We used scAAV/DJ shRNA to knockdown in cystic MOCs derived from -null embryos. Downregulation of reduced cyst growth and decreased levels of c-MYC protein. These data suggest that suppression of activity in autosomal dominant polycystic kidney disease might reduce cyst growth.