Small hairpin inhibitory RNA delivery in the metanephric organ culture identifies long noncoding RNA Pvt1 as a modulator of cyst growth.

Kara Eckberg, Ivan Weisser, Daniel Buttram, Nikunj Somia, Peter Igarashi, Karam S Aboudehen

American journal of physiology. Renal physiology 2022 Sep 01

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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 (PKD1) and polycystin-2 (PKD2). 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 Pvt1, a long noncoding RNA, which was upregulated in kidneys from Pkd1 and Pkd2 mutant mice and humans with ADPKD. shRNA delivery by scAAV/DJ downregulated expression of Pvt1 by 45% and reduced the cyst index by 53% in wild-type MOCs and 32% in Pkd1-null MOCs. Knockdown of Pvt1 decreased the level of c-MYC protein by 60% without affecting Myc mRNA, indicating that Pvt1 regulation of c-MYC was posttranscriptional. These results identify Pvt1 as a long noncoding RNA that modulates cyst progression in MOC.NEW & NOTEWORTHY This study identified scAAV/DJ as effective in transducing epithelial cells of the metanephric organ culture (MOC). We used scAAV/DJ shRNA to knockdown Pvt1 in cystic MOCs derived from Pkd1-null embryos. Downregulation of Pvt1 reduced cyst growth and decreased levels of c-MYC protein. These data suggest that suppression of Pvt1 activity in autosomal dominant polycystic kidney disease might reduce cyst growth.

Citation

Kara Eckberg, Ivan Weisser, Daniel Buttram, Nikunj Somia, Peter Igarashi, Karam S Aboudehen. Small hairpin inhibitory RNA delivery in the metanephric organ culture identifies long noncoding RNA Pvt1 as a modulator of cyst growth. American journal of physiology. Renal physiology. 2022 Sep 01;323(3):F335-F348