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

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)F335-F348
JournalAmerican Journal of Physiology - Renal Physiology
Volume323
Issue number3
DOIs
StatePublished - Sep 2022

Bibliographical note

Funding Information:
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant R01DK042921 (to P.I.). K.S.A. is supported by NIDDK Grant K01DK11693.

Publisher Copyright:
Copyright © 2022 the American Physiological Society.

Keywords

  • Pvt1
  • c-MYC
  • metanephric organ culture
  • polycystic kidney disease
  • self-complementary adeno-associated virus

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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