Glis3 Is a Modifier of Cyst Progression in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Background: ADPKD is caused by mutations affecting polycystin-1 (PC1) or -2 (PC2). The existence of a ‘cilia-dependent cyst activation’ (CDCA) pathway has been identified by demonstrating that structurally intact primary cilia are crucial for cyst growth following loss of polycystins. We used translating ribosome affinity purification (TRAP)-RNAseq on precystic mouse kidney cyst cells to determine the translatome that meet the criteria for CDCA and identified Glis2 as an early effector of polycystin signaling. Here, we investigate the potential role of Glis3 which, while not transcriptionally altered, encodes a cilia-localized transcription factor belonging to the same family of proteins as Glis2. Methods: We used Glis3-EGFP and live cell imaging to study the subcellular localization of Glis3 under ciliated conditions and in the presence or absence of Pkd1. We generated Glis3fl conditional allele and crossed it with Pkd1fl/fl; Pax8rtTA; TetOcremice to investigate the potential genetic interaction in early (postnatal day 14, P14) and adult (14 weeks) ADPKD models. Results: Live cell confocal imaging of wild type or Pkd1 knockout mIMCD3 cells stably expressing Glis3-EGFP and the Nphp31-200-mApple cilia marker shows that Glis3 is localized in both the nucleus and primary cilium, and its localization does not change upon inactivation of PC1. In the P14 early onset ADPKD model, compared to Pkd1-only knockouts, Glis3;Pkd1double knockouts have increased kidney-to-body weight ratio, cystic index, and blood urea nitrogen level, indicating a significantly worsening polycystic phenotype. Histological analysis of Glis3-only knockouts shows very occasional and sporadic tubule dilation at P14, which does not affect renal function. Immunofluorescence shows intact cilia in all four genotypes. Consistent with the early model, adult inactivation of both Glis3;Pkd1 also exacerbates cyst progression. Glis3-only knockouts evaluated at 14-, 18- and 24-weeks are all normal. Transcriptional targets of Glis3 are under investigation through multiple approaches (RNAseq, Cut&Run, and ATACseq). Conclusion: As a primary cilium localized transcription factor, Glis3 is genetically interacting with Pkd1. Loss of Glis3 exacerbates cystic progression in both early and adult onset ADPKD mouse model. Funding: NIDDK Support