Determine the Downstream Effectors of Polycystins in Autosomal Dominant Polycystic Kidney Disease

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Somlo, Stefan

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human monogenetic diseases that occurs 1 in 500-1000 people, affecting over 12 million population worldwide. The genetic causes of ADPKD are identified to be mutations in either the PKD1 or the PKD2 gene, encoding polycystin-1 (PC1) or polycystin-2 (PC2) protein, respectively. ADPKD is mainly characterized by cyst formation originating from the epithelia of kidney tubules. To date, the precise functions of polycystins and the exact molecular mechanisms of ADPKD still remain unknown. However, there is a growing consensus that the primary cilia is the most essential component driving cyst formation. The genetic relationship between polycystins and primary cilia has been elucidated in the lab by showing that structurally intact cilia is a critical requirement for cyst growth following the loss of polycystins in vivo, which suggested the existence of a polycystin dependent “cilia-dependent cyst activation (CDCA)” pathway. Subsequently, using TRAP RNA-seq on pre-cystic mouse kidneys, our lab generated a transcriptional profile that provided a robust dataset of translatome changes associated with CDCA, which was then termed as the CDCA signature genes. Glis2, a transcription factor belonging to the Gli-similar (Glis) Krüppel-like zinc finger family, was selected for further biological validation and was demonstrated to be an early effector of polycystin signaling and a therapeutic target for ADPKD.The first part of my thesis research aims to study a transcription factor called Glis3, another member of the Glis transcription factor family and was reported to have a ciliary location. Based on the similarities between the Glis and the Gli transcription factors, I hypothesized to use Hedgehog signaling as the model paradigm to investigate the potential role of Glis3 in ADPKD. By introducing a novel Glis3 conditional knockout allele to the ADPKD mouse model, I found that in both early- and adult-onset models, loss of Glis3 in concomitant with loss of Pkd1 exacerbates polycystic kidney phenotype, suggesting that Glis3 is a modifier of cyst progression. A transcriptomic approach coupled with chromatin accessibility analysis suggests Glis3 may be involved in a transcriptional network of Hnf1b, Hnf4a, Dbp, and Klf15 and indirectly perturb kidney metabolic and circadian programs, which results in the worsening phenotype. In the second part of my thesis research, I identified Anks3 from the transcriptomic analysis of TRAP RNA-seq as a candidate modulator of polycystin signaling. Anks3 is a scaffolding protein that has partially defined roles in genetic cilia-related kidney diseases. Using a combination of multiple in vitro approaches, I illustrated that Anks3 can regulate Glis2 nuclear expression in the context of ADPKD. Next, by crossing a novel Anks3 conditional knockout allele to the ADPKD mouse models, I found that loss of Anks3 in concomitant with Pkd1 inactivation significantly rescue cyst progression in both early- and adult-onset models. However, loss of Anks3 itself leads to acute tubular injury (ATI) and the associated inflammatory and fibrotic response, indicating that Anks3 not only plays an essential role in the molecular mechanism of ADPKD but is also critical in maintaining normal renal epithelial homeostasis. A subsequent study by combining the Anks3 and the Ift88 conditional alleles showed that the signaling pathway regulated by Anks3 is not dependent on intact primary cilia, suggesting Anks3 is downstream of cilia. Furthermore, I found that Anks3 is phosphorylated in a PKD genotype dependent manner and identified the phosphorylation sites that are differentially regulated by polycystins. Lastly, transcriptional analysis suggested global changes in extracellular matrix, cytokine receptor activity, and immune response that are associated with the loss of Anks3. Detailed examination of the expression of CDCA pattern translatome reveals that in addition to Glis2, inactivation of Anks3 also results in the normalization of a broader range of CDCA signature genes. Together, this study provided a context dependent role in the kidney for Anks3 as a novel mediator of polycystin signaling in ADPKD and as a central coordinator in maintaining renal homeostasis.

This document is currently not available here.

Share

COinS