Cilia-Localized Counterregulatory Signals as Drivers of Renal Cystogenesis

Abstract

Primary cilia play counterregulatory roles in cystogenesis-they inhibit cyst formation in the normal renal tubule but promote cyst growth when the function of polycystins is impaired. Key upstream cilia-specific signals and components involved in driving cystogenesis have remained elusive. Recent studies of the tubby family protein, Tubby-like protein 3 (TULP3), have provided new insights into the cilia-localized mechanisms that determine cyst growth. TULP3 is a key adapter of the intraflagellar transport complex A (IFT-A) in the trafficking of multiple proteins specifically into the ciliary membrane. Loss of TULP3 results in the selective exclusion of its cargoes from cilia without affecting their extraciliary pools and without disrupting cilia or IFT-A complex integrity. Epistasis analyses have indicated that TULP3 inhibits cystogenesis independently of the polycystins during kidney development but promotes cystogenesis in adults when polycystins are lacking. In this review, we discuss the current model of the cilia-dependent cyst activation (CDCA) mechanism in autosomal dominant polycystic kidney disease (ADPKD) and consider the possible roles of ciliary and extraciliary polycystins in regulating CDCA. We then describe the limitations of this model in not fully accounting for how cilia single knockouts cause significant cystic changes either in the presence or absence of polycystins. Based on available data from TULP3/IFT-A-mediated differential regulation of cystogenesis in kidneys with deletion of polycystins either during development or in adulthood, we hypothesize the existence of cilia-localized components of CDCA (cCDCA) and cilia-localized cyst inhibition (CLCI) signals. We develop the criteria for cCDCA/CLCI signals and discuss potential TULP3 cargoes as possible cilia-localized components that determine cystogenesis in kidneys during development and in adult mice.

Keywords: cilia-dependent cyst activation; cilia-localized cyst inhibition; cystogenesis; intraflagellar transport; polycystic kidney disease; polycystin 1 and 2; primary cilia; tubby-like protein 3.

FIGURE 1

Complex roles of cilia in cystogenesis. Cysts from PC1 loss are severe and only partially suppressed from cilia loss. Images adapted from (Ma et al., 2013) with permission. Postnatal day 24 (P24) kidneys from (A) Pkd1 cko (Pkhd1-cre;Pkd1 ^fl/fl ), (B) Pkd1 cko; cilia cko (Pkhd1-cre;Kif3a ^fl/- ;pkd1 ^fl/fl ), and (C) cilia cko (Pkhd1-cre;Kif3a ^fl/- ) mice. Abbreviations: cko, conditional knockout.

FIGURE 2

Cilia-dependent cyst activation (CDCA) model for cystogenesis in ADPKD (adapted from Ma, 2021). (A) The CDCA in the wild-type cell is suppressed by polycystins and is activated through an unknown mechanism, possibly ligand binding to polycystins or flow bending the cilium. (B) In Pkd1 cko, CDCA is derepressed and constitutively activated leading to severe cystogenesis. (C) If Pkd1 and cilia are co-ablated, the CDCA cannot be upregulated. The cystogenic signal is thus diminished compared with single Pkd1 cko, resulting in reduced cystogenesis. In this model, the CDCA signal must retain a partial “leaky” function in the cytoplasm to explain how Pkd1-cilia double mutants display significant cystic changes. (D) In cilia single mutants, there is persistent mild cystogenesis. Here the CDCA must have a leaky function despite the presence of intact polycystins and lack of cilia (“?”). Created in BioRender.

FIGURE 3

Model for TULP3 and IFT-A—mediated trafficking of cargoes into cilia. TULP3 tubby domain is anchored to the plasma membrane by PI(4,5)P_2. The tubby domain captures short CLS (cilia localization signal) peptide regions in diverse cargoes. The N-terminus (NT) of TULP3 binds to the IFT-A core subunits (IFT140, IFT122, IFT144) and recruits the tubby domain-bound cargoes to cilia. After reaching cilia, the lack of PI(4,5)P₂ in the ciliary membrane could dislodge the cargoes from the tubby domain. The cargoes are very diverse and include transmembrane proteins and membrane-associated proteins such as ARL13B. The N-terminus amphipathic helix in ARL13B that binds the tubby domain in TULP3 is shown in blue. ARL13B is anchored to the membrane by palmitoylation (green) inside the helix. Abbreviations: Ax, Axoneme; TF, transition fiber; BB, Basal body; CM, ciliary membrane; PM, plasma membrane. Created in BioRender.

FIGURE 4

Tulp3 and Pkd1 double knockout models of PKD. (A) Kidneys from postnatal day 5 (P5). Tulp3 cko (HoxB7-cre; Tulp3 ^fl/fl ), Pkd1 cko (HoxB7-cre; Pkd1 ^fl/fl ), Tulp3 cko;Pkd1 cko (HoxB7-cre; Tulp3 ^fl/fl ;Pkd1 ^fl/fl ). cko: conditional knockout. Scale bar, 1 mm. Images adapted from (Hwang et al., 2019). (B) Adult-onset models. Pax8 ^rtTA ; tetO-cre doxycycline inducible mice were given doxycycline (Dox) starting at P28 for 2 weeks and analyzed at 18 weeks. Control (mice without tetO-cre), Tulp3 cko (Pax8 ^rtTA ; tetO-cre; Tulp3 ^fl/fl ), Pkd1 cko (Pax8 ^rtTA ; tetO-cre; Pkd1 ^fl/fl ), Tulp3 cko;Pkd1 cko (Pax8 ^rtTA ; tetO-cre; Tulp3 ^fl/fl ;Pkd1 ^fl/fl ). Images adapted from (Legue and Liem, 2019) with permission.

FIGURE 5

Dual roles of cilia in cystogenesis. (A) The genetic data (Figures 1, 4) implies (i) cilia-localized cilia-dependent cyst activation (cCDCA) signal(s) inhibited by PC1/2 in both the cell body and cilium and (ii) independent cilia-localized cyst inhibition (CLCI) signal(s). (B) In the embryonic stages, the cCDCA is normally weak and suppressed by polycystins. However, in the absence of polycystins, cCDCA is enhanced. TULP3 likely traffics components of the CLCI. (C) In the adult stage, the inhibitory arm is weaker and a lack of polycystins is sufficient to lead to strongly activated cCDCA. TULP3 likely traffics components of the cCDCA. Created in BioRender.

FIGURE 6

Potential lipidated cargoes of TULP3. TULP3 directs the trafficking of ARL13B to cilia. ARL13B regulates the release of farnesylated and myristoylated cargoes in the ciliary compartment via ARL3. Created in BioRender.