Analysing the role of ciliary cAMP signalling in PKD development and progression

Abstract

Ciliopathies comprise a group of severe human diseases characterised by primary cilia dysfunction and are associated with a broad spectrum of clinical manifestations. Primary cilia are non-motile, finger-like protrusions of the plasma membrane, which are present on most mammalian cells. They function as cellular antennae, which receive environmental signals and transduce them into an intracellular response. The most common hereditary renal disease, autosomal-dominant polycystic kidney disease (ADPKD), is also a ciliopathy. Our lab has shown that chronic ciliary cAMP signalling in renal epithelial cells is a key driver for disease development. <br/> I hypothesized that ciliary cAMP signalling reprograms renal epithelial cells by altering intracellular signalling pathways. To investigate this, I performed phosphoproteomic analysis of renal epithelial cells undergoing ciliary cAMP stimulation. To gain mechanistic insight, I conducted kinase motif and 1D annotation enrichment analysis, followed by topological analysis of protein-protein association networks. I revealed extensive regulation of the phosphoproteome, identifying PKA as the principal integrator. Multiple signalling modules were affected through a single hub effector protein, &beta;-catenin. By examining the subcellular location, total abundance, transcriptional activity, and PKA-regulated phospho-forms of β-catenin, I validated the proteomic findings and demonstrated a direct contribution of β-catenin-mediated signalling to cystogenesis using a 3D culture model. <br/> How these intracellular alterations in aberrant renal epithelial cells influence the renal microenvironment and shape the fate of other cells in the renal tissue, particularly tissue-resident macrophages (TRMs), the largest immune cell population in the kidney, remained elusive. To address this, I performed an in-depth analysis of renal macrophage ontogeny and phenotype and investigated their function in an ADPKD mouse model (Pkd1^RC/RC) which showed that the numbers of TRMs were already increased before cyst formation. I further characterized the TRM population via flow cytometry and could identify novel markers that can be potentially used to determine TRM remodelling at different stages of disease progression. To investigate the functional interaction between TRMs and renal epithelial cells, I established a 3D co-culture system, demonstrating that TRMs from <em>Pkd1^RC/RC</em> mice, isolated at a stage before cysts were observed, already showed enhanced cystogenesis <em>in vitro</em>. To determine how macrophage remodelling is induced, I performed secretome analysis of renal epithelial cells following ciliary cAMP stimulation, which identified CCL5 as key candidate mediator of paracrine TRM remodelling. <br/> Taken together, I demonstrated that ciliary cAMP stimulation remodels renal epithelial cells through PKA-dependent activation of β-catenin signalling. The resulting aberrant epithelial behaviour alters the renal microenvironment, with CCL5 being secreted, and promotes accumulation and functional reprogramming of TRMs via paracrine signalling, ultimately enhancing cystogenesis.