Harnessing the potential of GPCRs localized to the primary cilium for the treatment of renal disease

Va Boston Health Care System, Boston MA

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Abstract

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease of renal dysfunction marked by cyst formation and gross enlargement of the kidney. The condition affects an estimated 40,000 U.S. Veterans. Although cysts are sometimes evident early in life, ADPKD generally has an "adult" onset. Military personnel are not screened for this disorder prior to entering the armed services and are thus often diagnosed only during or after their military duty. Half of these individuals will experience complete renal failure by the age of 60, requiring life-long dialysis and/or eventual organ transplantation for survival. These and other complications of the disease (e.g. liver failure, hypertension, arrhythmia, and propensity for cerebral aneurysm) are expected to exacerbate illnesses in an aging Veteran patient population already compromised by other service-connected conditions. ADPKD is defined as a "ciliopathy", a disease resulting from defective structure/function of the primary cilium, a tiny organelle that in the kidney tubule extends from the apical plasma membrane into the tubule lumen. Two proteins that are mutated in ADPKD, the "polycystins" (PKD1 and PKD2), form a heterodimer in the cilium. This localization is somehow essential for preventing the cellular proliferation, fibrosis and architectural remodeling of the kidney tubule responsible for generating the massive enclosed cysts characteristic of the disease. An ADPKD drug (tolvaptan) was approved in 2018 but provides only symptomatic relief for patients, principally by reducing the cyclic AMP (cAMP)-dependent fluid and electrolyte secretion that drives renal cyst enlargement. New studies published last year indicate that a ciliary signaling pathway prominent in development and tumor formation, Hedgehog (Hh), is aberrantly activated in ADPKD. Even more recently, our lab showed that Hh stimulation promotes the activity of specific G-protein-coupled receptors (GPCRs) confined exclusively to the ciliary membrane. This allows agonist-dependent cAMP signals to be generated solely within the microdomain of the cilium. Because the ciliary Hh pathway is inhibited by cAMP, these GPCRs present a unique opportunity to selectively intervene on Hh signaling without stimulating the global increases in cAMP that drive fluid secretion. The overall purpose of the present project is to evaluate the potential of this newly described ciliary GPCR­ dependent cAMP signaling circuit to alter PKO progression. First, using optical approaches developed in our lab for measuring intraciliary cAMP, we will determine why Hh stimulation leads to an apparent upregulation of GPCR function and cAMP signaling exclusively in the cilium and how this is affected by relevant polycyst in mutations. Next, considering that this new signaling system produces cAMP signals only in the ciliary microdomain, we will define the transcriptional pathways that become activated specifically by these local changes in second messenger. Finally, harnessing the potential of GPCRs in the cilium to control renal pathology requires additional knowledge about their identity and location within cyst-lining epithelia, in adjacent normal renal tubules and in the surrounding stromal cells. We will therefore use single molecule FISH (smFISH) to image copy number and spatial distribution of transcripts for prospective ciliary GPCRs in ADPKD renal specimens. The overall goal is to identify novel and potentially druggable GPCR s that may be used to selectively elicit cAMP signals within the cilium without affecting second messenger levels in the rest of the cell.