PTH1R Signaling Instigates a Cystogenic Program in Autosomal Recessive PKD Organoids

Background: Autosomal recessive polycystic kidney disease (ARPKD) is an inherited developmental cystic kidney disease caused by biallelic mutations in PKHD1. ARPKD confers high morbidity and mortality due to a lack of directed therapies. Unfortunately, the various Pkhd1 mutant mouse models (Pkhd1LSL/LSL, Pkhd1Δ3-4/Δ3-4, and Pkhd1Δ67/Δ67) fail to recapitulate the human developmental phenotype, manifesting limited renal disease. To overcome this limitation and to stimulate drug discovery, human pluripotent stem cell (hPSC)-derived ARPKD organoids have manifested well-described cystogenic mechanisms suggesting value for therapeutic screens. In these studies, PKHD1-/- organoids have been challenged with forskolin to induce cyclic AMP, a hallmark of PKD. While these studies validate genotype-phenotype correlation and the faithfulness of cystic responses, novel cystogenic mechanisms that expand potential therapeutic targets are yet to be reported. Methods: We use parental wild-type human PGP1 and isogenic CRISPR PKHD1-/- iPSCs, directed differentiation to kidney organoids, longitudinal brightfield microscopy, wholemount and cryosection confocal microscopy, native kidney and organoid single cell RNAseq, ARPKD tissue, and targeted mechanistic studies to describe PTH1R as a stimulatory G-protein coupled receptor (sGPCR) which instigates a cystogenic signaling cascade in developmental cystic kidney disease. Results: PTHR1 loss was confirmed in ARPKD organoids by immunostaining. Chemical cAMP induction in ARPKD organoids induced a dose-dependent cystic response, unlike their isogenic wild-type control, prompting a search for upstream sGPCRs as a driver for cAMP. Unbiased transcriptomic screen of native human kidney for sGPCRs identified PTH1R, which co-localized with LTL+ and CDH1+ in proximal and distal tubule cysts of ARPKD patient kidney tissue. ARPKD organoids subject to endogenous ligands of differential affinity for parathyroid hormone receptors recognized PTH1R agonism as a novel cystogenic stimuli, with cysts having apical-facing primary cilia. Targeted inhibition of adenylyl cyclase and Protein Kinase A demonstrated that the stimulatory GPCR, PTH1R, drives a cystogenic cAMP-PKA-CREB axis in developmental PKD. Conclusion: Our findings demonstrate the utility of kidney organoids as an in vitro model to explore pathomechanisms of rare diseases which lack faithful animal models. Funding: NIDDK Support