Determining the Role of GPR161 in PKD

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of life-threatening monogenic kidney disease. Abnormal cAMP increase in renal epithelial cells are correlated with the formation of kidney cysts. Recent evidence suggested a notion that the compartmentalized cAMP elevation in primary cilia triggers renal cystogenesis. Yet, how the ciliary cAMP is regulated remains vague. The cystic kidney phenotype is observed in INPP5E-null mice. Loss of INPP5E increases the TULP3-dependent ciliary trafficking of GPR161. It was shown recently that GPR161 undergoes a constitutive self-activation, raising a possibility that GPR161 may contribute to the cAMP elevation in INPP5E-null cilia that may underlie the renal cystogenesis in INPP5E-null kidneys. Methods: We constructed a ciliary cAMP indicator by fusing NPHP3 (aa 1-203) to the cAMP indicator Pink Flamindo (cPF). The stable expression of cPF was introduced in the IMCD3Flp-In cell line. We utilized CRISPR/Cas9 to knockout Inpp5e, Gpr161, or both. The self-activation deficient GPR161 mutants are made using site-directed mutagenesis and re-expressed back into Gpr161-null cells. 3D-Matrigel culture is employed for in vitro cystogenesis of IMCD3 cells. Inpp5ef/f, Gpr161f/f, and double conditional knockout mice are generated. Results: GPR161 and cAMP activity in cilia are both strongly increased in Inpp5e-null IMCD3 cells. Loss of GPR161 suppresses both the basal level and increase of ciliary cAMP in parental and Inpp5e-null IMCD3 cells, respectively. Notably, re-expression of the wild-type GPR161 back in Gpr161-null IMCD3 cells completely recues the cPF intensity in cilia. On the other hand, Inpp5e-null IMCD3 cells develop cystogenesis in 3D-Matrigel. Interestingly, the enlargement of Inpp5e-null IMCD3 cysts is insensitive to forskolin treatment and can be suppressed by co-depletion of GPR161. Studies on testing whether non-self-activatable variants of GPR161 can rescue the ciliary cAMP and cystogenesis in Inpp5e/Gpr161 dko IMCD3 cells are ongoing, as well as the animal studies. Conclusion: Our current results suggest an intriguing possibility that GPR161 via self-activation constitutively produces the basal level of cAMP in primary cilia, which may elevate the ciliary cAMP to a level that triggers cystogenesis when PKD mutations cause abnormal increase of GPR161 or weaken certain counteracting mechanisms in cilia.