A novel model of autosomal recessive polycystic kidney questions the role of the fibrocystin C-terminus in disease mechanism

Abstract

Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes Fibrocystin/Polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope “knocked-in” to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location in kidney, biliary duct and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model.

Analysis of the polycystin complex (PCC) in human urinary exosome–like vesicles (ELVs)

“…The region between the PPC site and the TM appears to be underrepresented (58% of the level of the extracellular portion <3616 aa), and the cytoplasmic C-terminal tail was at even lower levels and was probably not present at all (<5.1%), implying that it was removed prior to packaging into the ELV. This was compatible with the finding that nephrogenesis and hepatogenesis are completely normal in mice lacking the C-terminal cytoplasmic domain, the Pkhd1 Δ67 mice 30 . The evolutionarily close fibrocystin-L protein (PKHD1L1 Q86WI1 (PKHL1_HUMAN)) has only nine C-terminal tail amino acids suggesting that some members of the fibrocystin family can function without the cytoplasmic portion of the protein 31 .…”

Comprehensive genetic testing in children with a clinical diagnosis of ARPKD identifies phenocopies

“…It also contains the ciliary targeting sequence (p.3876_3893CLVCCWLKRSKSRKTKPE), that remains unaffected in the Gly4013Alafs*24 fibrocystin [40]. Along the same lines as the hypomorphic nature of this C-terminal truncation, mice lacking the last exon (exon 67), which encodes the nuclear localization signal and the polycystin 2 binding domain, develop a normal phenotype [41].…”

Targeted deletion of the AAA-ATPase Ruvbl1 in mice disrupts ciliary integrity and causes renal disease and hydrocephalus

“…We used a slightly modified protocol of this method that has previously been described 36 . A whole kidney was minced using a scalpel and then digested with Collagenase II (Worthington) and RNaseI (Applichem).…”