Background: PKHD1, the autosomal-recessive polycystic kidney disease (ARPKD) gene, encodes multiple alternatively spliced transcripts predicted to generate membrane-bound and secreted proteins. The longest open reading frame encodes polyductin (fibrocystin), a putative 4074 amino acid protein with a single transmembrane domain and an intracellular C-terminus.
Methods: To characterize the PKHD1 products and their expression profile, we raised polyclonal antibodies against different portions of polyductin and analyzed different organs using various methods.
Results: Western blot analyses demonstrated specific bands of >440 kD in human adult kidney, liver, and pancreas and approximately 230 kD in kidney and liver, predominantly observed in membrane fractions. The >440-kD putative membrane protein was immunoprecipitated from kidney and subsequently detected by Western blotting using two distinct antisera. An additional product of approximately 140 kD was specifically recognized by affinity-purified antisera predominantly in soluble fractions. Immunohistochemistry studies revealed specific staining in cortical and medullary collecting ducts and thick ascending limbs of Henle (TALH). Serial sections were stained with antibodies against aquaporin-2 and Tamm-Horsfall protein to confirm the nephron segment localization. Positive staining was also detected in biliary and pancreatic duct epithelia. Analyses of mouse developing tissues showed specific staining in the ureteric bud branches, intra- and extrahepatic biliary ducts, pancreatic ducts, and salivary glands. Immunofluorescence studies in inner medullary collecting duct cultured cells and immunoelectron microscopy analysis of medullary collecting ducts demonstrated that the protein localizes to the primary cilium. Positive signal was also detected in the apical membrane and in cytoplasm.
Conclusion: The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.