Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia

Abstract

Autosomal recessive polycystic kidney disease (ARPKD) is an infantile form of PKD characterized by fusiform dilation of collecting ducts and congenital hepatic fibrosis. The ARPKD gene, PKHD1, is large (approximately 470 kb; 67 exons) with a 12222 bp longest open reading frame, although multiple different splice forms may be generated. The predicted full-length ARPKD protein, fibrocystin, is membrane bound with 4074 amino acids (447 kDa molecular weight). To characterize the pattern of fibrocystin expression we have generated four monoclonal antibodies (mAb) to the cytoplasmic tail of the protein. Western analysis of human kidney membrane protein showed an identical pattern with each mAb; a strongly expressing large product (>450 kDa), consistent with the predicted protein size, and a weaker approximately 220 kDa band. The same large product was detected in rat and mouse kidney with lower level expression in liver. To further show that these mAbs recognize fibrocystin, tissue from ARPKD patients was analyzed and no fibrocystin products were detected. Immunohistochemical analysis of the developing kidney showed expression in the branching ureteric bud and collecting ducts, expression that persisted into adulthood. Biliary duct staining was found in the liver, plus staining in the pancreas and developing testis. Immunofluorescence analysis of MDCK cells showed a major site of expression in the primary cilia. Recent studies have associated the disease protein in various human and animal forms of PKD with cilia. The localization of fibrocystin to cilia further strengthens that correlation and indicates that the primary defect in ARPKD may be linked to ciliary dysfunction.

Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

“…FPC was demonstrated to localize to the primary cilium and/or basal bodies of renal tubular epithelia, [27][28][29][30][31] and malformation of the cilia was shown to induce cyst formation in the kidneys 34,35 ; therefore, we began to determine whether the lack of FPC also disrupts ciliogenesis in Pkhd1-deficient mice. We used IF with an anti-acetylated ␣-tubulin antibody to examine the number and morphology of renal primary cilia in 6-moold littermates.…”

“…The longest open reading frame is predicted to include 66 exons and to encode the 4074 -amino acid membrane-associated receptor-like protein fibrocystin/polyductin (FPC). [23][24][25][26] It was shown that FPC is associated with the basal bodies/primary cilia of epithelial cells [27][28][29][30] and co-localizes with PC2 within the cell. 31 These observations suggest the possibility that FPC and PC2 may function in a common molecular pathway in vivo.…”

Cholangiocyte Cilia Detect Changes in Luminal Fluid Flow and Transmit Them Into Intracellular Ca2+ and cAMP Signaling

“…When these genes are mutated, the absence of their protein products results in ciliary dysfunction and cyst formation. [6][7][8][9][10][11] Development of cysts in the liver is the most frequent extra-renal manifestation in both ADPKD and ARPKD. 10-13 Although in ADPKD the liver generally functions normally, the progressive increase in size of the polycystic liver causes abdominal pain, early postprandial fullness and/or shortness of breath, and thus significantly affects quality of life.…”

Cholangiocytes with Mesenchymal Features Contribute to Progressive Hepatic Fibrosis of the Polycystic Kidney Rat

“…6,7 Developing and mature intrahepatic bile ducts express the PKHD1 protein, fibrocystin, whereas bile ducts of ARPKD patients lack its expression. 8 Mice with targeted mutation of Pkhd1 develop cystic biliary dysgenesis and portal fibrosis. 9 In the PCK rat, cholangiocytes possess short and malformed cilia that do not express fibrocystin.…”