Inhibition ofPkhd1Impairs Tubulomorphogenesis of Cultured IMCD Cells

Abstract

Fibrocystin/polyductin (FPC), the gene product of PKHD1, is responsible for autosomal recessive polycystic kidney disease (ARPKD). This disease is characterized by symmetrically large kidneys with ectasia of collecting ducts. In the kidney, FPC predominantly localizes to the apical domain of tubule cells, where it associates with the basal bodies/primary cilia; however, the functional role of this protein is still unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which FPC was silenced by short hairpin RNA inhibition (shRNA). We showed that inhibition of FPC disrupted tubulomorphogenesis of IMCD cells grown in three-dimensional cultures. Pkhd1-silenced cells developed abnormalities in cell-cell contact, actin cytoskeleton organization, cell-ECM interactions, cell proliferation, and apoptosis, which may be mediated by dysregulation of extracellular-regulated kinase (ERK) and focal adhesion kinase (FAK) signaling. These alterations in cell function in vitro may explain the characteristics of ARPKD phenotypes in vivo.

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

“…Lack of FPC downregulates Pkd2-channel activities in either the Pkhd1-knockout renal epithelial cells in primary culture or the Pkhd1-knockdown IMCD cells that we generated previously. 39 Given that FPC and PC2 physically interact and that the lack of FPC downregulates PC2 expression in vivo but PC2 does not downregulate FPC, we speculate that PC2 may function immediately downstream of FPC.…”

“…48 Our previous in vitro study also demonstrated that renal epithelial IMCD cells with downregulated FPC exhibit aberrant migratory polarity and lose the ability to drive collective cell migration, suggesting that the planar cell polarity also might be disrupted. 39 Aberrant planar cell polarity seen in cells with Pkhd1 defects suggests that ciliary defects in our Pkhd1 mutant mice might be caused by impeding orientally centriole arrangement and disabling the establishment of epithelial polarity. 39,49 Because our Pkhd1 mutant mice bear an in-frame GFP reporter, immunostaining with anti-GFP antibodies provided an FPC expression profile in affected tissues.…”

“…Next, we used the same approach to test the Pkhd1-silenced inner medullary collecting duct (IMCD) cells (IMCD shRNA3e23 ) and WT control cells (IMCD sh15 ) that we had generated previously. 39 It is interesting that Pkhd1-silenced IMCD cells exhibited a similar channel reduction, indicating that inhibition of FPC also reduces Pkd2-channel activities (P Ͻ 0.001; Figure 9, Bb versus Bc). For verification that the functional channel changes were induced by missing Pkd2, the anti-PC2 polyclonal antibody hPKD2-Cp, which recognizes the intracellular COOH-terminal region of PC2, was transferred into WT control IMCD sh15 cells via pipette solution.…”

“…For functional validation of these findings, Pkd2-channel activities were examined both in primary cultures of renal epithelia derived from Pkhd1 mutant mice and in Pkhd1-silenced IMCD cells. 39 Pkd2-channel activities were significantly dysregulated in Pkhd1-deficient cells, indicating that FPC and PC2 reside in the same molecular pathway. During submission of our article, another group reported that there are genetic interactions between Pkhd1 and Pkd1.…”

“…Although the gene responsible for ARPKD, PKHD1, has been identified [23][24][25] and its gene product, FPC, has been initially characterized, 27,28,30,31,39,40 the mechanisms by which PKHD1 causes disease phenotypes remain largely unknown. To study the disease mechanism and pathogenesis of ARPKD, we created a mouse that allows manipulation of Pkhd1, an animal model that recapitulates the human ARPKD phenotype.…”