A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X) in polycystin-1.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X) in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells

Cyst formation by PKDQ4004X cells grown in Matrigel.

<p>Normal and PKDQ4004X cells were plated at a density of 3000 cells per ml of Matrigel with culture media and grown in culture for 14 days. A: Phase contrast image of PKDQ4004X cells forming cysts in Matrigel matrix. B: Phase contrast image of normal kidney cells in Matrigel matrix. Bar = 50 microns. C: Fluorescence photomicrograph of PKD Q4004X cell cyst. Extended focus image of 5 image planes covering a z distance of 5 microns. Actin cytoskeleton is labeled with Bodipy phalloidin (green) and nuclei are labeled with Hoechst 33342 (blue). A clear lumen (L) is surrounded by a layer of epithelial cells to form a cyst. Bar = 10 microns.</p

Characterization of PKD Q4004X growth and selection.

<p>A: Telomerase activity assay and immune blot analysis of transduced and untransduced cells. Left panel: Telomerase activity detected as labeled telomerase product telomeric DNA ladders. DNA ladders are only observed in cells transduced with hTERT. Untransduced control cells or cells transduced with empty vector (pLXSN) have no detectable telomerase activity. Right panel: Exogenous telomerase expression confirmed by immune blot analysis. A 120 kDa band is observed in lysates made from PKD cells transfected with hTERT. <b>B: </b><b>Mutation detection in the PKD cell line.</b> A C to T mutation resulting in a premature truncation at amino acid 4004 is shown. <b>C:</b> Phase contrast image of untransduced cells (upper image) and hTERT transfected PKD cells (lower image) maintained in selection media. <b>D: </b><b>Fluorescence activated cell sorting of LTL and DBA labeled HK2 cells.</b> Fluorescence intensity at 575 nM/unit cell area versus fluorescence intensity at 530 nM/unit cell area are plotted on the Y and X axis respectively. Since DBA was tagged with rhodamine, cells labeled with the DBA marker are expected to register in the region demarcated by the circle. <b>E: </b><b>Fluorescence activated cell sorting of LTL and DBA labeled MDCK-II cells.</b> Fluorescence intensity at 575 nM/unit cell area versus fluorescence intensity at 530 nM/unit cell area are plotted on the Y and X axis respectively. Since DBA was tagged with rhodamine, cells labeled with the DBA marker are expected to register in the region demarcated by the circle and shown in green. MDCK-II cells are a mixed population basted on this assay (compare red and green populations). <b>F: </b><b>Fluorescence activated cell sorting of LTL and DBA labeled PKD Q4004X cells.</b> Fluorescence intensity at 575 nM/unit cell area versus fluorescence intensity at 530 nM/unit cell area are plotted on the Y and X axis respectively. Since DBA was tagged with rhodamine, cells labeled with the DBA marker are expected to register in the region demarcated by the circle. Few cells have DBA labeling with greater than 99% of the cells showing LTL positive labeling.</p