Autosomal Dominant Polycystic Kidney Disease Cells Research Articles

In vitro fluid secretion by epithelium from polycystic kidneys.

The size of the kidneys in patients with autosomal dominant polycystic kidney disease (ADPKD) is due in large measure to the accumulation of secreted fluid within thin-walled epithelial sacs. We measured the net transepithelial movement of liquid in response to forskolin in isolated, intact cysts excised from the surface of human ADPKD kidneys and in cultured, polarized monolayers of epithelial cells derived from ADPKD cysts. 10 excised cysts bathed symmetrically in control culture medium secreted fluid at a rate of 0.19 +/- 0.03 microliter/cm2 per hour after stimulation with forskolin (10 microM). Ouabain (100 microM) addition to the cavity fluid did not change the rate of fluid secretion of 10 forskolin-treated cysts, but addition of the glycoside to the external bathing medium fluid of nine cysts decreased secretion to -0.004 +/- 0.05 microliter/cm2 per hour. 24 monolayers absorbed fluid (range -0.029 to -0.412 microliter/cm2 per hour); by contrast, fluid was secreted (range 0.074 to 1.242 microliters/cm2 per hour) after stimulation with forskolin (10 microM). Ouabain (0.1 microM) in the basolateral but not in the apical medium inhibited fluid secretion. Forskolin increased the intracellular cyclic AMP content of ADPKD and MDCK monolayers by 236 and 196%, respectively. Six ADPKD monolayers had stable lumen negative transepithelial electrical potential differences (PDte) of -1.4 +/- 0.3 mV, positive short circuit currents (SCC) of 11.9 +/- 2.1 microAmp/cm2 and a tissue resistance (Rte) of 116 +/- 14 ohm.cm2. Forskolin increased SCC to 15.5 +/- 1.9 microAmp/cm2 (P < 0.005) and decreased Rte to 95 +/- 13 ohm.cm2 (P < 0.05); PDte remained stable at -1.4 +/- 0.3 mV. Ouabain (10 microM) had no effect when added to the apical medium, but in the basolateral medium decreased SCC to 1.7 +/- 0.3 microAmp/cm2 and PDte to -0.2 +/- 0.1 mV. We conclude that ADPKD cells in surface cysts have the potential to absorb or to secrete solutes and fluid. cAMP-mediated fluid secretion from the basolateral medium into the lumen of surface ADPKD cysts may be driven by anion transport.

Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia

Human autosomal dominant polycystic kidney disease (ADPKD) epithelia were grown in primary monolayer cultures and their properties compared with intact kidney epithelial cultures derived from individually microdissected normal human kidney proximal convoluted tubules (PCT), proximal straight tubules (PST), and cortical collecting tubules (CCT). In vivo, ADPKD cyst epithelia exhibited a thickened basement membrane, and immunofluorescence demonstrated the presence of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan in basement membranes and type I collagen in the interstitium. ADPKD epithelia grown in culture synthesized and secreted basally a unique, extracellular matrix that took the form of proteinaceous spheroids when the cells were grown on dried, type I collagen. Incorporation of H2[S35O4] into basement membrane extracts was increased more than ten-fold in ADPKD epithelia by comparison to normal PST and CCT. In addition to incorporation into the normal tubular basement membrane 220 kD band, radioactivity was also seen at 175 kD and 150 kD in ADPKD extracts. Growth in culture of cyst-lining ADPKD epithelia was more rapid than normal tubules, and was abnormal since there was no absolute requirement for added extracellular matrix. However, when ADPKD epithelia were grown on different, exogenous matrix protein components, a profound influence on both structure and epithelial cell proliferation was seen. Growth on a complete basement membrane three-dimensional gel derived from the Engelbreth-Holm-Swarm (EHS) sarcoma led to a reduction in the numbers of spheroids and increase in amorphous filaments. Incorporation of [3H]-thymidine into ADPKD epithelia was greater than into normal PCT, PST, and CCT and was also greatly modified by the type of extracellular matrix components provided. In studies using single matrix components, the strongest proliferative response was seen when ADPKD epithelia were plated on type I collagen greater than type IV collagen greater than fibronectin greater than laminin. These findings suggest that the excessive growth of cyst-lining epithelia may be, at least in part, a result of abnormal basement membrane and extracellular matrix production by ADPKD cells.

Aberrant Epithelial Cell Growth in Autosomal Dominant Polycystic Kidney Disease

Renal cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) is characterized by increased epithelial cell proliferation and fluid accumulation. Using monolayer epithelial cultures derived from individually microdissected human ADPKD cysts and immunolocalization studies in vivo, the roles of matrix and growth factors in aberrant ADPKD cell proliferation have been studied. Abnormal ADPKD basement membrane ultrastructure was associated with increased turnover of 35S-labeled heparan sulfate proteoglycans (HSPG) by comparison to normal renal tubule epithelia in vitro. Mitogenic assays demonstrated significant increase in 3H-thymidine incorporation into ADPKD epithelia grown on type I and type IV collagen by comparison to normal proximal straight tubules (PST), collecting ducts, and thick ascending limbs of Henle (TAL). Proliferation on laminin or fibronectin matrices was unchanged and immunolocalization of matrix proteins was polarized and restricted to basal membranes of both ADPKD cysts and renal tubule epithelia in vivo. ADPKD epithelia in vitro were hypersensitive to the mitogenic action of epidermal growth factor (EGF) and EGF immunoreactivity was detected in ADPKD cyst lining epithelia, in cyst fluid, and in conditioned media from confluent ADPKD cultures, suggesting an autocrine mechanism of growth regulation. In addition, inhibition of epithelial proliferation by transforming growth factor-beta (TGF-beta), which was 100% in normal renal tubule epithelia, was reduced to 41% in ADPKD epithelia.

Intra- and extracellular proteins in human normal and polycystic kidney epithelial cells

A tissue culture method was established for the continuous growth of epithelial cells from the cortex of human normal kidney (HNC) and from the epithelial layer of kidney cysts from autosomal dominant polycystic kidney disease (ADPKD) patients. Primary cells were grown to 80 to 90% confluency from 1 mm2 slices of tissue, and subcultured up to 10 times. The subcultured HNC and ADPKD cells retained characteristic epithelial polygonal and elongated shape and positive immunofluorescent staining for cytokeratin. The cell doubling time for both HNC and ADPKD epithelia was three to four days at a fetal calf serum (FCS) concentration of 5%. Using these culturing procedures 1 to 5 x 10(9) epithelial cells could be obtained from each kidney specimen. Profiles of 35S-methionine radiolabeled intracellular proteins of HNC and ADPKD cells qualitatively demonstrated a high degree of similarity, thus confirming a similarity of epithelial origin and protein biosynthesis. Both the underexpression of three proteins (a) protein p2, Mr approximately 47 kDa, pI approximately 6.0; b) protein p3, Mr approximately 50 kDa, pI approximately 5.9; and c) protein p4, Mr approximately 44 kDa, pI approximately 5.8) and the overexpression of several proteins (including: a) p5, Mr approximately 56 kDa, pI approximately 7.3; b) protein p6, Mr approximately 32 kDa, pI approximately 7.3; c) protein p7, Mr approximately 33 kDa, pI approximately 5.3; d) protein p8, Mr approximately 45 kDa, pI approximately 6.9; e) protein p9, Mr approximately 35 kDa, pI approximately 6.7; and f) protein p10, Mr approximately 30 kDa, pI approximately 6.6) were found in ADPKD cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyst-derived cells do not exhibit accelerated growth or features of transformed cells in vitro

Progressive renal enlargement is a prominent feature in autosomal dominant polycystic kidney disease (ADPKD), suggesting that the disease is due to hyperplasia and/or preneoplastic transformation of renal epithelial cells. In this study in vitro methods were developed to grow and propagate large numbers of cyst-derived epithelial cells from ADPKD kidneys and cortical epithelial cells from normal human kidneys (NK). In order to study their biologic features during early cell passages, cells were grown on Vitrogen (bovine dermal collagen)-FCS (fetal calf serum) coated dishes and fed a basic medium (DME:F12) supplemented with 10% FCS or a defined medium (Sens) containing insulin, transferrin, selenium, hydrocortisone, tri-iodothyronine and epidermal growth factor (EGF). Both ADPKD and NK cells grew as monolayers, were positive for keratin by immunohistochemistry and flow cytometry and had ultrastructural features of renal epithelial cells. Confluent NK and ADPKD monolayers formed domes. In contrast to NK cells, the growth and propagation of ADPKD cells were not supported by defined medium alone but required serum supplementation and ADPKD cells did not respond to growth factors (insulin, transferrin, EGF) that promoted the growth of NK cells. In serum supplemented media, the growth rate, cell doubling time and end cell number of ADPKD and NK cells were the same. Moreover, ADPKD cells did not exhibit any in vitro features of transformed cells: they were not immortal, they were sensitive to contact inhibition, they were anchorage dependent and they were not tumorigenic in nude mice. These findings do not support an increased rate of cell growth or cell transformation as causative factors in ADPKD.