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Calcium Restores a Normal Proliferation Phenotype in Human Polycystic Kidney Disease Epithelial Cells

Tamio Yamaguchi^*,, Scott J. Hempson^*,, Gail A. Reif^*,, Anne-Marie Hedge^*, and Darren P. Wallace^*,,

^* Kidney Institute, Departments of Medicine and Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas

Address correspondence to: Dr. Darren P. Wallace, Kidney Institute, Department of Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, MSN 3018, Kansas City, KS 66160. Phone: 913-588-3889; Fax: 913-588-9251; E-mail: dwallace{at}kumc.edu

Received for publication June 21, 2005. Accepted for publication October 3, 2005.

	Abstract

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Polycystic kidney disease (PKD) is a lethal disorder characterized by progressive expansion of renal cysts. Genetic mutations associated with PKD are thought to disrupt intracellular Ca²⁺ regulation, leading to abnormal proliferation of tubule epithelial cells. cAMP stimulates the B-Raf/MEK/extracellular signal-regulated kinase (B-Raf/MEK/ERK) pathway and accelerates the proliferation of cells that are cultured from PKD cysts. By contrast, cAMP inhibits the proliferation of cells from normal human kidneys (NHK) and M-1 mouse collecting duct cells. Previously, it was found that a sustained reduction of intracellular Ca²⁺ levels in NHK and M-1 cells that were treated with Ca²⁺ entry blockers allowed cAMP activation of the B-Raf/MEK/ERK pathway, switching the cells to a cAMP-growth stimulated phenotype. In this study, primary cultures of cyst epithelial cells from autosomal dominant (ADPKD) and recessive (ARPKD) PKD kidneys were used to determine whether controlled addition of Ca²⁺ could reverse the aberrant mitogenic response to cAMP. Steady-state intracellular Ca²⁺ levels were found to be 20 nM lower in cyst-derived ADPKD cells (57 ± 2 nM) compared with NHK cells (77 ± 2 nM). Treatment of ADPKD cells or ARPKD cells with either Bay K8644, a Ca²⁺ channel activator, or A23187, a Ca²⁺ ionophore, caused sustained increases in intracellular Ca²⁺ levels and completely reversed the mitogenic response to cAMP. Elevation of intracellular Ca²⁺ levels in ADPKD cells increased Akt activity and blocked cAMP-dependent B-Raf and ERK activation. Thus, increases in [Ca²⁺]_i are able to restore the normal anti-mitogenic response to cAMP in cells that are derived from two genetically distinct forms of PKD.

	Introduction

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In polycystic kidney disease (PKD), the aberrant growth of renal tubule epithelial cells leads to the formation of fluid-filled cysts, massive enlargement of kidneys, and a decline in renal function (1). Mutations in the genes associated with human PKD (PKD1, PKD2, and PKHD1) transform tubule epithelial cells into benign hyperplastic cysts. Renal failure develops as cysts progressively enlarge and replace the normal parenchyma.

Autosomal dominant PKD (ADPKD) is caused by genetic mutations in either the PKD1 or the PKD2 gene (2–4). It now is thought that renal epithelial cell hyperplasia in ADPKD is a consequence of dysfunctional Ca²⁺ metabolism owing to mutations that affect the polycystin proteins. Polycystin-1 (PC-1) and PC-2, the protein products of the PKD1 and PKD2 genes, respectively, have been shown to assemble at the plasma membrane to regulate a Ca²⁺ entry mechanism (5). PC-1 may act as a receptor that gates Ca²⁺-permeant PC-2 channels. PC-2 has also been reported to be a Ca²⁺ release channel in the endoplasmic reticulum (6). Defects in either PC-1 or PC-2 are thought to disrupt intracellular Ca²⁺ homeostasis or Ca²⁺ signaling leading to cellular dedifferentiation and hyperproliferation.

Autosomal recessive PKD (ARPKD) is caused by mutations in PKHD1, a gene that encodes a novel protein, fibrocystin (polyductin) (7,8). The function of fibrocystin is unknown; however, in common with other proteins associated with PKD pathogenesis, fibrocystin localizes to the primary cilium on renal epithelial cells (9), a structure that is thought to transduce a Ca²⁺ signal in response to mechanical or chemical stimulation (10,11). The specific roles of PC-1, PC-2, and fibrocystin in [Ca²⁺]_i regulation and the processes by which mutations in their genes cause epithelial cell hyperplasia and renal cyst formation remain unclear.

cAMP has a central role in cystogenesis by stimulating both transepithelial fluid secretion (12,13) and the proliferation of cyst epithelial cells (14–16). In vitro studies have demonstrated that cAMP agonists, including arginine vasopressin (AVP), promote the proliferation of epithelial cells derived from human ADPKD (14–16) and ARPKD (17) kidneys. By contrast, cAMP agonists inhibit the proliferation of normal human kidneys (NHK) cells. Recently, AVP and cAMP were shown to have primary roles in the progressive enlargement of cysts in animal models of ARPKD (PCK rat), ADPKD (Pkd2^ws25/– mouse), nephronophthisis (pcy mouse), and a unique recessive cystic disorder (cpk mouse) (18–21). Vasopressin V2 receptor blockade decreased renal cAMP levels and strikingly reduced the size of the cystic kidneys. Thus, it seems that renal cyst growth in hereditary cystic disorders depends on a common cellular pathway incorporating cAMP.

The molecular mechanism for the phenotypic differences in the cAMP mitogenic response between NHK and ADPKD cells is linked to cAMP-dependent B-Raf signaling to MEK, a kinase that stimulates extracellular signal-regulated kinases (ERK) (15). In ADPKD cells, cAMP activates B-Raf to stimulate the MEK/ERK pathway and cell proliferation. In NHK and M-1 cells, cAMP decreases the rate of cell proliferation through inhibition of the Ras/Raf-1/MEK/ERK pathway by decreasing Raf-1 activity. Normally, B-Raf activity is repressed by Akt (protein kinase B) involving a phosphatidylinositol 3-kinase (PI 3-K)-dependent pathway (22). Restriction of intracellular Ca²⁺ with Ca²⁺ channel blockers is able to relieve B-Raf inhibition by Akt, thereby allowing cAMP activation of the B-Raf/MEK/ERK pathway and cell proliferation. Inhibition of Akt or PI 3-K also allowed cAMP-dependent activation of B-Raf and ERK and increased proliferation. Those studies demonstrated that restriction of intracellular Ca²⁺ in normal cells can induce a switch such that cAMP activates B-Raf/MEK/ERK and cell proliferation, mimicking the PKD phenotype (22).

Because Ca²⁺ restriction can be shown to switch normal cells to the PKD phenotype, we reasoned that Ca²⁺ elevation may be able to rescue PKD cells. Thus, in this study, we sought to determine whether an elevation in Ca²⁺ in ADPKD and ARPKD cells can repress cAMP activation of the B-Raf /MEK/ERK pathway and cell proliferation and restore a normal cellular phenotype.

	Materials and Methods

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Primary Cultures of Human Kidney Epithelial Cells
Normal regions of human kidneys, confirmed by histologic examination, were collected from nephrectomy specimens that were removed for the treatment of renal carcinomas. Normal kidneys that were withheld from transplantation as a result of anomalous vasculature were also obtained from the Midwest Transplant Network (Kansas City, KS). ADPKD kidneys are obtained from hospitals that participate in the Polycystic Kidney Research Retrieval Program with the assistance of the PKD Foundation (Kansas City, MO). An ARPKD nephrectomy specimen was obtained from a 2-wk-old enfant at a local hospital. A protocol for the use of discarded human tissues complies with federal regulations and was approved by the Institutional Review Board at the University of Kansas Medical Center.

Tissues were minced and digested overnight in DMEM/F12 (1:1) mixture that contained 220 IU/ml type IV collagenase and 100 IU/ml penicillin G and 0.1 mg/ml streptomycin (P/S) (13). Collagenase digestion was stopped by the addition of FBS. Cells were rinsed in medium and propagated in DMEM/F12 supplemented with 5% FBS, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite (ITS) and P/S. At 70 to 80% confluence, cells were lifted from the plastic and either frozen in medium that contained 10% DMSO for storage in liquid N₂ or seeded directly onto supports for experiments.

Measurement of Intracellular Ca²⁺
For preparing NHK, ADPKD, and ARPKD cells for Ca²⁺ imaging, cells were cultured on glass coverslips that were coated with fibrillar type I collagen in DMEM/F12 that contained 5% FBS. After 2 d, serum was reduced to 1% for an additional day. Then, the cells were incubated in 0.002% FBS for 24 h before the experiment to reduce serum factors that alter basal Ca²⁺ levels and to limit variations in resting [Ca²⁺]_i. The morphology of the NHK, ADPKD, and ARPKD cell monolayers (approximately 80% confluent) was similar. The steady-state effect of Bay K8644 on intracellular Ca²⁺ was determined in paired ADPKD monolayers that were treated with control medium or 1, 10, or 20 µM Bay K8644 (Sigma Chemical, St. Louis, MO) for 24 h. In other experiments, the effect of 10 nM A23187 was determined in paired monolayers of ADPKD cells. Cells were loaded with 1 µM Fura-2/AM (Teflabs, Austin, TX) at 37°C for 30 min, rinsed with a HCO₃^–-Ringer’s solution that contained 2 mM CaCl₂, and then the coverslips were mounted in a thermal-controlled chamber on the stage of a Nikon inverted microscope equipped with a monochromator (22). The chamber was perfused continuously at 3 ml/min with Ringer’s solution equilibrated with 5% CO₂/95% air at 37°C to ensure exchange of the 1-ml bath volume.

Fura-2 measurements were made with dual excitation wavelengths of 340 and 380 nm, and the emitted light at 510 nm was measured with a digital photomultiplier detection system (Photon Technology International, South Brunswick, NJ). Felix 32 analysis software (PTI) controlled the monochromator and data acquisition to generate the 340/380 fluorescence ratio (23). For each monolayer, 340/380 excitation ratios (collected over a 2.5-min interval) were measured at five different locations. At the end of each experiment, cells were permeabilized with 1 µM ionomycin in Ringer’s solution that contained 2 mM Ca²⁺ to determine the maximum 340/380 ratio (R_max), and then 10 mM EGTA was added to determine R_min. The 340/380 ratios were converted to [Ca²⁺] using the equation [Ca²⁺] = K_dx [(R – R_min)/(R_max – R)] x (S_f380/S_b380), where the dissociation constant (K_d) of Fura-2 for Ca²⁺ is 224 nM, R_max and R_min are 340/380 ratios for Ca²⁺-saturating and Ca²⁺-free conditions, and S_f380 and S_b380 are fluorescence signals at 380 nm for free Ca²⁺ and bound Ca²⁺, respectively (24). A significant difference in [Ca²⁺]_i between three NHK and three ADPKD cell preparations (n = 3 cell monolayers each) was determined using a parametric unpaired t test. Comparisons in steady-state Ca²⁺ levels among ADPKD cells that were treated in control medium or 1, 10, and 20 µM Bay K8644 were determined using an ordinary ANOVA.

Cell Proliferation Measurements
NHK, ADPKD, and ARPKD (4 x 10³/well) cells were seeded in a 96-well culture plate and incubated for 24 h in DMEM/F12 with 1% FBS, ITS, and P/S. The FBS concentration was reduced to 0.002% and ITS was removed, and the cells were incubated for 24 h before the addition of 100 µM 8-Br-cAMP (cAMP), 1 µM Bay K8644, 0.01 µM A23187, 0.1 µM nifedipine, 1 µM verapamil, or 5 µM Akt inhibitor (6 wells/condition). After 72 h, cell proliferation was determined by the Promega Cell Titer 96 MTT assay method (14).

Immunoblot Assay
Cells (0.5 to 1 x 10⁶) were seeded onto plastic petri dishes (100 mm) that contained DMEM/F12 medium with 1% FBS. The serum was reduced to 0.002% at approximately 75% confluence, and the cells were allowed to grow for an additional 24 h. The cells then were treated with 1 µM Bay K8644, 0.01 µM A23187, and/or 5 µM Akt inhibitor for 24 h, and cAMP was added for the final 15 min before cell lysates were prepared (15). Immunoblots were probed with antibodies for ERK-1 (C-16), ERK-2 (C-14), Akt (C-20), and phospho-ERK (P-ERK; E-4) from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Akt (Ser 473) and phospho-MEK were purchased from Biosource (Camarillo, CA). Secondary anti-rabbit, -mouse, -rat, or -goat IgG-conjugated horseradish peroxidase secondary antibodies were from Santa Cruz.

B-Raf Immune Complex Kinase Assay
Clarified cell lysates were immunoprecipitated for 2 h with gentle rotation at 4°C with anti–B-Raf antibody (C-19; Santa Cruz) bound to agarose beads (15,22). Precipitates were rinsed and incubated for 30 min at 30°C in a nonradioactive B-Raf kinase assay that contained ATP and MEK-1 fusion protein (Santa Cruz) as substrates for the reaction. Phosphorylated MEK, a measure of B-Raf kinase activity, was determined by immunoblot using an antibody to phospho-MEK. Phospho-MEK was detected and quantified with a Fluor-S MAX imager.

Growth Measurement of Cultured ADPKD Cysts
Primary cultures of ADPKD cells (4 x 10³ cells/well) were dispersed within an ice-cold type I collagen matrix (Vitrogen; Collagen Corp., Palo Alto, CA) in wells of a 96-well culture plate (13). Warming the plate to 37°C caused polymerization of the collagen, trapping the cells within the gel. A defined medium (DMEM/F12 with ITS, 5 x 10^–8 M hydrocortisone, and 5 x 10^–5 M triiodothyronine) supplemented with 5 µM forskolin and 5 ng/ml EGF was added for 3 d to initiate cyst growth. The diameters of cysts after 3 d were <100 µm. EGF was removed, and then 5 µM forskolin and 10 µM Bay K8644 were added for an additional 9 d. Outer diameters of cross-sectional images of spherical cysts with distinct lumens were measured using an inverted microscope with a digital camera and video analysis software. The total surface area (SA) of the cysts within each well was calculated from the individual cyst diameters (cysts 100 µm). These experiments were repeated with a total of three cell preparations from different ADPKD kidneys.

	Results

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Measurement of [Ca²⁺]_i in NHK, ADPKD, and ARPKD Cells
If cyst development is a consequence of dysfunctional intracellular Ca²⁺ homeostasis, then cells that are derived from these cysts might be expected to have altered basal Ca²⁺ levels. Primary epithelial cell cultures were prepared from the cortex (1 to 3 mm from the surface) of NHK or multiple surface cysts of ADPKD kidneys as described previously (13–15,25). Cells appeared pleomorphic and stained with Arachis hypoaea (PNA) and Dolichos biflorus agglutinin (DBA) lectins, suggesting distal tubule or collecting duct origin (15). Subconfluent monolayers lacked detectable surface cilia, in contrast to the presence of cilia on 4-d postconfluent monolayers as assayed with an antibody to acetylated tubulin (data not shown). For measurement of [Ca²⁺]_i, cells that were derived from ADPKD and NHK kidneys (n = 3 each) were loaded with Fura-2. We found that steady-state [Ca²⁺]_i in cultured ADPKD cells (56.5 ± 2.1 nM) was 20 nM lower than in cultured NHK cortex cells (76.5 ± 1.8 nM; Figure 1).

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Intracellular Ca2+ concentrations are lower in cultured autosomal dominant polycystic kidney disease (ADPKD) cells compared with normal human kidney (NHK) cells. Subconfluent monolayers (n = 9 per group) of cells that were derived from three NHK (filled symbols) and three ADPKD (open symbols) kidneys were grown on glass coverslips that were coated with type I collagen. Cells were incubated in a DMEM/F12 growth medium that contained 0.002% FBS for 24 h before the experiment to reduce factors that alter [Ca2+] i and limit variations in resting Ca2+ levels. Cells were loaded with Fura-2/AM for Ca2+ measurements of 340/380 excitation ratios. At the end of the experiments, 340/380 ratios were converted to concentration using calibration solutions. Each value represents an average intracellular Ca2+ concentration determined at five random locations on each monolayer. Filled and open diamonds represent the means ± SE of each group (*P < 0.0001 versus NHK cells). Similar results were obtained in a blinded study in which the cell types were unknown to the investigator until the completion of the experiment.

View larger version (72K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effects of cAMP and Ca2+ channel blockers on cells that were obtained from cystic and noncystic regions from the same ADPKD kidney. Hematoxylin and eosin (H&E)-stained sections of a renal cyst (A) and noncystic tissue (B) from the same ADPKD kidney. Bar = 100 µm. Inset in A shows a higher magnification of the cyst epithelial cells. Primary cultures of epithelial cells were prepared from the cystic (C and E) and noncystic (D and F) cells for measurement of phospho-extracellular signal-regulated kinase (P-ERK), total ERK, and cell proliferation. In ADPKD cystic cells, 8-Brc-AMP (cAMP) increased P-ERK (C) and cell proliferation (E). Ca2+ channel blockers nifedipine (nifed) and verapamil (verap) amplified the effects of cAMP. By contrast, cAMP decreased P-ERK (D) and cell proliferation (F) of noncystic ADPKD cells. Ca2+ entry blockers caused a reversal of the cAMP effect such that cAMP stimulated ERK and cell proliferation in the noncystic cells. Endogenous cAMP production by ADPKD cells during the 72-h incubation period may account for the increase in cell proliferation in the presence of verapamil (E). Similar results were obtained from two additional ADPKD kidneys. *P < 0.001 versus control; #P < 0.01 versus control; P < 0.05 versus control; P < 0.001 versus cAMP alone.

Proliferation Response to cAMP of Cystic and Noncystic Cells from ADPKD Kidneys
Our observation that noncystic cells from ADPKD kidneys have steady-state Ca²⁺ levels comparable to NHK cells suggests that these cells might have a normal cellular response to cAMP. Indeed, in noncystic ADPKD cells, cAMP inhibited ERK activity (Figure 2D) and decreased the rate of cell proliferation (Figure 2F), a phenotype similar to NHK and M-1 cells. By contrast, in cells that were derived from the cysts, cAMP stimulated ERK activity (Figure 2C) and increased the rate of cell proliferation (Figure 2E). Together, these data indicate that cells from normal-appearing tubules in ADPKD kidneys have normal intracellular Ca²⁺ levels and an anti-mitogenic response to cAMP. Thus, germline mutations in the PKD genes alone seem to be insufficient to disrupt Ca²⁺ levels to the degree that can initiate the phenotypic switch to a cAMP-mitogenic response.

Treatment of noncystic ADPKD cells with Ca²⁺ channel blockers verapamil and nifedipine reversed the cAMP response, allowing cAMP to stimulate ERK (Figure 2D) and cell proliferation (Figure 2F), thus mimicking cyst-derived cells. It is interesting that L-type Ca²⁺ channel inhibition enhanced cAMP-dependent ERK activation (Figure 2C) and proliferation of cyst-derived cells (Figure 2E). These observations strongly implicate a role for Ca²⁺ in determining the proliferative response to cAMP of renal epithelial cells, consistent with previous results in NHK and M-1 cells (22).

Elevation in [Ca²⁺]_i Inhibits cAMP-Dependent Proliferation of PKD Cells
Bay K8644 (20 µM), a dihydropyridine compound that is known to activate L-type Ca²⁺ channels, increased [Ca²⁺]_i in ADPKD cells during a 7-min incubation period (Figure 3A). By contrast, an inhibitory stereoisomer of Bay K8644, designated as R(+)-Bay K8644, caused a slight decrease in [Ca²⁺]_i, demonstrating the specificity of Bay K8644 to activate these channels. In ADPKD cells, Bay K8644 treatment for 24 h caused a concentration-dependent increase in steady-state [Ca²⁺]_i (Figure 3B). In other experiments, the Ca²⁺ ionophore A23187 (0.01 µM) also induced a sustained increase in [Ca²⁺]_i (22.3 ± 2.9 nM; n = 3 kidney preparations; P < 0.02). These data demonstrate that chronic activation of L-type Ca²⁺ channels with Bay K8644 or enhanced membrane Ca²⁺ permeability with A23187 can cause a sustained increase in [Ca²⁺]_i in ADPKD cells.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Bay K8644, an L-type Ca2+ channel activator, induces a sustained increase in [Ca2+]i in ADPKD cells. (A) L-type Ca2+ channel agonist Bay K8644 (filled symbols) increased [Ca2+]i in ADPKD cells. By contrast, R(+)-Bay K8644 (open symbols), a Bay K8644 stereoisomer that antagonizes L-type Ca2+ channels, caused a slight decrease in intracellular Ca2+ levels. (B) Incubation of ADPKD cell monolayers in Bay K8644 for 24 h caused a concentration-dependent increase in steady-state [Ca2+]i in ADPKD cells (*P < 0.05 versus control).

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Elevation of [Ca2+]i prevents cAMP-dependent proliferation of cultured ADPKD and autosomal recessive PKD (ARPKD) cells. Cells that were derived from the cysts of ADPKD kidneys (A) and an ARPKD kidney (B) were cultured in medium that contained 1% FBS + ITS for 24 h, and then the serum was reduced to 0.002% for an additional 24 h before treatment. Cells were incubated with 1 µM Bay K8644 (Bay K) or 0.01 µM Ca2+ ionophore A23187 and/or 100 µM 8-BrcAMP (cAMP) for 72 h. Cell proliferation was determined by the Promega Cell Titer 96 MTT assay. In minimal growth medium, cells continued to grow over the experimental period. cAMP stimulated the proliferation of ADPKD and ARPKD cells. Elevations in [Ca2+]i with Bay K8644 or A23187 reversed the proliferative response to cAMP. Values are means ± SE for the percentage change in control proliferation rate (set to zero) of cells from six ADPKD kidneys and one ARPKD kidney. *P < 0.05 versus control; #P < 0.05 versus Bay K8644 alone; P < 0.05 versus A23187 alone.

View larger version (55K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Bay K8644 inhibits cAMP-dependent enlargement of ADPKD cysts growing within collagen gels. (A) Images of ADPKD cells that were cultured within collagen gels. Cysts formed from individual ADPKD cells that were treated with EGF and forskolin, a cAMP agonist. Cells first were grown in the presence of 5 ng/ml EGF and 5 µM forskolin for 3 d to allow the development of microscopic cysts. Then, EGF and forskolin were removed, and forskolin ± 10 µM Bay K8644 was added for an additional 9 d. Cysts were found to expand in the presence of forskolin alone. Bay K8644 arrested or reversed cyst growth. Bar = 500 µm. (B) Total surface area (SA) was determined from the measurement of cysts 100 µm in diameter in each well (n = 6 wells). *P < 0.05 versus control; #P < 0.05 versus forskolin alone. In a composite of similar experiments, Bay K8644 inhibited forskolin-stimulated cyst formation by 74 ± 13%, P < 0.05, n = 3 ADPKD cell preparations.

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Elevation of [Ca2+]i blocks cAMP-dependent stimulation of ERK and B-Raf. (A) ERK activity in cystic cells from an ADPKD kidney (K207) was determined by Western blot using an antibody to P-ERK. (B) Relative P-ERK levels in cells that were cultured from six ADPKD kidneys. Control cells were grown in minimal growth medium. 8-Br-cAMP (cAMP) was added with or without pretreatment with Bay K8644 or Ca2+ ionophore A23187 for 24 h. Bay K8644 and A23187 prevented cAMP activation of ERK. (C) B-Raf activity in ADPKD cells was determined from immunocomplex kinase assay using MEK as a substrate for activated B-Raf kinase. The level of phosphorylated MEK (P-MEK) was determined by Western blot analysis. (D) Composite of the effects of cAMP ± Bay K8644 on B-Raf activity in cultured cells from five ADPKD kidneys. cAMP stimulated B-Raf activity approximately 100% above control levels in ADPKD cells. Bay K8644 decreased basal levels of B-Raf and completely blocked cAMP activation. Bars are means ± SE. *P < 0.001 versus control; #P < 0.001 versus cAMP alone.

To determine whether the difference in the mitogenic response to cAMP between NHK and ADPKD cells is associated Akt activity, we measured the levels of P-Akt (active) and total Akt by immunoblot analysis. The average ratio of P-Akt/total Akt was 63% higher in NHK cells compared with ADPKD cells (n = 4 kidneys each; Figure 7A). To determine whether changes in intracellular Ca²⁺ alter Akt activity, we treated ADPKD cells with Bay K8644, A23187, verapamil, or nifedipine for 15 min. Elevation of Ca²⁺ with Bay K8644 and A23187 increased P-Akt levels by 66.0 and 64.8%, respectively (Figure 7B), whereas the Ca²⁺ channel blockers decreased P-Akt by 21 and 22%, respectively.

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Basal Akt activity is diminished in ADPKD cells compared with NHK cells. (A) Levels of phosphorylated Akt (P-Akt) and total Akt in cells that were derived from NHK (NHK1 through NHK4) and ADPKD kidneys (PKD1 through PKD4) were determined by Western blot. Numbers above the bands indicate the P-Akt/total Akt. Basal Akt activity was lower in ADPKD cells (1.91 ± 0.10) compared with NHK cells (3.12 ± 0.28, P < 0.01, n = 4 each). (B) Levels of phosphorylated Akt (P-Akt) in ADPKD cells that were treated with Bay K8644, A23187, verapamil, or nifedipine. Relative changes in P-Akt from four ADPKD cell preparations. Decreasing [Ca2+]i with Ca2+ channel blockers decreased P-Akt levels, whereas elevation of [Ca2+]i with Bay K8644 or A23187 increased the levels of P-Akt (means ± SE; *P < 0.001). These data demonstrate that [Ca2+]i regulates Akt activity in ADPKD cells.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Inhibition of Akt enhances cAMP-dependent proliferation of ADPKD cells. Rates of cell proliferation were determined by the MTT assay. Inhibition of Akt with 5 µM 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Calbiochem, San Diego, CA) enhanced cAMP-dependent cell proliferation in ADPKD cells. Bars are means ± SE. *P < 0.001 versus control; #P < 0.001 versus cAMP alone.

	Discussion

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Abnormal epithelial cell proliferation underlies cyst formation and renal enlargement in both ADPKD and ARPKD. EGF, an agonist for the receptor tyrosine kinase EGFR, activates the Ras/Raf-1/MEK/ERK pathway and proliferation of both PKD cells and NHK cells (Figure 9). The discovery that cAMP stimulates the proliferation of ADPKD cells but inhibits the proliferation of NHK cells demonstrates a unique phenotypic difference between PKD and NHK cells. In NHK cells, cAMP has been shown to inhibit the Ras/Raf-1/MEK/ERK pathway at the level of Raf-1 and decrease cell proliferation (Figure 9A). By contrast, in PKD cells, cAMP has been shown to stimulate B-Raf and activate the MEK/ERK pathway to increase cell proliferation (Figure 9B).

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Proposed signal transduction pathways for Ca2+ regulation of cAMP-dependent B-Raf signaling to MEK/ERK and cell proliferation. Normal cell proliferation is controlled by growth factors binding to receptor tyrosine kinase with sequential activation of RasRaf-1MEKERK to induce cell proliferation. There is a phenotypic difference between normal kidney cells and PKD cells in the cAMP effect on proliferation. (A) In NHK cells, basal intracellular Ca2+ levels, controlled by a variety of Ca2+ entry mechanisms, maintain the activity of phosphatidylinositol 3-kinase (PI 3-K) and Akt, preventing cAMP-dependent activation of B-Raf. B-Raf kinase activity is inhibited by Akt phosphorylation of an inhibitory site. cAMP agonists, e.g., arginine vasopressin, inhibit Raf-1 through a protein kinase A–dependent mechanism. Thus, ERK activation and cell proliferation are controlled by a balance of signals (positive and negative) that affect Raf-1 in NHK cells. (B) In PKD cells, a reduction in intracellular Ca2+ levels, as a result of a loss of PC1/PC2 function, decreases PI 3-K activity, relieving B-Raf inhibition by Akt. cAMP then signals through B-Raf to activate MEK and ERK and to stimulate cell proliferation.

In our study, cells that were derived from ADPKD cysts were found to have basal [Ca²⁺]_i approximately 20 nM lower than either NHK cells or cells that were derived from noncystic regions of ADPKD kidneys. This observation that noncystic ADPKD cells and NHK cells have similar [Ca²⁺]_i suggests that a "second-hit" mechanism may be necessary to decrease intracellular Ca²⁺ levels in renal epithelial cells. Human ARPKD cells also were found to have reduced basal Ca²⁺ levels compared with NHK cells, suggesting that mutations in either the polycystins or fibrocystin lead to a Ca²⁺ defect in renal epithelial cells.

The data presented in this study provide evidence that intracellular Ca²⁺ is a central regulator of the mitogenic response to cAMP in human renal epithelial cells. In the noncystic ADPKD cells, cAMP decreased ERK activity and inhibited cell proliferation. In contrast, in cyst-derived ADPKD cells, cAMP stimulated ERK and cell proliferation (Figure 2). Thus, cyst-derived cells, which presumably have both germline and somatic mutations in the PKD gene, had a lower [Ca²⁺]_i and a cAMP-dependent proliferative phenotype, whereas noncystic cells from ADPKD kidneys had a normal [Ca²⁺]_i and a normal anti-proliferative phenotype. Reduction of [Ca²⁺]_i in noncystic cells with Ca²⁺ channel blockers reversed the mitogenic response to cAMP such that cAMP stimulated ERK and cell proliferation, mimicking the cystic cells. These types of reversal experiments strongly implicate a relationship between intracellular Ca²⁺ and cAMP-dependent cell proliferation and cyst formation.

In this study, we did not determine the specific mutations in either ADPKD or ARPKD cells. It is interesting to note that the steady-state Ca²⁺ levels were decreased in both ADPKD and ARPKD cells. Moreover, the addition of Ca²⁺ to ADPKD and ARPKD cells reversed the capacity of cAMP to stimulate cell proliferation. We think that the reduced steady-state intracellular Ca²⁺ levels, common to both ADPKD and ARPKD cells, and the reversal of the cAMP-dependent proliferation by increasing cell Ca²⁺ support the view that reduced intracellular Ca²⁺ alters the cAMP-proliferative response irrespective of the underlying mutations in either ADPKD or ARPKD.

Previously, the mitogenic response to cAMP had been induced experimentally by reducing [Ca²⁺]_i in M-1 cells with Ca²⁺ channel blockers or EGTA (22). Restriction of intracellular Ca²⁺ switched these cells from cAMP-growth inhibited to cAMP-growth stimulated, mimicking the PKD phenotype. On the basis of those studies, we proposed that Ca²⁺ acts to maintain Akt activity, which suppresses B-Raf signaling to the mitogen-activated protein kinase pathway in normal renal epithelial cells. Akt phosphorylation of B-Raf can block the activity of B-Raf, preventing cAMP activation of ERK and cell proliferation (27). Ca²⁺ restriction in M-1 cells decreased Akt activity, allowing cAMP-dependent activation of B-Raf and cell proliferation (22).

If the loss of polycystin in ADPKD cells were to diminish [Ca²⁺]_i, then one might expect that the basal Akt activity would be lower. In our study, relative levels of P-Akt/total Akt were found to be significantly lower in ADPKD cells compared with NHK cells (Figure 7A). Bay K8644 and A23187 caused a sustained increase in [Ca²⁺]_i (Figure 3A) and completely reversed the cAMP proliferative response in both ADPKD and ARPKD cells (Figure 4). This phenotypic switch in the cAMP-mitogenic response was associated with an increase in Akt activity (Figure 7) and decreased B-Raf and ERK activities (Figure 6).

In conclusion, these data support the hypothesis that intracellular Ca²⁺ has an important role in determining the proliferative response to cAMP agonists in renal epithelial cells (Figure 9). The reduction in [Ca²⁺]_i in cyst epithelial cells, secondary to mutations in the PKD genes, relieves Akt inhibition of B-Raf, allowing cAMP-dependent cell proliferation and cyst growth. We found that an increase in Ca²⁺ in PKD cells can increase Akt activity to repress cAMP stimulation of B-Raf, ERK, and cell proliferation, thus restoring a normal anti-mitogenic response to cAMP. Therapeutic approaches to reduce renal cAMP accumulation by blocking the vasopressin V2 receptors are being considered for the treatment of PKD (14,17). We propose that mechanisms that selectively increase [Ca²⁺]_i within renal cystic epithelial cells might provide an alternative or complementary treatment to retard cAMP-dependent cyst progression.

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health DK064756 (D.P.W.) and P20-RR-17686 (D.P.W.) and the PKD Foundation (D.P.W.).

We are grateful to Dr. J.J. Grantham and Dr. J.P. Calvet for helpful suggestions and reading of the manuscript and to Megan Quante for technical assistance.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

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