ADPKD cell proliferation and Cl−-dependent fluid secretion

ADPKD cell proliferation and Cl−-dependent fluid secretion

CHAPTER ADPKD cell proliferation and Cl-dependent fluid secretion 4 Gail A. Reif, Darren P. Wallace* Departments of Internal Medicine and Molecula...
Download PDF
2MB Sizes 1 Downloads 34 Views
Report

CHAPTER

ADPKD cell proliferation and Cl-dependent fluid secretion

4

Gail A. Reif, Darren P. Wallace* Departments of Internal Medicine and Molecular and Integrative Physiology, and The Jared Grantham Kidney Institute, University of Kansas Medical Center, Kansas City, KS, United States *Corresponding author: e-mail address: [email protected]

Chapter outline 1 Introduction........................................................................................................70 2 Thawing and passaging ADPKD cells.....................................................................72 2.1 Medium preparation.............................................................................72 2.1.1 DMEM/F12 reagents (2 L)..................................................................72 2.1.2 Preparing DMEM/F12 medium..........................................................72 2.1.3 Supplies and media supplements......................................................72 2.1.4 Preparing cell culture media..............................................................73 2.2 Thawing ADPKD cells...........................................................................73 2.3 Passaging cells for in vitro cell assays....................................................73 3 Cell proliferation.................................................................................................74 3.1 MTT assay...........................................................................................74 3.1.1 Supplies...........................................................................................74 3.1.2 Seeding cells in 96-well plates...........................................................74 3.1.3 Performing the experiment................................................................75 3.2 Cell count assay...................................................................................75 3.2.1 Supplies...........................................................................................75 3.2.2 Seeding cells in 24-well plates...........................................................76 3.2.3 Performing the experiment................................................................76 3.3 Presenting cell proliferation data...........................................................76 4 Cl2 secretion assay............................................................................................76 4.1 Method of short-circuit current..............................................................76 4.2 Plating ADPKD cells on snapwell supports..............................................77 4.2.1 Supplies...........................................................................................77 4.2.2 Seeding cells on Snapwell supports....................................................77 4.3 Equipment, supplies and solutions.........................................................79 4.3.1 Equipment and supplies....................................................................79 4.3.2 Ringer’s solution...............................................................................80 4.3.3 Preparing Ringer’s solution (500 mL).................................................81 Methods in Cell Biology, Volume 153, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.06.001 © 2019 Elsevier Inc. All rights reserved.

69

70

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

4.4 Measuring anion transport and presenting the data..................................82 4.4.1 Setting up the voltage clamp device...................................................82 4.4.2 Setting up the LabChart software.......................................................82 4.4.3 Mounting cell monolayers in Ussing chambers...................................83 4.4.4 Performing the experiment................................................................83 5 Fluid secretion assay...........................................................................................84 5.1 Supplies.............................................................................................85 5.2 Plating cells on transwell supports.........................................................85 5.3 Preparing the water-saturated mineral oil................................................87 5.4 Calibrating the capillary tubes...............................................................87 5.5 Preparing the fluid secretion assay.........................................................88 5.6 Collecting the secreted fluid..................................................................88 5.7 Measuring the fluid and presenting the data............................................90 6 Conclusions........................................................................................................90 Acknowledgments....................................................................................................90 References..............................................................................................................91

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by bilateral fluid-filled cysts, renal inflammation and extensive fibrosis, leading to the progressive decline in kidney function. Renal cyst formation begins in utero from aberrant proliferation of tubule epithelial cells; however, the mechanisms for cystogenesis remain unclear. Cell proliferation and Cl-dependent fluid secretion, which drives the accumulation of cyst fluid, are responsible for inexorable growth of cysts and the remarkable appearance of massively enlarged ADPKD kidneys. Investigators have used in vitro assays to explore cellular and molecular mechanisms involved in ADPKD cyst epithelial cell proliferation and Cl-dependent fluid secretion in experimentally controlled environments. These assays have been used to evaluate potential therapeutic approaches to inhibit cellular pathways involved in cyst growth. This chapter discusses methods for measuring ADPKD cell proliferation, transepithelial Cl secretion, and net fluid transport across cyst epithelial cell monolayers.

1 Introduction In the early 1980s, autosomal dominant polycystic kidney (ADPKD) was characterized as a “predominance of giant nephrons” (Grantham, 1983); however, causes of these pathologic changes in the kidney architecture were unclear. Mechanisms being considered included “the obstruction of tubule fluid flow by hyperplastic tubule cells, increased compliance of the tubule basement membranes, and/or increased radial growth of cells in specific portions of the renal tubule.” Scarcity of clinical samples from early stage ADPKD kidneys hindered the pathologic characterization

1 Introduction

of initial cyst formation. Studies using tubule specific lectins to identify the histological origin of renal cysts in end-stage ADPKD kidneys suggested that cysts were derived from renal tubules and glomeruli. Dilated tubules and small cysts still connected to the nephron are thought to fill with glomerular filtrate; however, as these cysts enlarge they detach from the tubule segment from which they originated and become isolated from the urinary space. A central question to progressive enlargement of these cysts was how fluid continuously accumulates within the cyst cavity once the cyst is no longer connected to the nephron. Grantham made the unexpected discovery that isolated kidney tubules have the capacity for net fluid secretion (Grantham, 1976), leading to the hypothesis that solute and fluid secretion by the cyst-lining epithelium is responsible for fluid accumulation within cysts and the massive enlargement of ADPKD kidneys. In vitro cell models have been used to characterize the cellular mechanisms responsible for cyst formation. Factors that increased intracellular cAMP, including permeable forms of cAMP, prostaglandin E1, forskolin (a direct activator of adenylyl cyclase), and 1-methyl-3-isobutylxanthine (a phosphodiesterase inhibitor) stimulated in vitro cyst formation of Madin-Darby canine kidney (MDCK) cells and primary human ADPKD cyst epithelial cells cultured in a collagen matrix (Mangoo-Karim & Grantham, 1990; Mangoo-Karim, Uchic, Grant, et al., 1989; Mangoo-Karim, Uchic, Lechene, & Grantham, 1989). Yamaguchi et al. showed that cAMP agonists promote human ADPKD cell proliferation using a MTT cell proliferation assay (Yamaguchi et al., 2000), which measures a mitochondrial reaction product that correlates directly with cell number (Rankin, Ziemer, Maser, Foo, & Calvet, 1996). The mitogenic effect of cAMP on ADPKD cells was confirmed by [3H]-thymidine incorporation and by counting the number of cells at various times after cAMP stimulation (Hanaoka & Guggino, 2000). In ADPKD cells, cAMP stimulates BRAF, a kinase that activates MEK, which subsequently stimulates the extracellular signal-regulated kinase (ERK/MAPK) to increase cell proliferation. This mitogenic effect of cAMP was blocked by H-89, an inhibitor of protein kinase A; sorafenib, a BRAF inhibitor; and PD98059, a MEK inhibitor (Yamaguchi et al., 2003; Yamaguchi, Reif, Calvet, & Wallace, 2010). By contrast, cAMP does not stimulate the MEK/ERK pathway and proliferation of normal human kidney (NHK) cells, providing the first functional difference between normal kidney cells and ADPKD cells. Subsequent studies determined that the mitogenic effect of cAMP was due to abnormal Ca2+ homeostasis in ADPKD cells. Ca2+ restriction with Ca2+ channel blockers de-repressed BRAF in NHK cells, allowing cAMP-dependent activation of ERK and cell proliferation, mimicking the ADPKD phenotype (Yamaguchi et al., 2004). Conversely, a graded increase in intracellular Ca2+ using a L-type Ca2+ channel activator, blocked BRAF and restored a normal phenotype in ADPKD cells (Yamaguchi, Hempson, Reif, Hedge, & Wallace, 2006). Elevation in intracellular cAMP also stimulated Cl-dependent fluid secretion by MDCK and ADPKD cystic cells (Davidow, Maser, Rome, Calvet, & Grantham, 1996; Hanaoka & Guggino, 2000; Jansson et al., 2015; Mangoo-Karim, Ye,

71

72

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

Wallace, Grantham, & Sullivan, 1995; Reif et al., 2011; Sullivan, Wallace, & Grantham, 1998). This chapter discusses experimental approaches for the measurement of cell proliferation, cAMP-dependent transepithelial Cl secretion and net fluid transport across ADPKD cell monolayers.

2 Thawing and passaging ADPKD cells Techniques for generating primary cultures of human ADPKD and normal human kidney (NHK) cells are described in chapter “Generation of primary cells from ADPKD and normal human kidneys” by Wallace and Reif. ADPKD and NHK cells (2  106 per vial) are frozen in cryotubes containing 1:1 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and Ham’s F12 Nutrient Mixture, referred to as DMEM/F12, supplemented with 10% fetal bovine serum (FBS), insulin, transferrin and selenium (ITS), penicillin-streptomycin (P/S), and 10% dimethylsulfoxide (DMSO) and stored in liquid nitrogen. This section describes thawing ADPKD cells for in vitro assays.

2.1 Medium preparation 2.1.1 DMEM/F12 reagents (2 L) 10.0 g 10.6 g 4.0 g 30 mL

Dulbecco’s Modified Eagle’s Medium (DMEM); Sigma-Aldrich #D5523 (St. Louis, MO) Ham’s F12; Sigma-Aldrich #N6760 NaHCO3; Sigma-Aldrich #S6014 HEPES (1M); Sigma-Aldrich #H0887

2.1.2 Preparing DMEM/F12 medium 1. Add the medium components to 1800 mL of water in a graduated cylinder, while stirring on a magnetic stirrer. 2. Titrate the medium to pH 7.23 and then add water to bring up the final volume to 2 L. 3. Sterile filter the medium using a 0.22 μm sterile filtration unit, transfer to 500 mL sterile bottles and store refrigerated. This medium contains L-glutamine, an unstable essential amino acid that is required for cell culture; therefore, we recommend that the medium be used within 4 weeks. This formulation provides a starting point for working with primary ADPKD cell cultures; however, premixed DMEM/F12 and other media may work as well or possibly better.

2.1.3 Supplies and media supplements • • •

50-mL centrifuge tubes T75 flasks Penicillin-Streptomycin (P/S; 100); Sigma-Aldrich #P0781

2 Thawing and passaging ADPKD cells

• •

Insulin, transferrin and selenium (ITS; 1000 ); Corning #354351 (Tewksbury, MA) Fetal bovine serum (FBS); Atlantic Biologicals #S11550 (Flowery Branch, GA)

2.1.4 Preparing cell culture media 1. Prepare a 1000  stock solution of ITS, a supplement for serum-reduced cell culture media. This product comes as a lyophilized powder and will need to be dissolved with sterile water. Freeze the stock in 0.5–1.0 mL aliquots to reduce the number of freeze/thaws. 2. Make up 100 mL DMEM/F12 containing 10% FBS, ITS and P/S for thawing cells. 3. Make up 250 mL DMEM/F12 containing 5% FBS, ITS and P/S for growing cells.

2.2 Thawing ADPKD cells 1. Rapidly thaw the vial of cells by warming in a 37 °C water bath. Gently rotate the tube until the ice in the vial has thawed. Do not exceed 37 °C. 2. Spray the vial and cap with 70–80% EtOH before taking it to the hood. 3. Transfer the cells to a 50-mL centrifuge tube and slowly add 20 mL DMEM/F12 +P/ S +ITS +10% FBS, allowing time for DMSO to diffuse out of the cells. This should reduce an osmotic shock due to the hypertonicity of DMSO within the cells. 4. Centrifuge the tube at 1100 rpm to pellet the cells. 5. Gently resuspend the cell pellet in 30 mL of DMEM/F12 + P/S + ITS + 10% FBS. 6. Transfer the cells into two T75 flasks containing a final volume of 15 mL of DMEM/F12 + P/S + ITS + 10% FBS and allow the cells to attach overnight. 7. The next morning, replace media with DMEM/F12 + 5% FBS + P/S + ITS and allow the cells to grow until 70–80% confluent (3–4 days). This procedure should provide a total of 6–8  106 cells for experimentation. An alternative approach is to directly plate the cells in flasks containing DMEM/F12+ P/S + ITS + 10% FBS without removing the DMSO. After allowing the cells to attach overnight, replace the medium with DMEM/F12 + P/S + ITS + 5% FBS. This eliminates the centrifugation step, which may be harmful to cells in a fragile state; however, DMSO may affect cell attachment and survival.

2.3 Passaging cells for in vitro cell assays 1. Discard the media from the flasks and rinse the cells with 10 mL Dulbecco’s PBS. 2. Add 5 mL trypsin-EDTA to each flask and return the flasks to the incubator. After 15 min, check the cells to determine if they have detached. 3. Once cells have detached, add 10 mL DMEM/F12 + 10% FBS + P/S + ITS and gently pipette up and down to generate a uniform single cell suspension. 4. Determine the total number of cells using a cell counter, such as a Bio-Rad TC20, or a classical hemocytometer.

73

74

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

5. Centrifuge the cells at 1100 rpm for 5 min. 6. Discard the supernatant and gently resuspend the cell pellet in the appropriate volume of DMEM/F12 + 5% FBS + P/S + ITS to yield a working concentration of 1  106 cells/mL.

3 Cell proliferation 3.1 MTT assay The MTT 96-well Cell Proliferation Assay is a nonradioactive, high-throughput method to measure cell proliferation and viability. The assay involves the measurement of a mitochondrial reaction product that correlates directly with cell number. NAD(P)H-dependent oxidoreductase enzymes reduce MTT, a yellow tetrazole dye, to an insoluble formazan product, which has a dark purple color. The absorbance of this product can be quantified by measuring the optical density at 570 nm using a spectrophotometer. While results obtained from a comparison between the MTT assay and 3[H]thymidine incorporation showed no significant differences, it is important to recognize that the assay measures cell metabolism and not cell number.

3.1.1 Supplies • • • • • •

Promega CellTiter 96 Nonradioactive Cell Proliferation Assay; Promega #G4000 (Madison, Wisconsin) Sterile 96-well plates; Corning #353075 DMEM/F12 + 1% FBS + P/S + ITS for attachment DMEM/F12 + 0.05% FBS + P/S Sterile water 96-well plate reader capable of reading O.D. at 570 nm

3.1.2 Seeding cells in 96-well plates 1. Prepare 96-well culture plates by filling the exterior wells with 300 μL sterile room temperature water. 2. Dilute the cell suspension to 4  104 cells/mL. For each 96-well plate, add 320 μL of the 1  106 cells/mL suspension to 7.68 mL of DME/F12 + 1% FBS + P/S + ITS for a final volume of 8 mL in a 50 mL tube. 3. Add 100 μL of the 4 104 cells/mL cell suspension to each well to seed 4  103 cells per well. Plate the 60 wells (10 by 6) in the center of the plate, moving horizontally across the plate. Gently swirl the tube to keep the cells in suspension. 4. Allow the cells to attach for 24 h in DMEM/F12 + 1% FBS + P/S + ITS. 5. Change the medium to DMEM/F12 + 0.05% FBS + P/S (no ITS) for an additional 24 h.

3 Cell proliferation

3.1.3 Performing the experiment 1. Prepare experimental conditions using DMEM/F12 + 0.05% FBS + P/S. One plate can accommodate 10 conditions with 6 wells/condition. Include both a positive (i.e., epidermal growth factor) and negative control for cell proliferation. 2. Aspirate off growth media without disturbing the cells and add 100 μL of experimental media for 24–72 h. It may be necessary to replenish the experimental media every day or every 2 days, depending on the stability of the test compounds 3. Monitor the cells daily to determine if there is higher cell proliferation in the positive control group compared to the negative control group. Stop the assay before the cells reach confluency. 4. Prepare dye solution in Dulbecco’s PBS following the instructions provided with the Promega assay. Each well of the 96-well plate will receive 100 μL PBS + 15 μL dye solution. Prepare enough solution for 70 wells, by mixing 7 mL PBS + 1.05mL dye solution. 5. Remove the treatment media from the wells and replace with 115 μL of the PBS + dye solution mixture. Also, replace the PBS in the first column of 6 wells (no cells) with the PBS + dye solution, which will be used as blanks when reading absorbance. 6. Return the plate to the incubator for 4 h. 7. After 4 h, add 100 μL “Stop solution” provided in the assay to each well (including the blanks). 8. Keep the plate at room temperature for a minimum of 1 h, then measure absorbance at 570 nm using a 96-well plate reader. The plate can be read several days after the experiment if the plate is sealed and stored at 4 °C.

3.2 Cell count assay The MTT assay, described above, measures the level of a mitochondria reaction product. Since some compounds may alter cell metabolism, it is important to confirm cell proliferation results by direct cell counting. This can be done using a classical hemocytometer or an automatic cell counter.

3.2.1 Supplies • • • • • •

Sterile 24-well plates; Corning #3524 DMEM/F12 + 1% FBS + P/S + ITS DMEM/F12 + 0.05% FBS + P/S (no ITS) Dulbecco’s PBS Trypsin-EDTA Automated cell counter; Bio-Rad TC20

75

76

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

3.2.2 Seeding cells in 24-well plates 1. Dilute the cell suspension to 4  104 cells/mL. For each 24-well plate, add 320 μL of the 1  106 cells/mL suspension to 7.68 mL of DME/F12 + 1% FBS + P/S + ITS for a final volume of 8 mL. 2. Add 0.25 mL to each well of a 24-well plate for a seeding density of 1  104 cells/well. There will be 6 experimental groups with 4 wells per group. 3. Allow cells to attach for 24 h in DMEM/F12 + 1% FBS + P/S + ITS. 4. After 24 h, reduce the serum concentration to 0.05% FBS (no ITS) for an additional 24 h.

3.2.3 Performing the experiment 1. Prepare experimental conditions in DMEM/F12 + 0.05% FBS + P/S (no ITS), including positive and negative control groups. 2. Replace growth media with experimental media and incubate for 24–72 h, depending on the growth rate of the cells. 3. Monitor the cells for increased proliferation in the positive control. Stop the assay before the cells reach confluency. 4. To stop the assay, remove the treatment media and rinse the cells with Dulbecco’s PBS. 5. Completely aspirate off the PBS. 6. Add 150 μL trypsin-EDTA/well and incubate at 37 °C for 30 min or until the cells are in a single-cell suspension. 7. Once the cells have detached, count total cells (live and dead) per well using an automated cell counter or hemocytometer.

3.3 Presenting cell proliferation data Data obtained from MTT assays can be expressed in O.D. values, and actual cell number per well can be presented from cell counting assays; however, there may be variability between experiments due to differences in the basal proliferation rates, particularly in studies involving primary cells from different ADPKD kidneys. To account for these differences, it may be necessary to normalize the data to the negative control group (set to 100%). Therefore, changes in proliferation with each treatment groups are expressed as percentage to basal proliferation.

4 Cl2 secretion assay 4.1 Method of short-circuit current Epithelial cells form a barrier separating luminal and serosal environments with apical tight junctions that prevent many substances, including ions, from diffusing through the paracellular pathway between adjacent cells. Transcellular transport is

4 Cl secretion assay

possible because of the coordinated actions of transporters and channels that are specifically localized in the apical (mucosal) and basolateral (serosal) membranes. Basolateral Na/K-ATPase, a primary active transporter, and the conductance of K+ channels establish transmembrane electrical and chemical gradients for Na+, which are utilized by secondary active transporters. Net ion movement across the epithelium, either absorption or secretion, can generate a transepithelial voltage (VTE), a potential difference proportional to the current (I) and transepithelial resistance (RTE), or V ¼ IR. The investigation of epithelial ion transport was facilitated by the development of a chamber system designed by Dr. Hans Ussing more than 40 years ago (Larsen, 2002). The Ussing chamber is an important scientific tool that allows the measurement of ion transport across native tissues, such as the gut mucosa. Recent designs of the Ussing chamber have allowed the measurement of ion transport across cultured cells grown on permeable supports (Fig. 1A). Ussing chambers consist of two halves that are clamped together, isolating the apical and basolateral surfaces of the cell monolayer. Medium of identical composition is placed in each chamber half so that there are no chemical gradients across the monolayer and the medium is circulated by a bubble-lift system. One pair of electrodes connected to a voltmeter is placed near the monolayer to measure the VTE. A second pair of electrodes connected to an electrical circuit provides a current sufficient to drive VTE to zero (Fig. 2). This is referred to as the short-circuit current (ISC or SCC). The magnitude of ISC equals the sum of all active ion transport across the epithelial cell monolayer and is largely independent of RTE. Since both anion secretion and cation absorption increase the positive ISC, it is important to use ion channel inhibitors and/or ion substitution to identify the transported ion (Fig. 1B). Additional information on electrical measurements in the Ussing chamber system can be found in review articles (Li, Sheppard, & Hug, 2004) and on-line information on (https://www.warneronline.com/introduc tion-to-ussing-systems-from-warner)

4.2 Plating ADPKD cells on snapwell supports 4.2.1 Supplies • • • •

Snapwells; Corning #3407 6-well culture plates DMEM/F12 + 5% FBS + P/S + ITS DMEM/F12 + 1% FBS + P/S + ITS

4.2.2 Seeding cells on Snapwell supports 1. Prepare a 6.25  105 cells/mL working cell suspension. For 6 Snapwells, mix 1.88 mL of 1  106 cells/mL and 1.12 mL of DMEM/F12 + 5% FBS + P/S + ITS (total volume 3 mL). 2. Seed 2.5  105 cells per Snapwell by adding 0.4 mL of the working cell suspension to the top of each Snapwell.

77

78

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

FIG. 1 Measurement of ion transport across a cell monolayer. (A) Schematic of an Ussing chamber demonstrating the position of a cell monolayer between the two chamber halves, separating the apical and basolateral compartments. Two sets of electrodes are placed in the chamber. One pair of electrodes is positioned near the cell monolayer to measure transepithelial voltage (VTE, mV). A second pair of electrodes in the back of the chamber applies a current that is necessary to drive VTE to zero. This is called short-circuit current (ISC or SCC, μA), which reflects the sum of ion transport across the monolayer. (B) An apical (lumen) negative VTE and positive ISC can be generated by anion (Cl) secretion and/ or cation (Na+) absorption across the monolayer. It is necessary to use specific channel inhibitors or ion replacement to identify the transported ion.

3. Pipet 5 mL of DMEM/F12 + 5% FBS + P/S + ITS to the lower chamber. 4. Allow the cells to grow for 2 days, and then switch the medium to DMEM/F12 + 1% FBS + P/S + ITS for an additional 7–9 days. ADPKD cell monolayers typically develop RTE suitable for Ussing chamber studies. RTE of monolayers can be monitored using an Epithelial Volt/Ohm meter (EVOM) with chopstick electrodes (World Precision Instruments; Sarasota, FL); however, measurements may not be accurate and may change depending on the placement of the electrodes.

4 Cl secretion assay

FIG. 2 Images of an Ussing chamber system and voltage clamp device. (A) NaviCyte Vertical Ussing Chamber, caps, air lines, retaining rings and clamp tool, and Snapwells with the 6-well plate. (B) An assembled Ussing chamber with electrodes. (C) EC-825A Dual Channel Voltage Clamp and (D) one of the headstages that connects the voltage and current electrodes to the EC-825A. (E) An enlarged view of the control panel for one of the channels on the voltage clamp device.

4.3 Equipment, supplies and solutions 4.3.1 Equipment and supplies • •

EC-825A Dual Channel Voltage Clamp (for 2 chambers; Fig. 2C); Warner Instruments #64-1605 (Hamden, CT), including two headstages (Fig. 2D) 6-Chamber heater block assembly containing the gas manifold (for 2–6 chambers) and retaining ring clamp tool; Warner Instruments #66-0075

79

80

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

Water bath set to 37 °C NaviCyte Vertical (Ussing) Chamber Systems, Snapwell, standard volume (Fig. 2A and B); Warner Instruments #66-0008 Electrode caps for Snapwell chamber, pair L/R (for 1 chamber); Warner Instruments #66-0020 Electrode cap screws (pack of 12); Warner Instruments #66-0050 Ag/AgCl electrodes with KCl, 4 pack (for 1 chamber); Warner Instruments #66-0023 Glass barrel for Ag/AgCl electrode, with ceramic tip, 8 pack (for 2 chambers; Fig. 2B); Warner Instruments #66-0024 Retaining rings (pack of 12); Warner Instruments #66-0007 Spacers (Snapwell with the membrane removed) PowerLab 4/30; AD Instruments (Colorado Springs, CO) LabChart data analysis software (Fig. 3); ADInstruments 5 cc syringes with tubing to empty and fill each half of the Ussing chambers Ringer’s solution 5% CO2/95% O2 tank with regulator, gas-washing bottle, and appropriate tubing

• • • • • • • • • • • • •

4.3.2 Ringer’s solution Ringer’s solution is an isotonic salt solution that mimics the extracellular fluid of animals, and is commonly used for the study of solute and fluid transport across epithelial cells. Ringer’s solutions can be purchased from various vendors; however, the following formulation allows the investigator to make specific changes to the composition for experimental purposes. For example, Cl can be replaced with the molar equivalent of cyclamate or nitrate to demonstrate the Cl dependence of cAMP-induced fluid secretion (Wallace, Grantham, & Sullivan, 1996). We recommend making the following stock solutions:

Stock

Compounds

Formula weight

I II

K2HPO4 CaCl22H2O MgSO47H2O Glucose Na Acetate3H2O L-Alanine Na3 Citrate NaCl Na Lactate (Sigma)

174.18 147.00 246.48 180.2 136.2 89.1 294.1 58.44 112.02

III

IV V

Amount (g/500 mL)

Stock conc.

Final conc. in Ringer’s

4.35 g 1.47 g 1.48 g 9.01 g 6.81 g 5.35 g 2.94 g 67.2 g

50 mM 20 mM 12 mM 100 mM 100 mM 120 mM 20 mM 2300 mM 60% w/w

2.5 mM 2.0 mM 1.2 mM 5.0 mM 5.0 mM 6.0 mM 1.0 mM 115.0 mM 4.0 mM

4 Cl secretion assay

FIG. 3 Screenshots of the LabChart software. Example templates for (A) the Chart View with four channels and (B) the Data Pad View that provides a record of the experiment time, SCC (ISC, μAmps), comment time and text for each channel. The software allows the investigator to determine the frequency for recording ISC.

Stocks I, II and IV are stored at room temperature. Stock V is stored at 4 ° C and Stock III is stored frozen at 20 °C. It is necessary to bubble the Ringer’s solution with high CO2 before the addition of Stock II to prevent a calcium phosphate precipitate. Stock V is viscous; therefore, it helps to warm the bottle before pipetting.

4.3.3 Preparing Ringer’s solution (500 mL) 1. Add 0.84 g NaHCO3 (for a final concentration of 20 mM) to a volumetric flask 2. Add 4.2 g raffinose (for a final concentration of 14.1 mM)

81

82

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

3. 4. 5. 6. 7. 8. 9.

Add 300 mL water Pipet 25 mL of stocks I, III and IV into the flask Add water to 70% of final volume Bubble with 10% CO2 for 20 min Add 50 mL of stock II Add 0.285 mL of stock V Bring up to 500 mL final volume and mix well

4.4 Measuring anion transport and presenting the data 4.4.1 Setting up the voltage clamp device 1. Turn on power to the EC-825A with the mode set to AMPLIFY 2. Set the toggle switch to VOLTAGE, CLAMP RESPONSE to SLOW, and MEMBRANE RES. to OHMS (Fig. 2C and E). 3. Warm the heating block and water bath to 37 °C. 4. Place a bottle containing Ringer’s solution in the water bath and bubble the solution with 5% CO2/95% O2. 5. Assemble the chambers with a spacer (instead of a Snapwell) and connect the electrodes (Fig. 2B). 6. Use syringes with tubing to fill each chamber with 10 mL Ringer’s solution. 7. Connect the CO2/O2 lines to the chamber and adjust the gas flow for a gentle and consistent airlift circulation of the chamber fluid. 8. Turn on the headstages. 9. On the EC-825A, use the COMMAND potentiometer to set the voltage to zero in order to offset the electrode potential differences (Fig. 2E). 10. Adjust the fluid resistance to zero by pushing the FLUID RESISTANCE button while turning the potentiometer. 11. Allow the system to equilibrate. Re-zero the potential as needed until the potential remains at zero (0.1) for a 10-min period. Typically, the fluid resistance does not need to be readjusted; however, make the adjustment if needed.

4.4.2 Setting up the LabChart software 1. The software and templates will need to be set up before the experiment since it may take time to complete. LabChart manual provides the necessary information on setting up templates and is beyond the scope of the chapter. 2. The Chart View template will need to contain the correct number of channels (chambers) for your experimental setup. An example of the chart recording for four channels is shown in Fig. 3A. The scale of the individual charts can be adjusted during the experiment. 3. A Data Pad worksheet will also need to be prepared to record experiment time, SCC (ISC) and user comments for each of the channels (Fig. 3B). Warner Instruments also offers the UssingChart DAQ, which is a complete package that comes in 2, 4, 6, or 8 channel versions. The DAQ includes the EC-825A amplifier, digitizer and software.

4 Cl secretion assay

4.4.3 Mounting cell monolayers in Ussing chambers 1. 2. 3. 4.

5. 6.

7. 8. 9.

10. 11. 12.

Make sure the voltage on the meter displays zero for all channels. Turn off the headstages and stop the flow of 5% CO2/95% O2 to the chambers. Remove the Ringer’s solution from the first chamber using the 5 cc syringes. Remove the retaining rings from the chamber and gently pull the two halves apart being careful not to alter the position of the electrodes. Remove the spacer and replace with the first Snapwell containing a cell monolayer. When removing the Snapwell from the plastic support avoid bending or buckling the membrane. Reassemble the chamber, replace the retaining rings, and return the assembled chamber to the heating block. Slowly fill both sides of the chamber from the bottom with 5 mL fresh Ringer’s solution using the syringes. Keep a slightly higher fluid volume on the apical side to avoid a positive pressure on the basolateral side of the monolayer which may dislodge the cells. Turn on the headstages. In the AMPLIFY mode, the meter will display the VTE. Check the RTE by turning the mode to MEMBRANE RES. Set the mode to V. CLAMP to clamp the voltage to zero. The ISC will appear on chart during the experiment; however, to view the current, move the toggle switch to CURRENT. The toggle switch should remain on VOLTAGE during the experiment. Repeat steps 4–8 for remaining Snapwells. Once all the chambers have been reassembled, turn on the flow of CO2/O2 to the chambers. Allow the cells to equilibrate for approximately 30 min in the chambers before beginning the experiment.

4.4.4 Performing the experiment 1. Turn on the PowerLab. 2. Open LabChart software and load your template from the list of recent files in the LabChart Welcome Center. 3. Start recording and establish a stable baseline for ISC. If the baseline is not stable or there is considerable drift in the reading, it may be necessary to replace the monolayer. 4. Check the VTE by turning to AMPLIFY and RTE by turning to MEMBRANE RES for each monolayer. You will need to type this information as a comment, using the comment bar at the top of the Chart View window. This provides a record of the basic electrical properties of the monolayers. 5. After recording the baseline ISC, proceed with the experiment. 6. Use the comment bar to record all additions of agonists, antagonists or drugs to the chambers. 7. Data (time, ISC) and comments are automatically added to the Data Pad worksheet. 8. At the end of the experiment, save the file with a unique filename. The file contains both the chart and worksheet.

83

84

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

FIG. 4 Representative tracings of the effect of arginine vasopressin (AVP) and CFTR Cl channel inhibitor CFTRinh-172 on ISC across ADPKD cell monolayers. (A) ISC was recorded every 10s and normalized to surface area of the cell monolayer (μA/cm2). Benzamil (10 μM), a potent inhibitor of the epithelial Na+ channel ENaC, was added to the apical medium to inhibit Na+ absorption. Basolateral addition of 1 and 10 nM AVP increased anion secretion. Forskolin (10 μM), a direct activator of adenylyl cyclases that maximally stimulates cAMP production, caused a further increase in ISC. (B) An ADPKD monolayer was pretreated with benzamil and ISC was recorded every minute after the addition of 10nM AVP and 100 μM CFTRinh-172, demonstrating that the AVP-induced anion secretion was mediated by CFTR Cl channels.

9. Copy the information from the Data Pad to an Excel worksheet, which is easier to work with and can be opened by most computers. In Excel, ISC can be normalized to the surface area of the Snapwell (μAmps/cm2; Fig. 4).

5 Fluid secretion assay The proposed mechanism for cAMP-dependent fluid secretion is shown in Fig. 5. Briefly, the Na+/K+-ATPase, acting in concert with K+ channels in the basolateral membrane, establishes and maintains the electrical and chemical gradients for

5 Fluid secretion assay

Na+ that are utilized by secondary active transporters. Cl enters the cell across the basolateral membrane via an electrically neutral Na+-K+-2Cl cotransporter NKCC1, using the transmembrane Na+ gradient to bring the ions into the cell. This maintains intracellular Cl above its electrochemical gradient, allowing Cl efflux across the luminal membrane upon cAMP activation of CFTR Cl channels. Chloride efflux is the initial step in cAMP-dependent Cl secretion, causing a transepithelial potential for paracellular transport of Na+. The net addition of NaCl to the luminal fluid drives the osmotic movement of fluid. This section discusses a method to measure transepithelial fluid movement across polarized cell monolayers grown on Transwell-COL permeable supports.

5.1 Supplies • • • • • • • • •

• • • •

6-well culture plates Transwell-COL (TW-COL; 24 mm diameter, 4.52 cm2); Corning #3491 (Cambridge, MA) Heavy mineral oil Single channel 1 mL pipettor Sterile 1 mL, clear, low fluid retention pipet tips Sterile gauze pads Glass capillary tube with a uniform inner diameter Plastic round-bottom clear centrifuge tubes, 3–5 mL Capillary aspirator system, including a mouthpiece, such as the one used on a nasal aspirator or a similar device to draw up the oil/fluid into the capillary tube. It may be necessary to use adapters to connect the tubing, filter and capillary tube Metric ruler Forceps (sterilized with 70% ethanol) Cell culture hood aspirating system with vacuum pump, needle and sterile tips Centrifuge with appropriate rotor for the round-bottom tubes

5.2 Plating cells on transwell supports 1. Prepare and label 6-well plates with the experimental information (3 wells per condition). 2. Transfer the TW-COL supports to the 6-well plates. 3. Prepare a working cell suspension of 5.9  105 cells/mL in DMEM/F12 + 5% FBS + P/S + ITS. For a 6-well plate, make up a 12 mL of the working cell suspension. 4. Add 1.7 mL of the cell suspension to the upper surface of each Transwell for 1  106 cells per Transwell. 5. Pipet 2.5 mL of the DMEM/F12 + 5% FBS + P/S + ITS below the Transwell. It is important to use the appropriate fluid volumes in the upper and lower chambers to prevent a hydrostatic pressure that may dislodge the cells from the support. To check that the appropriate fluid volumes are being used, add

85

86

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

FIG. 5 Proposed mechanism for transepithelial Cl secretion by ADPKD cells. The Na+/K+-ATPase, a primary active transporter that utilizes cell energy to pump 3Na+ out of the cell in exchange for 2K+, is responsible for establishing and maintaining the chemical gradients for Na+ and K+ across the membrane. NKCC1, an electrically neutral transporter, utilizes the Na+ gradient to bring Na+, K+, 2Cl into the cell. Na+ that enters the cell is transported back out via the Na+/K+-ATPase. K+ efflux via basolateral K+ channels, including KCa3.1, is primarily responsible for the negative membrane potential. These transport mechanisms raise the intracellular Cl concentration above its electrochemical gradient favoring Cl efflux. Binding of arginine vasopressin (AVP) to the V2 receptor (V2R), a G-protein couple receptor, increases cAMP synthesis by adenylyl cyclases (ACs). Basal cAMP and the magnitude of the cAMP response are largely determined by the activity of phosphodiesterases (PDEs), enzymes that degrade cAMP to AMP. In ADPKD, functional loss of the polycystins is thought to reduce intracellular Ca2+, increasing the activity of AC6, which is normally inhibited by Ca2+, and decreasing the activity of PDE3, a PDE isoform that is activated by Ca2 + . Elevated cAMP levels stimulate protein kinase A, leading to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel on the apical membrane. Chloride efflux is the initial step in cAMP-dependent Cl secretion that is maintained by basolateral Cl entry (Wallace et al., 1996). Active Cl secretion creates a transepithelial potential driving paracellular Na+ transport and the net addition of NaCl to the luminal fluid drives the osmotic movement of fluid.

fluid to the upper and lower chambers of a Transwell without cells and measure the volumes of fluid after equilibration. 6. Replenish the medium the next day and then every 2–3 days, allowing the cells to grow to confluency. To replenish media, transfer the Transwells to an empty

5 Fluid secretion assay

7.

8. 9. 10. 11.

6-well plate using sterile forceps. Remove each Transwell, blot the bottom surface on a sterile gauze pad to remove residual medium. Place the Transwell into the well of the empty plate that corresponds to the position in the original plate. This will be important when keeping track of the experimental conditions. After the Transwells are transferred, tip the plate slightly and gently remove the apical fluid by lowering the tip of the aspiration needle down the side of the Transwell until it is above the monolayer. Avoid contact with the cell monolayer. Once the apical media are removed, quickly remove all media from original “experimental” 6-well plate. Slowly add 1.7 mL of the medium to the upper part of each Transwells. Avoid pipetting the fluid directly onto the cell monolayer. Add 2.5 mL of the appropriate medium to each of the wells of the original “experimental” plate. Carefully transfer each Transwell back to the experimental plate, avoid trapping air bubbles under the Transwell.

5.3 Preparing the water-saturated mineral oil 1. Filter 100–120 mL of mineral oil with a 250 mL Sterile-Filter System. This is a slow process because of the viscosity of the oil. 2. Add 50 mL of sterile de-ionized water and mix vigorously. 3. Incubate the mineral oil/water at 37 °C overnight on a rocker, or mix several times throughout the day to hydrate the oil. 4. Let the mixture sit at room temperature until needed. 5. To use, mix well and then pour the oil into 50-mL centrifuge tubes. 6. Spin the oil at high speed for 5 min. 7. Recover the mineral oil (upper layer) and store in sterile bottles or specimen cups at 37 °C.

5.4 Calibrating the capillary tubes 1. Fluid volume is measured using standard diameter glass capillaries with a uniform internal diameter (I.D.). The volume per length (μL/mm) of the capillary tube can be determined from the I.D. using the volume of a cylinder (V ¼ πr2h). For example, a capillary tube with an I.D. of 1.0 mm (or 0.1 cm), the volume (V) for each 1 mm length (0.1 cm), will be V ¼ π (0.10 cm/2)2 0.1 cm ¼ 0.0007854 cm3 (mL). Therefore, the fluid volume will be 0.7854 μL per mm capillary length. Select a capillary tube I.D. that is appropriate for the working volume of the apical fluid. 2. To validate fluid volume and recovery from the monolayer, pipet 200 μL of medium on the monolayer, add 0.15 mL of water-saturated mineral oil, collect the fluid/oil and measure the fluid volume as described below.

87

88

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

5.5 Preparing the fluid secretion assay 1. Remove the basolateral media from the Transwells by tilting the culture plate and submerging the aspiration tip into lower medium where the crevice between the inner Transwell and the supporting ring. Gently tilt the Transwell using the aspiration tip so the fluid clinging to the bottom can be collected. 2. Remove all the medium from the upper surface of each Transwell, avoiding contact with the monolayer. 3. Carefully pipet 1.7 mL of water-saturated mineral oil to the apical part of the Transwell. Layer the oil over the monolayer slowly by pipetting to the side of the well. 4. Aspirate off the oil and any residual fluid from the monolayer. Repeated aspirations may be necessary for completely remove the oil/fluid. 5. Add 1.7 mL of mineral oil to the upper part of the Transwell. 6. Add 2.5 mL of experimental media to the wells of the plate, below the Transwells. Gently remove any bubbles below the Transwells by tilting the plate or by lifting the Transwell. 7. Return the plate to the incubator for the desired experimental period, typically 24 h. For assays involving the measurement of fluid absorption, add 0.2 mL of experimental medium and 1.5 mL of mineral oil on the apical side of the Transwell.

5.6 Collecting the secreted fluid 1. The collection of the fluid and oil from the apical chamber should be done one Transwell at a time (Fig. 6A). 2. Remove the Transwell with forceps, blot the bottom on sterile gauze and place it in an empty 6-well plate. 3. Collect the fluid and oil from above the monolayer using a 1 mL pipet. It may be best to pipet a small amount of oil before drawing up the secreted fluid, and then the remaining oil. 4. Transfer the oil and fluid to the plastic centrifuge tube. 5. Repeat until all the oil and fluid is removed from the monolayer. 6. Add additional oil to the monolayer and collect any residual fluid to ensure that all fluid has been collected. 7. Repeat steps 2–6 for the other Transwells. 8. Spin down the tubes (i.e., 2000 rpm) for 10 min forcing the fluid to collect in the bottom of the test tube below the oil. 9. Using a mouthpiece (with in-line filter), aspirate up a small amount of oil into the capillary tube, and then lower the capillary tube to the bottom of the tube to aspirate up all the fluid into a single droplet.

5 Fluid secretion assay

FIG. 6 Measurement of fluid transport across human renal cell monolayers. (A) Schematic drawing of an ADPKD cell monolayer grown on a Transwell permeable support in a fluid secretion assay. The monolayer is covered with sterile mineral oil to prevent evaporation of the apical fluid. Fluid secreted by the cells accumulates below the oil. Both the secreted fluid and oil are collected using a pipet with a low retention pipet tip. (B) Secreted fluid and a small amount of oil are drawn into a calibrated capillary tube for measurement of apical fluid volume. (C) In fluid transport experiments, a known volume of fluid can be added to the apical surface below the oil prior to the experiment to measure fluid absorption, as well as secretion. In this experiment, monolayers (N ¼ 3 per group) of human renal cells, enriched in collecting duct cells, absorbed fluid in the absence of a cAMP agonist. Net fluid transport reversed from absorption to secretion with the addition of the cAMP agonist forskolin.

89

90

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

10. After the fluid has been collected, aspirate a small amount of oil so that the column of fluid is within oil (Fig. 6B). 11. Carefully, remove the tubing from the end of the capillary tube without losing oil and/or fluid, and place it on a paper towel.

5.7 Measuring the fluid and presenting the data 1. Measure the column of fluid with the metric ruler positioned with the center of the meniscus (Fig. 6B). If the fluid is in multiple droplets, it will be necessary to measure the volume of each droplet and combine the volumes. 2. In experiments examining fluid absorption and secretion, subtract out the volume of fluid added to the monolayer (typically 200 μL) from the measurements. 3. Net fluid transport can be normalized to the surface area of the Transwell (4.52 cm2) and expressed per hour. In Fig. 6C, normal human kidney cells, enriched in collecting duct cells, absorbed fluid in control medium and secreted fluid in the presence of forskolin, a direct activator of adenylyl cyclase.

6 Conclusions Cellular assays provide a straightforward approach to study signaling pathways involved in renal cyst growth and to evaluate new therapeutic targets in carefully controlled experimental conditions. The use of renal cell lines, i.e., MDCK cells, and primary human ADPKD and normal human kidney cells were critical for establishing our current understanding of the mechanisms involved in ADPKD cell proliferation and Cl-dependent fluid secretion, key aspects of renal cyst enlargement. These in vitro assays also contributed to the rationale for the use of tolvaptan to reduce renal cAMP levels in ADPKD patients. Cell culture experiments in combination with PKD animal models will continue to play central roles in the discovery and development of new therapies for ADPKD patients.

Acknowledgments Methods for the investigation of cAMP-dependent cell proliferation and Cl-dependent fluid secretion using primary cultures of human ADPKD cells were first described by Drs. Jared Grantham and Lawrence Sullivan at the University of Kansas Medical Center. Preparation of this chapter was supported by grants from the National Institutes of Health (DK081579 to D.P.W.) and the PKD Biomarkers and Biomaterials Core of the Kansas PKD Research and Translational Core Center (DK106912 to D.P.W).

References

References Davidow, C. J., Maser, R. L., Rome, L. A., Calvet, J. P., & Grantham, J. J. (1996). The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro. Kidney International, 50, 208–218. Grantham, J. J. (1976). Fluid secretion in the nephron: Relation to renal failure. Physiological Reviews, 56, 248–258. Grantham, J. J. (1983). Polycystic kidney disease: A predominance of giant nephrons. The American Journal of Physiology, 244, F3–10. Hanaoka, K., & Guggino, W. B. (2000). cAMP regulates cell proliferation and cyst formation in autosomal polycystic kidney disease cells. Journal of the American Society of Nephrology, 11, 1179–1187. Jansson, K., Venugopal, J., Sanchez, G., Magenheimer, B. S., Reif, G. A., Wallace, D. P., et al. (2015). Ouabain regulates CFTR-mediated anion secretion and Na,K-ATPase transport in ADPKD cells. The Journal of Membrane Biology, 248, 1145–1157. Larsen, E. H. (2002). Hans H. Ussing—Scientific work: Contemporary significance and perspectives. Biochimica et Biophysica Acta, 1566, 2–15. Li, H., Sheppard, D. N., & Hug, M. J. (2004). Transepithelial electrical measurements with the Ussing chamber. Journal of Cystic Fibrosis, 3(Suppl. 2), 123–126. Mangoo-Karim, R., & Grantham, J. J. (1990). Transepithelial water permeability in an in vitro model of renal cysts. Journal of the American Society of Nephrology, 1, 278–285. Mangoo-Karim, R., Uchic, M. E., Grant, M., Shumate, W. A., Calvet, J. P., Park, C. H., et al. (1989). Renal epithelial fluid secretion and cyst growth: The role of cyclic AMP. The FASEB Journal, 3, 2629–2632. Mangoo-Karim, R., Uchic, M., Lechene, C., & Grantham, J. J. (1989). Renal epithelial cyst formation and enlargement in vitro: Dependence on cAMP. Proceedings of the National Academy of Sciences of the United States of America, 86, 6007–6011. Mangoo-Karim, R., Ye, M., Wallace, D. P., Grantham, J. J., & Sullivan, L. P. (1995). Anion secretion drives fluid secretion by monolayers of cultured human polycystic cells. The American Journal of Physiology, 269, F381–F388. Rankin, C. A., Ziemer, D. M., Maser, R. L., Foo, I., & Calvet, J. P. (1996). Growth characteristics of cells cultured from two murine models of polycystic kidney disease. In Vitro Cellular & Developmental Biology. Animal, 32, 100–106. Reif, G. A., Yamaguchi, T., Nivens, E., Fujiki, H., Pinto, C. S., & Wallace, D. P. (2011). Tolvaptan inhibits ERK-dependent cell proliferation, Cl() secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin. American Journal of Physiology. Renal Physiology, 301, F1005–F1013. Sullivan, L. P., Wallace, D. P., & Grantham, J. J. (1998). Epithelial transport in polycystic kidney disease. Physiological Reviews, 78, 1165–1191. Wallace, D. P., Grantham, J. J., & Sullivan, L. P. (1996). Chloride and fluid secretion by cultured human polycystic kidney cells. Kidney International, 50, 1327–1336. Yamaguchi, T., Hempson, S. J., Reif, G. A., Hedge, A. M., & Wallace, D. P. (2006). Calcium restores a normal proliferation phenotype in human polycystic kidney disease epithelial cells. Journal of the American Society of Nephrology, 17, 178–187.

91

92

CHAPTER 4 ADPKD cell proliferation and Cl-dependent fluid secretion

Yamaguchi, T., Nagao, S., Wallace, D. P., Belibi, F. A., Cowley, B. D., Jr., Pelling, J. C., et al. (2003). Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomaldominant polycystic kidneys. Kidney International, 63, 1983–1994. Yamaguchi, T., Pelling, J. C., Ramaswamy, N. T., Eppler, J. W., Wallace, D. P., Nagao, S., et al. (2000). cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway. Kidney International, 57, 1460–1471. Yamaguchi, T., Reif, G. A., Calvet, J. P., & Wallace, D. P. (2010). Sorafenib inhibits cAMPdependent ERK activation, cell proliferation, and in vitro cyst growth of human ADPKD cyst epithelial cells. American Journal of Physiology. Renal Physiology, 299, F944–F951. Yamaguchi, T., Wallace, D. P., Magenheimer, B. S., Hempson, S. J., Grantham, J. J., & Calvet, J. P. (2004). Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype. The Journal of Biological Chemistry, 279, 40419–40430.