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Department of Physiology and Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Correspondence to Dr. William B. Guggino, Department of Physiology, School of Medicine, The Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205. Phone : 410-955-7166 ; Fax : 410-955-0461 ; E-mail : wguggino{at}bs.jhmi.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Genes associated with the disease (PKD1 and PKD2) have been cloned (9,10,11), and proteins encoded by these genes (polycystins) have been investigated. Polycystins are widely expressed in fetal tissues (12,13). Recently, mouse models of PKD1 and PKD2 were developed and showed an embryonic lethal phenotype and massive cyst formation in the kidney (14,15). These results indicate that PKD1 and PKD2 probably play an important role during normal renal tubular development. However, their precise role in development and the cellular mechanisms of cyst formation induced by mutations in PKD1 and PKD2 is not fully understood.
Two key features associated with cyst formation in ADPKD are cell proliferation and fluid secretion, both of which are stimulated by cAMP. The effect of cAMP on cell proliferation varies among different cell types (16). For example, in smooth muscle cells, fibroblasts, and mesangial cells, elevation of intracellular cAMP blocks growth factor-stimulated cell growth by inhibiting the mitogen-activated protein (MAP) kinase cascade (16,17,18). On the other hand, in cell types such as thyroid cells, hepatocytes, and PC12 cells, cAMP activates cell proliferation (16,19). Although it is known that cAMP stimulates cyst enlargement, the exact mechanism of how cAMP stimulates cell proliferation in ADPKD is still unclear.
Maneuvers that raise intracellular cAMP such as addition of forskolin, which activates adenylate cyclase, addition of the membrane-permeable cAMP analogue 8-bromo-cAMP, and addition of prostaglandin E2 stimulate fluid secretion both in monolayers of cultured epithelial cells established from ADPKD patients and in isolated ADPKD cysts (20,21,22). Increasing intracellular cAMP also enhances cyst formation and enlargement in an in vitro culture system (23,24). These studies have led to the hypothesis that fluid secretion into ADPKD cysts may occur via a secretory mechanism similar to that found in airway epithelia (25).
Because cell proliferation and fluid secretion are key determinants of cyst formation in ADPKD, we designed this study to investigate the role of cell proliferation in cyst formation in ADPKD and to further explore the role of fluid secretion in cyst growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially, HRCE cells were cultured in the culture media, REGM, with the following supplements : fetal bovine serum (FBS) (0.5%), human transferrin (10 mg/ml), hydrocortisone (0.5 mg/ml), insulin (5 mg/ml), triiodothyronine (5 x 10-12 M), epinephrine (0.5 mg/ml), epidermal growth factor (EGF) (10 mg/ml), and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, and 50 mg/ml gentamicin). Initially, HRCE cells were cultured either in Click-RPMI 1640 media or REGM media to check cell growth and cyst formation. No differences were noted in growth conditions using the two different media. Therefore, all experiments were conducted with Click-RPMI 1640 media.
Primary cultures were established from ADPKD cysts. Techniques used here have been described in detail elsewhere (27). In brief, 12 ADPKD cysts were isolated from four human ADPKD, end-stage kidneys. Each cyst was stripped clean of fibrous tissue, cut open, and chopped into 1-mm2 pieces. Cyst-lining segments were plated onto type I collagen-coated plastic culture dishes and grown at 37°C in the culture media (Collaborative Research, Bedford, MA). Culture medium for ADPKD cells consisted of the following components : Click-RPMI 1640 media (Quality Biological, Rockville, MD) supplemented with FBS (1% ; Mediatech, Herndon, VA), human transferrin (5 mg/ml), dexamethasone (5 x 10-8 M), insulin (5 mg/ml), triiodothyronine (5 x 10-12 M), and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). All supplements described here except FBS were from Life Technologies-BRL (Gaithersburg, MD). When ADPKD cells and HRCE cells reached confluence, the cells were subcultured for the following experiments and incubated with the Click-RPMI 1640 media.
Thymidine Incorporation
DNA synthesis was measured as the amount of [3H]-thymidine
incorporated into TCA-precipitable material as described in detail previously
(28). Cells were subcultured
into 48-well culture plates at a density of 2 x 104/well and
incubated with the culture media described above, Click-RPMI 1640 media with
the supplements. When the cells reached 80% confluence, they were incubated
with the hormone-free Click-RPMI 1640 media, which contained 0.2% FBS (basal
medium) for 48 h, followed by exposure to the relevant agonist for 20 h.
[3H]-Thymidine (1 µCi/ml) (NEN Life Science Products, Boston,
MA) was added 10 h before harvest. At the end of a 20-h exposure period,
medium was removed by aspiration, washed twice with Ca2+-,
Mg2+-free phosphate-buffered saline (Life Technologies-BRL), and
exposed to 10% TCA. After the cells were incubated with TCA at 4°C for 1
h, TCA-precipitated material was washed twice with 5% TCA and solubilized with
a mixture of 1N NaOH and 1% sodium dodecyl sulfate. Radioactivity was
determined using liquid scintillation counting.
Cell Proliferation
To assess cell replication, ADPKD cells and HRCE cells were subcultured
into 48-well culture plates with a concentration of 2 x
104/well and incubated with Click-RPMI 1640 media with the
previously mentioned supplements. When the cells reached 60% confluence, the
culture medium was replaced by basal medium, which is necessary to maintain
cell viability for the experimental period. After 48 h, ADPKD cells were
stimulated with the relevant agonists. ADPKD cells were counted at days 0, 4,
and 8. Cells were washed with phosphate-buffered saline and then incubated in
0.05% trypsin (Life Technologies-BRL) with 0.53 mM ethylenediaminetetra-acetic
acid for 15 min at 37°C. Detached cells were then suspended in the culture
media containing 0.2% trypan blue (Life Technologies-BRL), and the number of
viable cells was determined in a counting chamber.
Cyst Formation in Vitro
Before subculturing cells into a collagen gel, the gel mix was prepared and
kept on ice. Gel mix was made from 0.7 vol of type I collagen, 0.2 vol of
Click-RPMI 1640 media, and 0.1 vol of Hepes buffer solution. Cells were added
into the gel mix with a concentration of 1 x 104/well and
incubated at 37°C. When the gel mix became solid, culture medium was added
and cells were cultured at 37°C with 5% CO2. To promote cyst
formation, normal culture medium with 1% FBS and hormones was used for 24 h.
After cells were washed with Click-RPMI 1640 media three times, culture medium
was changed to basal medium. After 24 h, relevant agents and/or blockers were
applied into the culture medium and cyst size was determined with an inverted
light microscope.
Materials
All of the cAMP analogues and antagonists, and 8-bromo-cGMP were purchased
from Calbiochem (San Diego, CA).
Statistical Analyses
Data were presented as means ± SEM and analyzed by one-way ANOVA
with the unpaired t test. A value of P < 0.05 was
considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracellular cGMP activates protein kinase G, another serine/threonine kinase. To check whether cGMP affects cell proliferation, 50 µM 8-bromo-cGMP, a membrane-permeable cGMP analogue, was tested (Table 1). The 8-bromo-cGMP did not change thymidine uptake, suggesting that an increase of cGMP does not play a role in stimulating thymidine uptake in ADPKD cells.
Although cAMP activated DNA synthesis in ADPKD cells, the response of HRCE cells to cAMP was quite different. As with ADPKD cells, addition of either 1% FBS or EGF stimulated thymidine uptake in HRCE cells by nearly twofold (Table 1). On the other hand Sp-cAMP did not change thymidine uptake in HRCE cells. We tested six different concentrations of Sp-cAMP (0.05 to 100 µM). In all cases, thymidine uptake was similar to that obtained from basal medium (Figure 1C). To exclude the possibility that Sp-cAMP could not effectively increase intracellular cAMP in HRCE cells, we also tested CPT-cAMP and forskolin. However, none of those agents changed thymidine uptake. Inhibiting protein kinase A by Rp-cAMP also did not change thymidine uptake (Table 1). Furthermore, the effect of EGF on thymidine uptake was not modified by adding either Sp-cAMP or Rp-cAMP (Figure 1D and Table 1). Thus, the HRCE cells respond quite differently to growth stimulation.
cAMP Activates Cell Proliferation
To confirm that the enhanced [3H]-thymidine incorporation was
associated with cell proliferation, we counted the number of cells at days 0,
4, and 8 after stimulation by cAMP and/or EGF
(Figure 2 and
Table 2). Hormone-free
Click-RPMI 1640 basal media, which contained 0.2% FBS, did increase the cell
number of both ADPKD cells and HRCE cells. The effect of 0.2% FBS on the
increase of cell number was much smaller than 1% FCS or 5 ng/ml EGF
(Table 2).
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ADPKD cells proliferated when exposed to 5 ng/ml EGF. When exposed to 50 µM Sp-cAMP, cell proliferation in ADPKD cells was stimulated to an extent comparable to 5 ng/ml EGF (Figure 2). The effects of EGF and Sp-cAMP were additive when applied together onto ADPKD cells. As shown in Table 2, 50 µM of the membrane-permeable cAMP antagonist Rp-cAMP alone or in the presence of EGF neither enhanced nor inhibited cell proliferation in ADPKD cells.
HRCE cells proliferated in the presence of either 5 ng/ml EGF or 1% FCS. In sharp contrast, 50 µM Sp-cAMP did not change the rate of cell proliferation in HRCE cells. Similar to ADPKD cells, 50 µM Rp-cAMP by itself did not show any effect on cell proliferation in HRCE cells (Table 2). These results demonstrate that cAMP activates cell proliferation in ADPKD cells but not in HRCE cells.
Cyst Formation and Effect of cAMP
We tested whether ADPKD cells and HCRE cells form in vitro cysts.
In initial studies, cells (1 x 104/well) were cultured in
type I collagen gel matrix (for details, see Materials and Methods) with basal
medium ; however, neither ADPKD cells nor HRCE cells formed a cyst (n
= 12). When cells were incubated in Click-RPMI 1640 medium with a supplement
of 1% FBS, HRCE cells formed a few microcysts in 24 h (10.4 ± 5.4/well
at day 2 ; n = 8 wells). However, cyst formation could not be
observed from ADPKD cells under this condition
(Figure 3A). Addition of
hormones, transferrin, dexamethasone, insulin, and triiodothyronine induced
formation of cysts in ADPKD cells as well as in HRCE cells
(Figure 3B for ADPKD cyst). At
day 2, although average cyst volume was almost identical (0.0043 ±
0.0005 nl for ADPKD versus 0.0049 ± 0.0007 nl for HRCE ;
P > 0.05 ; n = 8 wells), the number of cysts was
significantly different (125.4 ± 28.6/well for ADPKD versus
25.4 ± 8.3/well for HRCE ; P < 0.05 ; n = 8
wells). Although both ADPKD and HRCE cells do form cysts, the number of cysts
that are formed from HRCE cells is less. Because both 1% FBS and hormones were
essential for cyst formation in ADPKD cells, in subsequent studies cells were
cultured in the presence of 1% FBS and hormones for an initial 24-h period,
followed by basal medium for 24 h.
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After 2 d, the lumen diameter of ADPKD cyst was approximately 20 µm (Figure 3B). ADPKD cysts did not change their volume further when cultured only in basal medium for a period of 6 d (Figure 4A). In contrast, cyst volume increased when treated with 50 µM Sp-cAMP. Figure 3C shows a representative ADPKD cyst cultured with Sp-cAMP for 14 d. At 14 d the lumen diameter had increased to approximataely 200 µm, indicating that when Sp-cAMP is applied, ADPKD cysts grow both by proliferation of cells lining the cyst wall and by fluid secretion into the cyst lumen. CPT-cAMP and forskolin also had a similar effect to increase cyst volume. Treatment of cysts with Rp-cAMP alone did not affect cyst volume. When H-89 was added into the culture medium, the effect of Sp-cAMP was completely inhibited. Additional acceleration of cyst enlargement was observed when both EGF and Sp-cAMP were applied (Figure 5). When FBS was removed from the culture medium, ADPKD cysts reduced in size by about 13% (P < 0.05 ; n = 4 wells) in 1 d. Beyond 1 d, cyst cells began to die in the absence of FBS. The demonstrates that cyst fluid is absorbed in the absence of stimulants in ADPKD cysts and that stimulation is needed to maintain cyst integrity.
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After microcysts were formed from the control HRCE cells, cysts were cultured in basal medium or in the presence of 50 µM Sp-cAMP (Figure 4B). Unlike cysts formed from ADPKD cells, HRCE cysts continued to enlarge when treated with only basal medium lacking cAMP, but at a slower rate than ADPKD cysts activated by cAMP. The rate of HRCE cyst enlargement was not changed when 50 µM Sp-cAMP (Figure 4B) was added. HRCE cyst enlargement could be enhanced by higher concentrations of FBS or EGF (data not shown). Complete removal of FBS from culture medium reduced HRCE cysts in size by nearly 15% (P < 0.05 ; n = 4 wells) in 1 d.
We also tested purified proximal tubular epithelial cells (RPTEC) isolated
from normal human kidney, for their ability to form cysts. RPTEC cells were
purchased from Clonetics. Ninety-five percent of the RPTEC cells react to an
antibody against 
-GTP, one of the markers of proximal tubule cells.
When RPTEC cells were cultured in type I collagen gel matrix, the cells did
not form any cysts in the presence of 1% FBS and hormonal supplements (0 cysts
were observed in 12 dishes). Unlike HRCE cells, higher concentrations of FBS
did not enhance cyst formation. This suggests that cyst formation is not a
universal property of all renal cells in culture.
Effects of Cl- Channel Blockers
cAMP-activated Cl- transport plays an important role in fluid
transport in ADPKD cysts (27,
30,
31). To study whether channel
blockers could inhibit cyst enlargement, cysts were cultured with 50 µM
Sp-cAMP beginning at day 2, and different Cl- channel blockers were
added into the culture medium at day 4. Increase of cyst volume was then
measured at day 8 (Figure 6).
DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic aicd) is a drug
that blocks several types of Cl- channels and cotransporters but
not cystic fibrosis transmembrane conductance regulator (CFTR)
(32,33,34).
A total of 200 µM DIDS had no effect on the volume of either ADPKD or HRCE
cysts. Higher concentrations of DIDS (500 µM) were also examined, but cyst
volume could not be measured because cells died during the experimental
period. Diphenylamine carboxylic acid (DPC), an arylaminobenzoate, is also a
potent Cl- channel blocker and is known to block CFTR
(32,
33). A total of 200 µM DPC
significantly inhibited cyst enlargement both in ADPKD and HRCE cysts.
Glibenclamide, a sulfonylurea compound used for the treatment of diabetes
mellitus, is known to block both ATP-sensitive K+ channels
(35) and CFTR Cl-
channels (32,
36). Cyst growth was
significantly inhibited in ADPKD cysts but not in HRCE cysts when 100 µM
glibenclamide was applied. The inhibitory effect of glibenclamide on cyst
enlargement in ADPKD cysts was reversible. When glibenclamide was taken out of
the culture medium, ADPKD cysts again grew in the presence of 50 µM Sp-cAMP
(data not shown). These results indicated that cAMP-activated cyst growth was
decreased by ion channel blockers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our work, we measured both thymidine incorporation and cell proliferation directly. We show that only EGF activates proliferation in a mixed population of cells isolated from human renal cortex (Figures 1 and 2). Similar to the results of Heasley et al. in RIMCT (37), the proliferation of a mixed population of human renal cortical cells is unaffected by cAMP. In contrast, however, we show that both EGF and cAMP activate cell proliferation in ADPKD cells grown in tissue culture and that the effect of both agonists is additive. We also show that EGF-stimulated cell proliferation is not inhibited by cAMP antagonists, whereas cAMP-activated proliferation is inhibited by these agents (Table 1 and 2). Thus, our results indicate that stimulation of proliferation by EGF and cAMP in ADPKD cells occurs via separate signal transduction cascades. Clearly, ADPKD cells have changed their ability to respond to signals that control cell proliferation when compared to a population of cells isolated from the cortex of a normal human kidney.
Cyst Formation
When a mixed population of normal human cells isolated from the renal
cortex (HRCE) was cultured in collagen gels, cells formed cysts and secreted
fluid into the cyst lumen in the presence of FBS and hormonal supplements (see
Materials and Methods) (23,
40). Although HRCE cells did
form cysts under these conditions, the number of cysts formed was less than
that noted for cells isolated from patients with ADPKD. In our experiments,
cyst growth in HRCE cells was not dependent on cAMP. These results were
consistent with the lack of effect of cAMP on thymidine incorporation and cell
proliferation in these HRCE cells.
Although cells isolated from normal kidneys will form cysts albeit with properties different from those isolated from ADPKD patients, cyst formation is not a universal property of renal cells. For example, we showed that purified proximal tubular cells from normal human kidney, RPTEC cells, do not form cysts. Mangoo-Karim et al. reported that LLC-PK1 cells, derived from porcine proximal tubules, also do not develop cysts (23). Finally, Neufeld et al. conducted a detailed immunohistologic investigation of a mixed population of human cells isolated from the cortex and showed that most cysts grown in collagen gels were of distal tubular origin (40). Given that under our experimental conditions, HRCE cells can form cysts but a purer population of proximal tubule cells, RPTEC cells, cannot, suggests that most of the cysts grown from mixed populations of normal renal tubular cells in vitro are probably of distal tubular origin. Why can some cells isolated from normal kidneys form cysts and others cannot ? One explanation is that the levels of expression of polycystin 1 or 2 may be altered in some cells in culture allowing them to form cysts.
In contrast to normal cells, cysts established from cells from ADPKD patients grow readily in three-dimensional culture and expand vigorously in the presence of cAMP (Figures 3 and 4). After cysts are formed, they require continued stimulation with hormonal supplements to maintain cyst growth (23, 34), and without supplements fluid is actually absorbed (this study and reference (24). This suggests that the hormonal environment within the in vivo kidney must be conductive to the formation and growth of cysts. In cells isolated from patients, cAMP stimulates cell proliferation, but in vivo the precise mechanism of that stimulation is not understood. Yamaguchi et al. measured cAMP concentrations in cystic kidney tissue, urine, and cyst fluid in the murine polycystic kidney disease model, the pcy mouse (41). They showed that cAMP production and secretion into urine are increased compared with control animals, indicating that cAMP does play a role in cystic kidney in vivo. In normal human kidney, many hormones increase intracellular cAMP concentrations in different nephron segments. Given that cultured ADPKD cells enlarge in response to cAMP, it is feasible that hormones that operate by enhancing cAMP production, such as AVP, parathyroid hormone, and calcitonin, may play a role in enhancing cyst production in ADPKD in vivo (42). On the other hand, it is also possible that the enhanced cAMP production noted in cystic kidneys arises through an autocrine process, whereby each cyst stimulates its own growth.
Fluid Secretion
Fluid secretion also plays a key role in cyst formation in ADPKD. A key
question is what mechanisms are involved in cyst formation ? Cysts form from
various nephron segments and are comprised of a heterogeneous population of
cells that vary within and among individual cysts. Some cysts, especially
those early in their development, may display some structural characteristics
of proximal or distal tubule cells. These cysts may still transport solutes,
using mechanisms similar to those found in the intact nephron. On the other
hand, the majority of the cells within cysts do not have characteristics
associated with their origin
(6,
43,44,45).
These cysts are comprised of a heterogeneous population of cells. CFTR protein
and channel activity can be detected in the apical cell membrane of renal
cysts (27,
30,
31). Furthermore, there is
some evidence that in patients with both cystic fibrosis and ADPKD, the
severity of ADPKD is lessened. This suggests that fluid secretion into cysts
may occur via a mechanism similar to that found for other CFTR-containing
tissues (46). Although CFTR
clearly plays a role, it is only expressed in about half of the cells in some
cysts, and other cysts within a kidney do not express CFTR. This suggests that
other ion channels and transporters are also involved
(27,
31). Clearly, the mechanism
and the rate of cyst enlargement may vary greatly among individual cysts,
depending on the origin of the cells and the variety of transporters expressed
in an individual cyst.
Grantham and colleagues have studied the effect of blockers of ion channels and transporters in ADPKD cells (20, 24, 34). In our study, we cultured ADPKD and HRCE cysts in the presence of channel blockers in the culture medium for 4 d and evaluated the effect of these agents. Both glibenclamide and DPC inhibited cAMP-activated enlargement of cysts grown from ADPKD cells, whereas only DPC inhibited growth in cysts established from HRCE cells. DIDS did not block cyst enlargement either in HRCE or in ADPKD cysts (Figure 6).
Glibenclamide is a known blocker of ATP-sensitive K+ channels (35) and can also block CFTR (32, 36). Although the presence of CFTR has been established, the existence of ATP-sensitive K+ channels in ADPKD cells has not been determined. Given the importance of ROMK channels in renal K+ transport, it is clearly possible that ATP-sensitive K channels, such as ROMK channels, exist and function during fluid secretion in ADPKD cells. Thus, it might be possible that glibenclamide is blocking both ROMK and CFTR and ultimately inhibiting fluid secretion in ADPKD cysts. The compound DPC is a general blocker of several types of Cl- channels. We show that cyst enlargement is also inhibited by DPC. Because several transport pathways are involved in the formation of cysts and because of the propensity of blockers such as DPC and glibenclamide to block more than one channel, our experiments do not pinpoint the exact characteristics of ion channels present in the cysts. However, the demonstration that blocking transport pathways in cysts inhibits enlargement raises the possibility that this strategy may be a useful treatment for ADPKD patients suffering from renal dysfunction.
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