 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, and Atlanta Veterans Affairs Medical Center, Decatur, Georgia.
Correspondence to Dr. Harold A. Franch, Renal Division, Emory University School of Medicine, W.M.B., Room 338, 1639 Pierce Drive, N.E., Atlanta, GA 30322. Phone: 404-727-9217; Fax: 404-727-3425; E-mail: hfranch{at}emory.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two mechanisms have been proposed to explain the cell growth that occurs in renal tubular hypertrophy. In the first mechanism, growth factors induce entry into the cell cycle, increasing the synthesis and limiting the degradation of proteins (6). Hypertrophy occurs when a second signal, such as transforming growth factor-ß (TGF-ß), blocks progression of the cell cycle before entry into S phase, so that cells do not divide but continue to synthesize protein and suppress protein degradation; this response requires the activity of the retinoblastoma family of proteins (pRB) (6,7,8,9,10).
The second mechanism yielding renal hypertrophy is linked to increased ammonia production, as occurs with metabolic acidosis or hypokalemia (11). Ammonia inhibits lysosomal proteolysis, leading to protein accumulation without cell cycle entry (11,12). It is not clear whether growth factors and ammonia interact to augment hypertrophic responses, but activation of growth factors (either increased insulin-like growth factor-1 or immediate-early gene responses) occurs in the renal cortex in two separate in vivo models of hypertrophy associated with elevated ammonia levels, hypokalemia and chronic acidosis (9,10).
We found that ammonia or the related weak base methylamine suppressed proliferation of the NRK-52E renal epithelial cell line (12,13). A similar antiproliferative effect of ammonia was observed in primary cultures of rabbit proximal tubule cells (14). Because we also noted that methylamine does not block protein synthesis induced by growth factors (13), ammonia might suppress growth factor-induced proliferation without blocking protein accumulation. If this was the case, the hyperplasia induced by epidermal growth factor (EGF) would be converted to hypertrophy, just as TGF-ß1 changes EGF-induced hyperplasia to hypertrophy (6). Consequently, I tested the ability of NH4Cl to convert EGF-induced hyperplasia to a hypertrophic response, and I examined the effect of inactivation of the retinoblastoma family of proteins on this response in NRK-52E cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture
NRK-52E cells (a rat kidney epithelial cell line)
(15) were obtained from the
American Type Culture Collection (Bethesda, MD) at passage 15, subcultured,
and grown in high-glucose DMEM supplemented with 25 mM Hepes, 25 mM glutamine,
and 5% calf serum. Studies were performed on cells from passages 19 to 29.
Cells in six-well plates were grown to confluence and rendered quiescent by
serum removal 48 h before experimental treatments. The cell culture medium was
refreshed every 24 h during quiescence and experimental treatments, to prevent
growth factor-induced changes in cell pH. Medium pH values did not differ
between control and treatment groups at any time point.
To study the role of the retinoblastoma family of proteins, NRK-52E cells stably expressing the human papilloma virus 16 (HPV16) E7 protein, which had been produced with the assistance of Dr. Jerry Shay and Dr. Patricia Priesig (University of Texas Southwestern, Dallas, TX), were used. These cells had been produced using a retroviral construct of the HPV16 E7 gene and a neomycin resistance gene, both inserted into the vector pLXSN (6,16,17). Control cells were infected with the pLXSN vector containing only the neomycin resistance gene (6).
Recombinant human TGF-ß1 was reconstituted in 4 mM HCl containing 0.1% heat-treated bovine serum albumin. Recombinant human EGF was reconstituted in phosphate-buffered saline (PBS) containing 0.1% heat-treated bovine serum albumin. In all studies, TGF-ß1 was used at 10-10 M and EGF was used at 10-8 M (6); the appropriate vehicle was used for control cells.
Measurement of Hypertrophy
After exposure to an experimental variable, cells were washed with PBS,
incubated with 0.05% trypsin/0.5 mM EDTA for 5 min (NRK-52E cells),
centrifuged at 1500 x g for 5 min, and washed with PBS. The
final pellet was resuspended in 1 ml of lysis buffer (50 mM
Na2PO4, pH 7.4) and then lysed on ice by repeated
passage through a 27-guage needle. The lysate was divided and stored at
-70°C. Protein concentrations were determined by the method of Lowry
et al. (18), using
bovine serum albumin as a standard. DNA was measured using the fluorescent
compound H33258, which was detected using a Shimadzu (Kyoto, Japan) NK-1540
fluorophotometer, as described
(6,19).
Cell hypertrophy was expressed as an increase in the cell protein/DNA
ratio.
Protein Turnover
Protein synthesis was measured as the incorporation of
L-[14C]phenylalanine, after correction for intracellular specific
activity
(20,21).
The medium of quiescent cells was changed to DMEM with 1.1 mM unlabeled
L-phenylalanine plus growth factors for 20 h. For the final 4 h, 0.5 µCi of
L-[14C]phenylalanine was added to each well
(20). After 4 h, the cells
were rapidly washed twice with ice-cold PBS, and the protein was precipitated
by the addition of 10% TCA (1 ml/well). The plates were incubated on ice for 1
h, after which the cells were scraped, transferred to 1.5-ml tubes, and
centrifuged. Each pellet was washed twice with 1 ml of 10% TCA, vortex-mixed,
and centrifuged for 5 min, followed by rinsing with 1 ml of ethanol/ether.
After removal of the supernatant, the precipitate was solubilized overnight in
1 ml of 0.3 M NaOH, with vigorous shaking, and radioactivity was measured.
Protein synthesis was expressed as counts per milligram of protein after
correction for the specific radioactivity of phenylalanine in the medium,
because the medium equilibrates rapidly with the intracellular fluid
(20).
Protein degradation was measured as the release of L-[14C]phenylalanine from cells that had been prelabeled as described (20,21). Unlabeled phenylalanine (5 mM) was added to the medium to minimize the reuse of released phenylalanine. An initial 4-h washout period was used to eliminate short-lived proteins and unincorporated L-[14C]phenylalanine (20). Four aliquots of medium were removed at various intervals and counted, after TCA precipitation to remove protein. At the end of the experiment, cell protein was solubilized in 1 ml/well 1% sodium dodecyl sulfate, and radioactivity remaining in the cells was measured. The protein degradation rate was determined as the slope of the logarithm of [14C]phenylalanine remaining in cell protein versus time (20). Total radioactivity recovered (a marker of cell loss from the monolayer) was calculated from the amount of radioactivity appearing in the medium and the amount remaining in the cell monolayer. Total radioactivity recovered did not change with any experimental treatment.
Cell Counts
Cells were washed twice in PBS and removed from the plates using 0.05%
trypsin/0.5 mM EDTA for 5 min; 0.2% trypan blue was added before the cell
numbers were counted using a hemocytometer. The percentages of cells taking up
trypan blue did not significantly vary among treatments.
Bromodeoxyuridine (BrdU) Enzyme-Linked Immunosorbent Assay
To measure DNA synthesis, a BrdU assay (Boehringer Mannheim, Indianapolis,
IN) was used. Growth factors were added to cells in 96-well microtiter plates
for 17 h. BrdU was then added for 3 h, because the 17- to 20-h period includes
peak EGF-induced DNA synthesis in NRK-52E cells
(22). The cells were fixed
with ethanol for 30 min, and an anti-BrdU antibody was added for 90 min. After
washing, the anti-BrdU antibody was detected using a microtiter reader.
Results are presented after subtraction of the values for blank wells (without
cells added); blank wells and negative control samples yielded readings of
<5% of values measured in quiescent cells.
Statistical Analyses
Results are expressed as mean ± SEM. Because there was variation in
the magnitudes of responses among experiments, results are presented as
percentages of the control value measured on the same day. Differences between
two groups were analyzed using the t test and multiple comparisons
were analyzed using ANOVA, with the Student-Newman-Keuls test for multiple
comparisons. Comparisons of slopes of lines representing the release of
L-[14C]phenylalanine were performed using analysis of
covariance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
To examine the mechanism responsible for the lower level of DNA with NH4Cl, I measured DNA synthesis as BrdU uptake. Figure 3 demonstrates that NH4Cl suppressed both basal and EGF-stimulated DNA synthesis. Counting of cells that excluded trypan blue demonstrated no increase in cell death when NH4Cl was added (in either the presence or absence of EGF). These results support the hypothesis that NH4Cl decreases the DNA content in cells treated with EGF plus NH4Cl by inhibiting proliferation, compared with cells treated with EGF alone.
|
To determine how NH4Cl and/or EGF stimulates protein accumulation, I measured changes in protein synthesis and degradation. EGF caused a predictable increase in protein synthesis, but NH4Cl did not significantly alter protein synthesis (Figure 4). Although the rate of protein synthesis with EGF plus NH4Cl was slightly, but not significantly, less than with EGF alone, the percentage increase in EGF-induced protein synthesis was equivalent in cells with and without added NH4Cl. EGF decreased the rate of protein degradation by approximately 30%, and NH4Cl suppressed proteolysis to a greater extent (Figure 5). In contrast, the combination of EGF plus NH4Cl did not decrease protein degradation more than did NH4Cl alone. Therefore, EGF stimulates proliferation and increases cellular protein contents by increasing the rate of protein synthesis and suppressing protein degradation. With ammonia, however, proteolysis was lower than with EGF alone and EGF-induced protein synthesis was not significantly impaired.
|
|
To examine the effect of lysosome alkalinization on EGF-induced growth, I used another weak base, methylamine, or the specific vacuolar proton pump inhibitor bafilomycin A1. These agents also reversed EGF-induced proliferation and increased the protein/DNA ratio (Figure 6). Because both agents may affect acidic intracellular compartments in addition to lysosomes (23,24), I also examined the effect of directly inhibiting lysosomal enzymes with the combination of leupeptin plus E64 [a combination that inhibits approximately two thirds of the lysosomal proteolysis in NRK-52E cells (13)]. Leupeptin plus E64 converted hyperplasia to hypertrophy more effectively than did ammonia (Figure 6).
|
Because TGF-ß1 converts EGF-induced hyperplasia to hypertrophy by activating the retinoblastoma family of proteins (6,9,10), NH4Cl might act to suppress proliferation through this family of proteins. To test this hypothesis, I studied NRK-52E cells expressing the HPV16 E7 protein, which blocks the activity of the retinoblastoma family of proteins by binding to their active cleft (6,16,17). As previously reported (6,12), expression of the E7 protein did not block the increase in the protein/DNA ratio associated with NH4Cl treatment alone (Figure 7A) but did block that associated with the combination of EGF plus TGF-ß1 (Figure 7B). Expression of the E7 protein inhibited the increase in the protein/DNA ratio that occurred when cells were incubated with EGF plus NH4Cl (Figure 7B). Expression of HPV16 E7 did not block protein accumulation (Figure 7C) but, rather, led to an increase in DNA content (Figure 7D). Thus, inactivation of the retinoblastoma family of proteins by expression of the HPV16 E7 protein partially reverses the effect of NH4Cl to inhibit proliferation. This response leads to more hyperplasia and less hypertrophy.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
I confirmed that NH4Cl exerts an antiproliferative effect when it converts hyperplasia to hypertrophy (Figures 1, 5, and 6), as reported for NRK-52E cells and other cultured tubular cells (11,12,14,32,34,35). Of those reports, only one (35) included evidence (increased thymidine incorporation) that NH4Cl stimulated DNA synthesis. My results using the BrdU uptake method confirm the published findings and are consistent with the observation that NH4Cl does not change the levels of cell cycle mediators such as c-Fos mRNA and cyclin E protein (12). Although others have noted the antiproliferative effect of NH4Cl, it has never been demonstrated that the result of inhibiting hyperplasia is hypertrophy.
Evidence that the conversion of hyperplasia to hypertrophy by NH4Cl is not an artifact caused by cell death includes the following observations. First, I observed no change in the number of cells that were permeable to trypan blue. Second, total radioactivity recovered in the protein degradation experiments did not change, indicating no loss of cells from the monolayer. Third, Rabkin et al. (14) demonstrated that the inhibition of proliferation with NH4Cl in cultured renal tubular cells was reversible when the ammonia was removed. Fourth, EGF-induced protein synthesis was normal in NH4Cl-treated cells (Figure 4), indicating that NH4Cl does not produce generalized toxic effects. Finally, expression of the HPV16 E7 protein reversed both the antiproliferative effect and the hypertrophy.
The retinoblastoma gene family of proteins regulates cell cycle progression through the G1-S interface (5,36,37,38) and, in cultured renal epithelial cells, the retinoblastoma family plays a role in converting hyperplasia to hypertrophy (6,25). In NRK-52E cells, TGF-ß converts hyperplasia to hypertrophy by inhibiting growth factor-induced phosphorylation of retinoblastoma family members through its actions on cyclin E/cdk2 kinase (22). Inactivation of the retinoblastoma family of proteins by expression of the HPV16 E7 protein prevents TGF-ß-induced hypertrophy (6). In contrast, NH4Cl-induced hypertrophy, which does not seem to involve cell cycle entry, is unaffected by HPV16 E7 protein (12) (Figure 7A). However, when NH4Cl and EGF are combined, expression of HPV16 E7 blocks the antiproliferative effect of NH4Cl and decreases the degree of hypertrophy (Figure 7, B and D). Therefore, these data are the first evidence that NH4Cl can affect growth factor responses in much the same manner as TGF-ß1; the result is a conversion of hyperplasia to hypertrophy.
NH4Cl seems to convert hyperplasia to hypertrophy by affecting lysosomal function. Because EGF-induced protein synthesis is not inhibited (Figure 4), early EGF signaling is probably unaffected. In Rat-1 fibroblasts, the weak base methylamine does not impair EGF-induced early signaling or protein synthesis but does block proliferation (39). In lower eukaryotes, ammonia can act as a signaling molecule. The slime mold Dictyostelium completes its development ("culmination") when exposed to ammonia, and this response is dependent on the alkalinization of acidic intracellular compartments (40). The novel finding that all classes of lysosomal inhibitors (weak bases, proton pump inhibitors, and protease inhibitors) can mimic the effects of ammonia (Figure 6) suggests that ammonia acts by inhibiting lysosomes, rather than by affecting other acidic intracellular compartments.
How inhibition of lysosomal function might act as a signal to inhibit proliferation is unclear. In human fibroblasts, it has been demonstrated that ammonia can block the degradation of at least one signaling protein, namely the MARCKS protein (41). Ammonia decreases the activity of the hsc73-dependent lysosomal import pathway (42) and the expression of lgp96 (43), which acts at the hsc73 binding site to allow transport of cytosolic proteins into the lysosome (44). These findings suggest that ammonia could act by altering the half-lives of signaling proteins that are degraded in lysosomes. It is unlikely that the regulators of retinoblastoma family phosphorylation, i.e., the cyclin-dependent kinases, could themselves be regulated by lysosomal proteolysis, because the cyclins are primarily degraded by the ubiquitin-proteasome proteolytic system and lack the characteristic lysosomal import consensus sequence (45,46,47,48). Therefore, ammonia is more likely to activate an antiproliferative signaling pathway upstream of these kinases. This inhibition could occur directly or through the autocrine release of a cytokine, such as TGF-ß (8,49).
In conclusion, the antiproliferative effect of NH4Cl results in conversion of growth factor-mediated hyperplasia to hypertrophy in cultured renal epithelial cells. In animals with acidosis or hypokalemia, the relatively small increase in cell number that occurs in the renal cortex is consistent with a substantial role for ammonia (9,10). Because there is little hyperplasia and a large amount of hypertrophy in most renal tubular growth responses (38), it is tempting to speculate that ammonia may play a role in controlling hyperplasia in other settings. For example, when there is nephron dropout, both the reduction in the concentration of ammonia and the stimulation of growth factors could lead to unchecked tubular cell proliferation and cyst formation (4). Verification of this hypothesis will require evidence from animal models of renal disease.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  W. Shen, N. S. Brown, P. F. Finn, J. F. Dice, and H. A. Franch Akt and Mammalian Target of Rapamycin Regulate Separate Systems of Proteolysis in Renal Tubular Cells J. Am. Soc. Nephrol., September 1, 2006; 17(9): 2414 - 2423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Franch, S. Raissi, X. Wang, B. Zheng, J. L. Bailey, and S. R. Price Acidosis impairs insulin receptor substrate-1-associated phosphoinositide 3-kinase signaling in muscle cells: consequences on proteolysis Am J Physiol Renal Physiol, October 1, 2004; 287(4): F700 - F706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Cheval, J. P. Duong Van Huyen, P. Bruneval, J.-M. Verbavatz, J.-M. Elalouf, and A. Doucet Plasticity of mouse renal collecting duct in response to potassium depletion Physiol Genomics, September 16, 2004; 19(1): 61 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. A. Franch, X. Wang, S. Sooparb, N. S. Brown, and J. Du Phosphatidylinositol 3-Kinase Activity Is Required for Epidermal Growth Factor to Suppress Proteolysis J. Am. Soc. Nephrol., April 1, 2002; 13(4): 903 - 909. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Franch, S. Sooparb, J. Du, and N. S. Brown A Mechanism Regulating Proteolysis of Specific Proteins during Renal Tubular Cell Growth J. Biol. Chem., May 25, 2001; 276(22): 19126 - 19131. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
), the DNA level (
), and the protein/DNA ratio
([UNK]) in NRK-52E cells. Cells were treated with 10-8 M EGF alone,
10 mM NH4Cl, or both. Results are expressed as percentage changes,
compared with values measured in untreated cells. (A) 48 h (n = 12);
(B) 72 h (n = 18). *P < 0.05 versus
control; +P < 0.05 versus EGF alone and
NH4Cl alone.
