Sustained activation of MAPK/ERKs signaling pathway in cystic kidneys from bcl-2 -/- mice

Christine M. Sorenson1 and Nader Sheibani2,3

Departments of 1 Pediatrics, 2 Ophthalmology and Visual Sciences, and 3 Pharmacology, University of Wisconsin-Madison, Madison, Wisconsin 53792


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell proliferation, survival, and differentiation are carefully orchestrated processes during nephrogenesis that become aberrant during renal cyst formation. Signaling through focal adhesion kinase (FAK) impacts these processes, although its role during nephrogenesis requires further delineation. We previously demonstrated that phosphorylation of FAK and paxillin is not downregulated in cystic kidneys from B cell lymphoma/leukemia-2 (bcl-2) -/- mice. Here we examine whether FAK downstream signaling pathways are affected in these cystic kidneys. Cystic kidneys from bcl-2 -/- mice exhibited sustained phosphorylation of Src and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK, ERK1). However, similar levels of expression were noted for phosphorylated c-Jun NH2-terminal kinase, phosphatidylinositol-3-kinase, and its target protein kinase B/ATP-dependent tyrosine kinase in kidneys from postnatal day 20 bcl-2 +/+ and bcl-2 -/- mice. We also examined expression of the adapter protein Shc, implicated in growth and apoptosis. Expression of p66Shc decreases to low levels in postnatal kidneys, whereas p52/p46Shc was constitutively expressed during nephrogenesis. Shc expression was similar in normal and cystic kidneys. Therefore, sustained activation of MAPK/ERKs through the Src/FAK pathway may contribute to the hyperproliferation observed in cystic kidneys from bcl-2 -/- mice.

mitogen-activated protein kinases; renal cysts; signal transduction


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

B CELL LYMPHOMA/LEUKEMIA-2 (bcl-2) is a death repressor

that plays an important role during nephrogenesis. Mice deficient in bcl-2 (bcl-2 -/-) develop renal hypoplasia/cystic dysplasia (16). Renal cystic disease occurs in these mice by postnatal day 20 (P20) and is characterized by hyperproliferation in the cortex and medulla, increased amounts of apoptosis, and epithelial cells that have not fully differentiated (15). Cystic kidneys from these mice display altered signaling through focal adhesion kinase (FAK). We have observed sustained phosphorylation of FAK and paxillin and altered distribution and decreased expression and activity of the tyrosine phosphatases Src homology-2 domain phosphatase (SHP-2) and protein tyrosine phosphatase (PTP) 1B in cystic kidneys from these mice (17, 18). Bcl-2 forms a complex with paxillin/FAK, potentially influencing cell adhesion-mediated signaling (18). Thus the early loss of bcl-2 during nephrogenesis may result in an inability to complete renal differentiation/maturation as a consequence of not receiving the appropriate signals or not forming appropriate signaling complexes.

The role of the FAK/paxillin signal transduction pathway(s) in nephrogenesis and in the pathogenesis of kidney disease has not been elucidated. Signaling through FAK can prevent apoptosis and promote cell proliferation and migration. Our studies indicate that proper regulation of FAK/paxillin during renal maturation is important (18). FAK plays a pivotal role in integrin and growth factor signaling pathways. It interacts with signaling and cytoskeletal proteins such as paxillin, Src kinase, phosphatidylinositol-3 (PI3)-kinase, and p130cas. Src's association may protect FAK from dephosphorylation by phosphatases and allow Src to phosphorylate FAK on additional tyrosine residues, keeping it in an active state. However, continued signaling through FAK could result in the aberrant activation of downstream signaling pathways, including mitogen-activated protein kinase (MAPK), PI3-kinase, and protein kinase B (PKB)/ATP-dependent tyrosine kinase (Akt) (3).

MAPKs, including extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinase (JNK), and p38, play important roles in the cell by transmitting extracellular signals from the cell membrane to the nucleus (9). MAPKs are activated by various stimuli, influencing cell proliferation, differentiation, and apoptosis. Altered regulation of MAPKs can have profound cellular effects. Constitutive activation of MAPK/ERKs is observed in an organ-specific manner in primary tumors and tumor cell lines, with kidney, colon, and lung having the highest frequencies (4). We recently demonstrated that sustained activation of ERKs results in dedifferentiation of Madin-Darby canine kidney epithelial cells, affecting cell adhesive mechanisms (13). Activation of FAK can also result in signal transduction through PI3-kinase. PI3-kinase contributes to cell growth and survival. PI3-kinase can influence cell survival by activation of PKB/Akt. PKB/Akt, a serine-threonine kinase, inhibits apoptosis by a variety of stimuli (2). Thus, altered regulation of cell proliferation and differentiation observed in renal cystic disease would be consistent with aberrant regulation of MAPKs and/or PI3-kinase signaling pathways.

In the studies described here, we examined FAK downstream signal transduction in kidneys from P20 bcl-2 +/+ and bcl-2 -/- mice. Cystic kidneys from P20 bcl-2 -/- mice display sustained activation of Src and MAPK/ERK (ERK1), although their levels of expression are similar. In contrast, JNK, PI3-kinase, and its target PKB/Akt were not affected. The adapter protein Shc has been implicated in growth and apoptosis. Consistent with their described roles, expression of p66Shc decreases to low levels in postnatal kidneys, whereas p52/p46Shc was constitutively expressed during nephrogenesis. Shc expression was similar in normal and cystic kidneys. Thus sustained activation of MAPK/ERKs (ERK1) through the Src/FAK pathway may contribute to the hyperproliferation observed in cystic kidneys from bcl-2 -/- mice.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal breeding. Bcl-2 heterozygote animals were interbred, and the genotypes were determined as previously described (16). For expression studies, mice were interbred and the kidneys were surgically dissected at the times noted. Embryos were removed from anesthetized pregnant female mice on day 15 of pregnancy. The protocols were performed with approval from the University of Wisconsin Animal Care and Use Committee.

Protein lysate preparation, immunoprecipitation, and Western blot analysis. The protein lysates were prepared in a modified RIPA buffer [(in mM) 142.5 KCl, 5 MgCl2, 10 HEPES, pH 7.4, 2 orthovanadate, and 2 sodium difluoride, as well as 1% Nonidet P-40 and a complete protease inhibitor cocktail (Boehringer Mannheim)], and 20 µg of total protein were electrophoresed in a 4-20% polyacrylamide gel, transferred to a Hybond ECL nitrocellulose membrane (Amersham, Arlington Heights, IL), blocked, and incubated with anti-PKB-alpha /Akt (Transduction Laboratories; 1:1,000), phosphorylated Akt (New England Biolabs; 1:1,000) Src (Santa Cruz Biotechnology; 1:1,000), ERK (Santa Cruz Biotechnology; 1:1,000), phosphorylated ERK (Santa Cruz Biotechnology; 1:1,000), JNK (Promega; 1:2,000), phosphorylated JNK (Santa Cruz Biotechnology; 1:1,000), or Shc (Transduction Laboratories; 1:2,000) overnight at 4°C. The membranes were then washed, incubated with the appropriate secondary antibody (Pierce, Rockford, IL), washed again, and developed with ECL (Amersham). For immunoprecipitations, 600 µg of kidney lysate were incubated with 3 µg of antiphosphotyrosine (PY20, Transduction Laboratories) or mouse IgG (control), incubated with GammaBind Plus Sepharose (Pharmacia Biotech, Piscataway, NJ), and washed as previously described (18). Western blot quantitation was performed using a Molecular Dynamics PhosphorImager.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sustained Src phosphorylation in kidneys from P20 bcl-2 -/- mice. Renal cystic disease in bcl-2 -/- mice is characterized by hyperproliferation in the cortex and medulla, increased apoptosis, and sustained phosphorylation of FAK and paxillin (15, 18). Src phosphorylates FAK, and signal transduction through this pathway impacts proliferation. To determine whether Src could potentially contribute to sustained phosphorylation of FAK in P20 bcl-2 -/- mice, we examined Src expression and phosphorylation. Kidney protein lysates prepared from postnatal day 10 (P10) mice (before renal maturation) and P20 mice (after renal maturation in normal mice; cystic kidneys from bcl-2 -/- mice) were immunoprecipitated with mouse IgG (negative control) or PY20 (antiphosphotyrosine) and blotted with anti-Src to determine Src tyrosine phosphorylation. Levels of phosphorylated Src were similar before renal maturation (P10) in bcl-2 +/+ and bcl-2 -/- mice (Fig. 1A). In contrast, Src phosphorylation was sustained in lysates from P20 bcl-2 -/- mice compared with P20 bcl-2 +/+ littermates (after renal maturation; Fig. 1, A and C). Similar results were obtained when lysates were immunoprecipitated with anti-Src and blotted with 4G10 (antiphosphotyrosine; data not shown).


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Fig. 1.   Src is highly phosphorylated (active) in kidneys from mice at postnatal day 20 (P20) lacking B cell lymphoma/leukemia (bcl-2 -/-). A: protein lysates (500 µg) prepared from postnatal day 10 (P10) and P20 bcl-2 +/+ and bcl-2 -/- mice were immunoprecipitated (IP) with antiphosphotyrosine (PY20) and subjected to Western blot analysis. Membrane was incubated with antibodies to Src. B: lysates from P10 and P20 mice were subjected to Western blot and incubated with antibodies to Src. C: relative ratio of phosphorylated Src to total Src. Western blots were quantitated by scanning densitometry, and ratio was calculated relative to P10 bcl-2 +/+, which was set to 1. Statistical differences between groups were evaluated with Student's unpaired t-test (2-tailed). Values are means ± SE. Western blots are representative of protein lysates from 4 individual animals.

Figure 1B demonstrates that total Src expression was similar in kidneys from P10 and P20 bcl-2 +/+ and bcl-2 -/- mice by Western analysis. We did observe a modest decline (~15%) in the level of total Src expression in kidney lysates from P20 bcl-2 -/- mice compared with wild-type mice by scanning densitometry of the blots. Figure 1C illustrates the quantitative changes observed in Fig. 1, A and B, determined by scanning densitometry, as a bar graph representation of the relative ratio of phosphorylated Src to total Src. The relative ratio of phosphorylated Src to total Src is similar in lysates from P10 bcl-2 +/+ and bcl-2 -/- mice. The relative ratio of phosphorylated Src to total Src in lysates from bcl-2 -/- mice at P20 was similar to that observed at P10. In contrast, this ratio decreases significantly in lysates from P20 bcl-2 +/+ mice. Thus Src phosphorylation decreases significantly after renal maturation in normal mice but is sustained in kidneys from P20 bcl-2 -/- mice.

Sustained activation of ERKs but not JNK or PKB/Akt is observed in cystic kidneys. FAK can activate downstream signaling pathways, including MAPK/ERKs and PI3-kinase (3). We next examined the expression and phosphorylation of MAPKs. The level of total ERK1 and ERK2 expression was similar at all times (Fig. 2B). Levels of phosphorylated ERK1 were similar before renal maturation (P10) in bcl-2 +/+ and bcl-2 -/- mice (Fig. 2A). After renal maturation (P20) in normal mice, the level of ERK1 phosphorylation declines significantly. In contrast, ERK1 phosphorylation lysates from P20 bcl-2 -/- mice remained at higher levels, similar to those observed at P10. Expression of activated (phosphorylated) ERK2 was similar at all times in bcl-2 +/+ and bcl-2 -/- mice.


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Fig. 2.   Analysis of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-kinase) pathways. Kidney protein lysates (20 µg) prepared from bcl-2 +/+ and bcl-2 -/- mice were analyzed by Western blot. Phosphorylation (A) and expression (B) of MAPK/extracellular signal-regulated kinases (ERKs) were examined in lysates from P10 and P20 mice. C: relative ratio of phosphorylated ERK1 to total ERK1. D: relative ratio of phosphorylated ERK2 to total ERK2. Western blots were quantitated by scanning densitometry. Relative ratios were calculated as described in Fig. 1 legend. Statistical differences between groups were evaluated with Student's unpaired t-test (2-tailed). Values are means ± SE. E: phosphorylated c-Jun NH2 kinase (JNK) expression. F: total JNK expression. G: expression of PI3-kinase (p85 subunit), protein kinase B (PKB)-alpha /ATP-dependent tyrosine kinase (Akt), and phosphorylated PKB-alpha /Akt (p-PKB-alpha -Akt) in lysates from P20 bcl-2 +/+ and bcl-2 -/- mice. Western blots are representative of protein lysates from 4 individual animals.

Changes in the relative ratio of phosphorylated ERKs to total ERKs in Fig. 2, A and B, are shown quantitatively in Fig. 2, C and D. Figure 2C shows that the relative ratio of phosphorylated ERK1 to total ERK1 is similar in lysates from P10 bcl-2 +/+ and bcl-2 -/- mice. The relative ratio of phosphorylated ERK1 to total ERK1 in lysates from bcl-2 -/- mice at P20 was similar to that at P10. In contrast, this ratio decreases significantly in lysates from P20 bcl-2 +/+ mice. The relative ratio of phosphorylated ERK2 to total ERK2 is similar at all times. Thus ERK1 activation decreases significantly after renal maturation in normal mice but is sustained in kidneys from P20 bcl-2 -/- mice.

To determine whether other MAPK family members are activated in cystic kidneys from bcl-2 -/- mice, we examined the expression and phosphorylation of JNK in kidneys from P20 bcl-2 +/+ and bcl-2 -/- mice. Figure 2, E and F, demonstrates that the expression of phosphorylated JNK (Fig. 2E) and total JNK (Fig. 2F) was similar in lysates from P20 bcl-2 +/+ and bcl-2 -/- mice. We also observed similar levels of expression of p38 and phosphorylated p38 in lysates from P20 bcl-2 +/+ and bcl-2 -/- mice (data not shown). Thus sustained activation of Src and MAPK/ERKs is consistent with the hyperproliferative cystic phenotype observed in kidneys from P20 bcl-2 -/- mice (15).

The FAK survival signal is mediated through PI3-kinase/Akt-dependent and -independent pathways (5). Bcl-2 can negate the necessity of matrix attachment for survival in endothelial cells and fibroblasts (7). However, it is not known whether this occurs through a PI3-kinase/PKB-alpha /Akt-dependent or -independent pathway. PI3-kinase has an 85-kDa regulatory subunit and a 110-kDa catalytic subunit and plays an important role in cell survival by activation of PKB-alpha /Akt. We examined the expression of PI3-kinase (p85 subunit), PKB-alpha /Akt, and phosphorylated PKB-alpha /Akt in kidney lysates from P20 bcl-2 +/+ and bcl-2 -/- mice by Western blot analysis. Figure 2G demonstrates that the expression of PI3-kinase, PKB-alpha /Akt, and phosphorylated PKB-alpha /Akt was similar in kidney lysates from P20 bcl-2 +/+ and bcl-2 -/- mice.

Expression of adapter proteins Shc and Grb2. Shc isoforms (p46, p52, and p66) are SH2-containing proteins implicated in the regulation of apoptosis and life span (p66) and growth (p52/p46) (8). The loss of p66Shc expression confers resistance to H2O2-induced apoptosis (11). In Fig. 3A, we examined Shc expression in kidneys from mice at embryonic day 15, postnatal day 0 (birth), P10, and P20 to delineate expression during normal nephrogenesis and after renal maturation (P20). In addition, we examined Shc expression in cystic kidneys from P20 bcl-2 -/- mice. The expression of p66Shc decreased to low levels in kidneys from postnatal mice. This suggests that decreased expression of p66Shc may aid cell survival in the postnatal kidney. In contrast, expression of p46Shc and p52Shc was similar throughout nephrogenesis. Expression of Shc isoforms was similar in kidneys from P20 bcl-2 +/+ and bcl-2 -/- mice. We also observed constitutive expression of the adapter protein Grb2 during nephrogenesis and in cystic kidneys (Fig. 3B).


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Fig. 3.   Expression of adapter proteins, Shc and Grb2, during nephrogenesis. Kidney protein lysates (20 µg) prepared from normal mice from embryonic day 15 (E15) through P20 and kidneys from P20 bcl-2 -/- mice were analyzed by Western blot. Membrane was incubated with antibodies to Shc (A) or Grb2 (B). Arrows, forms of Shc (p66, p52, p46). Note similar levels of Shc in lysates from bcl-2 +/+ and bcl-2 -/- mice. Western blots are representative of protein lysates from 4 individual animals.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Signal transduction through FAK impacts cell proliferation, survival, and differentiation. Altered regulation of this pathway can lead to abnormal growth and differentiation. We previously demonstrated that sustained phosphorylation of FAK and paxillin in cystic kidneys from bcl-2 -/- mice correlated with an increased rate of cell proliferation and an inability of renal epithelial cells to terminally differentiate (15, 18). Here, we have further investigated FAK signal transduction in cystic kidneys from bcl-2 -/- mice. Our major findings are as follows: 1) sustained phosphorylation of Src is observed in cystic kidneys, 2) MAPK/ERKs (ERK1) remains activated in cystic kidneys, and 3) PI3-kinase/Akt, JNK, and Shc expression is not affected in cystic kidneys from P20 bcl-2 -/- mice. Thus our data indicate that sustained phosphorylation of Src and FAK may initiate downstream signaling, resulting in sustained activation of MAPK/ERKs in cystic kidneys from P20 bcl-2 -/- mice.

FAK binding to paxillin or p130cas can activate MAPK/ERKs via Crk (3). FAK is required for transducing survival signals from a complex extracellular matrix. The FAK survival signal is mediated through PI3-kinase/Akt-dependent and -independent pathways. Our data suggest that sustained phosphorylation of FAK leads to sustained activation of MAPK/ERKs, disruption of renal epithelial cell terminal differentiation, and formation of renal cysts. Activated ERKs are observed in samples from patients with autosomal-dominant polycystic kidney disease (1, 19). Sustained activation of ERKs in renal cystic disease is consistent with our in vitro data demonstrating an inability of kidney epithelial (Madin-Darby canine kidney) cells with activated ERKs to form stable adherens junctions (13). These cells also have downregulated adherens junction proteins, including E-cadherin, alpha -catenin, and gamma -catenin. We have demonstrated that inhibition of MAPK/ERKs in these cells is sufficient to reestablish a normal epithelial phenotype and formation of adherens junctions (13). Sustained activation of MAPK/ERKs in these cells also inhibits tube formation in collagen gels. These in vitro data are consistent with our observation that cell-cell adhesive interactions are aberrant in kidneys from bcl-2 -/- mice (14, 18).

We believe that, in the absence of bcl-2, focal adhesion complexes do not form properly in early development and are not properly regulated at maturation. Apoptosis, proliferation, and differentiation are affected in cystic kidneys. Continued signaling through FAK (phosphorylated FAK) may lead to signaling through MAPK/ERKs and/or PI3-kinase, affecting apoptosis, proliferation, and differentiation. However, the data presented here suggest that sustained activation of Src, FAK, and MAPK/ERKs (ERK1) plays a causal role in the hyperproliferation we previously reported in the cortex and medulla of cystic kidneys from bcl-2 -/- mice (15). Figure 4 summarizes the potential cellular effects of signaling through MAPK/ERKs. Aberrant signal transduction through this pathway is consistent with described renal cystic phenotype.


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Fig. 4.   Schematic diagram of MAPK pathway activated in cystic kidneys. In cystic kidneys from P20 bcl-2 -/- mice, sustained activation of Src contributes to sustained activation of focal adhesion kinase (FAK) and paxillin (PAX). Resulting downstream effect is sustained activation of MAPK/ERKs (ERK1), increased expression of c-myc (data not shown), and increased proliferation in cortex and medulla.

Protein tyrosine phosphorylation is a dynamic reversible process in which the level of phosphorylation, at any time, is the result of kinase and/or phosphatase activity. Altered regulation of kinases and/or phosphatases can lead to abnormal growth and differentiation. Here we show that sustained activation of Src and MAPK/ERKs (ERK1) may contribute to the aberrant phosphorylation/activation of signaling proteins in cystic kidneys from bcl-2 -/- mice. However, the important role of tyrosine phosphatases in this process should not be neglected. We recently showed that cystic kidneys from P20 bcl-2 -/- mice have reduced expression, a sixfold decrease in activity, and altered distribution of SHP-2 and PTP 1B, whereas PTP-proline, glutamate, serine, and threonine (PEST) sequence expression and distribution were similar in bcl-2 +/+ and bcl-2 -/- mice. Therefore, our data indicate that the hyperproliferation observed in cystic kidneys from bcl-2 -/- mice may be due to an inability to complete the final stages of terminal differentiation, aberrantly keeping signal transduction pathways active that are normally downregulated.

Signal transduction through Src/FAK is one way to affect proliferation. However, growth and apoptosis can also be influenced by Shc isoforms. Shc isoforms regulate apoptosis and life span (p66Shc) as well as growth (p52/p46Shc). p52/p46Shc isoforms are ubiquitously expressed. However, p66Shc expression varies in different cell types and is lacking in some cells, including hematopoietic cells (12). In the kidney, p66Shc is highly expressed embryonically, and its expression decreases to low levels postnatally, whereas p52/p46Shc is constitutively expressed. Similarly, p66Shc is highly expressed in the embryonic lung, becomes restricted late in gestation, and is nearly absent in the postnatal lung (6). The decline in p66Shc expression postnatally may give us an insight into its function. Mice lacking p66Shc have increased resistance to paraquat and increased life span, whereas fibroblasts lacking p66Shc have enhanced resistance to apoptosis due to H2O2 or ultraviolet light (10). Thus, reducing p66Shc expression in the postnatal kidney or lung may provide additional protection from apoptosis in a stressful/harsh environment. We observed similar expression of p66Shc in kidneys from P20 bcl-2 +/+ and bcl-2 -/- mice, perhaps suggesting redundancy of this pathway.

In summary, the appropriate regulation of FAK and its downstream signal transduction pathway(s) plays an important role during terminal differentiation of renal epithelial cells. Aberrant activation of these pathways may contribute to sustained activation of MAKP/ERKs and hyperproliferation in cystic kidneys from bcl-2 -/- mice.


    ACKNOWLEDGEMENTS

The authors thank R. Gordon and J. Oswald for preparation of the figures.


    FOOTNOTES

This research and C. M. Sorenson were supported in part by the Solomon Papper, M.D., Young Investigator Grant of the National Kidney Foundation and the Polycystic Kidney Foundation. N. Sheibani was funded by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-45599.

Address for reprint requests and other correspondence: C. M. Sorenson, Dept. of Pediatrics, University of Wisconsin-Madison, H4/444 CSC, 600 Highland Ave., Madison, WI 53792-4108 (E-mail: cmsorenson{at}facstaff.wisc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 26, 2002;10.1152/ajprenal.00380.2001

Received 27 December 2001; accepted in final form 19 June 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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