Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, Brazil
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ABSTRACT |
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It is well established that after removal of renal mass, the remaining tissue undergoes compensatory growth. Several laboratories have reported that the activity of the apical membrane Na+-H+ exchanger (NHE3) is increased after a reduction in renal mass. These studies were designed to determine whether NHE3 expression is altered early after loss of renal mass and to investigate the possible role of NHE3 activation in the compensatory tissue growth response. Experiments were performed in young male Wistar rats submitted to left nephrectomy or sham operation. At either 4 or 24 h after the surgery, the right kidney from each animal was removed and weighed. Significant increases in the wet weight of the remaining kidney were only observed 24 h after uninephrectomy (UNX). Western blot analysis of brush-border membranes and Northern blot analysis of cortex RNA showed that NHE3 protein abundance and NHE3 mRNA were greatly enhanced 4 and 24 h after UNX in relation to the sham kidney. To identify which growth pattern was mostly responsible for the enlargement of the remained kidney in our experimental models, we measured 5-bromo-2-deoxyuridine incorporation (BrdU) and protein-to-DNA ratio (protein/DNA ratio). The number of BrdU-positive nuclei increased and protein/DNA ratio slightly decreased, indicating that a hyperplastic response was the main component involved in the early compensatory renal growth in our animals. BrdU incorporation and protein/DNA were also assessed in rats treated with S3226, a selective blocker of NHE3. Neither the number of BrdU-positive nuclei nor the protein/DNA ratio was significantly altered 4 and 24 h after UNX in rats treated with S3226. In conclusion, UNX induced an upregulation of NHE3, which was evidenced at both functional and expression levels. The compensatory growth response in young UNX rats could be blocked by inhibiting NHE3 activity, suggesting that NHE3 activation may result in a facilitator state for the cell growth response in the renal proximal tubule.
type 3 sodium/hydrogen exchanger; uninephrectomy; hyperplasia; cell proliferation; S3226; renal proximal tubule.
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INTRODUCTION |
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THE RENAL EPITHELIUM IS COMMONLY used as an in vivo model for the investigation of events that trigger cellular growth. In the adult kidney, differentiated nephrons are relatively quiescent, with few cells undergoing mitosis. However, a proliferative response can be induced by acute injuries to the kidney. Cell growth is then reactivated in the form of hyperplasia to repair the damaged tissue. Conversely, compensatory growth after ablation of renal mass in adult rats occurs predominantly by hypertrophy (14, 15, 18), a condition in which both cell size and cellular protein content increase without an increment in cell number. Also of interest is the fact that the pattern of cell growth is dependent on the age of the animal. Young rats, sexually immature, show a pattern of kidney growth with significant DNA synthesis and cell division (6, 17, 38).
Na+/H+ exchange is the predominant mechanism for absorption of Na+ and secretion of H+ across the apical membrane of the proximal tubule. A number of studies have indicated that the type 3 Na+/H+ exchanger (NHE3) is the Na+/H+ exchanger (NHE) isoform responsible for most, if not all, apical membrane Na+/H+ exchange in this segment of the nephron (2-3, 20, 35, 39-41). Several laboratories have reported that the activity of the apical membrane NHE is increased after a reduction in renal mass (8, 9, 28, 32). However, these reports have not addressed the question of whether the increase in the activity of this transporter was due to an increase in transporter number or an increased turnover rate per transporter.
Activation of NHEs, especially the ubiquitous isoform NHE1, has been
identified as an important facilitator event in the
hyperplastic/hypertrophic response in different cell types
(37). This role seems to be mediated by an increase in
intracellular pH (pHi), because growth of fibroblast clones
lacking the NHE was found to be pH conditional in an
HCO
In the present study, we examined whether NHE3 expression is rapidly altered after removal of a contralateral kidney. We also investigated the possible role of NHE3 upregulation as one of the primary events responsible for tissue growth in young UNX rats.
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METHODS |
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Reagents and antibodies.
Reagents were obtained from Sigma (St. Louis, MO) unless otherwise
specified. Primary antibodies comprised the following. A monoclonal
antibody (mAb) raised to the renal brush-border NHE3 was a gift from
Drs. Peter S. Aronson and Daniel Biemesderfer (Yale School of Medicine,
New Haven, CT) (3). An mAb (mouse IgG) to villin was
purchased from AMAC (Westbrook, ME). Horseradish peroxidase-conjugated
goat anti-mouse (-chain specific) was purchased from Zymed
Laboratories (San Francisco, CA). S3226 was kindly provided by Dr.
Jurger Punter (Aventis Pharma, Frankfurt, Germany). Slow-releasing
S3226 pellets were purchased from Innovative Research of America.
Animals and surgical procedures. Studies were conducted in male Wistar rats weighing 90-110 g with free access to tap water and standard laboratory chow. The rats were anesthetized by an intraperitoneal injection of 100 mg/kg Inactin and then submitted to either left nephrectomy or sham operation, the latter consisting of left hylum manipulation. Either 4 or 24 h after these procedures, the animals were killed by heart incision under ether anesthesia, and the right kidney from each animal was removed and weighed.
In another series of experiments, rats submitted to the same conditions described above were treated with 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride (S3226), a selective inhibitor of NHE3 (36). The compound S3226 was dissolved in saline and slowly infused through the caudal vein at the dose of 150 µg/100 g body wt. After the infusion, rats also received interscapular subcutaneous (sc) implantation of slow-release S3226 pellets (250 µg · 100 g body wtClearance studies. Rats were anesthetized as described above and placed on a heated surgical table to maintain body temperature at 36-37°C. Polyethylene catheters were placed in the bladder, and urine was collected during 4 h for clearance determination. Plasma was collected from the left carotid artery at the time of death for measurement of creatinine.
Brush-border membrane preparation. Immediately after kidney removal, cortices were separated at 4°C and homogenized in HEPES buffer containing the protease inhibitors (Sigma) pepstatin A (0.7 µg/ml), leupeptin (0.5 µg/ml), PMSF (40 µg/ml), and K2EDTA (1 mM). Brush-border membrane (BBM) obtained from our experimental models described elsewhere was prepared using a method based on Mg2+ precipitation and differential centrifugation, essentially as previously described (23).
Sodium uptake studies. Uptake of 22Na into the membrane vesicles was assayed at room temperature using a rapid filtration technique (23).
SDS-PAGE and immunoblotting.
Protein samples were solubilized in SDS sample buffer, and proteins
were separated by SDS-PAGE using 7.5% polyacrylamide gels according to
Laemmli (19). For immunoblotting, proteins were transferred to polyvinylidene difluoride (PVDF; Immobilon-P; Millipore, Bedford, MA) from polyacrylamide gels at 500 mA for 5 h at 4°C with a Transphor transfer electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA) and stained with Ponceau S in 0.5%
trichloroacetic acid. Entire sheets of PVDF membranes containing transferred proteins were incubated first in Blotto (5% nonfat dry
milk and 0.1% Tween 20 in PBS, pH 7.4) for 1-3 h to block nonspecific binding of antibody, followed by overnight incubation in
primary antibody (anti-NHE3) diluted 1:1,000 in Blotto. The membranes
were then washed five times in Blotto and incubated for 1 h with
horseradish peroxidase-conjugated IgG (-chain specific) from Zymed.
Bound antibody was detected with ECL enhanced chemiluminescence (Amersham) according to the manufacturer's protocols. The membranes were stripped and reprobed with an additional primary antibody (anti-villin). The stripping procedure consists of incubating the PVDF
membranes in 2% SDS, 50 mM Tris buffer (pH 6.9), and 100 mM
-mercaptoethanol for 60 min at 70°C. The visualized bands were
digitized using a scanner (STORM, Molecular Dynamics) and quantified by
the ImageQuant program (Molecular Dynamics).
Northern blot analysis. Total RNA was isolated from renal cortices as previously described by Puissant and Houdebine (33). Aliquots of 10 µg of total RNA from renal cortex were size fractionated in 1% agarose gel with 0.66 M formaldehyde. After electrophoresis, gels were stained with SYBR Green II (Molecular Probes) and laser scanned (STORM 840, Molecular Dynamics) to assess RNA integrity. RNA was fixed to the nylon membranes (Hybond N, Amersham) by irradiation with ultraviolet light (UV-crosslinker, Amersham). Prehybridization (4 h at 65°C) and hybridization (18 h at 65°C) of the RNA blots were performed with a buffer consisting of 5× SSC, 25 mM K2PO4, 5× Denhardt's solution (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum albumin, fraction V), 50 µg/ml denatured salmon sperm DNA, and 50% deionized formamide containing 10% dextran sulphate. For DNA probe synthesis, we used a partial-length cDNA of rat NHE3, obtained in our laboratory as previously described (11). pTRI 18S rRNA from Ambion was used as template for the 18S-RNA probe. We used T7 RNA polymerase from Ambion (StripEZ T7/T3 kit) to synthesize 18S-cRNA probe labeled with [32P]UTP (NEN-DuPont). After hybridization, blots were washed twice for 30 min in 2× SSC+0.1% SDS at 65°C and twice for 15 min in 0.1× SSC+0.1% SDS at 65°C. The membranes were exposed to phosphor screen (Molecular Dynamics) for 24 h and scanned using a STORM 840 (Molecular Dynamics). NHE3 mRNA levels and 18S-rRNA levels were quantified using the ImageQuant program (Molecular Dynamics).
Detection of cell proliferation. Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Rats were intraperitoneally injected with 1 ml/100 g of body wt of BrdU (Cell Proliferation Kit, Amersham Pharmacia Biotech, Bucks, UK) 2 h before death. The animals were deeply anesthetized with ketamine (10 mg/100 g body wt im) and xylazine (5 mg/100 g body wt im) and perfused through the left ventricle and aorta with buffered saline and 4% paraformaldehyde in phosphate buffer. Kidneys were removed, postfixed for 4 h, cryoprotected with 30% sucrose in phosphate buffer for 48 h, and sectioned in different planes (30 µm) on a cryostat. Sections were then submitted to a standard immunoperoxidase protocol (4). The material was initially treated with 2N HCl for 1 h. After a washing with phosphate- buffered saline with 0.1% Triton X-100 (PBST, 3 × 10 min), the sections were treated with 0.1 M sodium tetraborate for 10 min and washed with PBST. The mouse monoclonal anti-BrdU antibody was diluted 1:1,000 in PBST with 5% normal goat serum and applied to the sections for 12-16 h at room temperature (~24°C). After additional washes with PBST, the sections were sequentially incubated with a biotinylated anti-mouse goat antiserum for 2 h at room temperature and the avidin-biotin-peroxidase complex (ABC Elite Kit, Vector Labs) for 1 h at room temperature. After incubation with 0.05% 3-3'-diaminobenzidine and 0.3% hydrogen peroxide in phosphate buffer and intensification with 0.05% osmium tetroxide in water, the sections were dehydrated, cleared, and coverslipped with Permount (Fisher). Controls included the omission of the primary antibody and its substitution for normal mouse serum. Both procedures completely eliminated the staining. The labeled nuclei were electronically counted in several areas measuring 400 × 400 µm from at least 10 sections of the kidney from each rat, by using an image-analysis system with the National Institutes of Health Image software.
DNA and protein determination. The remaining kidney was removed and cut in half. One-half was homogenized in TE buffer, pH 8.0, and digested overnight with 1 mg/ml of proteinase K (GIBCO BRL) for further DNA quantification. The other half was homogenized in HEPES buffer containing the protease inhibitors pepstatin A (0.7 µg/ml), leupeptin (0.5 µg/ml), PMSF (40 µg/ml), and K2EDTA (1 mM) for protein analyses.
PicoGreen dsDNA quantitation reagent (Molecular Probes) was used to measure total DNA. Protein concentration was determined by the method of Lowry et al. (21). All measurements were performed in triplicate.Statistical analyses. Data are expressed as means ± SE, unless otherwise specified. Statistical tests were performed using analysis of variance with Tukey's posttest. P < 0.05 was considered statistically significant, and n represents the number of experiments performed or the number of measurements.
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RESULTS |
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Animal model characteristics.
The mean weights of the remaining kidney as well as the functional
parameters in each group of rats are summarized in Table 1. Significant increases of kidney weight
were observed 24 h after UNX compared with sham-operated animals.
Renal clearance per kidney measured in 12 rats from each group is also
shown in Table 1. During the fourth hour after UNX, renal clearance did not differ from that of the same kidney in sham-operated control rats.
However, renal clearance was significantly greater (30 ± 3%)
(P < 0.05 UNX vs. control) 24 h after UNX.
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Effect of UNX on NHE activity.
In agreement with previous studies, we found that the activity of the
apical membrane NHE is increased after removal of renal mass (28,
32). NHE activity was increased 41 ± 7% after 4 h
and 72 ± 11% 24 h after UNX. Figure
1 illustrates the proton-dependent uptake
of 22Na in BBM vesicles from each experimental group (sham
4 h = 1.74 ± 0.17; UNX 4 h = 2.46 ± 0.15; sham
24 h = 1.82 ± 0.22; UNX 24 h = 3.13 ± 0.09 nmol · mg protein1 · min
1).
There was no significant difference among the groups when BBM vesicles
were assayed in the presence of 100 µM EIPA.
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Effect of UNX on NHE3 protein abundance.
To determine whether alterations in NHE activity in Fig. 1 result from
changes in the NHE3 protein abundance, BBM vesicles were subjected to
SDS-PAGE and immunoblotting by using a monoclonal antibody raised to
NHE3. As indicated in Fig. 2, UNX led to
increased abundance of BBM NHE3. Densitometric analyses, corrected to
villin expression used as an internal control, revealed an increase of 154 ± 37% on NHE3 protein expression 4 h after UNX and an
increase of 267 ± 47% 24 h after UNX compared with the
sham-operated group.
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Effect of UNX on mRNA NHE3 abundance.
NHE3 expression after UNX was also determined at the mRNA level.
Northern blot analyses of total RNA from renal cortex from each group
is shown in Fig. 3. These data show that
UNX results in a marked increase in NHE3 mRNA 106 ± 15% 4 h
after surgery and 230 ± 33% 24 h after surgery in relation
to the expression observed in sham-operated rats. When the blot was
stripped and rehybridized with an 18S-cRNA probe, the signals were
similar in all groups, indicating minimal differences in RNA loading.
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Renal tubular epithelial cell proliferation.
In adult rats, the increment of the contralateral kidney mass is due
mainly to an increase in cell size, with little increase in cell
number. However, contrasting models of adaptation may develop depending
on the age patterns. As we have used young rats, with body weights
between 90 and 110 g, we decided to analyze the extension of the
proliferative response in our experimental models. As seen in Fig.
4, the removal of the contralateral
kidney induced an evident increase in cell proliferation, especially in
proximal tubules. The number of BrdU-positive nuclei, expressed as
percentages of control, was 79 ± 16 and 157 ± 25%, 4 and
24 h after UNX, respectively (P < 0.05, n = 17). Cell division was also present in control
rats, but to a much lesser extent.
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Effect of UNX on protein-to-DNA ratio.
The protein-to-DNA ratio (protein/DNA ratio) was used to determine
whether part of the renal compensatory response was due to hypertrophy.
As shown in Fig. 5, there was a small
decrement in protein/DNA ratio at both 4 (22 ± 3%) and 24 h
(28 ± 4%) after UNX (P < 0.05; n = 4/group). This
small decrement in the protein/DNA ratio shows that the early
compensatory growth after UNX in young rats is not attributable to
hypertrophy.
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Effect of S3226 on compensatory cell proliferation.
To investigate the possible role of NHE3 activation in the compensatory
renal tissue growth response, we performed a second series of
experiments in which both sham-operated and UNX rats were treated with
S3226, the selective NHE3 inhibitor. Compared with sham-operated
animals, there was no significant increase in the remaining kidney
weight 4 and 24 h after UNX (Table
2), suggesting that the compound S3226
may suppress cell proliferation.
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DISCUSSION |
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After a reduction in renal mass, the remaining kidney undergoes structural as well as functional adaptations (18). Increments of tubular transport capacity are observed in many segments of the nephron but occur primarily in the proximal tubule (7). One of the most prominent findings reported by researchers in this field is the increase in NHE activity in BBM of proximal tubules (13, 28). In the present study, we showed that in addition to increments in apical NHE activity, apical NHE expression, mainly due to NHE3, is also altered after UNX. This overexpression occurs as early as 4 h after UNX and precedes measurable increments in glomerular filtration rate.
We have observed an increase in NHE activity of 41 and 72% 4 and
24 h after UNX. These data are consistent with previous studies using micropuncture techniques, pHi recordings, and sodium
uptake by brush-border vesicles. The activation of NHE activity occurs in parallel to the Na+-K+-ATPase and
Na+-HCO
The findings in the present study show that activation of Na+/H+ exchange in the remaining kidney was accompanied by increases in both protein and mRNA content. The NHE3 mRNA levels are significantly increased 4 h after UNX (106%), and even more (230%) after 24 h. The increase in mRNA levels is closely related to the increase in NHE3 protein levels, 153 and 266%, 4 and 24 h after contralateral nephrectomy, respectively. The parallel changes observed in NHE3-specific mRNA suggest control at the transcriptional level or on mechanisms that define the stability of this message. However, mobilization of preexisting NHE3 transporters from subapical vesicles to the plasma membrane, a well-known mechanism of NHE3 modulation, cannot be ruled out.
Both hyperplastic and hypertrophic growth processes involve an increase in the physical size of the cell. However, unlike hyperplastic cells, hypertrophic cells do not duplicate their DNA or undergo mitosis (30). We observed that the removal of the contralateral kidney induced an evident increase in cell proliferation, especially in proximal tubules. The protein/DNA ratio was slightly smaller after UNX. We found increases in both DNA and protein contents. However, the increment in DNA synthesis was more pronounced. One possible explanation of this fact could be that the majority of the cells in the population we studied have recently exited mitosis. In this situation, cells need to accumulate a full complement of cytoplasm before they achieve a mature cell size (31).
We also undertook Western blot analyses of total renal homogenates incubated with an antibody against the inhibitor of cyclin-dependent kinases p21. The expression of p21 was not detectable in our blots (data not shown). p21 Effectively stops cell cycle progression and is known to regulate hypertrophy in response to renal ablation (1, 24). Cells must complete all phases of the cell cycle for hyperplasia to occur, and the lack of p21 that we observed is necessary for cell proliferation. We conclude from these results that a hyperplastic response was the main component involved in the early compensatory renal growth in our experimental models.
Several studies have determined that the type of compensatory renal growth after UNX is time and age related (17, 26, 27, 34). Mulroney et al. (26, 27) have reported that the early, accelerated remaining kidney growth after UNX in young rats occurs mainly by hyperplasia. Karp et al. (17) observed that in rats that were not completely sexually mature, the increase in kidney mass immediately after nephrectomy was mainly due to hyperplasia, whereas after 2 wk both hyperplastic and hypertrophic processes had equal participation (17). In our rats, the epithelial tissue growth is largely due to hyperplasia. This pattern of proliferative response can be explained by the fact that we have analyzed the early predominant type of renal growth response in young rats, body wt 90-110 g, which corresponds to an age of 4-5 wk.
NHE stimulation that we and others have found could result from an
increment in the capacity of the proximal tubule to secrete protons due
to an enhanced filtered load of HCO
We found that the compensatory cell proliferative response in young UNX rats is greatly impaired by the specific NHE3 inhibitor S3226. Several studies support the concept that cell proliferation is associated with an increased pHi. Our findings suggest that stimulation of NHE with consequent cytoplasmic alkalinization plays a key role in cell growth. Because of the importance of pHi alkalinization in cell growth, inhibition of NHE3 activity by S3226 may prevent the compensatory growth response by decreasing pHi in proximal tubule cells. This hypothesis can be supported by the observation of Hropot et al. (16) that NHE3 inhibition by S3226 results in a significantly lower pHi in the animal's proximal tubule cells.
We conclude from these studies that UNX induced an upregulation of NHE3, which is evidenced at both functional and expression levels. The compensatory growth response in young UNX rats can be blocked by inhibiting NHE3 activity, suggesting that NHE3 activation may result in a facilitator state for the cell growth response in renal proximal tubules. NHE3 activation could be triggered by the same cellular events that culminate in tissue growth, a process that represents a cell cycle-dependent mechanism. However, the initial triggering events of the renal growth response remain unknown.
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ACKNOWLEDGEMENTS |
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We thank Adilson S. Alves and Ana Lúcia Faria for technical assistance.
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FOOTNOTES |
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This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo.
This work was presented in part at the Annual Meeting of the American and International Society of Nephrology, San Francisco, California, October 2001, and has been published in abstract form (10).
Address for reprint requests and other correspondence: N. A. Rebouças, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Professor Lineu Prestes 1524, 05508-900 São Paulo, Brazil (E-mail: nancy{at}fisio.icb.usp.br).
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.
August 6, 2002;10.1152/ajprenal.00010.2002
Received 9 January 2002; accepted in final form 26 July 2002.
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