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*
Department of Internal Medicine, Chonnam National University Medical
School, Kwangju, Korea.
Department of Physiology, Chonnam National University Medical School,
Kwangju, Korea.
Department of Anatomy, Chonnam National University Medical School,
Kwangju, Korea.
Professional Graduate School of Oriental Medicine, Wonkwang University,
Iksan, Korea.
Correspondence to Dr. Jong-Un Lee, Department of Physiology, Chonnam National University Medical School, 5 Hak-dong, Kwangju 501-746, Korea. Phone: +82-62-220-4262; Fax: +82-62-232-1242; E-mail: julee{at}chonnam.chonnam.ac.kr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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proteins in the outer
medulla and the inner medulla. These results suggest that a reduced expression
of AQP water channels accounts at least in part for the cisplatin-induced
urinary concentration defect. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been established recently that the epithelial water transport occurs through distinct channels called aquaporins (AQP) (5, 6). Among multiple isoforms of the AQP family, AQP1 is present in the cells of the proximal tubule and the descending limb of Henle's loop (7). Recent evidence from AQP1 gene knockout mice demonstrated that 80 to 90% of osmotic permeability in the proximal tubule and descending thin limb can be attributed to AQP1 channels (8, 9). This points to an important role of AQP1 in concentrating the urine. A decreased expression of AQP1 may then result in a decreased medullary hypertonicity that is produced by countercurrent multiplication.
Conversely, AQP2 is expressed on the subapical vesicles as well as the apical membranes of the collecting duct principal cells (10). It is regulated short term and long term by the AVP/cAMP pathway. A short-term regulation of AQP2 represents trafficking of cytoplasmic vesicles to the apical membrane (11, 12), and a long-term regulation is to augment the abundance of its protein in the cell (13). On the contrary, AQP3 is localized mainly to the basolateral membrane of collecting duct principal cells (14). Recent studies demonstrated a long-term regulation of AQP3 with a marked increase in its expression in the collecting duct in response to water restriction or AVP infusion but not of AQP1 or AQP4 (13, 14). Certain pathophysiologic conditions associated with an altered urinary concentration have been related causally to an altered regulation of AQP water channels (15,16,17,18).
The present study aimed to investigate whether the cisplatininduced urinary concentration defect may be related to an altered regulation of AQP channels. Rats were treated with cisplatin, and their expression of AQP1, AQP2, and AQP3 proteins in the kidney was determined by Western blot analysis and immunohistochemistry. To specify further the point of primary impairment in the pathway that leads to the stimulation of AVP-mediated AQP channels, the expression of stimulatory G proteins and the activity of adenylyl cyclase were also determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the experimental day, the rats were anesthetized with thiopental sodium
(50 mg/kg intraperitoneally). The urinary bladder was cannulated with PE 50
tubings through a low abdominal incision to collect urine samples. At the end
of urine collection, the arterial blood was taken. Creatinine clearance was
determined. Free water reabsorption (TcH2O) was
calculated by the following formula:
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Protein Preparation and Western Blot Analysis
The kidneys were isolated rapidly after the rats were decapitated under a
conscious state. They were kept at -70°C until assayed. The cortex, outer
medulla, and inner medulla were dissected and homogenized at 3000 x
g in a solution containing 250 mM sucrose, 1 mM
ethylenediaminetetraacetate, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mM
Tris-HCl buffer at pH 7.6. Large tissue debris and nuclear fragments were
removed by two low-speed spins in succession (1000 x g, 10 min;
10,000 x g, 10 min). Protein samples were loaded and
electrophoretically size-separated with a discontinuous system consisting of
12.5% polyacrylamide resolving gel and 5% polyacrylamide stacking gel. The
proteins then were electrophoretically transferred to a nitrocellulose
membrane at 20 V overnight. The membranes were washed in Tris-based saline
buffer (pH 7.4) containing 0.1% Tween-20 (TBST), blocked with 5% nonfat milk
in TBST for 1 h, and incubated with affinity-purified anti-rabbit polyclonal
AQP1 (diluted 1:750), AQP2 (1:750), and AQP3 (1:200) antibodies (Alomone
Laboratories, Jerusalem, Israel), or heteromeric G-protein subunit Gs
(1:1000) antibody (Calbiochem-Novabiochem, San Diego, CA) in 2% nonfat
milk/TBST for 1 h at room temperature. The membranes then were incubated with
a horseradish peroxidase-labeled goat anti-rabbit IgG (1:1200) in 2% nonfat
milk in TBST for 2 h. The bound antibody was detected by enhanced
chemiluminescence (Amersham, Little Chalfont, Buckinghamshire, UK) on
hyperfilm. The relative protein levels were determined by analyzing the
signals of autoradiograms using the transmitter scanning
videodensitometer.
Immunohistochemistry
The expression of AQP1, AQP2, and AQP3 was also determined by
immunohistochemistry using an immunoperoxidase procedure (Vector Laboratories,
Burlingame, CA). The rat was anesthetized with thiopental sodium (50 mg/kg,
intraperitoneally), and the kidneys were fixed by in vivo perfusion
of the abdominal aorta with 4% paraformaldehyde for 10 min. The kidneys then
were excised and cut into 2-mm coronal slices, which were immersed in the same
fixative overnight at 4°C. The slices were washed in phosphate-buffered
saline (PBS), dehydrated in a graded series of ethanol washes, and embedded in
paraffin. Tissue sections were made at 6 µm and mounted on gelatin-coated
glass slides.
The tissue sections were deparaffinized in xylene, rehydrated in a graded series of ethanol, rinsed twice in PBS, and then treated with 3% H2O2 in 60% methanol for 30 min to quench endogenous peroxidase activity. After the sections were washed twice (5 min each) in PBS, they were blocked in PBS containing 5% goat serum for 1 h. The sections were incubated for 12 to 14 h with AQP1, AQP2, or AQP3 antibodies diluted 1:1000 in PBS with 0.3% bovine serum albumin. For a negative control, the sections were incubated in PBS containing 5% goat serum only. The sections were then rinsed thrice in PBS and incubated sequentially for 30 min each with biotinylated secondary antibody and ABC reagents, followed by a 6-min incubation with the peroxidase substrate solution diaminobenzidine. The sections were examined and photographed on a light microscope.
Differential Centrifugation
To differentiate the apical membrane-enriched versus subapical
vesicle-enriched fractions, we conducted centrifugation as described by
previous investigators (19).
The homogenates of the cortex, outer medulla, and inner medulla were
centrifuged at low-speed spins (1000 x g for 10 min) to remove
cell debris and nuclear fragments. The supernatant was centrifuged at 17,000
x g for 20 min to yield apical membrane-enriched pellets
(high-density fraction [HD]). The supernatant was centrifuged again at 100,000
x g for 1 h to obtain a vesicle-enriched pellet (low-density
fraction [LD]). Comparing the magnitude of its expression in the two fractions
assessed the trafficking of AQP2. A decrease in HD/LD represents an inhibited
trafficking.
Membrane Preparation and Adenylyl Cyclase Activity
The membrane preparation was obtained as described previously
(15). The cortex, outer
medulla, and inner medulla were separated. They were homogenized in ice-cold
homogenizing buffer (50 mM Tris-HCl [pH 8.0] containing 1 mM
ethylenediaminetetraacetate, 0.2 mM phenylmethylsulfonyl fluoride, and 250 mM
sucrose) and centrifuged at 1000 x g and 100,000 x
g in succession. The resulting pellet was used as membrane
preparation. Protein concentrations were measured by bicinchonic acid assay
kit (BioRad, Hercules, CA).
Adenylyl cyclase activity was assayed by the method of Bar (20), with a slight modification. Segments of adenylyl cyclase complex were examined separately using different drugs. AVP was used to activate V2 receptor, sodium fluoride was used to stimulate adenylyl cyclase in a receptor-independent but G-protein-dependent manner (21), and forskolin was used to provoke directly the catalytic unit of adenylyl cyclase complex (22). The reaction was started by adding the membrane fraction, of which protein contents were 20, 10, and 10 µg for the cortex, outer medulla, and inner medulla, respectively, in 100 µl of working solution (50 mM Tris-HCl [pH 7.6], containing 1 mM ATP, 20 mM phosphocreatine, 0.2 mg/ml creatine phosphokinase, 6.4 mM MgCl2, 1 mM 3-isobutyl-1-methylxanthine, 0.02 mM GTP). After 15 min, the reaction was stopped by cold application of solution consisting of 50 mM sodium acetate (pH 5.0) and centrifuged at 1000 x g for 10 min at 4°C.
cAMP was measured in the supernatant by equilibrated RIA. Iodinated
2'-O-monosuccinyl-adenosine 3',5'-cyclic monophosphate
tyrosyl methyl ester (125I-ScAMP-TME) was prepared as described by previous
investigators (23). Standards
or samples were taken up in a final volume of 100 µl of 50 mM sodium
acetate buffer (pH 4.8). Dilute cAMP antiserum (Calbiochem-Novabiochem) and
125I-ScAMP-TME (10,000 cpm/100 µl), 100 µl each, were added and
incubated for 15 h at 4°C. The bound form was separated from the free form
by charcoal suspension, and the supernatant was counted in a 
counter
(Packard Instrument, Meriden, CT). All samples in one experiment were analyzed
in a single assay. Nonspecific binding was <2.0%. The 50% intercept was at
16.5 ± 0.8 fmol/tube (n = 10). Intra- and interassay
coefficients of variation were 5.0 ± 1.2 (n = 10) and 9.6
± 1.9% (n = 10), respectively. Results were expressed as moles
of cAMP generated per milligram of protein per minute. Drugs were purchased
from Sigma Chemical Company (St. Louis, MO), unless stated otherwise.
Statistical Analyses
Results are expressed as mean ± SEM. The statistical significance of
differences between the groups was determined using ANOVA or unpaired
t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of AQP Water Channels
The expression of AQP proteins was determined in the cortex, outer medulla,
and inner medulla of the kidney. The anti-AQP1 antibody recognized 29-kD and
35- to 50-kD bands, corresponding to nonglycosylated and glycosylated AQP1,
respectively. The anti-AQP2 antibody recognized 29-kD and 35- to 50-kD bands,
corresponding to nonglycosylated and glycosylated AQP2, respectively. The
anti-AQP3 antibody recognized 27-kD and 33- to 40-kD bands, corresponding to
nonglycosylated and glycosylated AQP3, respectively. After treatment with
cisplatin (8 mg/kg), the expression of AQP1 was decreased significantly in the
cortex, outer medulla, and inner medulla
(Figure 1). The AQP2 expression
was also decreased in the cortex, outer medulla, and inner medulla
(Figure 2). The expression of
AQP3 was decreased in the outer medulla and the inner medulla but not in the
cortex (Figure 3). By the use
of a lower dose of cisplatin (4 mg/kg), the expression of AQP1 and AQP2 was
decreased only in the outer medulla while that of AQP3 was not affected
significantly (Figures
1,2,3).
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Cellular Distribution of AQP2
The expression of AQP2 channels was determined differentially in the apical
membrane-enriched and the subapical vesicle-enriched fractions to examine
whether their targeting was changed. Cisplatin decreased the expression of
AQP2 proteins in the apical membrane-enriched fraction in parallel with that
in the subapical vesicle-enriched fraction, so HD/LD remained unchanged
(Figure 4).
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Immunohistochemistry of AQP Channels
Histologic changes induced by cisplatin were most prominent in the outer
stripe of the outer medulla. Partial to complete desquamation and necrosis of
individual cells or short sections of the tubular epithelium was variably
present along the straight portion of the proximal tubule. In contrast, the
glomerulus, proximal convoluted tubule, loop of Henle, and collecting duct
were well preserved. The immunoreactivity for AQP1 was expressed prominently
in the apical membrane of the S3 segment of proximal tubules but was markedly
decreased by cisplatin (Figure 5, A and
B). The abundance of AQP2 labeling was observed in the principal
cells of collecting duct, both in the apical region of the cell and throughout
the cytoplasm (Figure 5C).
Cisplatin decreased the AQP2 labeling, and the residual AQP2 was expressed in
both the cytoplasmic region and the apical membrane
(Figure 5D). The expression of
AQP3, which was localized to the basolateral membrane of principal cells in
the outer medullary collecting duct, was decreased by cisplatin
(Figure 5, E and F).
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Expression of Stimulatory G Proteins
Figure 6 shows immunoblots
of Gs expressed in the cortex, outer medulla, and inner medulla. The
anti-Gs antibody recognizes a doublet at 50 kD and 45 kD. The
expression of Gs proteins was decreased significantly in the outer
medulla and inner medulla after cisplatin treatment but was not affected in
the cortex.
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Adenylyl Cyclase Activity
Figures 7 and
8 show cAMP generation in the
cortex, outer medulla, and inner medulla. The generation in response to AVP
was blunted by the cisplatin treatment and the effect was most prominent in
the outer medulla. Conversely, the cAMP generation evoked by forskolin was not
affected by cisplatin treatment, whereas that stimulated by sodium fluoride
was attenuated significantly (Figure
8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the different regions of the kidney, the outer medulla may be most vulnerable to cisplatin. There was a dose-dependency in the degree of polyuria and in the extent of the reductions of AQP channels. By the use of a lower dose (4 mg/kg), the expression of AQP1 and AQP2 was decreased only in the outer medulla, whereas that of AQP3 was not affected. This finding is in line with that in a previous investigation (24), in which cisplatin-induced histopathologic features were limited primarily to the S3 segment of the proximal tubule located in the outer medulla.
AQP2 channels are regulated short term as well as long term (10). Although the expression of AQP2 was reduced, the reductions were in parallel in the apical membrane-enriched and subapical vesicle-enriched fractions, suggesting a preserved targeting. Similar findings have been noted in several acquired nephrogenic diabetes insipidus syndromes, such as urinary tract obstruction (16), lithium-induced nephropathy (17), and chronic renal failure induced by surgical renal mass reduction (18). However, there may have been a transient impairment of AQP2 targeting during the initial stage after the cisplatin treatment, and only a reduced expression may prevail in the long run.
AQP1 is present in the cells of the proximal tubule and the descending limb of Henle's loop (7). Histopathologically, partial to complete desquamation and necrosis of the tubular epithelium were variably present along the straight portion of the proximal tubule after treatment with cisplatin. Therefore, the decreased abundance of AQP1 proteins may be attributed primarily to a structural damage in the proximal nephron.
It has been suggested that AVP has a regulatory role on the expression of AQP2 and AQP3 (13, 14). Therefore, an abnormality in the AVP/cAMP pathway may result in an altered regulation of AQP2 and AQP3. The biologic effects of AVP determining the water permeability in the collecting duct are initiated by membrane-bound V2 receptors that are coupled to adenylyl cyclase complex. The adenylyl cyclase consists of three major parts: receptor, G protein, and catalytic unit. G protein acts as a transducer and sends a signal from the hormone-occupied receptor to the catalytic unit (25). The catalytic unit then induces final enzymatic activity responsible for ATP hydrolysis to generate cAMP.
Our study showed that the cAMP generation in response to AVP was diminished
after cisplatin treatment. On the contrary, the cAMP generation in response to
direct activation of the catalytic unit of adenylyl cyclase was not affected,
whereas that stimulated by a receptor-independent and G-protein-dependent
manner was significantly blunted. Therefore, the cisplatin-induced defect
seems to lie primarily at the G protein. This is substantiated further by the
reduced expression of Gs proteins as assessed by Western blot analysis.
The decreases in the abundance and activity of Gs proteins may result
in a diminished generation of cAMP and hence AQP2 and AQP3 proteins.
One may argue that there may also be some derangement upstream affecting
the Gs, such as the expression of V2 receptors or the binding of AVP to
the receptor. In addition, Gs may not be a rate-limiting factor because
its expression is relatively high compared with that of V2 receptor. Further
studies will be needed to examine these possibilities in the cisplatin-induced
nephropathy.
In summary, our results suggest that a reduced expression of AQP water channels accounts at least in part for the cisplatininduced urinary concentration defect.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, control; [UNK], cisplatin 4 mg/kg; [UNK], cisplatin 8 mg/kg;
*, P < 0.05; **, P < 0.01
versus control.
, control; [UNK], cisplatin
(8 mg/kg)-treated rats. Each point represents mean ± SEM of six
experiments. *, P < 0.05; **, P
< 0.01 versus control.