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Division of Nephrology, Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong.
Correspondence to Professor Kar N. Lai, Department of Medicine, University of Hong Kong, Room 409, Professorial Block, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong. Phone: + 852-28554251; Fax: +852-28162863; E-mail: knlai{at}hkucc.hku.hk
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aquaporins (AQP), a family of newly discovered water channel proteins, provide a pathway for water movement across cell membranes that could not be accounted for solely by simple diffusion through the lipid bilayer of the cell membrane. AQP belong to the major intrinsic protein family of channel-like proteins, named after the first member of the family to be identified, the major intrinsic protein of the eye lens (5). In a previous study, only AQP1 was found in human peritoneal endothelial cells in both uremic and CAPD patients (6). As human peritoneal mesothelial cells (HPMC) play an important role in dialysis adequacy and fluid balance in CAPD patients, we examined in this study whether AQP-type water channels are present in cultured HPMC and in the mesothelial lining of peritoneal tissue. We confirmed the expression of AQP1 in peritoneal mesothelium. The expression of AQP1 in cultured HPMC and endothelial cells induced by increasing hyperosmotic milieu supports its role in in vivo osmotic water transport across the peritoneal barrier.
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Materials and Methods |
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Electrophoresis and Immunoblot Analysis for Studying the Specificity
of Anti-AQP1
Crude tissue or cell extracts were prepared from human kidney cortex, human
omentum, cultured peritoneal mesothelial cells, and an immortalized human
endothelial cell line (EA.hy 926; a gift from Dr. S. H. Lin, Baker Institute,
Melbourne, Australia) using a protease inhibitor cocktail (Sigma P2714) to
minimize protein degradation. The extracts (5 µg) were electrophoresed
through 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and
transferred to polyvinylidenedifluoride membrane. After the membrane was
blocked for 1 h at room temperature in blocking buffer (1% gelatin in
phosphate-buffered saline [PBS] with 0.05% Tween 20), it was incubated for 16
h with anti-AQP (1:2000) in PBS-Tween. The membrane was washed and incubated
for 2 h at room temperature with a peroxidaselabeled goat anti-rabbit Ig and
detected with ECL chemiluminescence (Amersham Pharmacia Biotech, Arlington,
IL).
Cell Culture of Human Peritoneal Mesothelial Cells and E.A.hy
926
HPMC were obtained from omental tissues of patients who were undergoing
elective abdominal surgery. Full consent was obtained from patients for
removing small pieces of omentum from resected abdominal tissues for
experimental studies. The cells were isolated and characterized using
procedures described previously
(7). Mesothelial cells showed
typical cobblestone appearance at confluence. Immunohistochemical staining was
performed with monoclonal antibodies (mAb) for human cytokeratin 18 and
vimentin, as well as a polyclonal antibody for human factor VIII.
Visualization was done with a fluorescein-conjugated secondary antibody. All
cells were positive for cytokeratin and vimentin (excluding the presence of
fibroblasts), but factor VIII antigen was not detected (excluding the presence
of endothelial cells). The cells were maintained in medium 199 (Life
Technologies, Gaithersburg) supplemented with penicillin (100 U/ml),
streptomycin (100 µg/ml), L-glutamine (2 mM), transferrin (5 µg/ml),
insulin (5 µg/ml), and 10% vol/vol FCS. HPMC were incubated at 37°C in
a humidified atmosphere with 5% CO2. Once monolayers of HPMC
reached confluence, the culture medium was removed and medium 199 containing
0.1% (vol/vol) FCS was added to the cells for 48 h before further culture
experiments. Under these conditions, the HPMC remained in a nonproliferative
viable condition for up to 96 h
(8). Confluent cells were split
at a ratio of 1:3, and all experiments were performed with cells of first to
second passage.
The endothelial cell line (EA.hy 926) was maintained in Dulbecco's modified Eagle's medium supplemented with HAT (hypoxanthine 100 µM, aminopterin 0.4 µM, thymidine 16 µM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% FCS. The characterization of the cell line was reported previously by Edgell et al. (9).
AQP Protein Staining on Cultured HPMC and Human Peritoneal
Membrane
HPMC were grown to confluence in an eight-well chamber slide (Nalge Nunc
International, Naperville, IL). The confluent cells were fixed with 2%
paraformaldehyde in PBS for 5 min at 4°C and washed with PBS.
Paraffin-embedded human peritoneal membrane was obtained from four nonuremic
patients who were undergoing laparotomy (two men and two women aged 45 to 63
yr with diagnosis of colon cancer) and from four CAPD patients (two men and
two women aged 48 to 66 yr on dialysis for 12 to 25 mo with no evidence of
ultrafiltration failure) with removal of the peritoneal catheter after
repeated tunnel tract infection. The tissues were sectioned at a thickness of
4 µm, and the sections were deparaffinized with xylene and then rehydrated
through a descending gradient of ethanol. AQP1 expression on the HPMC
monolayer and on the paraffin sections was determined by immunohistochemical
staining using specific antibodies for AQP1. Briefly, the slides were
incubated with 0.5% hydrogen peroxide for removal of endogenous peroxidase
activity. Nonspecific binding was blocked by incubation of the slides for 30
min with blocking buffer (5% normal goat serum and 3% bovine serum albumin in
PBS). The sections were then incubated with anti-AQP1 antibody (0.5 µg/ml)
overnight. The bound rabbit anti-AQP1 antibody was visualized as brown color
using the Dako Envision Plus System. To ensure the specificity of the
staining, we performed the following labeling controls: (1) the
primary antibodies were substituted with preimmune rabbit immunoglobulins,
(2) staining was carried out without either the primary antibodies or
the peroxidase-labeled polymer, and (3) the primary antibodies were
preincubated with 50 mg/ml COOH-terminated peptides of AQP1. Some sections
were counterstained with hematoxylin before mounting.
Double immunohistochemical staining was performed on paraffin sections to confirm that the staining of AQP1 was located in mesothelial cells using techniques described previously (10). Briefly, sections first were stained for AQP1 as described above. After the first labeling and color development (brown color for positive AQP1 signal), sections were treated by 10 min of microwave heating in 0.01 M sodium citrate buffer (pH 6.0) at 2450 MHz and 800 W. The sections were blocked again with blocking buffer and were incubated with mouse anti-human mesothelial cell mAb (clone HBME-1, 1 µg/ml) for 1 h. The sections then were incubated sequentially with alkaline phosphatase-conjugated goat anti-mouse IgG and mouse alkaline phosphatase anti-alkaline phosphatase (Dako) and then developed with Fast Blue BB Salt, which gives a blue color for positive reaction.
AQP Protein Expression on Cultured HPMC and EA.hy 926 Exposed to
Different Osmotic Agents
HPMC and EA.hy 926 were grown to confluence in eight-well chamber slides
(Nalge Nunc International) and exposed to glucose, mannitol, or urea at
increasing concentrations (0, 50, 100, or 200 mmol) for a defined time period
at 37°C. To study whether AQP1 synthesis was inducible with extended
exposure to different osmotic agents, we performed similar experiments with
cells cultured for exposure to glucose (100 mmol) or mannitol (100 mmol) for
defined time periods (0, 3, 6, or 24 h) at 37°C. AQP1 expression on the
monolayer of cells was determined immunohistochemically as described
above.
RNA Extraction and cDNA Synthesis
Total RNA was extracted from HMPC or EA.hy 926 monolayers using QIAGEN
RNeasy kit (QIAGEN GmbH, Hilden, Germany). All RNA samples prepared from 1
x 106 cells were dissolved in 20 µl DEPC-H20 and were
stored at -70°C until assay. The quality of RNA was checked by
formaldehyde agarose gel electrophoresis, and RNA was quantified by absorbance
at 260 mm. Five µg of total RNA was reverse transcribed to cDNA with
Superscript II reverse transcriptase (Life Technologies, Paisley, UK) in a
20-µl reaction mixture containing 160 ng oligo (dT)12-18' 500
µM of each dNTP, and 40 U RNase inhibitor for 10 min at 37°C, 60 min at
42°C, and 5 min at 99°C. cDNA was stored at -20°C until further
use.
AQP1 Gene Expression by Cultured HPMC and EA.hy 926
HPMC or EA.hy 926 (1 x 106) were grown to confluence in
six-well cell culture plates (Falcon, Becton-Dickinson, Cowley, UK). The cells
then were exposed to glucose, mannitol, or urea for defined time periods (0,
3, 6, or 24 h) at 37°C. Total cellular RNA then was extracted and reverse
transcribed to cDNA as described above. Specific primers for AQP1 and
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were designed from known
GeneBank sequences (AQP 1 NM_000385; GAPDH J04038). The sequences of each
primer were as follows: (1) AQP1, sense primer
5'-AGATCAGCATCTTCCGTG and anti-sense primer 5'-AGTTGTGTGTGATCACCG;
(2) GAPDH, sense primer 5'-TGAAGG-TCGGAGTCAACGGATTTGGT and
anti-sense primers 5'-CATGTGGGCCATGAGGTCC-ACCAC. The PCR was carried out
in the following profile: 1st cycle: 94°C for 3 min, 55°C for 1 min,
72°C for 1 min; 2nd to 30th cycles: 95°C for 45 s, 55°C for 40 s,
72°C for 45 s. The final cycle was 94°C for 1 min and 72°C for 10
min. The PCR reactions from AQP1 and control (GAPDH) amplicons were mixed and
separated by 1.5% (wt/vol) agarose gels and stained with ethidium bromide, and
the gel image was captured and analyzed using the Gel Doc 1000 Gel
Documentation System and Quantity One software (Bio-Rad Laboratories Ltd.,
Hercules, CA). The result of AQP1 mRNA yield was expressed as a ratio of AQP1
amplicon to GAPDH amplicon. The following precautions were taken to ensure the
validity of the results. (1) Precautions to avoid carryover of PCR
product included physical separation of pre-PCR and post-PCR mixtures and
aliquots of reagents. (2) The results were considered valid only if
these were consistent in repeated independent experiments (four times).
(3) Positive control including cell lines (HK2; human kidney proximal
tubular cell) that are known to express AQP1 water channel. (4)
Negative controls including cells (HepG2, hepatocyte cell line) that are not
known to produce the AQP1 water channel, as well as a reaction mixture without
RNA or DNA were run in each experiment. (5) Negative controls for PCR
consisted of reagent control that RNA was replaced by DEPC-dH2O (6)
Confirmation of RNA PCR signal was performed by repeating the PCR without
reverse transcription. (7) Further validation of RNA PCR signal was
achieved by treating the extracted RNA with DNase free RNase before reverse
transcription and PCR.
Cellular Enzyme-Linked Immunosorbent Assay for Assay of AQP1 on
Cultured Cells
HPMC or EA.hy 926 (1 x 106) were grown to confluence in
96-well plates (Falcon, Becton-Dickinson). The cells were exposed in
quadruplicate to different concentrations of glucose, mannitol, or urea (0 to
200 mmol) for defined time periods (0, 3, 6, or 24 h) at 37°C. After
incubation, culture medium was removed and the cells were washed once with PBS
and fixed with 1.5% (vol/vol) paraformaldehyde in PBS (pH 7) for 30 min at
4°C. The cells were then washed with PBS, and nonspecific binding was
blocked by incubating with 5% normal goat serum and 3% bovine serum albumin in
PBS for 1 h. After the cells were washed with PBS, they were incubated with
100 µl of anti-AQP1 antibody (0.1 µg/ml) overnight. The cells were
washed thrice with PBS, and 100 µl of peroxidase-conjugated goat
anti-rabbit immunoglobulins (Dako) diluted 1:1000 was added and incubated for
another hour. After a final wash, 100 µl/well of ophenylenediamine (OPD)
was added and the plates were kept in the dark at room temperature for a
minimum of 30 min. Finally, the reaction was terminated by the addition of 50
µl/well 2 M sulfuric acid, and the absorbances were measured at 490 nm
using an enzymelinked immunosorbent assay (ELISA) plate reader (Titertek
Multiskan MCC/340, Labsystem, Helsinki, Finland). All experiments were
performed in the same batch to avoid interbatch variation.
Measurement of Osmolality
The osmolality of culture medium before and after the addition of various
doses of osmotic agents was measured by freezing point depression using the
Advanced Osmometer Model 3900 (Advanced Instruments, Norwood, MA), with an
interassay precision (coefficient of variation of < 1% at 281 mmol/kg). All
three osmotic agents (glucose, mannitol, and urea) led to a similar stepwise
increase of osmolality from 287 mOsm in culture medium alone to 313 mOsm, 396
mOsm, and 495 mOsm with 50 mM, 100 mM, and 200 mM osmotic agent,
respectively.
Statistical Analyses
All data (from four experiments) were expressed as means ± SD.
Intergroup differences for continuous variables were assessed by unpaired
t test. The AQP1 mRNA or AQP1 expressed on cultured cells after
exposure to different osmotic agents and at different time intervals were
analyzed with multivariate ANOVA for repeated measures. Statistical analysis
was performed using statistical software (Statview, SAS Intelligence, Cary,
NC). Significance was defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue Localization of AQP1 Proteins in the Peritoneal Membrane
To localize the in vivo site of AQP1 synthesis within the
peritoneal membrane, paraffin sections of omental tissue were
immunohistochemically stained using specific antibodies for AQP1 and
mesothelial cells as described previously. AQP1 protein was detected in
erythrocytes, mesothelial cells, and endothelial cells of venules but not in
the endothelial lining of arterioles
(Figure 2, A through C). The
localization of AQP1 protein in peritoneal mesothelial cells was confirmed by
double immunohistochemical staining (Figure
2, E through G). Examination at higher magnification revealed that
the AQP1 distribution was both luminal (apical) and abluminal (basolateral).
In omental tissues from patients who previously underwent CAPD, there was no
obvious difference in the detection of AQP1 in the lining mesothelial cells.
However, there were discrete areas with loss of mesothelium and disruption of
the interstitium in peritoneal biopsies from patients on CAPD. Not infrequent,
there was intimal thickening of vessels with increased interstitial thickening
of the peritoneum. AQP1 was not detected in peritoneal lining denuded of
mesothelial cells or replaced by fibrous tissue
(Figure 2, H and I).
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Gene and Protein Expression of AQP1 by HPMC and EA.hy 926
Reverse transcription-PCR (RT-PCR) with sequence-specific cDNA primers was
performed on growth-arrested, confluent HPMC or EA.hy 926 monolayers. Primary
human proximal tubule cells and HK-2 cell lines were used as positive
controls. Constitutive expression of AQP1 mRNA was detected in both types of
cells (Figure 3). The cDNA
product of AQP1 is identical to the known GeneBank sequence.
Immunohistochemical staining using specific anti-AQP antibody on quiescent
HPMC monolayers confirmed that AQP1 protein was expressed constitutively
(Figure 4A). Similarly, AQP1
protein was expressed constitutively in cultured EA.hy 926 (data not
shown).
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Effect of Glucose on AQP Gene Expression
To mimic the peritoneal milieu in acute peritoneal dialysis, glucose
adjusted to a concentration of 100 mM (1.8% wt/vol) was added to the culture
medium in which cells were incubated for different time periods. AQP1 gene
expression was significantly upregulated in both types of cells after 3 h of
incubation with glucose. By 24 h, the normalized AQP1/GAPDH ratio had
increased by 31% and 15% compared with control (constitutive) values in HPMC
and EA.hy 926, respectively (Figure
5A). Similarly, incubating either cell type with increasing
concentration of glucose for 16 h enhanced AQP1 gene expression
(Figure 6A). Upregulation in
AQP1 mRNA expression was significant even in HPMC or EA.hy 926 exposed to a
lower concentration of glucose (50 mM), rising to 128% and 12% above that of
control (constitutive) values, respectively. In all experiments under
identical conditions, the upregulation in AQP1 mRNA in HPMC was significantly
higher than that of EA.hy 926 (P < 0.001).
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Effect of Glucose on AQP1 Protein Synthesis
To confirm that the AQP transcripts led to protein biosynthesis, we grew,
growth arrested, and incubated with glucose (100 mM) HPMC or EA.hy 926 to
confluence in eight-well chamber slides, for various time points. The
monolayer of cells then was stained immunohistochemically for AQP1 antigen as
described previously. Positive control for AQP1 protein was confirmed by
immunohistochemical studies on cultured human proximal tubule cells, HK-2 cell
lines, and proximal renal tubules in paraffin sections of renal cortex from
human. Treatment with glucose led to a time-dependent progressive increase in
the number of cells that stained positively for AQP1 (data not shown). After 3
h of incubation, an appreciable proportion of HPMC displayed positive AQP1
staining (Figure 2D). Treatment
with increasing concentration of glucose led to a progressive increase in the
number of cells that stained positively for AQP1
(Figure 4). Similarly,
increasing staining for APQ1 protein was detected in cultured EA.hy 926 after
exposure to glucose (data not shown).
To quantify further the stimulatory effect of glucose on AQP1 synthesis by HPMC or EA.hy 926, we performed a cellular ELISA on monolayers of quiescent cells exposed to glucose (100 mM) for 3 to 24 h. As depicted in Figure 5B, there was a time-dependent increase in the synthesis of AQP1 on exposure to glucose. By 24 h, AQP1 synthesis had surged by more than 32% and 63% over baseline values in HPMC and EA.hy 926, respectively.
To quantify the dose response, we applied escalating doses of glucose (0 to 200 mM) to monolayers of quiescent cells for 16 h. A dose-dependent increase in the production of AQP1 was detected when HPMC or EA.hy 926 was incubated with progressively higher concentrations of glucose (Figure 6B). The dose-related rise in AQP1 production in EA.hy 926 seemed to plateau earlier than that in HPMC. There were detectable morphologic changes in the cells (shrinkage in cell volume) with higher than 50 mM glucose in the culture medium (data not shown).
Effect of Other Osmotic Agents on AQP1 Gene Expression
A stepwise upregulation in AQP1 mRNA expression was observed in HPMC or
EA.hy 926 exposed to increasing concentration of mannitol (from 50 to 200 mM;
Figure 7A). There was a
parallel increased in AQP1 synthesis when cells were incubated with increasing
concentration of mannitol (Figure
7B). The findings of HPMC or EA.hy 926 when exposed to urea were
different from those with glucose or mannitol. Although exposure to a lower
concentration of urea (50 mM) induced an upregulation in both the gene
expression and the protein synthesis of AQP1, exposure to a higher
concentration (
100 mM) led to a significant fall in the gene expression
and protein synthesis of AQP1 (Figure
8). At these concentrations, cells showed marked morphologic
changes in the volume and viability. These findings likely were due to cell
injury and apoptosis induced by a high concentration of urea
(11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rippe et al. (15),
using computer simulations, first suggested that the peritoneal membrane
permeability is best described by a three-pore model. Large pores (>150
) allow the transport of macromolecules and,
together with small pores (20 to 25 ), the
transport of low-molecular-weight solutes such as glucose, urea, and
creatinine. An "ultrasmall, water-only" pore (3 to 5
) has been postulated to be selective for
water and responsible for the majority of osmotically induced water transport
(16). Recently discovered AQP
were suggested to have a pore size comparable to that of the ultrasmall pores
and might provide the molecular route for water movement through apparent
ultrasmall pores in the peritoneal barrier
(17,18).
It is apparent that examination of the expression of AQP in resident cells of
the peritoneum will provide better understanding of the mechanism of
transcellular water transport.
AQP1 has been localized to the peritoneum by RT-PCR and in situ hybridization (19), as well as by immunocytochemistry (20). In rat peritoneum, AQP1 has been localized to capillary endothelia and "mesangium/mesothelium" near the peritoneal luminal surface (20,21). Using affinity-purified polyclonal antibodies from the same laboratory, Combet et al. (22) failed to detect AQP1 in the mesothelium in a rat model of acute peritonitis. AQP1 has also been detected in microvascular endothelial cells in peritoneal biopsies of uremic patients (6,23). Similarly, the signal for AQP1 was restricted to endothelial cells of peritoneal capillaries and venules but not in arterioles or mesothelial cells when immunocytochemistry was performed using polyclonal antibodies from the same laboratory. Recent studies showed a significant reduction of osmotically induced water transport upon AQP1 deletion in AQP1 knockout mice (21). In contrast, AQP1 deletion exerted little effect on slow isosmolar fluid absorption from the peritoneal cavity. It seems, therefore, that AQP1 is crucial in transendothelial water transport when driven by osmosis in peritoneal dialysis, yet the presence of AQP1 in peritoneal mesothelial cells is not well documented.
Mesothelial cells behave as perfect osmometers: they shrink when exposed to hypertonic medium and swell when exposed to medium with a lower osmolality. The change in cellular water content in mesothelial cells is immediate—within 5 min of exposure to the hyperosmotic medium (24). Volume of cultured mesothelial cells was reduced by 36% of control value when exposed to medium supplemented with 90 mM glucose for 90 min (24). It is important to clarify whether transmembrane water transport induced by osmotic agents in peritoneal dialysis is mediated by endothelial AQP1 alone or the mesothelial cells may also play a role. The latter possibility is raised by the murine experiment of Akiba et al. (25), who detected AQP1 transcripts from peritoneal cells removed by peritoneal lavage. The cells were identified as mesothelial in origin with strong staining of cytokeratin 18. The AQP1 signal in cultured rat mesothelial cells increased with higher concentration of glucose (2.1- and 3.7-fold increase in AQP1 expression versus 2.6- and 2.9-fold increase in our human data after incubation with 100 mM and 200 mM glucose). Unfortunately, as the AQP protein was not measured and AQP1 mRNA might be derived from other cell sediments, a definitive conclusion cannot be drawn.
In this study, we demonstrated that AQP1 mRNA is present in HPMC constitutively. With the use of a different peptidederived, affinity-purified polyclonal anti-AQP1 antibody and a peroxidase-labeled polymer for more sensitive visualization of signals, constitutive AQP1 protein was readily detected in resting HPMC. The localization of AQP1 protein in peritoneal mesothelial cells was confirmed by double immunohistochemical staining of mesothelial lining of human peritoneal membrane. The mesothelial cells, identified by their position and by a specific mouse anti-human mesothelial cell mAb (clone HBME-1), clearly expressed AQP1 constitutively. On the histologic examination, we confirmed the previous observation that AQP1 expression is restricted to endothelial cells of peritoneal capillaries and venules but not in arteriole (23). More important, the expression of AQP1 in HPMC not only is constitutive but also can readily be upregulated upon exposure to osmotic agents as in peritoneal dialysis leading to rapid transmembrane water transport. We developed a novel cellular ELISA for detecting AQP1 on the cell surface. The assay was confirmed by parallel flow cytometric analysis (data not shown). Our in vitro studies showed clearly that transcription and biosynthesis of AQP1 in HPMC is inducible by osmotic agents such as glucose. There was significant enhancement of AQP1 biosynthesis upon exposure to glucose in a time- and dose-dependent manner. Similar findings were observed in the AQP1 biosynthesis by an endothelial cell line, EA.hy 926. Of particular interest, the upregulation in AQP1 mRNA or biosynthesis in mesothelial cells was always significantly higher than that of endothelial cells when the experiments were conducted under identical conditions. The parallel experiments with mannitol adding to the medium suggest that our findings with glucose merely reflect its role as an osmotic agent in osmotically induced water transport. The experimental findings with lower concentration of urea adding to the medium also support our observation that HPMC readily upregulates its AQP1 biosynthesis upon exposure to any osmotic agent. A higher concentration of urea (raising the osmolality to 550 mOsm) readily induced apoptotic cell death in mouse renal collecting duct cells (11). It seems, from our findings in cell morphology and AQP1 expression, that both HPMC and endothelial cell line (EA.hy 926) are more susceptible to the cytotoxic effect of urea when the urea concentration exceeds 100 mM (raising the osmolality to 400 mOsm).
Our demonstration of constitutive AQP1 in HPMC, which is inducible by an osmotic agent, poses a question on the traditional understanding of endothelium of peritoneal capillaries and venules as being the major site of water transport across the peritoneal membrane (6,22). The luminal and abluminal distribution of AQPI in HPMC, shown in this study, affords a transcellular mechanism of water movement across the peritoneal mesothelial lining after the transport of water from the capillary lumen to the interstitium near the peritoneal luminal surface. The crucial question is how this new information of localization of AQP1 in peritoneal cells is related to ultrafiltration failure in long-term CAPD. Obviously, this can not be answered in our in vitro experiments with HPMC exposed to osmotic agents for a short period. However, the histologic examination of omentum from our patients on CAPD sheds some intriguing information. There was evidence of denudation of mesothelium, interstitial fibrosis, and luminal narrowing of vessels. Despite that there was no obvious difference in the detection of AQP1 in the lining mesothelial cells between control subjects and CAPD patients, AQP1 was not detected in peritoneal lining denuded of mesothelial cells or replaced by fibrous tissue. One may speculate that such peritoneal changes with long-term CAPD will lead to decreased expression of AQP1 on the peritoneal lining denuded of mesothelial cells. The effect of AGE accumulation and vascular sclerosis on the AQP1 expression by endothelium remains to be examined. Further in vitro studies with AGE and animal models of chronic peritoneal dialysis may provide answers to some of these questions.
In summary, we demonstrated for the first time AQP1 expression in HPMC. Osmotic agents such as glucose and mannitol upregulate both mRNA and protein expression of this AQP. Though the exact pathophysiologic sequel to transmembrane water transport in long-term CAPD remains to be elucidated, our findings may pave the way for future research into the mechanisms of ultrafiltration failure in CAPD.
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Acknowledgments |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Kidney Int 43:226
-233, 1993[Medline]
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