Water Channel AQPl, 3, and 4 in the Human Peritoneum and Peritoneal Dialysate


Takashi Akiba, Tomoko Ota, Kiyohide Fushimi, Hiroyuki Tamura, Toshihiko Hata, Sei Sasaki, Fumiaki Marumo

To clarify the mechanism of water transport driven by osmotic gradient through "ultrasmall pores " in the peritoneum, we tried to identify water channels in the peritoneum and cells in the peritoneal dialysate.

Peritoneum was surgically excised from uremic patients at the insertion or removal of a cathete1: Sediment was collected from 2 L of peritoneal dialysate by centrifugation at 1500 rpm. RNA was extracted and amplified by reverse transcription-polymerase

chain reaction (RT-PCR). Contamination of reticulocytes was tested by the presence of ankyrin mRNA. Peritoneal tissue expressed aquaporin (AQP) 1, 3, and 4 (AQP 1 > 3 > 4). Sediment of dialysate expressed mRNA ofAQP1 and AQP3 (AQP1 > AQP3). The ,\,ample did not express ankyrin mRNA, indicating that the A QP 1 in the sediment did not originate from reticulocytes.

These data indicate that aquaporins are present in the peritoneum and might participate in water transport. Further quantitative analysis of aquaporin message,\' in the dialysate might clarifY the pathogenesis of water removal.failure.

Key words

Aquaporins, water channels, peritoneum, dialysate

From:

Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.


Introduction

Removal of excess body fluid is one of the major objectives of dialysis therapy for end-stage renal disease (ESRD). The osmotic gradient between glucose-containing dialysate and blood is the main force of water movement in peritoneal dialysis. The pathway of this osmotic-gradient-driven water transport was suggested to be through "ultrasmall pores" in the peritoneum with a pore size of 2 4 A (1).

Recently identified water channels, called aquaporins (AQP) (2,3), were suggested to have a pore size comparable to that of the ultrasmall pores of peritoneum (4). These channels included APQl (channel-forming integral membrane protein 28 kD) (2), AQP2 (water channel of collecting ducts), AQP3 (5), AQP4 (mercurial-insensitive water channel) (6), and AQP5. Pannekeet et al. recently showed the immunohistochemical presence of AQPl in the peritoneal capillary endothelial cells of uremic and continuous ambulatory peritoneal dialysis (CAPD) patients (7). Hayakawa et al. preliminarily reported that APQl and APQ4 were present in the rat peritoneum, while AQP2 and AQP3 were not (8).

We tried to identify water channels in the peritoneum and cells in the peritoneal dialysate in ESRD patients.

Material and methods

After obtaining informed consent, peritoneal tissue was surgically obtained from a uremic patient at the time of catheter insertion and from 2 CAPD patients at the time of removal of displaced catheters. Sediment was collected at 1500 rpm from 2 L of 2.5% Dianeal PD-2, 4-hour dwell (Baxter Healthcare, Deerfield, IL).

Total RNA was extracted from the homogenate of the peritoneal tissue with TRlzol Reagent (Gibco, Grand Island, NY). Cells in drained peritoneal dialysate were collected by centrifugation at 1500 rpm for 10 minutes. mRNA of the sediment was extracted with a QuickPrep Micro mRNA Purification Kit (Pharmacia, Uppsala, Sweden). Presence of reticulocytes in the peritoneal drainage was tested by examining ankyrin (ANK) mRNA (9).

RNA was subjected to RT -PCR using the followlUg prImers:

human AQPI sense primer

(AGATCAGCATCTTCCGTG),
antisense primer
(AGTTGTGTGTGATCACCG) (2),
human AQP3 sense primer
(TTTGCCATGTCCTTCCTG),
antisense primer
(GGCAAGGGCTGTAAAAAG) (5),
human AQP4 sense primer
(CTCAGCATTGCAACCATG),
antisense primer
(CACAGCTGGCAAAGATAG) (6),
human ANK sense primer
(TGTGAAGAAAGCTGCCCA),
antisense primer
(TTCATCTCTGCCTGCTCT) (9),
human B actin sense primer
(TGGACTTCGAGCAAGAGA),
and antisense primer
(CTGCTTGCTGATCCACAT).

Electrophoresis (using 2.0% agarose gel and ethidium bromide stain) was performed on PCR products, which were then observed under ultraviolet light.

Results

None of the patients showed any signs of tunnel infection, peritonitis, or ultrafiltration failure. AQP1, AQP3, and AQP4 were expressed in the peritoneal tissue of all patients (Figure 1 ). The relative strengths of their aquaporin expressions were AQPI > AQP3 > AQP4 in the same PCR condition. There was no difference in aquaporin expression between the patient who had no history of peritoneal dialysis and those who were on peritoneal dialysis for 12 or 29 months (data not shown).

Sediment of dialysate expressed mRNA of AQP 1 and AQP3, but not AQP4 (Figure 2). The expression of AQPl appeared stronger than that of AQP3 in the same PCR condition. The sample did not express ankyrin mRNA (Figure 3), indicating that the AQPl in the sediment did not originate from reticulocytes.

Discussion

These data are the first to demonstrate the presence of AQP1, AQP3, and AQP4 in the peritoneum from uremic patients, as well as the presence of AQPl and AQP3 in the sediment of peritoneal dialysate from uremic patients. The presence of aquaporins in human peritoneal tissue supports the hypothesis that a part of the water could pass through the ultrasmall pores in the peritoneum.

The presence of AQPl confirmed by our study is compatible with the immunohistochemical identification of AQPl in the peritoneum from uremic and CAPD patients (7). As Pannekeet et al. mentioned in their paper, their data did not exclude the possible presence of other members of the aquaporin family. In contrast, the message of AQP3 had not been detected in normal and uremic rat peritoneal tissue (8). This disagreement might be due to the species difference or the different sensitivities of the methods used.

The presence of aquaporins in the peritoneum raises the possibility of functional impairment of aquaporins leading to ultrafiltration failure.

The AQPl detected in the sediment of dialysate did not originate from reticulocytes, as shown by the absence of ankyrin message. The clinical significance of aquaporins in the sediment of dialysate is not clear. Kanno et al. showed that urinary excretion of AQP2 in patients with diabetes insipidus is a good index of the action of the antidiuretic hormone on the kidney (10). As an analogy to this, we can hypothesize that a quantitative estimation of aquaporin message in the dialysate may facilitate analysis of transport of water through the ultrasmall pores in clinical practice. Further experiments should be performed to test this hypothesis.

In summary, we confirmed the presence of AQP1 , AQP3, and AQP4 in the peritoneum from uremic patients as well as the presence of AQPl and AQP3 in the sediment of peritoneal dialysate from uremic patients. These aquaporins might be the equivalents of the ultras mall pores of peritoneum. The clinical value of quantitative analysis of aquaporin messages in the dialysate in the pathogenesis of water' removal failure in peritoneal dialysis patients awaits further study.

Acknowledgment

This study was partly supported by the research fund from the Ministry of Culture and Education (No. 06557087).

References

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Corresponding Author:
Takashi Akiba, ~11), Department of Internal Medicine, Tokyo Medical and Ocntal lJniversity, 1-5-45 Yushima Bunkyoku Tokyo II J. .Iapan.