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【关键词】 tubules
1Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo
2Division of Stem Cell Regulation Research, Osaka University, Osaka, Japan
3Department of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California
ABSTRACT
The aquaporin-7 (AQP7) water channel is known as a member of the aquaglyceroporins, which facilitate the transport of glycerol as well as water. Although AQP7 is abundantly expressed on the apical membrane of the proximal straight tubules in the kidney, the physiological role of AQP7 is still unknown. To investigate this, we generated AQP7 knockout mice. The water permeability of the proximal tubule brush-border membrane measured by the stopped-flow method was slightly but significantly reduced in the AQP7 knockout mice compared with that of wild-type mice (AQP7, 18.0 ± 0.4 x 103 cm/s vs. wild-type, 20.0 ± 0.3 x 103 cm/s). Although AQP7 solo-knockout mice did not exhibit a urinary concentrating defect, AQP1/AQP7 double-knockout mice had a reduction in urinary concentrating ability compared with AQP1 solo-knockout mice, suggesting that the amount of water reabsorbed through AQP7 in the proximal straight tubules is physiologically substantial. On the other hand, AQP7 knockout mice showed marked glyceroluria (AQP7, 1.7 ± 0.34 mg/ml vs. wild-type, 0.005 ± 0.002 mg/ml). This identified a novel glycerol reabsorption pathway in the proximal straight tubules. In two mouse models of proximal straight tubule injury, the cisplatin-induced acute renal failure (ARF) model and the ischemic ARF model, an increase in urine glycerol was observed (pretreatment, 0.007 ± 0.005 mg/ml; cisplatin, 0.063 ± 0.043 mg/ml; ischemia, 0.076 ± 0.02 mg/ml), suggesting that urine glycerol could be used as a new biomarker for detecting proximal straight tubule injury.
aquaporin-7; kidney; urea; biomarker
AQUAPORINS (AQPS) ARE MEMBRANE proteins that allow rapid water transport across the cell membrane (8, 25). At present, at least 13 AQPs have been identified and cloned in mammals (17). Some AQPs are classified as part of the aquaglyceroporin family, the members of which facilitate the transport of glycerol as well as water (7). AQP7 is an aquaglyceroporin that is abundantly expressed in kidney, testis, and adipocytes (6, 9). Moreover, AQP7 is also known to allow rapid urea and arsenite transport (6, 12). In the kidney, AQP7 is expressed on the apical membrane of the proximal straight tubules (S3 segment) (6), where AQP1 is also present (19). Studies of AQP1 knockout mice have shown that AQP1 plays a major role in proximal tubular water transport (15, 22). In fact, AQP1 knockout mice were found to develop polyuria due to both a defective water reabsorption mechanism in the proximal tubules and a dysfunctional countercurrent mechanism in the inner medulla (26). Other renal AQPs, such as AQP2, AQP3, and AQP4, are all present in the collecting ducts and are directly involved in water reabsorption to produce the final concentrated urine (2, 13, 14). Very recently, Maeda et al. (16) generated AQP7 knockout mice and reported on the phenotype characteristics only in the adipocytes, where AQP7 constitutes a glycerol-releasing pathway without which hypoglycemia occurs after starvation. We also found adipocyte hypertrophy in AQP7 knockout mice (3).
In this study, we focused on the renal phenotypes of AQP7 knockout mice to determine the physiological roles that AQP7 has in the kidneys. Although the contribution of AQP7 to the water permeability of the brush-border membrane vesicles of proximal straight tubules was identified to be minimal compared with that of AQP1, we identified a novel glycerol reabsorption pathway that may be important for preventing glycerol from being excreted into urine. Based on this new finding, we explored the possibility that the measurement of urine glycerol could be a new marker for the in vivo detection of proximal straight tubule injury.
MATERIALS AND METHODS
Generation of AQP7 knockout mice. The mouse genomic clones containing the AQP7 gene were isolated from a mouse genomic library (Stratagene) by plaque hybridization using mouse AQP7 cDNA as a probe. As shown in Fig. 1A, the construct was designed to result in the insertion of the three loxP sequences that flank exon 2 and a LacZ-Neo-selective marker. D3 embryonic stem (ES) cells (129/Sv) were electroporated with the targeting construct and selected for resistance to G418. Diphtheria toxin A was used for negative selection. To screen homologous recombinant ES clones, we used PCR with the following primers: AQP7 flanking primer (5'-ATCCTGTGGTATGCTGGGGTG-3'; R-Wild) and neo-specific primer (5'-CGTGATATTGCTGAAGAGCT-3'; F-Neo) (Fig. 1A). Additional Southern blot analysis was used to verify the results of PCR screening. Following sequence verification, targeted ES cells were injected into C57BL/6 blastocysts to generate chimeric mice. The resulting male chimeras were mated with female C57BL/6J mice. Tail biopsies from agouti-pigmented F1 animals were genotyped by PCR as mentioned above. To delete exon 2 of the aqp7 gene, aqp7(flox/+) offspring were mated with the Cre recombinase transgenic mice (CAG Cre mouse). Confirmation of the deletion of exon 2 and Neo cassette was done using PCR with a common forward primer (5'-CGTGTCTCATGTCATGTGAC-3'; F1) paired with a reverse exon 2-specific primer (5'-CAGAGCCACCTGAAGTGAGG-3'; R1) to detect the wild allele and a reverse intron 3-specific primer (5'-AGACAATCCAGGAAGACTGC-3'; R2) to detect the mutant allele (Fig. 1A). To produce AQP1/AQP7 double-knockout mice, AQP1 (15) and AQP7 knockout mice were intercrossed. The protocols for these studies were approved by the Institutional Animal Care and Use Committees of Tokyo Medical and Dental University (no. 0050119).
Water permeability in brush-border membrane vesicles from the outer medulla. Sealed brush-border vesicles from the outer medulla of the kidney were isolated by a magnesium aggregation procedure (1, 27). To obtain the outer medulla vesicles, the cortex and the inner medulla were removed from kidneys sliced 1 mm thick. Next, the isolated outer medullas were homogenized and centrifuged to remove unbroken cells. MgCl2 was added to the homogenate to a final concentration of 30 mM. Vesicles were isolated by 48,000-g centrifugation and suspended in 50 mM mannitol-Tris buffer, pH 7.4. Osmotic water permeability was measured by stopped-flow light scattering. Briefly, vesicles were suspended in 50 mM mannitol-2 mM Tris (55 mosmol/kgH2O) and mixed in <1 ms with an equal volume of hyperosmolar buffer (155 mosmol/kgH2O) to give a 50 mosmol/kgH2O inwardly directed osmotic gradient. Ninety-degree light scattering was measured at a wavelength of 520 nm by a Stopped-Flow Reaction Analyser (Applied Photophysics, Leatherhead, UK) and analyzed by SX.18MV Spectrometer Software (Applied Photophysics). The time course of the vesicle volume is given by
(27)
where V(t) is the vesicle volume, VW is the molar volume of water, Pf is the osmotic water permeability, S/V0 is the initial vesicle surface-to-volume ratio, Ci(t = 0) is the initial intravesicular osmolality, and C0 is the constant solution osmolality. To calculate the S/V0, the diameter of the vesicles was measured by the electron microscopic negative stain method. Water permeability was computed using a vesicle surface-to-volume ratio of 2.5 x 105 cm1.
Immunoblot analysis and immunohistochemistry. For immunoblot analysis, tissues were homogenized in a solution containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, and a protease inhibitor cocktail (Roche Diagnostics). The tissue homogenate was centrifuged twice at 7,000 rpm at 4°C for 15 min each, and the supernatant was centrifuged at 50,000 rpm at 4°C for 10 min. The resultant pellet was resuspended and denatured in an SDS sample buffer at 70°C for 10 min, separated by SDS-PAGE, and transferred to an Hybond-ECL membrane (Amersham). The membranes were incubated with a rabbit anti-AQP7 antibody. The signal was detected by anti-rabbit IgG (Fc) AP conjugate (Promega, Madison, WI) and Western blue (Promega). Immunohistochemical analysis was performed on cryostat sections of the mouse kidney using AQP7 antibody and Alexa Fluor 568-conjugated anti-rabbit secondary antibody. Immunofluorescent images were obtained using a LSM510 laser-scanning confocal microscope system (Carl Zeiss, Jena, Germany). AQP7 antibody was generated as follows: the COOH-terminal cytosolic portion of AQP7 (784861) was cloned into the pGEX6p-1 vector (Amersham), and a glutathione-S-transferase (GST) and AQP7 fusion protein was obtained using a Prepacked Glutathione Sepharose 4B column (Pharmacia Biotech) and used for immunization. Antiserum was preabsorbed by GST protein to eliminate the anti-GST antibody. Specificity of the antibody was confirmed by the disappearance of signals following preincubation with the immunizing fusion protein.
Animal study. Serum and urine glycerol levels were measured by a Glycerol Assay Kit (Megazyme, Wicklow, Ireland). Urea levels were measured using an Urea N B Kit (Wako, Osaka, Japan). Urine osmolality was measured with a Fiske One-ten Osmometer (Fiske Associates, Norwood, MA). To generate cisplatin-induced acute renal failure, cisplatin (30 mg/kg) was intraperitoneally injected into wild-type mice. A kidney ischemia-reperfusion model was generated by clamping bilateral renal arteries for 23 min and subsequently reperfusing them. In these models, the mice do not eat or drink. Therefore, as control samples, we measured the urine and plasma glycerol levels in mice deprived of food and water for 24 h. In addition, to obtain pretreatment data, we measured the urine and plasma glycerol levels before the injection of cisplatin or before the operation to induce ischemia.
Statistical analysis. Results obtained in the knockout mice were compared with those in the wild-type mice by Student's t-test. When more than three groups were compared, one-way ANOVA was used followed by Fisher's post hoc test.
RESULTS
Generation of AQP7 knockout mice. We crossed chimeric mice from two independent recombinant ES clones with C57BL/6 mice to produce AQP7 (flox/+) mice. The generation of AQP7 (flox/+) mice was verified by PCR (Fig. 1B). Then, to delete exon 2 from the aqp7 gene, we crossed AQP7 (flox/+) mice with the Cre recombinase mice. The Cre-mediated excision of exon 2 and Neo cassette was verified by PCR, as shown Fig. 1C. The absence of AQP7 protein was confirmed by immunoblotting and immunohistochemistry (Fig. 1, D and E).
Water permeability in the brush-border membrane vesicles obtained from the outer medulla of AQP7 knockout mice. To investigate whether AQP7 contributes to the water permeability of the brush-border membranes in proximal straight tubules, we measured water permeability of the brush-border membrane vesicles obtained from the outer medulla of the kidney. Osmotic water permeability was measured over the time course of 90° scattered light intensity in response to a rapidly imposed osmotic gradient. There was no significant difference among the diameters of vesicles in each preparation (data not shown). As shown in Fig. 2B, the water permeability of the vesicles obtained from the outer medulla of AQP7 knockout mice (18.0 ± 0.4 x 103 cm/s, n = 4) was slightly but significantly lower than that of wild-type mice (20.0 ± 0.3 x 103 cm/s, n = 4, P < 0.001). These results indicate that AQP7 makes a small contribution to the water permeability of the proximal straight tubules. To compare the contribution of AQP7 with that of AQP1, we also measured the water permeability of the brush-border membrane vesicles from AQP1/AQP7 double-knockout mice. Before this experiment, we used Western blotting to confirm that there was no compensatory increase in AQP1 and AQP7 expression in AQP7 and AQP1 knockout mice, respectively (data not shown). As shown in Fig. 2B, the water permeability of the vesicles was found to be decreased more in the AQP1/AQP7 knockout mice than in the AQP1 knockout mice. Compared with AQP1 knockout mice, AQP1/AQP7 double-knockout mice showed significantly lower water permeability (AQP1, 3.3 ± 0.11 x 103 cm/s vs. AQP1/AQP7, 2.4 ± 0.14 x 103 cm/s, n = 5, P < 0.001). Based on these results and previously published data (15), the estimated contribution of AQP7 to the water permeability in the proximal straight tubules is one-eighth that of AQP1.
Urinary concentrating defect of AQP1/AQP7 double-knockout mice. As anticipated from the small decrease in water permeability of the AQP7 solo-knockout mice, these mice did not show a urinary concentrating defect, which was observed in AQP1 knockout mice (15) (data not shown). Thus we compared the urinary concentrating ability of AQP1/AQP7 double-knockout mice with that of AQP1 knockout mice to investigate whether the additional decrease in water permeability of the brush-border membranes caused by AQP7 deletion resulted in any additional functional consequences. We wished to determine whether the decrease in water permeability caused by AQP7 was physiologically important. As shown in Fig. 3A, under normal conditions urine volume increased significantly in AQP1/AQP7 double-knockout mice compared with AQP1 knockout mice (AQP1/AQP7, 7.3 ± 0.46 ml vs. AQP1, 5.7 ± 0.31 ml, n = 6, P < 0.03). Similarly, before and after a 36-h water-deprivation period, urine osmolality was significantly reduced in AQP1/AQP7 double-knockout mice compared with AQP1 knockout mice (AQP1/AQP7, 597 ± 18 mosmol/kgH2O vs. AQP1, 715 ± 39 mosmol/kgH2O, before water deprivation, P < 0.03; AQP1/AQP7, 626 ± 65 mosmol/kgH2O vs. AQP1, 810 ± 16 mosmol/kgH2O, after water deprivation, P < 0.03, n = 6) (Fig. 3B). Furthermore, after dehydration significantly greater weight loss was observed among AQP1/AQP7 double-knockout mice compared with AQP1 knockout mice (AQP1/AQP7, 31.0 ± 0.54% vs. AQP1, 28.2 ± 0.46%, n = 6, P < 0.01) (Fig. 3C).
Reabsorption of glycerol through AQP7 in vivo. To investigate whether AQP7 has an important role to play in the transport of glycerol in the kidney, we measured the serum and urine glycerol concentrations of AQP7 knockout mice. The serum glycerol level in AQP7 knockout mice was lower than that of wild-type mice, although this difference was not significant (AQP7, 0.036 ± 0.007 mg/ml vs. wild-type, 0.042 ± 0.004 mg/ml, n = 10) (Fig. 4A). However, the urine concentration of glycerol in AQP7 knockout mice was much higher than in wild-type mice (AQP7, 1.7 ± 0.34 mg/ml vs. wild-type, 0.005 ± 0.002 mg/ml, n = 10, P < 0.0001) (Fig. 4B). These results indicate that glycerol is reabsorbed through AQP7 in the proximal straight tubules and that there might be no other glycerol-reabsorbing system to compensate for this defect in the more distal nephron segment. We also found elevated urine glycerol levels in AQP1/AQP7 double-knockout mice, whereas AQP1 solo-knockout mice did not show glyceroluria (AQP1/AQP7, 0.22 ± 0.10 mg/ml vs. AQP1, 0.004 ± 0.003 mg/ml, n = 4, P < 0.001).
Urine leakage of glycerol in mouse models of proximal straight tubule injury. The identification of a novel glycerol-reabsorbing pathway restricted to the proximal straight tubules suggested the hypothesis that glyceroluria could be a new biomarker that indicates proximal straight tubular injury. To investigate whether glycerol can leak into urine due to proximal tubule dysfunction, we measured urine glycerol levels in two mouse models of proximal straight tubule injury: the cisplatin-induced acute renal failure (ARF) model and the ischemic ARF model. As shown in Fig. 5A, in both of these models of S3 injury, the mice showed significantly higher urine glycerol levels compared with that of pretreatment mice (pretreatment, 0.007 ± 0.005 mg/ml; cisplatin, 0.063 ± 0.043 mg/ml; ischemia, 0.076 ± 0.02 mg/ml; control, 0.010 ± 0.007 mg/ml; P values and sample sizes are shown in Fig. 5A). We also measured the urine glycerol levels of mice that had fasted for 24 h as controls, because these mice are known to show higher plasma glycerol levels than wild-type mice under normal conditions (9, 23). These control mice showed significantly lower urine glycerol levels compared with S3 injury model mice (Fig. 5A), although their plasma glycerol levels were similar to those of S3 injury model mice, as shown in Fig. 5B (pretreatment, 0.039 ± 0.013 mg/ml; cisplatin, 0.061 ± 0.035 mg/ml; ischemia, 0.055 ± 0.006 mg/ml; control, 0.071 ± 0.013 mg/ml; P values and sample sizes are shown in Fig. 5B).
Urea transport in the kidney of AQP7 knockout mice. To investigate the involvement of AQP7 in urea transport in the kidney, we first measured plasma and urine urea levels in AQP7 knockout mice. Because urea transport in the proximal straight tubules has been postulated to be involved in the countercurrent exchange of urea transport (5, 10) and this urea-recycling pathway may be a mechanism by which a high urea content is maintained in the inner medulla, we also measured the urea content in the papilla of AQP7 knockout mice. The plasma urea concentration in AQP7 knockout mice was not different from that in the wild-type mice (AQP7, 31.5 ± 5.3 mg/dl vs. wild-type, 30.7 ± 2.3 mg/dl, n = 4). Similarly, the ratio of urine urea to urine creatinine concentration in AQP7 knockout mice did not show a significant difference compared with wild-type mice (AQP7, 22 ± 9 vs. wild-type 28 ± 10, n = 4). There was also no difference between AQP7 knockout and wild-type mice in the urea content in the papilla under normal conditions (data not shown). To detect small differences in the ability to maintain urea accumulation, the same experiments were performed when the urea supply to the kidney was limited, as in the case of a low-protein diet (4% protein). The AQP7 knockout mice fed a low-protein diet for 20 days did not show an impairment in urea accumulation in the papilla under dehydration (AQP7, 2,470 ± 417 mg/g papilla wet wt vs. wild-type, 2,202 ± 430 mg/g papilla wet wt, n = 4). They also did not show a urine concentrating defect with the low-protein diet (data not shown).
DISCUSSION
In this study, we found that AQP7 made a small contribution to the water permeability of the brush-border membranes in the proximal straight tubules. As anticipated from the restricted distribution of AQP7 compared with AQP1, the role of AQP7 in water reabsorption in the proximal tubules is minor compared with that of AQP1. Therefore, we generated AQP1/AQP7 double-knockout mice, because we considered that the small contribution of AQP7 to water permeability would be more readily detected in the situation where the major water transport pathway through AQP1 was eliminated. In fact, AQP1/AQP7 double-knockout mice showed significantly increased urine excretion compared with AQP1 solo-knockout mice, which was accompanied by a proportional decrease in urine osmolality. This suggests that the total excretion of osmolar substances might not be altered in AQP1/AQP7 double-knockout mice compared with AQP1 solo-knockout mice, although AQP1/AQP7 double-knockout mice excreted more glycerol in their urine. However, the urine glycerol concentration in AQP1/AQP7 double-knockout mice was only 2.4 mM, which constitutes only a small portion of urine osmolarity (600 mosmol/kgH2O). In addition, urea excretion into the urine and urea accumulation in the papilla were not altered in AQP7 knockout mice. Taken together, these data suggest that the increased urine volume in AQP1/AQP7 double-knockout mice is most likely explained by an increased delivery of water to the distal nephrons Furthermore, this validates the notion that the amount of water reabsorbed through the effect of AQP7 in the proximal straight tubules is physiologically substantial.
Although we found that the role of AQP7 in water transport was minor compared with that of AQP1, we identified a new glycerol reabsorption pathway dependent on AQP7. AQP7 knockout mice showed glyceroluria, although their plasma glycerol level was not increased. According to the review by Lin (11), urine glycerol in the glomerular filtrate of mammals is completely reabsorbed in the tubules, but the reabsorption pathway has not been identified. Maeda et al. (16) recently reported that AQP7 knockout mice had lower plasma glycerol levels compared with wild-type mice. Our AQP7 knockout mice did not show a significant difference in serum glycerol level. This indicates that glyceroluria in AQP7 knockout mice was not due to a change in plasma glycerol levels but was due to the reduction or the elimination of the glycerol reabsorption pathway in the kidney. It is not clear at present whether AQP7 is the only glycerol reabsorption pathway present along the nephron, because, for example, AQP3, another aquaglyceroporin found in the kidney, is localized in the collecting ducts. Nevertheless, the fact remains that AQP7 effectively prevents glycerol from being excreted into the urine and that the other glycerol-reabsorbing pathway(s), if any, cannot completely compensate for the deletion of AQP7.
Although the physiological significance of the glycerol reabsorption pathway remains to be determined, we hypothesized that the failure of this pathway could be used as a marker for proximal straight tubule damage, because AQP7 is highly localized to this nephron segment. It has been reported that cells of the proximal straight tubule are especially sensitive to cisplatin and ischemia and undergo extensive necrosis when challenged (4, 18, 20, 24). Therefore, we measured urine glycerol levels in both of these proximal straight tubule injury models. As expected, the mice in these models showed higher urine glycerol levels than nontreated or starved control mice. As shown in Fig. 5B, plasma glycerol levels could not account for these changes, indicating that the urine leakage of glycerol was due to the failure of glycerol reabsorption. We also measured the urine N-acetyl--D-glucosaminidase level, a clinically useful marker for proximal tubular injury (21), and found that the urine N-acetyl--D-glucosaminidase level was not elevated in the early stages of our model (data not shown). Therefore, the measurement of urine glycerol levels could be used as a new method for detecting S3 segment injury that may be more sensitive than the conventional markers.
In summary, the present study clearly identified some physiological roles of AQP7 in the kidney. Although AQP7 plays a minor role in water transport, AQP7 constitutes a major glycerol-reabsorbing pathway in the kidney.
GRANTS
This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
ACKNOWLEDGMENTS
We thank S. Ichinose of the Tokyo Medical and Dental University for electron-microscopic measurement of the diameter of the vesicles obtained from the outer medulla.
FOOTNOTES
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.
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