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【关键词】 protein
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
Department of Nephrology, Graduate School, Tokyo Medical and Dental University, Yushima Bunkyo-Ku, Tokyo, Japan
Department of Physiology University of Arizona, Tuscon, Arizona
ABSTRACT
Aquaporin-1 is the major protein responsible for transport of water across the epithelia of the proximal tubule and thin descending limbs. Rapid water efflux across the thin descending limb is required for the normal function of the countercurrent multiplier mechanism. Therefore, urinary concentrating capacity is severely impaired in aquaporin-1 knockout (AQP1 /) mice. Here, we have investigated the long-term consequences of deletion of the AQP1 gene product by profiling abundance changes in transporters expressed in the inner medullas of AQP1 (/) mice vs. heterozygotes [AQP1 (+/)], which have a normal concentrating capacity. Semiquantitative immunoblotting demonstrated marked suppression of two proteins strongly expressed in the inner medullary collecting duct (IMCD): UT-A1 (a urea transporter) and AQP4 (a basolateral water channel). Furthermore, the urea permeability of the IMCD was significantly reduced in AQP1 (/) mice. In contrast, there was increased expression of three proteins normally expressed at higher levels in the cortical collecting duct (CCD) than in IMCD: AQP3 (another basolateral water channel) and the epithelial sodium channel subunits -ENaC and -ENaC. Changes in expression of these proteins were confirmed by immunocytochemistry. Messenger RNA profiling (real-time RT-PCR) revealed changes in UT-A1, -ENaC, -ENaC, and AQP3 transcript abundance that paralleled the changes in protein abundance. Thus, from the perspective of transport proteins, the IMCDs of AQP1 (/) mice have a significantly altered phenotype. To address whether these changes are specific to AQP1 (/) mice, we profiled IMCD transporter expression in a second knockout model manifesting a concentrating defect, that of ClC-nK1, a chloride channel in the ascending thin limb important for urinary concentration. As in the AQP1 knockout mice, ClC-nK1 (/) mice showed decreased expression of UT-A1 and increased expression of -ENaC and -ENaC vs. WT controls. In conclusion, the expression profile of IMCD transporters is markedly altered in AQP1 / mice and this manifestation is related to the associated concentrating defect.
ClC-nK1; urea
THE RENAL COLLECTING DUCT (CD) exhibits considerable physiological and histological heterogeneity along its length. The solute transport process and cellular composition of the CD change as the segment descends from the renal cortex, through the hypertonic medullary interstitium, down to the papillary tip. The phenotypic heterogeneity along the CD is readily appreciated by contrasting the cortical CD (CCD) with the inner medullary CD (IMCD). The principal cells of the rat, rabbit, and mouse CCD and IMCD have distinct transport characteristics that are a reflection of the differential expression of transporter proteins in these segments. For example, the CCD is well known as a site of active sodium reabsorption; a process dependent on the epithelial sodium channel, ENaC (12). In contrast, direct studies in isolated, perfused tubules from rats have detected little or no active sodium transport in the IMCD (unpublished observations), which may be related to the much lower expression of ENaC protein in this region (10). Additionally, the IMCD of the rabbit, rat, and mouse have significant urea permeability (Purea) that is absent in the CCD owing the restricted expression of specific urea transporters (7, 21). Finally, there is differential expression of the basolateral water channels, in the rat CD with AQP3 being more abundant in the CCD while AQP4 is the predominant basolateral water channel of the IMCD (24). In summary, although the entire CD is derived from a common embryological precursor, there is significant phenotypic diversity in this segment of the renal tubule resulting from a differential expression of solute transporters.
Here, we have addressed the role urinary concentrating ability may play in determining the expression pattern of IMCD transporters using aquaporin-1 (AQP1) null mice as well as another mouse line in which the major chloride channel of the thin ascending limb (ClC-nK1) has been deleted (16). AQP1 water channels and ClC-nK1 chloride channels are normally expressed in renal structures that participate in the formation of concentrated urine and are integral components of the counter-current multiplier mechanism. The genetic ablation of either of these transporters results in a severe urinary concentrating defect (13, 16). We hypothesize that the special characteristics of the IMCD (reviewed above) are to some extent dependent on the high tissue osmolality in the inner medulla. To test this, we have assessed the expression of several solute and water transport proteins in IMCD in AQP1 and ClC-nK1 knockout mice.
METHODS
Animals. AQP1 (/) breeder mice (13) in a CD1 background were kindly supplied by Dr. Alan Verkman (University of California, San Francisco, CA). A colony of CLC-nK1 (/) mice (16) was also established at NIH utilizing breeding pairs provided by Dr. Shinichi Uchida (Tokyo, Japan). All mice utilized in these studies were maintained on an ad libitum diet of normal rodent chow and water. For each experiment, mice of the appropriate genotype were selected from two separate litters of approximately the same age. Animal studies were approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee.
Semiquantitative immunoblotting. Immunoblotting procedures for comparing two sets of samples of kidney homogenates with regard to relative abundances of specific proteins were described in detail previously (11, 24). Preliminary gels were run for the entire set of samples in a given experiment on 12% polyacrylamide/SDS gels, which were stained with Coomassie blue dye to assess equality of loading as described (11, 24). We refer to this procedure as "semiquantitative immunoblotting" because the relative abundance, but not the absolute abundance, of the target proteins is determined.
Immunocytochemistry. Mouse kidneys were perfusion-fixed by cannulating the heart with a 26-gauge needle under isoflurane anesthesia and perfusing the mouse with 2% parafomaldehyde (PFA) in PBS (26). After perfusion fixation, kidneys were removed and postfixed overnight in 2% PFA in PBS. Kidneys were embedded in paraffin and 5 μM sections were obtained. Sections were labeled following the immunoperoxidase method described by Hager et al. (10).
Antibodies and terminology for apical Na transporters. Affinity-purified primary antibodies used for immunoblotting and immunocytochemistry were directed to the major transporter proteins in the IMCD: the -, -, and -subunits of ENaC (14), AQP1 (25), aquaporin-2 (5), aquaporin-3 (6), aquaporin-4 (24), and the urea transporter UT A-1 (18). The antibodies were prepared in our laboratory using carrier-coupled synthetic peptides as immunogens and were affinity purified. Specificity of each antibody has been demonstrated by a combination of immunoblotting showing appropriate peptide-ablatable bands, and immunocytochemistry showing localization in appropriate membrane domains.
Real-time RT-PCR. Quantitative, real-time RT-PCR was used to measure relative mRNA abundances in the renal inner medulla of AQP1 (+/) and (/) mice. Inner medullas were homogenized in a guanidine thiocyanate (Sigma) solution and RNA was isolated from the homogenate by CsTFA (ICN, Aurora, OH) centrifugation. Isolated RNA was DNase (DNA-free, Ambion, TX) treated and equal amounts of RNA (determined spectrophotometrically) were reverse transcribed using oligo dT and Superscript II (Invitrogen, Carlsbad, CA) according the manufacturer's specifications. Real-time PCR was performed on an ABI Prism 7900HT system using primers designed to amplify specific mouse cDNAs and the Quantitect Syber green PCR kit (Qiagen, Valencia, CA). Specificity of the reaction was determined by melting curve analysis. Relative quantitation of gene expression was determined using the comparative CT method (2) as outlined at: http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf. t-Tests were performed on transformed CT values (i.e., 2CT). The primer pairs used for the real-time PCR are listed in Table 1.
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Urea permeability measurements. The urea permeability (Purea) was measured from IMCD using the isolated tubule microperfusion technique. IMCD segments were microdissected from the region of the medulla 3070% of the distance from the inner-outer medullary junction to the papillary tip of the mouse kidney. The dissection solution contained (in mM): 125 NaCl, 25 NaHCO3, 2 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, and 5 creatinine. The tubules were transferred to a perfusion chamber mounted on an inverted microscope, cannulated by concentric pipets and perfused in vitro. The perfusate and the pertitubular bath solutions were identical to the dissection solution except that 5 mM creatinine was replaced by 5 mM urea in the bath solution. Therefore, the tubules were perfused with solutions of equal osmolality, but with a 5 mM bath-to-lumen urea gradient. The urea permeability was determined by measuring the urea flux resulting from the transepithelial urea gradient as described previously (4). The urea concentrations in the perfusate, bath, and collected fluid was measured fluorometrically using a continuous-flow ultramicrofluorometer (4). The Sigma BUN reagent (Kit number 6420) was used in the continuous-flow system.
Statistical analysis. Quantification of the band densities from immunoblots was carried out by densitometry using a laser densitometer (Molecular Dynamics, San Jose, CA) and ImageQuaNT software (Molecular Dynamics). Values from knockout animals were compared with controls using an unpaired t-test when standard deviations were the same, or by Welch t-test when standard deviations were significantly different (INSTAT; Graphpad Software, San Diego, CA). To facilitate comparisons, we normalized the densitometry values such that the mean for the control group is defined as 100. P < 0.05 was considered statistically significant.
RESULTS
Expression of ENaC subunits, aquaporins, and urea transporter in IMCD of AQP1 (/) vs. control AQP1 (+/) mice. Figure 1 shows immunoblots demonstrating the expression profile of the major inner medullary transport proteins in AQP1 (/) mice compared with control (+/) mice. The data in Fig. 1 are representative of two separate experiments. There was a marked increase in the band densities corresponding to the - and -subunits of the amiloride-sensitive sodium channel (ENaC) in the inner medulla of AQP1 (/) mice (P < 0.05 and P < 0.05, respectively); however, there was no significant change in the abundance of the -subunit. The 40-kDa glycosylated form of the basolateral water channel AQP3 (8) was significantly increased (P < 0.05) in AQP1 (/) inner medullas. In contrast, the band densities corresponding to the 28- and 51-kDa forms (19) of AQP4 were both significantly decreased in AQP1 (/) inner medullas (P < 0.05 and P < 0.05). AQP2 levels were not significantly different. Finally, the abundance of the urea transporter UT-A1 was markedly suppressed in AQP1 (/) inner medullas relative to controls (P < 0.05). In summary, there were pronounced changes in the expression levels of several of the major solute transporter proteins of IMCDs of AQP1 (/) mice relative to those of AQP (+/) mice and these changes altered the overall transporter abundance profile to more nearly resemble what is normally seen in the CCD as described earlier.
Immunocytochemistry. We next sought to confirm changes seen in the immunoblotting studies using immunocytochemistry. Figure 2 presents immunoperoxidase labeling of UTA-1, AQP3, and AQP4 in the renal inner medulla of AQP1 (/) and (+/) mice. Figure 2, A and B, demonstrates reduced immunoreactive UT A-1 in AQP1 (/) inner medullas (B) relative to the heterozygote controls (A). Additionally, there is a moderate increase in the basolateral AQP3 labeling in (/) inner medullas (Fig. 2D) relative to controls (Fig. 2C). Basolateral labeling of AQP4 is markedly reduced in the inner medulla of (/) mice (Fig. 2F) relative to control inner medullas (Fig. 2E) from heterozygotes. The results of the immunoperoxidase labeling demonstrate changes in UT A-1, AQP3, and AQP4, which are qualitatively similar to the changes identified in initial Western blotting experiments.
Real-time RT-PCR measurements of mRNA levels. Figure 3 demonstrates the transcript abundance of individual solute transporters in the inner medulla of AQP1 (/) mice relative to the transcript abundance found in control, AQP1 (+/), mice. The cumulative results of two separate studies are presented on a log linear scale to emphasize the broad range of changes in transcript abundance. Additionally, a reference line representing a ratio of 1, or no change in transcript abundance, is provided to facilitate the comparisons between groups. The only change in transcript abundance observed among the three water channel isoforms examined in this study (AQP2, AQP3, and AQP4) was a profound 20-fold increase in the abundance of AQP3 (P < 0.05) transcript in the inner medulla of AQP1 (/) mice. At the other extreme, there was a striking 90% reduction of UT A-1 and UT A-3 transcript abundance in the inner medulla of AQP1 (/) mice relative to controls (P < 0.05). Finally, there was a highly significant threefold increase in the abundance of each of the transcripts encoding the -, -, and -ENaC subunits (P < 0.05 for each transcript).
Urea permeability. Based on the marked differences in inner medullary urea transporter abundance between AQP1 (+/) and (/) animals, we hypothesized there would be differences in urea permeability (Purea) of the IMCDs. A comparison of basal and vasopressin stimulated Purea of isolated, perfused IMCDs from AQP1 (+/) and (/) mice are presented in Fig. 4. The basal Purea in control, AQP1 (+/), IMCDs (30.9 ± 2.8 x 105 cm/s) was significantly different from the basal Purea of (/) IMCDs (17.4 ± 8.1 x 105 cm/s) (n = 3, P < 0.05). Additionally, after exposure to 100 pM peritubular vasopressin for 30 min, the Purea of AQP1 (/) IMCDs was significantly less than (n = 5, P < 0.05) control (17.0 ± 3.7 vs. 88.1 ± 14.1, respectively). In summary, both the basal Purea and the vasopressin-dependent Purea were significantly lower in the AQP1 (/) mice compared with (+/) controls.
Expression of ENaC subunits, aquaporins, and urea transporter in IMCD of ClC-nK1 (/) vs. wild-type (+/+) mice. Finally, we examined the medullary transporter profile in the ClC-nK1 knockout mice (Fig. 5) which also have impaired urinary concentrating ability resulting from defective passive chloride transport out of the thin ascending limb of Henle. As was the case in the AQP1 (/) model, the band densities corresponding the the - and -ENaC subunits were significantly increased relative to wild-type controls (P < 0.05 and P < 0.05, respectively), whereas -ENaC abundance did not significantly change. Also, the levels of UT-A1 expression in ClC-nK1 (/) mice were significantly reduced (P < 0.05) compared with controls. In contrast to the findings in AQP1 (/) mice, AQP2 levels in the inner medulla of ClC-nK1 (/) were significantly increased compared with wild-type controls (P < 0.05) while levels of AQP3 in the inner medulla were significantly reduced in CLC-nK1 (/) mice (P < 0.05). In conclusion, the expression profile of transporters in the CLC model share some similarities with that found in the AQP1 (/) model, especially with regard to solute transporter expression.
DISCUSSION
The physiological properties of the CD change as the tubule descends from the renal cortex to the papillary tip. The factors influencing the expression of the proteins ultimately responsible for manifesting the different transport properties of the CCD and IMCD may include the nature of physical environment the tubule resides in. Here, we have examined the transporter expression profile of the IMCD in two mouse models with urinary concentrating defects.
AQP1 (/) and ClC-nK1 (/) mice have urinary concentrating defects of roughly the same magnitude (13, 16) with spontaneous urine osmolalities of 600700 mosmol/kgH2O and increasing minimally, if at all, on water deprivation. Beyond these phenotypic similarities, impaired counter-current multiplication underlies the concentrating defect in both models albeit through different mechanisms. In the case of AQP1 (/) mice, the severely diminished water permeability of the thin descending limb prevents extraction of water from this tubule segment. For CLC-nK1 (/) mice, the absence of the apical and basolateral chloride conductance of the thin ascending limb prevents NaCl efflux. In both instances, the inner medullary concentrating process is impaired thereby resulting in a lower inner medullary interstitial osmolalilty.
In both models, there were significant increases in - and -ENaC protein expression in the inner medulla. Earlier work has shown that - and -ENaC subunits facilitate the surface expression of functional ENaC complexes (3), suggesting sodium transport may be elevated in the IMCD of these mice. We propose that such an increase in sodium transport in the IMCD may help in maintaining extracellular fluid balance in the presence of polyuria by providing an alternative means of fluid reabsorption. Previous studies have demonstrated the rapid osmotic equilibration of urine in the micropuncture-accessible distal tubule (9) and the ability of the cortical collecting duct to continue to reabsorb isosmotic fluid in a process dependent on active sodium transport (22). The extension of this mechanism of fluid reabsorption from the cortical collecting duct into the inner medulla could ameliorate the urinary salt and water loss in these mice.
There was a significant decrease in the expression of the UT-A1 protein and mRNA in the AQP1 (/) mice. The isolated, perfused tubule measurements confirm that the low urea transporter expression levels are associated with low urea permeability. UT-A1 protein expression was also reduced in the IMCD of the CLC-nK1 (/) mice suggesting that the mechanism of suppression is somehow related to the concentrating defect present in both. Hypertonicity stimulates the UT-A1 promoter (17) implying the decreased expression of UT-A1 reported here may stem from a reduced tonicity of the inner medulla associated with the concentrating defects. Thus tonicity of the inner medulla may directly contribute to the altered expression of UT-A1 seen in these models.
AQP3 levels were significantly increased and AQP4 protein levels significantly decreased in the inner medulla of the AQP1 (/) model thus reversing their normal pattern of expression. It is not readily apparent what physiological consequences this would impart as the apical rather than the basolateral membrane of the IMCD is rate limiting for water transport (8). However, the changes in AQP3 (and AQP2) expression in the inner medulla of the CLC-nK1 (/) do not parallel the changes seen in the AQP1 model and suggest a concentrating defect per se is not the sole factor determining the final expression pattern of the transporters in the inner medulla of these models. Indeed, the dissociation of changes in protein levels and transcript abundance for AQP4 and -ENaC in the AQP1 (/) model also supports the idea there are multiple regulatory mechanisms shaping the final expression profile of the transport proteins.
The dissociation of the AQP4 and -ENaC transcript and protein levels has not, to our knowledge, been previously described. In general, reports of increases in -ENaC protein abundance are associated with elevations in -ENaC transcript (1, 14, 15). However, in the A6 model of the renal collecting duct, hypotonicty is known to 1) increase transepithelial sodium transport without an increase the ENaC protein levels (20) and 2) increase the abundance of -ENaC transcript (19). These effects of hypotonicity on ENaC regulation may be particularly relevant in the context of mouse models with chronic concentrating defects.
In conclusion, the expression pattern of many of the transport proteins known to play prominent roles in the physiology of the IMCD is altered in AQP1 (/) mice. Several, but not all of the changes in transporter expression seen in the IMCD of AQP1 (/) mice are also present in the IMCD of the CLC (/) mice; a model with a similar concentrating defect. Given the similarities between the expression profiles in AQP1 and CLC-nK1 mouse models, as well as the underlying similarities in the urinary concentrating defect, it is tempting to propose that many of the changes in transporter expression occur in response to a chronically diminished tonicity of the inner medulla. However, the changes in IMCD transporter expression in the knockout models may be present at birth and reflect differences in ontogeny rather than adaptation to, or resulting from, the concentrating defect. Furthermore, there are clearly many other possible mechanisms that may contribute to the altered expression of transporters in the IMCD of mice with concentrating defects, including urinary flow, glomerular filtration rate (GFR), and hormone levels, which have not been addressed in this study. For example, GFR is significantly reduced in the AQP1 (/) models (23). Further study will be required to establish the mechanism(s) responsible for the changes in IMCD transporter expression.
ACKNOWLEDGMENTS
The authors thank Dr. J. Wade (University of Maryland) for advice regarding mouse tissue fixation and Dr. R. A. Fenton (National Institutes of Health) for the UT-A1 and UT-A3 real-time PCR primers.
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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