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【摘要】 Defects in renal proximal tubule transport manifest in a number of human diseases. Although variable in clinical presentation, disorders such as Hartnup disease, Dent's disease, and Fanconi syndrome are characterized by wasting of solutes commonly recovered by the proximal tubule. One common feature of these disorders is aminoaciduria. There are distinct classes of amino acid transporters located in the apical and basal membranes of the proximal tubules 95% of filtered amino acids, yet few details are known about their regulation. We present our physiological characterization of a mouse line with targeted deletion of the gene collectrin that is highly expressed in the kidney. Collectrin-deficient mice display a reduced urinary concentrating capacity due to enhanced solute clearance resulting from profound aminoaciduria. The aminoaciduria is generalized, characterized by loss of nearly every amino acid, and results in marked crystalluria. Furthermore, in the kidney, collectrin-deficient mice have decreased plasma membrane populations of amino acid transporter subtypes B 0 AT1, rBAT, and b 0,+ AT, as well as altered cellular distribution of EAAC1. Our data suggest that collectrin is a novel mediator of renal amino acid transport and may provide further insight into the pathogenesis of a number of human disease correlates.
【关键词】 proximal tubule crystalluria osmotic diuresis
COLLECTRIN ( Tmem27, NX-17) was originally identified in a screen for genes that are upregulated during the hypertrophic phase of the remnant kidney after subtotal renal ablation ( 44 ). This gene putatively encodes a 222-amino acid transmembrane glycoprotein with sequence homology to ACE, a member of the angiotensin-converting enzyme superfamily ( 43 ). Collectrin shares 47.8% sequence identity with ACE2, primarily in the transmembrane and COOH-terminal regions, but lacks the carboxypeptidase catalytic domain of ACE2 ( 43 ). Its sequence is highly conserved among species and 80% identity between mouse, rat, and human ( 43 ). Collectrin's expression was originally described to be limited to the uropelvic epithelium of the embryonic mouse kidney and to the collecting duct of the adult mouse kidney; hence its present name collectrin was coined ( 43 ). Since its discovery, however, collectrin has been reported to be also expressed in abundance in extrarenal tissues including the liver and lung ( www.genecards.org/cgi-bin/carddisp.pl?gene=TMEM27 ).
More recently, two groups have described expression of collectrin in the mouse pancreas ( 1, 15 ) and postulate a role for collectrin in -cell physiology. Both groups independently identified that collectrin is a downstream target of the transcription factor hepatocyte nuclear factor-1 (HNF-1 ). In the kidney, HNF-1 localizes to the proximal tubule ( 29 ). Targeted deletion of HNF-1 in the mouse results in a renal phenotype consistent with Fanconi syndrome ( 29 ), characterized by glucosuria, phosphaturia, calciuria, and aminoaciduria. The proximal tubule dysfunction characteristic of Fanconi syndrome can occur in humans as isolated idiopathic cases or in connection with inheritable diseases ( 42 ). Additionally, more limited proximal tubule dysfunction with solute wasting is associated with other human disease states, such as Hartnup disease ( 3, 20, 35 ), Dent's disease ( 21, 24, 30 ), and X-linked recessive nephrolithiasis ( 21 ). Currently, little is known about the precise mechanisms of these disease processes.
We sought to determine the physiological role of collectrin by generating a mouse line with targeted deletion of the gene. We demonstrate that, contrary to original reporting, collectrin is also expressed in the proximal tubule of the mouse kidney. Loss of collectrin results in decreased urinary concentrating capacity as a consequence of an osmotic diuresis. This osmotic diuresis is due to isolated severe generalized wasting of amino acids, with accompanying tyrosine and glutamine crystalluria. Furthermore, we demonstrate that loss of collectrin is associated with altered expression of key amino acid transporters in the kidney: B 0 AT1 (neutral/aromatic amino acid transport), b 0,+ AT-rBAT (cationic amino acid transport), and EAAC1 (anionic amino acid transport). We suggest that collectrin represents a novel mediator of renal amino acid transport.
MATERIALS AND METHODS
Generation of collectrin null mouse line. Collectrin ( Tmem27 ) is located on the X chromosome of the mouse genome. A targeting construct containing thymidine kinase and a neomycin cassette flanked by 3-kb 5' and 3' homology regions was used to disrupt most of exon 4, intron 4, and exon 5 of the collectrin gene ( Fig. 1 A ). Embryonic stem cells derived from the 129/SvEv strain were used for electroporation. Targeting was confirmed by Southern blot analysis.
Fig. 1. Targeted deletion of collectrin. A : diagram of the targeting strategy. Probe for Southern blot verification of genotypes recognizing 12.3-kb wild-type (WT) allele and 5.8-kb null allele (KO) is shown. B : fluorescent immunostaining of paraffin-embedded kidney sections obtained from wild-type (+/Y; top ) and hemizygous knockout (KO; -/Y) males ( bottom ). In wild-type mice, collectrin (Alexa 488; bright green) is observed only at the apical membrane of proximal tubules in the cortex. Aquaporin-2 (AQP2; Alexa 568; red) is located in the collecting duct and does not colocalize with collectrin. Staining for collectrin was absent in knockout kidney tissue, demonstrating successful deletion of protein product.
Genotyping. Southern blot analysis was initially performed to verify genotypes. Genomic DNA was isolated from the tail using standard methods and digested overnight at 37°C with Eco RI restriction endonuclease (New England Biolabs). Digests were subjected to agarose gel electrophoresis, blotted to nitrocellulose, and incubated with a probe complementary to a region of DNA between exon 5 and an Eco RI site. Successful recombination with a neomycin cassette resulted in an addition of an extra Eco RI site, such that wild-type (WT) alleles generated a 12.3-kb band and knockout (KO) alleles generated a 5.8-kb band. (Southern blot analysis can be provided on request.) Genotyping was then performed using a PCR protocol with primers (Operon) that recognize sequence in exon 4 (forward: 5'-GGGGTAGGGGCAGAGCTCAA-3', reverse: 5'-TGCCCTCTTCCGGTTGTGTC-3') or the neomycin cassette (forward: 5'-GCAGGATCTCCTGTCATCTCACC-3', reverse: 5'-GATGCTCTTCGTCCAGATCATCC-3') at an annealing temperature of 66°C and using Taq polymerase (Qiagen). Genotyping was consistent using both methods, and thus PCR was used for subsequent large-scale screening.
Immunostaining. Immunostaining was performed on paraffin-embedded, formalin-fixed 4-µm longitudinal sections of kidney from WT and KO animals that were obtained using a microtome (RM2125, Leica Microsystems, Bannockburn, IL). The sections were rehydrated and the antigen was retrieved with microwave heat for 15 min in TEG buffer (10 mM Tris and 0.5 mM EGTA, pH 9.0). After neutralization with NH 4 Cl buffer, the sections were blocked with 1% BSA, 0.2% gelatin, and 0.05% saponin in PBS before incubation overnight with primary antibody diluted in 0.1% BSA and 0.3% Triton X-100 in PBS. The primary antibodies against aquaporin-2 ( 2 ) (generous gift of Dr. Mark Knepper, National Institutes of Health, Bethesda, MD) and collectrin ( 15 ) (kind gift of Dr. Kazuya Yamagata, Osaka University, Osaka, Japan) were previously characterized. After being rinsed with 0.1% BSA, 0.2% gelatin, and 0.05% saponin in PBS, the sections were reacted for 1 h with secondary antibody diluted in 0.1% BSA and 0.3% Triton X-100 in PBS. The secondary antibodies used were Alexa 488 or Alexa 568 conjugated (Invitrogen, Carlsbad, CA). After washes with PBS, the sections were mounted in Vectashield solution containing 4,6-diamidino-2-phenylindole to stain nuclei (H-1500, Vector Labs, Burlingame, CA). Confocal fluorescence images were taken using a Zeiss LSM 510 microscope and software (Carl Zeiss MicroImaging, Thornwood, NY).
Laser capture microscopy and RT-PCR. Total RNA was isolated from collecting duct cells selected using a combination of immunohistochemistry and laser capture microdissection and catapulting (Zeiss PALM Microbeam System). Frozen sections (5 µm) of kidney were immediately processed for rapid immunostaining using RNase-free reagents. Collecting duct cells were identified by incubation with a rabbit anti-aquaporin-2 antibody ( 2 ) at 1:100 dilution, followed by a biotinylated anti-rabbit secondary antibody (Vector Labs) at 1:200. Staining was accomplished with VECTASTAIN reagents (Vector Labs) and diaminobenzidine (Innovex Biosciences) as the chromagen. The total process was completed in 35 min. For each sample, 50,000 µm 2 of tissue were collected and processed using a RNeasy Micro Kit (Qiagen) with on-column DNA digestion. The entire eluted volume was used for cDNA synthesis using an iScript cDNA synthesis kit (Bio-Rad Laboratories). Five microliters of the cDNA reaction were then used for PCR, which was performed using fluorescence detection with Sybr Green and the iCycler system (Bio-Rad Laboratories).
Metabolic cage studies. Protocols for use of experimental animals were approved by the Duke University Medical Center/Veterans Affairs Medical Center Institutional Care and Use Committee. Animals were housed in metabolic cages (Hatteras Instruments) for 24 h. They were given free access to standard mouse chow (Harlan Teklad) and water. Urine was collected into flasks. At the completion of the collection period, blood was obtained from conscious animals using tail vein collection and collected into BD Microtainer SST tubes (Becton-Dickinson) for isolation of serum. Serum chemistries were obtained from a commercial lab (IDEXX Preclinical Research) or by flame photometry (Laboratory Instrumentation), and serum creatinine was determined by HPLC analysis ( 9, 41 ) (kindly performed by Dr. Steve Dunn, Thomas Jefferson University, Philadelphia, PA). Urine chemistries were performed using an autoanalyzer (IDEXX Preclinical Research and mouse core facility at the University of North Carolina at Chapel Hill). Serum and urine osmolality were determined using a vapor pressure osmometer (Wescor).
Vasopressin-response studies. Blood was drawn from the tail vein for measurement of serum vasopressin levels at baseline (ad libitum water intake) and after 18 h of water deprivation. This enabled paired statistical analysis and avoidance of effects of anesthesia on vasopressin levels. Aprotinin (Sigma-Aldrich) at 10.6 TIU/ml of whole blood was added to each sample to minimize vasopressin degradation. Serum was then immediately processed for vasopressin extraction. Four volumes of chilled absolute ethanol were added, samples were spun at 14,000 rpm for 15 min at 4°C, and the supernatant was collected into separate microfuge tubes. Samples were then evaporated to dryness using a SpeedVac (Savant). Reconstitution and measurement of vasopressin were done according to a commercially available enzyme immunoassay kit (Assay Designs). For DDAVP studies, lyophilized DDAVP (Sigma-Aldrich) was reconstituted in sterile normal saline before intraperitoneal injection.
Amino acid analysis. Urine samples were collected over 24 h from mice housed in metabolic cages. Serum samples were obtained immediately after the 24-h urine collection. Each sample was treated with 2 volumes of methanol to precipitate the insoluble particles. O -phthalaldehyde solution (Sigma-Aldrich) was added to the clear supernatant to reach a final concentration of 10 mM. After a 2-min incubation at room temperature, the sample was centrifuged for 10 min at 20,000 rpm, and the supernatant was filtered through a 0.2-µm-pore-size filter. A 10-µl portion of the filtered urine was injected into an Agilent 1100 HPLC system equipped with a calibrated C 18 Ultrasphere reverse-phase column (Beckman, Palo Alto, CA) and a fluorescence detector (Excitation = 348 nm; Emission = 450 nm). The column was eluted with a linear gradient of 20-40% acetonitrile. Peak and area analyses were done with software provided by Agilent. Amino acid standards (Sigma-Aldrich) were treated and analyzed similarly and used to calculate the absolute concentration of each amino acid.
Real-time RT-PCR of amino acid transporter transcripts. Total mRNA was isolated from 30 mg of kidney tissue using an RNeasy Kit with on-column DNase digestion (Qiagen). One microgram of mRNA was used for cDNA synthesis (iScript kit, Bio-Rad). Real-time RT-PCR was performed using primers specific for each transporter and for the GAPDH housekeeping gene. Fluorescence detection was accomplished using Sybr Green and the iCycler system (Bio-Rad).
Western blots of amino acid transporters. Mice were euthanized by approved methods, and kidney cortices were rapidly dissected and placed into ice-cold isolation buffer (10 mM Tris, 250 mM sucrose, and 5 mM EDTA, pH 7.4) with a protease inhibitor cocktail (Sigma-Aldrich). Cortical homogenates were then rapidly processed as described ( 37 ). Briefly, cortices were homogenized for 10 s and lysates were spun at 3,000 g for 10 min at 4°C. The supernatant was saved on ice, and the pellet was resuspended, homogenized, and centrifuged. The two supernatants were combined and spun at 16,000 g for 30 min at 4°C. The resulting supernatant represented the intracellular fraction. Pellets were resuspended in isolation buffer and represented the plasma membrane fraction. Protein concentrations of each fraction were determined by the Bradford assay (Bioassay Systems). Twenty micrograms of total protein were loaded onto 12% SDS-PAGE gels and then transferred to nitrocellulose membranes according to the manufacturer's instructions (X-Cell Blot Module, Invitrogen). Membranes were blocked using Odyssey Blocking Buffer (LI-COR) for 1 h at room temperature. Membranes were then incubated at 4°C overnight with primary antibody diluted in blocking buffer as follows: rabbit anti-B 0 AT1 at 1:2,000 ( 33 ) (generous gift of Dr. Francois Verrey); rabbit anti-rBAT ( 16 ), and anti-b 0,+ AT at 1:1,000 ( 12 ) (generous gift of Dr. Josep Chillaron); rabbit anti-EAAC1 at 1 µg/ml (Alpha Diagnostics); mouse anti-rhoGDI at 1:5,000 (BD Transduction Labs); and mouse anti-tubulin at 1:2,000 (Santa Cruz Biotechnology). Secondary antibody incubation was performed for 1 h at room temperature with anti-rabbit and anti-mouse conjugated with IRDye 700 or 800 (Rockland Immunochemicals) at 1:10,000. Blots were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).
Statistical analysis. Student's t -test was used for all comparisons between two groups, with paired analysis completed where possible. Statistical calculations were done using commercially available software packages (Minitab and NCSS).
RESULTS
Generation of collectrin-deficient mice. Two of eight male chimeras that were mated with C57BL/6 females transmitted the disrupted collectrin gene to their progeny producing (129 x C57BL/6) F 1 female mice heterozygous (+/-) for the mutation. Due to the X-linked inheritance, subsequent intercrosses produced the following genotypes: WT females ( Tmem27 +/+ ), heterozygous females ( Tmem27 +/- ), homozygous females ( Tmem27 -/- ), WT males ( Tmem27 +/Y ), and hemizygous males ( Tmem27 -/Y ). Expected Mendelian ratios for X-linked inheritance were observed, suggesting that loss of collectrin does not affect perinatal survival. Additionally, fertility rates were normal. Male mice were used in our experiments to enable littermate controls. Loss of functional collectrin gene product was verified by RT-PCR (data not shown) and immunostaining of formalin-fixed and paraffin-embedded kidney sections from WT and Tmem27 -/Y animals ( Fig. 1 B ).
Collectrin is expressed in the proximal tubule. Unexpectedly, in WT mice antibody staining was only observed on the apical surface of proximal tubule epithelial cells ( Fig. 1 B ). This is consistent with the localization of collectrin's upstream activator HNF-1 ( 1, 15, 29 ) but is contrary to the original reporting of its localization solely in the collecting duct ( 43 ). We queried the genome databases ( http://www.ncbi.nlm.nih.gov/IEB/Research ) and found that human collectrin mRNA sequence has a number of putative alternative splice sites. We postulate that variability in antibody recognition may depend on epitope availability. To confirm that collectrin is also expressed in the collecting duct, we utilized laser capture microscopy to isolate collecting duct cells from WT frozen kidney sections immunostained with anti-aquaporin-2 antibody ( 2 ) ( Fig. 2 ), a marker specific for collecting duct, and positively identified the presence of collectrin transcript using RT-PCR with fluorescence detection.
Fig. 2. Laser capture microscopy of collecting duct cells from frozen kidney sections from WT animals stained using rabbit anti-AQP2 antibody and horseradish peroxidase detection. Left: visualization of collecting duct cells at x 40 magnification. Right: cells marked for capture.
Initial phenotypic characterization. On gross examination, Tmem27 -/Y animals displayed normal body morphology, body weights, behavior, and renal ultrastructure. Furthermore, survival rates were similar to WT, even by 6 mo of age. On light microscopy, kidney tissues from WT and Tmem27 -/Y were indistinguishable ( Fig. 3 ).
Fig. 3. Histological examination by light microscopy. Hematoxylin- and eosin-stained paraffin-embedded kidney sections from WT and KO animals at x 40 magnification. A : WT cortex. B : KO cortex. C : WT inner medula. D : KO inner medulla.
Collectrin-deficient mice display a urinary concentrating defect. To determine the physiological role of collectrin in the kidney, we first examined the 24-h urine output of Tmem27 -/Y (KO) and their WT littermates. Tmem27 -/Y animals had significantly greater urine output (WT 1.1 ± 0.2 ml, n = 10 vs. KO 1.8 ± 0.2 ml, n = 10; P = 0.0061). To determine the etiology of the polyuria, urine osmolality was measured on spot urine samples obtained by bladder massage. At baseline, there was no statistically significant difference in urine osmolality between the two groups, ( Fig. 4 A; WT 1,712 ± 140 mosmol/kgH 2 O, n = 16 vs. KO 1,534 ± 77 mosmol/kgH 2 O, n = 22; P = 0.30). However, with repeated measurements, there was a trend toward more dilute urine in Tmem27 -/Y animals, suggesting a difference in renal water conservation. To more accurately assess the maximal urinary concentrating capacity, urine osmolality was measured in mice after 18-h water deprivation. In our experience, this is a sufficient period of deprivation to achieve maximal urine concentration. Tmem27 -/Y mice were unable to concentrate their urine to the same level as WT littermates ( Fig. 4 A; WT 3,880 ± 63 mosmol/kgH 2 O, n = 12 vs. KO 3,189 ± 97 mosmol/kgH 2 O, n = 13; P < 0.00001). Furthermore, after water deprivation, there was a corresponding increase in serum sodium in the Tmem27 -/Y animals ( Fig. 4 A; increase in serum Na: WT 0.83 ± 0.7 meq/l, n = 8 vs. KO 6.72 ± 1.5 meq/l, n = 10; P = 0.004), consistent with a greater degree of free water loss.
Fig. 4. Assessment of urinary concentrating capacity. For all data included here, error bars represent SE. A, left: urine osmolality measurements on spot urine samples obtained by bladder massage, at time 0 and 18 h post-water deprivation. * P < 0.00001 KO vs. WT at 18 h. Right: change in serum sodium measurements between time 0 and 18 h post-water deprivation. P = 0.004. B, left: serum vasopressin levels at baseline and 18 h post-water deprivation. No difference was observed between groups. Right: response to DDAVP. Paired analysis of urine osmolalities at time 0 and 2 h post-DDAVP administration. * P = 0.0016 KO vs. WT at 2 h. C : solute and water clearance studies using 24-h urine collections. Left: osmolar clearance (C osm ) represented as means ± SE. P = 0.0067. Right: free water reabsorption (T c H 2 O) represented as means ± SE. P = 0.018.
Impaired urinary concentrating capacity is renal in origin. Although collectrin is expressed in the kidney, urinary concentrating defects can result from deficiencies either in the central nervous system (CNS) or at the level of the kidney. We thus proceeded to exclude the possibility of a contribution from a CNS defect that may result in abnormal drinking behavior. Primary polydipsia is known to cause a urinary concentrating defect ( 10 ) by a resultant "washout" of the inner medullary interstitial solute gradient. Tmem27 -/Y mice tended to have higher fluid intake than WT, but the differences were not statistically significant (WT 1.5 ± 0.3 ml, n = 10 vs. KO 2.2 ± 0.4 ml, n = 10; P = 0.20). Additionally, Tmem27 -/Y mice have virtually identical serum sodium levels at baseline compared with WT [ Table 1; WT 147.5 ± 0.5 meq/l, n = 14 vs. KO 147.1 ± 0.4 meq/l, n = 15; P = not significant (NS)], arguing against primary polydypsia. To definitively rule out primary polydypsia, we next performed paired drinking studies to control the fluid intake of Tmem27 -/Y mice to the same volume as that of WT mice for 6 days' duration to allow reestablishment of the inner medullary gradient. As previously demonstrated ( 39 ), this is a sufficient period of time to reverse the inner medullary washout effects of polydipsia. At the end of the study, spot urine samples were collected by bladder massage, and urine osmolalities were measured. Despite paired drinking, Tmem27 -/Y mice failed to achieve a urinary concentration level similar to WT (WT: 3,831 ± 273 mosmol/kgH 2 O, n = 4 vs. 2,883 ± 218 mosmol/kgH 2 O, n = 6; P = 0.035). This suggests that the urinary concentrating defect cannot be attributable to primary polydipsia.
Table 1. Urine and serum chemistries under basal conditions
Next, we examined the response of the hypothalamic-pituitary axis to water deprivation by assessing serum vasopressin levels. Vasopressin release is triggered by increases in plasma osmolality and acts on the collecting duct to enhance free water reabsorption or water conservation ( 34 ). Starting from similar values at baseline, the vasopressin levels increased by 10-fold in both groups after 18-h water deprivation ( Fig. 4 B; WT: 40.3 ± 3.6 pg/ml, n = 6 vs. KO 36.0 ± 2.2 pg/ml, n = 6; P = NS). This excludes impaired vasopressin release as a cause of the urinary concentrating defect in Tmem27 -/Y mice. To determine whether Tmem27 -/Y mice have renal resistance to vasopressin, we administered a supraphysiological dose of DDAVP, 1 µg/kg, by intraperitoneal injection. Preliminary pharmacokinetic studies suggested that peak drug response occurs 2 h after dosing; therefore, we collected spot urine samples at this time point and determined the urine osmolality. Again, Tmem27 -/Y animals failed to achieve the same degree of urine concentration as WT ( Fig. 4 B; WT 3,477 ± 144 mosmol/kgH 2 O, n = 5 vs. KO 2,493 ± 66 mosmol/kgH 2 O, n = 5; P = 0.0016). The difference in magnitude between WT and Tmem27 -/Y urine osmolality after DDAVP was similar to that post-water deprivation. Taken together, our data support a mechanism of renal origin for the urinary concentrating defect in collectrin-deficient mice.
Urinary concentrating defect is secondary to an osmotic diuresis. To determine whether the urine concentrating defect is due to impaired free water reabsorption or to increased solute clearance with resultant obligatory free water loss (osmotic diuresis), we measured osmolar clearance (C osm ) using 24-h metabolic cage collection. Tmem27 -/Y mice had significantly higher osmolar clearance than WT ( Fig. 4C; WT: 7.1 ± 0.9 ml/24 h, n = 11, vs. KO: 11.2 ± 1.0 ml/24 h, n = 12; P = 0.007) and a corresponding increase in T c H 2 O (calculated as C osm - V), or free water absorption ( Fig. 4 C; WT 5.9 ± 0.8 ml/24 h, n = 11 vs. KO 9.0 ± 0.9 ml/24 h, n = 12; P = 0.018). These data are consistent with those observed in classic studies on renal concentrating mechanisms in osmotic diuresis ( 23, 32 ) in which both C osm and T c H 2 O are increased. Moreover, compared with WT, Tmem27 -/Y mice tended to have increased glomerular filtration rate (GFR), measured by creatinine clearance (WT 25.9 ± 3.4 ml/h, n = 8 vs. KO 36.2 ± 3.9 ml/h, n = 9; P = 0.07). Elevated GFR is associated with osmotic diuresis ( 23 ), as the presence of solutes leads to dilution of tubular sodium ( 23 ) and a tubuloglomerular feedback enhancement of filtration rate ( 4 ). Taken together, our data support that collectrin-deficient mice have impaired urinary concentration due to an osmotic diuresis.
Osmotic diuresis is associated with aminoaciduria. To determine the source of the osmotic diuresis, we examined 24-h urinary excretion of the main solutes reabsorbed by the renal tubules along with the corresponding serum electrolytes ( Table 1 ). As expected, concentrations of the main urine osmolytes, sodium and urea, are decreased in Tmem27 -/Y animals, consistent with a free water excess. Notably, initial HPLC data suggested a significant qualitative increase in amino acid excretion in Tmem27 -/Y animals. Furthermore, examination of the urine by light microscopy revealed the presence of numerous crystals that are morphologically consistent with amino acid crystalluria ( Fig. 5 A ). In contrast, crystals were absent from the urine of WT mice ( Fig. 5 A ). HPLC analysis of the crystals revealed that these were tyrosine (60%) and glutamine (40%) in composition. This suggests that the aminoaciduria is the source of the osmotic diuresis and that there is a proximal tubule defect in collectrin-deficient animals. This proximal tubule defect appears to be limited to amino acid transport as urinary excretions of glucose and phosphate are normal in Tmem27 -/Y animals ( Table 1 ). The aminoaciduria appears to be an X-linked recessive trait since no amino acid crystals were seen in urine samples from heterozygous females, but were present in those of homozygous females (data not shown).
Fig. 5. Assessment of aminoaciduria. A, left: light microscopy of WT urine ( x 20) demonstrating absence of crystals. Middle: crystals present in the urine of collectrin-deficient mice ( x 20). Right: amino acid crystals at x 80 magnification. By light microscopy, they are morphologically similar to tyrosine crystals. By HPLC analysis, these are predominantly composed of Gln (40%) and Tyr (60%). B : 24-h urine concentration of individual amino acids in WT and collectrin-deficient mice. For all comparisons, P < 0.05. A = Ala; E = Glu; F = Phe; G = Gly; H = His; I = Ile; K = Lys; L = Leu; M = Met; N = Asn; Q = Gln; S = Ser; T = Thr; V = Val; W = Trp; Y = Tyr. C : corresponding serum concentrations of individual amino acids in WT and collectrin-deficient animals. * P < 0.05.
We next examined the aminoaciduria quantitatively. Urine analysis by HPLC ( 31 ) revealed significantly enhanced excretion of 16 of 18 amino acids measured ( Fig. 5 B ). Urinary excretion of aspartic acid and arginine was also enhanced (1.5- and 6-fold, respectively) but did not reach statistical significance. Cysteine and proline were not examined due to assay limitations. Similar analysis of the serum ( Fig. 5 B ) demonstrated corresponding low-to-normal levels of most amino acids, suggesting that the aminoaciduria represents a true loss from the kidney, with decreased proximal tubular reabsorption rather than increased filtered load exceeding proximal tubular reabsorptive capacity. In support of this evidence, 24-h metabolic cage data reveal that Tmem27 -/Y mice consume twice the amount of food than WT (WT 0.7 ± 0.1 g, n = 10 vs. KO 1.4 ± 0.4 g, n = 10; P = 0.0018), yet have virtually identical body weights (WT 26.6 ± 0.7 vs. KO 26.5 ± 0.8 g, P = NS). These data suggest that the increased food intake represents a compensatory effort for nutritional losses, rather than primary polyphagia.
Of note, serum levels of two amino acids, tyrosine and methionine, were increased in Tmem27 -/Y animals despite enhanced urinary excretion. Targeted deletion of HNF-1, an upstream transcriptional regulator of collectrin, also results in elevation of serum tyrosine levels, among other amino acids, from probable liver dysfunction ( 29 ). We therefore investigated the possibility of an altered hepatic phenotype in Tmem27 -/Y animals by examination of liver mass, histology, and enzyme levels. By histology, no discernable differences were observed between WT and Tmem27 -/Y animals ( Fig. 6 ). There were no statistical differences in liver enzyme levels ( Table 2 ). There was a trend toward increased hepatic mass in Tmem27 -/Y mice ( Table 2 ), but this did not reach statistical significance ( P = 0.12). However, these findings do not exclude the possibility of altered hepatic amino acid metabolism in collectrin-deficient animals.
Fig. 6. Histological examination by light microscopy. Hematoxylin- and eosin-stained paraffin-embedded liver sections from WT and Tmem27 -/Y animals. A : WT liver section at x 20 magnification. B : WT liver section at x 40 magnification. C : KO liver section at x 20 magnification. D : KO liver section at x 40 magnification.
Table 2. Liver functional markers
Collectrin-deficient animals have decreased populations of several key amino acid transporter subtypes in the proximal tubule plasma membrane. We next sought to determine a possible mechanism for the aminoaciduria. As we have demonstrated, collectrin protein product localizes to the apical surface of proximal tubule epithelial cells, where a number of amino acid transporters are also located. These transporters can be broadly classified by the types of amino acids they transport: neutral/aromatic, cationic, and anionic ( 38 ). As collectrin-deficient animals appear to waste all classes of amino acids, we focused on the main transporters representative of each transporter subtype, B 0 AT1 (neutral/aromatic), rBAT-b 0,+ AT (cationic/cysteine), and EAAC1 (anionic) ( 38 ). The rBAT-b 0,+ AT transporter is heteromeric, and both subunits were examined. First, we assessed the kidney transcription level of each transporter using real-time RT-PCR to obtain qualitative determinations of transcript levels as normalized to GAPDH. Our data revealed no difference between WT and Tmem27 -/Y animals for B 0 AT1; however, there were increased transcript levels in Tmem27 -/Y mice for the other three transporters ( Fig. 7 A ). We next performed immunoblotting of plasma membrane ( Fig. 7 B ) and intracellular ( Fig. 7 C ) lysate fractions obtained from renal cortical homogenates. Here we observed markedly reduced levels of B 0 AT1 in the plasma membrane fractions from Tmem27 -/Y animals. Similarly, there were also decreases in rBAT and b 0,+ AT levels in the plasma membrane fraction. Because the differences in rBAT and b 0,+ AT were not as prominent, we used near infrared fluorescence quantitation to more accurately determine the differences and found that plasma membrane levels of rBAT and b 0,+ AT are reduced in Tmem27 -/Y mice by 40 ( P = 0.0036) and 20% ( P = 0.08), respectively. There was no difference in EAAC1 plasma membrane levels between the two groups. However, Tmem27 -/Y animals had significantly greater levels of EAAC1 in the intracellular fraction ( Fig. 7 C ). We speculate that this is due to intracellular sequestration of EAAC1 protein, and the lack of appreciable difference at the plasma membrane reflects the limitations of our technique to detect a difference given the intense signals in both groups. Taken together, our data suggest that the aminoaciduria in collectrin-deficient animals results from deficient levels of several amino acid transporter subtypes in the proximal tubular apical membrane. The increase in transcript levels of three of these transporters may represent a compensatory response to a defect downstream of transcription.
Fig. 7. A : real-time RT-PCR quantitation of transcript levels of amino acid transporters B 0 AT1 and EAAC1 ( left ) and b 0,+ AT and rBAT ( right ) normalized to GAPDH and expressed as a percentage. * P < 0.008; n = 6/group. B : Western blots of renal cortical plasma membrane homogenates with tubulin as a loading control. Twenty micrograms of protein were loaded onto each well. Expression levels of B 0 AT1, and b 0,+ AT, and rBAT are decreased in plasma membrane in Tmem27 -/Y mice. C : Western blots of renal cortical intracellular homogenates with rhoGDI loading control. Twenty micrograms of protein were loaded onto each well. Expression level of EAAC1 is increased in the cytosolic fractions in Tmem27 -/Y mice.
DISCUSSION
Here, we describe the renal phenotype in mice with targeted deletion of the gene collectrin, a recently discovered downstream target of the transcription factor HNF-1 ( 1, 15 ). HNF-1 is expressed in the polarized epithelium of the kidney proximal tubule, liver, pancreas, and intestine ( 29 ). We show that, in the kidney, collectrin is also localized in the epithelium of the proximal tubule, specifically in the apical brush border. Loss of HNF-1 from the kidney results in Fanconi syndrome with polyuria, glucosuria, phosphaturia, and aminoaciduria ( 29 ). HNF-1 -deficient animals have severe polyuria, with urine volumes equaling a daily average of 40% of body weight, and are attributed to glucosuria and loss of other solutes. Collectrin-deficient animals display more subtle polyuria with only 1.6 times the urine volume of WT mice. Additionally, they are able to recover glucose and phosphate appropriately but have similar generalized wasting of amino acids as in HNF-1 -deficient animals. The defect in amino acid transport in collectrin-deficient mice appears to be X-linked recessive, since heterozygous female mice have no urinary amino acid crystals that are seen in homozygous females and hemizygous males. As with the absence of HNF-1, targeted deletion of collectrin also results in an osmotic diuresis, but this is attributable to isolated aminoaciduria. Taken together, our data suggest that HNF-1 may act on the downstream target collectrin in regulating amino acid transport.
Aminoaciduria can occur when a transport defect of the proximal tubule decreases its reabsorptive capacity, or when the reabsorptive capacity of the proximal tubule is exceeded due to increased filtered load from abnormal amino acid metabolism. In collectrin-deficient animals, with the exception of methionine and tyrosine, which are 2.2- and 2.8-fold higher, all serum levels of other amino acids are normal or low compared with WT mice. Our data support that the aminoaciduria is due to impaired renal reabsorptive capacity. Both methionine and tyrosine are essential amino acids and must, therefore, be obtained through diet. We demonstrate that while both groups have the same body weights, collectrin-deficient mice consume twice the amount of food than WT mice, presumably in compensation for nutritional losses. However, it is unlikely that the increased food consumption can explain the elevated serum levels of these two amino acids, particularly in the setting of ongoing urinary losses. As previously mentioned, our searches of protein expression databases reveal that collectrin is also expressed in the liver. We have verified this by RT-PCR (data not shown). It is possible that collectrin is involved in the liver metabolism of methionine and tyrosine and that utilization of these two amino acids is impaired out of proportion to urinary loss. Consistent with this hypothesis, targeted deletion of HNF-1 results in increased tyrosinemia (1.5-fold), among other amino acids, and is associated with liver dysfunction, as evidenced by elevated liver enzymes and hepatomegaly ( 29 ). We do not observe the same hepatic phenotype in Tmem27 -/Y animals, but cannot exclude the possibility of altered hepatic amino acid metabolism. Nevertheless, our data are consistent with a primary renal wasting of amino acids resulting from collectrin deficiency.
The mechanism of amino acid wasting associated with HNF-1 deficiency is unknown. Based on our findings, we speculate that this may be mediated through collectrin, a downstream target of HNF-1. In collectrin-deficient animals, the aminoaciduria is nearly complete, with losses of 16 of 18 amino acids measured. To date, there has been identification of three main amino acid transporter families which localize to the apical brush border of the proximal tubule. B 0 AT1 transports neutral/aromatic amino acids in a sodium-dependent manner ( 22 ), and defects in this transporter result in Hartnup disorder ( 20, 35 ). EAAC1 exhibits sodium-dependent anionic amino acid transport ( 19 ) and is the main transporter for L -glutamic acid ( 38 ). b 0,+ AT-rBAT transports cationic amino acids and L -cysteine in exchange for neutral amino acids ( 5, 6, 8, 26 ). Both rBAT and b 0,+ AT subunits appear to be necessary for proper trafficking and functioning of this transporter ( 11, 26, 28 ). We have demonstrated that collectrin also localizes to the apical brush border of the proximal tubules. Furthermore, we have shown that, in the absence of collectrin, there are decreased protein levels of B 0 AT1, rBAT, and b 0,+ AT in the plasma membrane fraction of kidney cortical homogenates and little, if any, present in the intracellular fraction, despite elevated transcript levels for rBAT and b 0,+ AT and equal transcript levels for B 0 AT1 compared with WT. We postulate that loss of collectrin results in a defect downstream of transcription of these transporters, such as in translation, folding, or trafficking. Evidence in support of this comes from studies demonstrating that mutations in rBAT that are associated with human cystinuria result in errors of rBAT trafficking, with likely retention of this subunit in the endoplasmic reticulum ( 7, 28 ). Our observation of the absence of intracellular protein product by immunoblotting could suggest loss of protein through degradation pathways. With respect to EAAC1, we do not detect a difference in plasma membrane populations between WT and collectrin-deficient animals, but we do observe an increase in the intracellular pool in collectrin-deficient animals. Again, there is a corresponding increase in transcript levels, possibly as a compensatory response. This may suggest either transporter dysfunction or errors in trafficking, turnover, or folding. EAAC1 has been extensively studied in neuronal tissue, where it is known to play an important role in the regulation of neurotransmitter levels ( 25, 36 ). In neurons, EAAC1 has been demonstrated to be maintained in intracellular pools, from which it is redistributed to the plasma membrane when needed ( 13 ). We see a greater population of this transporter in the renal cortical intracellular fraction of collectrin-deficient mice, suggesting perhaps sequestration of protein from deficient trafficking or increases in protein product in compensation for insufficient functioning.
Interestingly, there is evidence that N -ethylmaleimide-sensitive fusion attachment receptor (SNARE) complex proteins are involved in EAAC1 trafficking ( 14 ). Numerous SNARE complexes are found in mammalian cells and are extensively involved in vesicular transport ( 18 ). Recently, in their study examining the role of collectrin in the pancreas, Yamagata and associates ( 15 ) provided data suggesting that collectrin may facilitate insulin exocytosis from -cells. Using the yeast two-hybrid system and GST-pulldown assays, they demonstrated that collectrin directly interacts with snapin, a SNAP-25 binding protein, important in SNARE complex formation. This suggests the possible involvement of collectrin in vesicular transport, and a possible role for collectrin in mediating the trafficking of amino acid transporters to the proximal tubule apical membrane. Studies are currently underway in our laboratory to examine this question.
Collectrin was originally identified in a search for genes upregulated during the hypertrophic phase of the remnant kidney after subtotal nephrectomy, a well-established model of chronic kidney disease. One common consequence of chronic kidney disease, regardless of the primary etiology, is abnormal amino acid handling by the kidney, presumably from loss of nephron mass, resulting in severe aminoaciduria and hence altered homeostatic maintenance of amino acids ( 17, 27, 40 ). We speculate that upregulation of collectrin in chronic kidney disease is an adaptive response to conserve amino acids.
A number of human diseases are associated with aminoaciduria, but little is known about the mechanisms involved or how amino acid transporters are regulated in these disease states. Here, we demonstrate that mice with targeted deletion of collectrin exhibit a urinary concentrating defect induced by an osmotic diuresis. Furthermore, we show that the osmotic diuresis results from generalized aminoaciduria, which is caused, at least in part, by insufficient levels of several amino acid transporter subtypes in the proximal tubule plasma membrane. Collectrin is a novel mediator of amino acid transport and may provide new insights into human disease correlates and the regulation of amino acid transport.
GRANTS
This work was supported by Duke Nephrology Training Grant 2T32-DK-007731-12 (to S. M. Malakauskas).
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. Mark Knepper for helpful discussions and critical review of the manuscript, as well as Drs. Robert Spurney and John Foreman for additional helpful insights. We further thank Anne Latour and Dr. Beverly Koller for embryonic stem cell injections and the generation of our collectrin-deficient mouse line, and Dr. Jay Snouweart for providing the targeting vector, pXena, and assistance with construct design.
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作者单位：1 Departments of Medicine and Pathology, Duke University, and Durham Veterans Affairs Medical Centers, Durham; 2 CIIT Centers for Health Research, Research Triangle Park, North Carolina; and 3 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institu