点击显示 收起
【摘要】 Genetic studies indicated that mutations of the chloride channel CLC-5 in the kidney are responsible for a group of clinical disorders, collectively called Dent's disease. In the kidney, CLC-5 was found to be expressed in the proximal tubule, medullary thick ascending limb (mTAL) of loop of Henle, and intercalated cells of the collecting tubule. In proximal tubular cells, CLC-5 was found to play an important role in receptor-mediated endocytosis. However, the functional roles of CLC-5 in mTAL and collecting tubules remain unclear. Because mTAL is normally exposed to a hypertonic environment, we aimed to examine the effect of hypertonicity on CLC-5 expression in this nephron segment. Our studies revealed that exposure to hypertonicity (up to 550 mosM) increased CLC-5 mRNA and protein levels in a murine mTAL cell line (MTAL) but not in an opossum kidney (OK) proximal tubular cell line. A similar effect was also found in mouse kidneys, where CLC-5 expression was enhanced in renal medulla, but not cortex, after 48 h of water deprivation. We also tested the effect of hypertonicity on endocytotic activity and found that exposure to hypertonicity caused a significant decrease in cellular uptake of FITC-labeled albumin in OK but not in MTAL cells. Our results suggest that CLC-5 expression is upregulated by hypertonicity in mTAL cells but not in proximal tubular cells. We speculate that the increased CLC-5 levels in mTAL may serve to maintain the endocytotic activity in a hypertonic environment.
【关键词】 chloride channels water deprivation endocytosis
OVER THE PAST DECADE, CHLORIDE channels have gained great attention for their roles in membrane excitability and transepithelial transport. Thus far, nine different mammalian chloride channels (CLC), including CLC-K1, CLC-K2, and CLC-1 to CLC-7, have been identified ( 1, 4, 15, 16, 18, 19, 30, 36 - 38, 40 ). Mutations of different chloride channels have been linked to a range of clinical conditions including CLC-1 in myotonia congenita, CLCNKB (human gene for CLC-K2) in Bartter's syndrome, and CLC-5 in Dent's disease, X-linked recessive nephrolithiasis (XLRN), X-linked recessive hypophosphatemic rickets, and low-molecular-weight (LMW) proteinuria/nephrocalcinosis ( 21, 26, 28, 34, 38 ). Clinical features reported among patients afflicted with various inactivating CLC-5 mutations include varying degrees of LMW proteinuria, hypercalciuria, hyperphosphaturia, nephrocalcinosis, nephrolithiasis, urinary concentrating defect, and renal failure.
In the kidney, CLC-5 has been localized to proximal tubules ( 6, 11, 22, 31, 38 ), medullary thick ascending limb (mTAL) ( 6, 22, 38 ), and intercalated cells ( 6, 11, 29, 31 ) in collecting tubules ( 38 ). In proximal tubular cells, CLC-5 colocalizes with subapical endosomes, where it plays an important role in receptor-mediated endocytosis (RME) through its function as an electrical shunt to neutralize the electrogenic hydrogen transport by V-H + -ATPase ( 11, 22, 31, 32, 43 ). More recent studies have further suggested that CLC-5 may also be involved in RME in other cell types including mouse collecting duct cells ( 33 ) and rat intestinal cells ( 42 ).
In addition to RME, the role of CLC-5 in calcium metabolism has also been an area of great interest due to its localization in nephron segments responsible for calcium reabsorption and the hypercalciuric phenotype noted in disorders associated with inactivating CLC-5 mutations. The possibility that CLC-5 may participate in renal calcium transport was suggested by the finding that CLC-5 expression in renal cortex is under the regulation of parathyroid hormone and that CLC-5 expression in the kidney inversely correlated with urinary calcium excretion ( 35 ). The notion that hypercalciuria is an abnormality intrinsic to the kidney was further supported by the finding that Dent's disease patients may be cured of their hypercalciuria by a successful kidney transplantation ( 8, 34 ). However, other reports have raised the possibility that CLC-5 may be linked to gastrointestinal absorption of calcium and that hypercalciuria observed in patients with inactivating CLC-5 mutations may represent a secondary event ( 23 ).
Although the functional role of CLC-5 in mTAL is unclear, we aimed in our current study to examine the effect of hypertonicity on CLC-5 expression in a murine mTAL cell line, the MTAL cells. We found that exposure to hypertonicity increased CLC-5 expression in MTAL cells but not in opossum kidney proximal tubular cells, the OK cells. We also found that exposure to hypertonicity suppressed endocytotic activity in proximal tubular cells but not in MTAL cells.
MATERIALS AND METHODS
Materials. The culture media (DMEM/Ham's F-12) were purchased from Irvine Scientific (Santa Ana, CA). Tissue culture plates were purchased from Nunc Interlab (Thousand Oaks, CA). Radioisotopes were purchased from ICN Biochemicals (Irvine, CA). The FITC-labeled BSA and other chemicals were purchased from Sigma (St. Louis, MO).
Cell cultures. Two cell lines were used in our studies: OK cells (obtained from Dr. D. M. Shoback, UCSF, San Francisco, CA) and mouse mTAL cells ( 25 ). Cells were cultured in 1:1 DMEM/Ham's F-12 at 37°C in a 95% air-5% CO 2 incubator and used when monolayer confluence was reached. To test the effect of hypertonicity, the cells were incubated in culture media made hypertonic by adding mannitol. To improve cell tolerance to tonicity changes, the tonicity of the culture media was increased in a stepwise fashion by adding 125 mM mannitol during the first 24 h. This was followed by the addition of another 125 mM mannitol, and the cells were further incubated for another 48 h. To avoid any potential complicating effects from humoral factors in the serum, serum-free culture media were used in our studies.
Animals. To examine the effect of hypertonicity in vivo, C57BL/6J male mice, weighing 20-30 g, were used. Mice were obtained from Jackson Laboratory (Bar Harbor, ME) and were housed in a climate-controlled vivarium and allowed free access to food (Purina mouse chow, Ralston-Purina, St. Louis, MO) and water. To study the effect of dehydration on CLC-5 expression in the kidney, the mice were deprived of water for 48 h. After euthanasia by neck dislocation under halothane anesthesia, the kidneys were immediately removed for the dissection of cortical and outer medullary tissues. The current study was approved by our institutional review board.
Northern blot analysis. Cell and tissue RNA were isolated using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX) according to the manufacturer's instructions. For Northern blot analysis, 5 µg of total cellular RNA were size-fractioned on a 1.0% formaldehyde/agarose gel in 1 x MOPS buffer (20 mM MOPS, 8 mM sodium acetate, 1 mM EDTA, pH 7.0) and transferred to nylon membranes (Pierce, Rockford, IL). The blots were probed with a biotinyl-labeled CLC-5 RNA probe and detected by chemiluminescence according to the manufacturer's instructions (Pierce). To make the CLC-5 RNA probe, a 2.1-kb PCR product of CLC-5 cDNA exon 12 was first obtained by using the ClC-5 sense (5'-ATTGGATCCGGCCGGCCTGGAGGAATA GGTTCTTCAAATAG-3') and antisense (5'-ACATATCCATGGTCTGTAATGTCC-3'). This PCR product was cloned into pGEM-T easy (Promega, Madison, MI), sequence verified, and then used to produce the RNA probe by using a North2South in vitro transcription kit according to the manufacturer's instructions (Pierce). A RNA probe for GAPDH was used to ensure consistent sampling. Scanning and analysis of the Northern blot for the calculation of ClC-5:GAPDH mRNA ratio were performed by the VersaDoc Imaging System (Bio-Rad, Hercules, CA).
Western blot analysis. Cell lysates were prepared from OK and mTAL cells in 0.25 M sucrose, 20 mM imidazole, 1 mM EDTA lysis buffer, pH 7.4, containing complete protease inhibitors (Roche Molecular Biochemicals, Brussels, Belgium) and briefly sonicated (3 pulses, 40% intensity; Branson Sonifier 250, Danbury, CT). A postnuclear supernatant was obtained after centrifugation at 16,000 g for 1 min at 4°C, transferred (200 µl) into fresh tubes containing 5 µl of 10% SDS, and heated at 95°C for 90 s ( 12 ). Protein concentrations were determined with the bicinchoninic acid protein assay (Pierce), using BSA as the standard.
Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose as previously described ( 6 ). After being blocked, membranes were incubated overnight at 4°C with the affinity-purified anti-CLC-5 antibodies (1:1,000 dilution), washed, incubated for 1 h at room temperature with anti-rabbit IgG peroxidase-labeled antibodies (Dako), washed again, and visualized with enhanced chemiluminescence (Amersham Pharmacia Biotech). Affinity-purified antibodies (SB-499) raised in rabbits against the NH 2 terminus of human CLC-5 have been extensively characterized previously in mammalian kidney and various renal cell lines ( 17, 43 ). Ponceau red (Sigma) staining and reprobing with monoclonal antibodies against -actin (Sigma) verified the transfer efficacy. The experiments were performed in duplicate.
Characterization of MTAL cell line. Total RNA was extracted from MTAL cells using Ultraspec RNA (Biotex). Ten micrograms of total RNA were reverse-transcribed using oligo (dT)20 and ThermoScript reverse transcriptase (Invitrogen, Merelbeke, Belgium) as recommended by the manufacturer. cDNA was amplified using the Expand Long Template PCR system (Roche Applied Science). Primers for amplification of Tamm-Horsfall protein (THP) and Na + -K + -2Cl - cotransporter (NKCC) were as follows: THP, sense 5'-TCTAGCTGGAGCCAGTAACTCA-3', antisense 5'-AGTCCTGGCTCACAGGAGCTT-3'; and NKCC, sense 5'-GTGCATTGTCTTAACAGGCG-3', antisense 5'-GTGTTTGGCTTCATTCTCCC-3'. The predicted lengths of the resulting PCR fragments were 417 bp (THP) and 279 bp (NKCC). PCR amplification was carried out at 94°C for 2 min followed by 40 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 7 min for final extension. The PCR products were separated on 2% agarose gel containing 5 µg/ml ethidium bromide and visualized over a UV light box (Versadoc Bio-Rad).
RT-PCR and semiquantitative real-time RT-PCR. In parallel with protein extraction, part of the MTAL cell pellets was homogenized in TRIzol (Invitrogen) and total RNA was extracted according to the manufacturer's instructions; 2.7 µg total RNA were treated with DNase I, Amp Grade (Invitrogen) according to the manufacturer's instructions and reverse-transcribed into cDNA using SuperScript II.
Changes in CLC-5 mRNA levels were determined by semiquantitative real-time RT-PCR (iCycler IQ System, Bio-Rad) using SYBR Green I detection of single PCR product accumulation.
Primers for amplification of CLC-5 GAPDH were as follows: CLC-5, sense 5'-ACCACGTACAGTGGCTTTCC-3', antisense 5'-AATGCTCGGAAGAAACAGGA-3'; and GAPDH sense 5'-tgcaccaccaactgcttagc-3', antisense 5'-ggatgcagggatgatgttct-3'. The predicted lengths of the resulting PCR fragments were 113 bp (CLC-5) and 176 bp (GAPDH).
Real-time semiquantitative PCR analyses were performed in triplicate with 200 nM of both sense and antisense primers in a final volume of 25 µl using 1 U of Platinum Taq DNA Polymerase, 2 mM MgSO 4, 400 µM dNTP and SYBR Green I (Molecular Probes, Leiden, The Netherlands) diluted 1:100,000.
The PCR mix contained 10 nM fluorescein for initial well-to-well fluorescence normalization. PCR conditions were 94°C for 3 min followed by 40 cycles of 30 s at 95°C, 30 s at 61°C, and 1 min at 72°C. The melting temperature of the PCR product was verified at the end of each PCR by recording the increase in SYBR Green fluorescence on slow renaturing of DNA (initial denaturation at 98°C for 1 min followed by stepwise decrease in the temperature by 10-s steps of 0.5°C).
To exclude amplification from contaminating genomic DNA, samples of RNA that had not been reverse-transcribed were run in a parallel PCR reaction: these controls always remained negative. For each assay, standard curves were prepared by serial fourfold dilutions of the MTAL cDNA. The relative changes in the CLC-5/GAPDH mRNA ratio between MTAL and MTAL high-osmolality cells were determined by 2 Ct after normalization to GAPDH, where Ct is the difference in Ct between MTAL high osmolality and MTAL control.
Endocytosis assay. Endocytotic activity was determined by measuring cellular uptake of FITC-labeled albumin. Uptake was performed at 37°C in a solution containing (in mM) 120 NaCl, 5.4 KCl, 1.2 CaCl 2, 0.8 MgCl 2, 0.8 Na 2 HPO 4, 0.2 NaH 2 PO 4, 5.5 glucose, 10 HEPES, pH 7.4, with FITC-labeled albumin 200 µg/ml and terminated by repeated washing at 4°C with the same solution except for albumin. When the effect of hypertonicity was tested, the cells were preexposed to hypertonicity for 48 h as previously described and the endocytosis assay was performed with the same uptake solution but with the addition of 250 mM mannitol. The cells were subsequently lysed by detergent (Triton X-100, 0.1% vol/vol in MOPS solution at pH 7.4), and the fluorescence intensity released from the cell was measured by a spectrofluorometer at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The protein content of each sample was quantitated by using Coomassie brilliant blue G250 with BSA as the standard. Nonspecific binding of albumin was determined in separate samples treated in a similar fashion, except for uptake being performed at 4°C, and was subtracted from other samples.
Statistical analyses. At least three determinations were obtained for each data point, and the experimental data are expressed as means ± SE. The significance of differences was analyzed by Student's t -test for unpaired data.
RESULTS
Characterization of MTAL cells. To ensure our cultured MTAL cells retain their characteristics, the expressions of the NKCC and THP were examined. The expected bands for THPs and NKCC at 417 and 279 bp, respectively, are shown in Fig. 1.
Fig. 1. RT-PCR characterization of medullary thick ascending limb (MTAL) cell line. Left, lane 1 : negative control for Na + -K + -2Cl - cotransporter (NKCC) [reverse transcriptase (RT) was substituted with water to ensure that the RNA sample was not contaminated with genomic DNA]; lane 2 : NKCC (279 bp). Right, lane 1 : negative control for Tamm-Horsfall protein (THP) (reverse transcriptase was substituted with water to ensure that the RNA sample was not contaminated with genomic DNA); lane 2 : THP (417 bp).
Effect of hypertonicity on CLC-5 expression in MTAL and OK cells. The exposure of MTAL cells to hypertonicity induced a significant upregulation of CLC-5 mRNA expression in MTAL compared with controlled cells with CLC-5/GAPDH mRNA ratios 3.58 ± 2.52 vs. 0.83 ± 0.48, respectively, n = 6, P < 0.01 ( Fig. 2 ). In contrast, incubation of OK cells in a similar hypertonic culture medium did not affect the CLC-5/GAPDH ratio (0.98 ± 0.34 vs. 0.79 ± 0.14, n = 6, P = not significant; Fig. 2 ). The increase in CLC-5 mRNA level in MTAL cells was further confirmed by semiquantitative and real-time PCR. Analyses performed in triplicate similarly revealed that culture of MTAL cells in hypertonic conditions induced a 2.5-fold (256 ± 19%) increase in CLC-5 expression vs. control conditions. As shown in Fig. 3, immunoblot analyses performed on lysates obtained from the same pellets used for mRNA extraction also showed that culture of MTAL cells in hypertonic conditions also induced an upregulation of CLC-5 at the protein level. In contrast, no such effect was observed in OK cells.
Fig. 2. Effect of hypertonicity (H) on CLC-5 mRNA levels in opossum kidney proximal tubular cells (OK) and MTAL cells. There was a significant increase in the expression of CLC-5 mRNA in MTAL but not in OK cells with exposure to hypertonic culture media. The CLC-5 probe hybridizes to a band of 9.5 kb as expected. C, control.
Fig. 3. Effect of hypertonicity on CLC-5 protein levels in OK and MTAL cells. The diffuse bands corresponding to CLC-5 and its glycosylated isoforms (Ref. 17 ) were clearly upregulated in MTAL cells cultured in hypertonic conditions but not in OK cells.
Effect of water deprivation on CLC-5 expression in mouse kidneys. To correlate the results from our in vitro model to an in vivo setting, we tested the effect of dehydration on CLC-5 expression in mouse kidney cortex and medulla. For these studies, mice were deprived of water intake for 48 h before kidney tissues were obtained. As shown in Fig. 4, the CLC-5 mRNA expression increased to a greater extent in outer medullary tissues (3.74 ± 0.58-fold) compared with a minimal increase in cortical tissues (1.47 ± 1.01-fold). The increase in CLC-5 expression in the outer medullary tissues was further confirmed by immunoblot analysis ( Fig. 5 ).
Fig. 4. Effect of water deprivation (WD) on CLC-5 mRNA levels in mouse cortex and outer medulla. There was a significant increase in CLC-5 mRNA expression in the mouse outer medulla but not in the cortex.
Fig. 5. Effect of water deprivation on CLC-5 protein levels in mouse cortex and outer medulla. There was an increase in CLC-5 protein levels in the mouse outer medulla but not in the cortex.
Effect of hypertonicity on endocytotic activity. Because CLC-5 has been suggested to play an important role in RME in proximal tubular cells ( 11, 22, 31, 32, 43 ) and other cell types ( 33, 42 ), studies were performed to test the effect of hypertonicity on endocytosis in MTAL and OK cells. In these studies, cellular endocytotic activity was determined by measuring the uptake of FITC-labeled albumin as previously described ( 10 ). At baseline, the endocytotic activity was significantly higher in OK cells than in MTAL cells (2.05 ± 0.34 vs. 0.77 ± 0.04 µg·mg protein -1 ·30 min -1, n = 6, P < 0.0001). As shown in Fig. 6, preincubation of OK cells to hypertonic culture media for 48 h caused a significant decrease in albumin uptake. In contrast, MTAL cells maintained the uptake activity at the baseline level over the same study period.
Fig. 6. Effect of hypertonicity on albumin uptake in MTAL and OK cells. Although there was a significant decrease in albumin uptake in OK cells ( n = 6, P < 0.0001) after 48 h of exposure to hypertonic media, MTAL cells ( n = 6, P = not significant) maintained their baseline albumin uptake activity over the same study period.
DISCUSSION
Direct studies on the functional role(s) of CLC-5 and its regulatory factors within the MTAL nephron segment are lacking. To our knowledge, this is the first study to demonstrate that hypertonicity enhances CLC-5 expression in MTAL cells. Raising the culture medium tonicity increased both CLC-5 mRNA and protein levels in MTAL cells but not in OK cells. Similarly, in water-deprived mice, where an increase in renal medulla tonicity is expected, CLC-5 expression was increased compared with the cortex, where the tonicity remains relatively constant. The mechanism whereby hypertonicity and water deprivation increase CLC-5 expression is unclear. It is not known whether the induction of CLC-5 is mediated by a tonicity-responsive enhancer-binding protein signaling mechanism. In addition, it is also unknown whether the upregulation of CLC-5 by water deprivation is directly related to increased medullary tonicity or to humoral mediators such as antidiuretic hormone and/or renin-angiotensin. The former possibility may be supported by our in vitro studies where only serum-free culture medium was used.
The physiological significance of enhanced CLC-5 expression with hypertonicity and dehydration is not known. Given the known function of CLC-5 in RME in proximal tubular cells ( 11, 22, 31, 32, 43 ) and possibly other cell types including collecting duct ( 33 ) and intestinal cells ( 42 ), it is possible that a similar role may exist in the mTAL nephron segment. Our albumin uptake studies revealed that endocytosis takes place in MTAL cells, albeit at a lower extent than that observed in OK cells. Our results also showed that exposure to hypertonicity had no effect on CLC-5 expression and reduced endocytotic activity in OK cells. In contrast, hypertonicity enhanced CLC-5 expression with no appreciable change in endocytotic activity in MTAL cells. Based on the presumed role of CLC-5 in RME in other cell types, we speculate that the observed increase in CLC-5 expression in MTAL cells may serve to maintain normal endocytotic activity and compensate for the reduction in endocytosis due to hypertonicity. Because MTAL cells are regularly exposed to a hypertonic environment, they may have developed a mechanism to upregulate and maintain adequate endocytosis activity under these conditions. Proximal tubular cells, on the other hand, do not require this regulatory mechanism because of their relatively constant isotonic environment and are hence susceptible to hypertonicity-induced suppression of endocytosis.
We recognize that despite our current findings, evidence for the involvement of CLC-5 in endocytosis in the mTAL segment remains circumstantial and speculative. However, one of the unique features of this nephron segment is the secretion of THP into the lumen through an exocytosis process ( 2, 14, 24 ). Similar to endocytosis, exocytosis also requires vacuolar acidification for a variety of functions, such as hydrolysis of macromolecules, release of ligands from receptors, and processing of preproproteins ( 9 ). Indeed, previous immunocytochemical studies have demonstrated the presence of vacuolar H + -ATPase pumps in subapical endosomes of mTAL segment ( 3 ). As other CLC isoforms have been implicated in playing a role in the exocytosis process ( 5, 7, 39 ), and in some cases, specifically in maintaining the electrical neutrality with endosomal acidification ( 39 ), it is conceivable that CLC-5 may play a similar role in the mTAL nephron segment. Inactivating mutations of CLC-5 in the mTAL segment may thus contribute to ineffective delivery of THP to the apical cell surface membranes, as suggested by preliminary data showing an increased expression of THP mRNA in the kidneys of CLC-5 knockout mice (Devuyst O, Guggino S, and Guggino WB, unpublished observations). These data support the hypothesis that the loss of CLC-5 in the mTAL segment could result in modified THP turnover due to its altered clearance and expression. Because THP has been implicated in rendering the thick ascending limb water impermeable and potentially inhibiting calcium stone formation ( 20 ), we speculate that any ineffective THP delivery to the mTAL apical surface may contribute to the urinary concentrating defect and facilitate the development of nephrolithiasis observed in patients with inactivating CLC-5 mutations. Another interesting aspect regarding THP is its potential autoantigenic role in inducing tubulointerstitial nephritis and renal tissue damage ( 13, 24, 41 ). The improper accumulation and distribution of THP within the kidney may promote diffuse nonspecific inflammatory responses and subsequent renal failure. Indeed, kidney biopsies from patients with different CLC-5 mutations showed nonspecific tubular atrophy, interstitial fibrosis, and glomerulosclerosis ( 27, 34 ).
In summary, we showed that hypertonicity upregulates the expression of CLC-5 at the transcriptional level in MTAL cells but not in OK cells. Water deprivation in mice similarly enhanced CLC-5 expression in renal outer medulla but not in renal cortex. Although the baseline endocytotic activity was lower in MTAL cells compared with that of OK cells, hypertonicity suppressed endocytosis in OK cells but not MTAL cells. We speculate that the upregulation of CLC-5 in MTAL cells under hypertonic conditions may play a role in maintaining their endocytotic and possibly exocytotic activity. In view of the possible association between CLC-5 and THP trafficking and the role of THP in inhibiting calcium stone formation, further studies on the functional role of CLC-5 in the mTAL nephron segment would be of great interest.
GRANTS
This study was supported by National Institutes of Health Grant RO1-DK-58886 to N. Yanagawa, Veterans Administration grant to N. Yanagawa, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47403 to A. M. Sun, and Fonds National de la Recherche Scientifique and Fondation pour la Recherche Scientifique Médicale grants (Belgium) and the Action de Recherches Concertées 00/05-260 to O. Devuyst.
ACKNOWLEDGMENTS
We thank Dr. A. Yu for comments and critical review of the manuscript and Dr. V. A. Luyckx for providing the CLC-5 antibody for the initial study.
【参考文献】
Adachi S, Uchida S, Ito H, Hata M, Hiroe M, Marumo F, and Sasaki S. Two isoforms of a chloride channel predominantly expressed in thick ascending limb of Henle's loop and collecting ducts of rat kidney. J Biol Chem 269: 17677-17683, 1994.
Bachmann S, Koeppen-Hagemann I, and Kriz W. Ultrastructural localization of Tamm-Horsfall glycoprotein (THP) in rat kidney as revealed by protein A-gold immunocytochemistry. Histochemistry 83: 531-538, 1985.
Bastani B. Immunocytochemical localization of the vacuolar H + -ATPase pump in the kidney. Histol Histopathol 12: 769-779, 1997.
Brandt S and Jentsch TJ. CLC-6 and CLC-7 are two novel broadly expressed members of the CLC chloride channel family. FEBS Lett 377: 15-20, 1995.
Denda M, Fuziwara S, and Inoue K. Influx of calcium and chloride ions into epidermal keratinocytes regulates exocytosis of epidermal lamellar bodies and skin permeability barrier homeostasis. J Invest Dermatol 121: 362-367, 2003.
Devuyst O, Christie PT, Courtoy PJ, Beauwens R, and Thakker RV. Intrarenal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease. Hum Mol Genet 8: 247-257, 1999.
Dietrich J and Lindau M. Chloride channels in mast cells: block by DIDS and role in exocytosis. J Gen Physiol 104: 1099-1111, 1994.
Frymoyer PA, Scheinman SJ, Dunham PB, Jones DB, Hueber P, and Schroeder ET. X-linked recessive nephrolithiasis with renal failure. N Engl J Med 325: 681-686, 1991.
Futai M, Oka T, Sun-Wada GH, Moriyama Y, Kanazawa H, and Wada Y. Luminal acidification of diverse organelles by V-ATPase in animal cells. J Exp Biol 203: 107-116, 2000.
Gekle M, Mildenberger S, Freudinger R, and Silbernagl S. Endosomal alkalinization reduces J max and K m of albumin receptor-mediated endocytosis in OK cells. Am J Physiol Renal Fluid Electrolyte Physiol 268: F899-F906, 1995.
Gunther W, Luchow A, Cluzeaud F, Vandewalle A, and Jentsch TJ. CLC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc Natl Acad Sci USA 95: 8075-8080, 1998.
Hanaoka K, Devuyst O, Schwiebert EM, Wilson PD, and Guggino WB. A role for CFTR in human autosomal dominant polycystic kidney disease. Am J Physiol Cell Physiol 270: C389-C399, 1996.
Hession C, Decker JM, Sherblom AP, Kurmar S, Yue CC, Mattaliano RJ, Tizard R, Kawashima E, Schmeissner U, Heletky S, Chow EP, Burne CA, Shaw A, and Muchmore AV. Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines. Science 237: 1479-1484, 1987.
Hoyer JR, Sisson SP, and Vernier RL. Tamm-Horsfall glycoprotein: ultrastructural immunoperoxidase localization in rat kidney. Lab Invest 41: 168-173, 1979.
Jentsch TJ, Gunther W, Pusch M, and Schwappach B. Properties of voltage-gated chloride channels of the CLC gene family. J Physiol 482, Suppl P: 19S-25S, 1995.
Jentsch TJ, Steinmeyer K, and Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348: 510-514, 1990.
Jouret F, Igarashi T, Gofflot F, Wilson PD, Karet FE, Thakker RV, and Devuyst O. Comparative ontogeny, processing, and segmental distribution of the renal chloride channel, CLC-5. Kidney Int 65: 198-208, 2004.
Kawasaki M, Uchida S, Monkawa T, Miyawaki A, Mikoshiba K, Marumo F, and Sasaki S. Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron 12: 597-604, 1994.
Kieferle S, Fong P, Bens M, Vandewalle A, and Jentsch TJ. Two highly homologous members of the CLC chloride channel family in both rat and human kidney. Proc Natl Acad Sci USA 91: 6943-6947, 1994.
Kokot F and Dulawa J. Tamm-Horsfall protein updated. Nephron 85: 97-102, 2000.
Lloyd SE, Pearce SHS, Fisher SE, Steinmeyer K, Schwappach B, Scheinman SJ, Harding B, Bolino A, Devoto M, Goodyer P, Rigden SPA, Wrong O, Jentsch TJ, Craig IW, and Thakker RV. A common molecular basis for three inherited kidney stone diseases. Nature 379: 445-449, 1996.
Luyckx VA, Goda FO, Mount DB, Nishio T, Hall A, Hebert SC, Hammond TG, and Yu AS. Intrarenal and subcellular localization of rat CLC5. Am J Physiol Renal Physiol 275: F761-F769, 1998.
Luyckx VA, Leclercq B, Dowland LK, and Yu AS. Diet-dependent hypercalciuria in transgenic mice with reduced CLC5 chloride channel expression. Proc Natl Acad Sci USA 96: 12174-12179, 1999.
Malagolini N, Cavallone D, and Serafini-Cessi F. Intracellular transport, cell-surface exposure and release of recombinant Tamm-Horsfall glycoprotein. Kidney Int 52: 1340-1350, 1997.
McManus M, Fischbarg J, Sun AM, Hebert SC, and Strange K. Laser light-scattering system for the study of cell volume regulation. Am J Physiol Cell Physiol 265: C562-C570, 1993.
Morimoto T, Uchida S, Sakamoto H, Kondo Y, Hanamizu H, Fukui M, Tomino Y, Nagano N, Sasaki S, and Marumo F. Mutations in CLCN5 chloride channel in Japanese patients with low molecular weight proteinuria. J Am Soc Nephrol 9: 811-818, 1998.
Moulin P, Igarashi T, Van Der Smissen P, Cosyns JP, Verroust P, Wrong O, Thakker RV, Scheinman SJ, Courtoy PJ, and Devuyst O. Altered polarity and expression of H + -ATPase without ultrastructural changes in kidneys of Dent's disease patients. Kidney Int 63: 1285-1295, 2003.
Nakazato H, Yoshimuta J, Karashima S, Matsumoto S, Endo F, Matsuda I, and Hattori S. Chloride channel CLCN5 mutations in Japanese children with familial idiopathic low molecular weight proteinuria. Kidney Int 55: 63-70, 1999.
Obermuller N, Gretz N, Kriz W, Reilly RF, and Witzgall R. The swelling-activated chloride channel CLC-2, the chloride channel CLC-3, and CLC-5, a chloride channel mutated in kidney stone disease, are expressed in distinct subpopulations of renal epithelial cells. J Clin Invest 101: 635-642, 1998.
Sakamoto H, Kawasaki M, Uchida S, Sasaki S, and Marumo F. Identification of a new outwardly rectifying Cl - channel that belongs to a subfamily of the CLC Cl - channels. J Biol Chem 271: 10210-10216, 1996.
Sakamoto H, Sado Y, Naito I, Kwon TH, Inoue S, Endo K, Kawasaki M, Uchida S, Nielsen S, Sasaki S, and Marumo F. Cellular and subcellular immunolocalization of CLC-5 channel in mouse kidney: colocalization with H + ATP-ase. Am J Physiol Renal Physiol 277: F957-F965, 1999.
Sasaki Y, Nagai J, Kitahara Y, Takai N, Murakami T, and Takano M. Expression of chloride channel, CLC-5, and its role in receptor-mediated endocytosis of albumin in OK cells. Biochem Biophys Res Commun 282: 212-218, 2001.
Sayer JA, Stewart GS, Boese SH, Gray MA, Pearce SH, Goodship TH, and Simmons NL. The voltage-dependent Cl - channel CLC-5 and plasma membrane Cl - conductances of mouse renal collecting duct cells (mIMCD-3). J Physiol 536: 769-783, 2001.
Scheinman SJ. X-linked hypercalciuric nephrolithiasis: clinical syndromes and chloride channel mutations. Kidney Int 53: 3-17, 1998.
Silva IV, Blaisdell CJ, Guggino SE, and Guggino WB. PTH regulates expression of CLC-5 chloride channel in the kidney. Am J Physiol Renal Physiol 278: F238-F245, 2000.
Simon DB, Karet FE, Hamdan JM, Di Pietro A, Sanjad SA, and Lifton RP. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2. Nat Genet 13: 183-188, 1996.
Steinmeyer K, Ortland C, and Jentsch TJ. Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel. Nature 354: 301-304, 1991.
Steinmeyer K, Schwappach B, Bens M, Vandewalle A, and Jentsch T. Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease. J Biol Chem 270: 31172-31177, 1995.
Thevenod F. Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins. Am J Physiol Cell Physiol 283: C651-C672, 2002.
Thiemann A, Grunder S, Pusch M, and Jentsch TJ. A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356: 57-60, 1992.
Thomas DB, Davies M, and Williams JD. Release of gelatinase and superoxide from human mononuclear phagocytes in response to particulate Tamm-Horsfall protein. Am J Pathol 142: 249-260, 1993.
Vandewalle A, Cluzeaud F, Peng KC, Bens M, Luchow A, Gunther W, and Jentsch TJ. Tissue distribution and subcellular localization of the CLC-5 chloride channel in rat intestinal cells. Am J Physiol Cell Physiol 280: C373-C381, 2001.
Wang SS, Devuyst O, Courtoy PJ, Wang T, Wang H, Wang Y, Thakker RV, Guggino S, and Guggino WB. Mice lacking renal chloride channel, CLC-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis. Hum Mol Genet 9: 2937-2945, 2000.
作者单位：1 Renal Division, Department of Medicine, Olive View-UCLA Medical Center, Sylmar 91342; 3 Kidney and Pancreas Transplantation, Department of Medicine, David Geffen UCLA Medical Center, Los Angeles 90095; 4 Renal Division, Department of Medicine, Sepulveda Veterans Administration Medical Center, Sepu