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【摘要】 Proximal tubular receptor-mediated endocytosis (RME) of filtered proteins prevents proteinuria. Pharmacological and genetic studies in cultured opossum kidney cells have shown that the apical Na + /H + exchanger isoform 3 (NHE3) supports RME by interference with endosomal pH homeostasis and endocytic fusion events. However, it is not known whether NHE3 also supports proximal tubular RME in vivo. We analyzed proximal tubular protein reabsorption by microinfusion experiments in rats and investigated renal protein excretion in NHE3 knockout (Nhe3 -/-) mice. Inhibition of NHE3 by EIPA or S-3226 reduced the fractional reabsorption of [ 14 C]cytochrome c by 50% during early proximal microinfusion. During early distal microinfusion, no protein reabsorption could be detected. Urinary protein excretion of Nhe3 -/- or heterozygous mutant mice was significantly higher compared with wild-type mice. SDS-PAGE analysis of urinary proteins revealed that Nhe3 -/- animals excreted proteins the size of albumin or smaller. Thus a reduction in NHE3 activity or abundance causes tubular proteinuria. These data show that NHE3 supports proximal tubular RME of filtered proteins in vivo.
【关键词】 urinary protein excretion pH homeostasis
RECEPTOR - MEDIATED ENDOCYTOSIS is an essential mechanism for reabsorption of filtered proteins in the proximal tubule. In recent years, it has been shown that megalin/cubilin-mediated endocytosis is one of the most important pathways in this context ( 5, 6, 9, 14, 17 ). An important process along the endocytic pathway is the proper pH homeostasis of endosomal compartments ( 21, 27, 29 ), which influences ligand-receptor dissociation, vesicle trafficking, endosomal fusion events, and recycling to the plasma membrane ( 18, 19, 27, 29, 37 ). Endosomal pH homeostasis is maintained, at least in part, by the vacuolar-type H + -ATPase ( 33 ). In addition, cell culture studies provided evidence for the involvement of Na + /H + exchanger isoform 3 (NHE3) in endosomal pH homeostasis and megalin/cubilin-mediated endocytosis ( 1, 11, 14 - 16, 23, 26 ). Renal proximal tubular protein reabsorption is of major importance because it prevents the loss of vitamins, hormones, and amino acids ( 8, 17 ), but at the same time enhanced tubular protein loads can induce tubulointerstitial inflammation and fibrosis ( 3, 13 ) and change proximal tubular gene expression ( 30 ). The mechanisms leading to protein-induced inflammation or fibrosis involve the expression of pathophysiologically relevant mediators such as RANTES and MCP-1, transcription factors such as NF- B, and activation of mitogenic pathways during enhanced protein reabsorption ( 3, 12, 22, 38 ). Using proximal tubule-derived opossum kidney cells, we showed previously that prevention of apical protein uptake by inhibition of NHE3 reduces the activation of inflammatory and fibrotic events ( 40 ). Thus, if the cell culture data were to hold true for the situation in vivo, then NHE3 would be a potential therapeutic target for the treatment of proteinuria-induced tubulointerstitial nephropathies. The present study was performed to test whether NHE3 supports proximal tubular protein endocytosis also in vivo. For this purpose, we performed micropuncture experiments in rats and determined urinary protein excretion in NHE3 knockout mice ( 34 ).
METHODS
Materials. [ 14 C]cytochrome c (850 kBq/mg) was obtained from Biotrend (Cologne, Germany). S-3226 and EIPA were kindly provided by Dr. H.-J. Lang (Aventis, Frankfurt, Germany). All other materials were obtained from Sigma (Deisenhofen, Germany), if not stated otherwise.
Preparation of rats for microinfusion experiments in vivo. Male Wistar rats (Charles River, Konstanz, Germany) weighing 210-290 g were fed an Altromin standard diet and had free access to water. The rats were anesthetized with 120 mg/kg body wt Inactin (Byk-Gulden, Constance, Germany). After a polyethylene tube was inserted into the trachea and two catheters were inserted into the jugular vein, the animals were infused intravenously with Ringer solution at a rate of 50 µl/min. The kidney was prepared for micropuncture as described elsewhere ( 20 ).
Microinfusion experiments. After identification of the nephron section by intravenous injection of lissamine green SF (Chroma-Gesellschaft, Köngen, Germany) at a bolus dose of 0.02 ml of a 100 g/l solution titrated with NaOH to pH 7.4, the tubule was micropunctured using glass capillaries. They had ground tips (outer tip diameter 10-12 µm) and were mounted on a microperfusion pump ( 36 ). Puncture sites were the earliest superficial loop of the proximal tubule ("early proximal"), which represents a distance from the glomerulus of 0.3-1.2 mm. The microinfused solution was a Ringer solution containing [ 14 C]cytochrome c and [ 3 H]inulin. Microinfusion at a rate of 10 nl/min lasted for 20 min. Starting shortly before microinfusion, the ipsilateral urine was collected from a uretheral catheter in 15-min fractions for 60 min, and radioactivity of each fraction was counted in a liquid scintillation spectrometer (Packard Instruments, Frankfurt, Germany). Urinary [ 14 C]label recovery [fractional excretion (FE)] was calculated from {[ 3 H]urine·[ 14 C]perfusion}/{[ 14 C]urine·[ 3 H]perfusion}. Fractional reabsorption (FR) is 1 - FE. As a control, the urine of the contralateral kidney was collected from a bladder catheter. Here, radioactivity did not exceed the background level.
Preparation of mice for clearance experiments. Wild-type, heterozygous, and homozygous NHE3-deficient mice were obtained from an established colony that was generated by targeted gene disruption as previously described ( 34 ). All animals were derived from heterozygous crosses, and genotypes were determined by polymerase chain reaction analysis of DNA from tail biopsies. Mice were maintained on normal rodent chow and tap water in a barrier facility until the time of the experiment.
Male mice of each genotype (Nhe3 +/+, n = 4; Nhe3 +/-, n = 7; and Nhe3 -/-, n = 4) weighing between 30 and 40 g were prepared for clearance studies according to the conventional techniques modified for use in the mouse as described ( 25 ). Mice were anesthetized with thiobutabarbital (100 µg/g body wt Inactin; Research Biochemicals International, Natick, MA) and placed on a thermally controlled surgical table. After a tracheostomy, the jugular vein was cannulated with polyethylene tubing hand-drawn to a fine tip over a flame (0.3- to 0.5-mm OD). The venous catheter was connected to a syringe pump for infusion. The bladder was also cannulated with flared PE-10 polyethylene tubing for the collection of urine. Body temperature was maintained at 37.5°C, and animals were provided with a steady stream of 100% O 2 to breath. Four to seven samples of urine were collected from each animal. At the end of the experiment, arterial blood was obtained by left cardiac puncture. All experiments were carried out according to the national legal guidelines for animal use in research.
Analytic procedures. Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Munich, Germany). Creatinine was determined by the Jaffé reaction. SDS-PAGE was performed as described previously ( 24 ).
Calculations and statistics. Data are presented as means ± SE; n represents the number of animal or micropuncture samples. Significance of difference was tested by Student's t -test or ANOVA as appropriate. Differences were considered significant if P < 0.05.
RESULTS AND DISCUSSION
Micropuncture studies in rats. To investigate the importance of NHE3 for proximal tubular protein reabsorption, we determined the FR of cytochrome c during microinfusion in rats. Cytochrome c is reabsorbed via the "megalin/cubilin pathway" ( 32 ). Thus FR-cytochrome c provides information about megalin/cubilin-mediated endocytosis. During early proximal microinfusion, FR was 45% ( Fig. 1 ). Because of the relatively low specific activity of [ 14 C]cytochrome c, we had to use a relatively high chemical concentration of 350 µmol/l in the infusion solution, resulting in partial saturation of the reabsorption capacity. During early distal microinfusion, no significant reabsorption was observed. Precipitation with trichloroacetic acid confirmed that all radioactivity was bound to protein.
Fig. 1. Micropuncture experiments in rats. A : fractional reabsorption (FR) of cytochrome c was 45% during early proximal (EP) microinfusion, whereas no reabsorption was observed during early distal (ED) microinfusion. Number of animals = 8 for EP and 6 for ED. B : effect of 100 µmol/l EIPA or 30 µmol/l S-3226 on FR expressed as a percentage. C : effect of 100 µmol/l EIPA or 30 µmol/l S-3226 on cytochrome c reabsorption expressed as µg/nephron. * P < 0.05 vs. control; n = 8-10 rats.
Next, we tested the effect of pharmacological NHE3 inhibition with EIPA or the NHE3-specific S-3226 on cytochrome c reabsorption. To achieve complete inhibition of NHE3 and taking into consideration that the infusion solution is diluted ( 5- to 6-fold) by endogenously formed ultrafiltrate, we used 100 µmol/l EIPA and 30 µmol/l S-3226 ( 31, 35 ). Figure 1 shows that both inhibitors reduced proximal reabsorption of cytochrome c by 50%. The possibility that the inhibition observed resulted from a weak base effect can be excluded because the concentrations used are too low, as we showed previously ( 14 ). In vivo, in addition, 10 mmol/l NH 4 Cl are necesarry to inhibit protein reabsorption ( 4 ). These data show, for the first time, that NHE3 supports proximal tubular receptor-mediated endocytosis of proteins in vivo, as observed previously in vitro. To further confirm this finding, we used NHE3 knockout mice as a second, independent experimental model.
Clearance studies in mice. We compared urinary protein excretion of Nhe3 +/+, Nhe3 +/-, and Nhe3 -/- animals ( 34 ). As shown in Fig. 2, total urinary protein excretion (µg/min) was significantly higher in Nhe3 -/- and Nhe3 +/- mice compared with Nhe3 +/+ mice. Serum protein concentrations did not differ significantly in the three groups (59 ± 1 g/l). The gene-dose effects observed for urinary protein excretion correspond to the gene-dose effect for proximal tubular fluid reabsorption ( 25 ). Thus increasing protein excretion in Nhe3 +/- and Nhe3 -/- animals most probably reflects the graded reduction in NHE3 expression. It is known from previous studies ( 2, 25 ) that glomerular filtration is reduced in Nhe3 -/- animals. A recent study revealed that this reduction results from renal compensatory mechanisms due to decreased proximal tubular salt reabsorption ( 41 ). This altered glomerular filtration is also reflected by the reduced creatinine clearance measured in the present investigation (129 ± 12 µl/min for Nhe3 +/+, 116 ± 8 µl/min for Nhe3 +/-, and 94 ± 4 µl/min for Nhe3 -/- mice). The values for creatinine clearance underestimate glomerular filtration by a factor of about three due to a nonspecific color formation mainly in serum ( 28 ). However, taking this factor into account, the values obtained here are in good agreement with filtration rates determined with inulin ( 41 ). To account for reduced filtration in Nhe3 -/- animals, we expressed protein excretion normalized to creatinine in Fig. 2 B. Here, the differences are even more pronounced. Total urinary protein excretion (µg/min) in Nhe3 -/- animals was 188% compared with that in Nhe3 +/+ mice. Creatinine-normalized protein excretion in Nhe3 -/- mice was 221% compared with Nhe3 +/+ mice. These data obtained from Nhe3 -/- animals confirm the hypothesis of NHE3-supported protein reabsorption.
Fig. 2. Clearance experiments in mice. A : urinary protein excretion, expressed as µg/min. B : creatinine-normalized protein excretion. * P < 0.05 vs. Nhe3 +/+; n = 4 wild-type (+/+), 7 Nhe3 heterozygous (+/-), and 4 Nhe3 homozygous mutant (-/-) mice.
Enhanced protein excretion can result from enhanced protein filtration or reduced protein reabsorption ( 10 ). Although glomerular filtration is reduced in Nhe3 -/- animals and there is no evidence for altered glomerular protein sieving, we characterized the urinary proteins by SDS-PAGE (gel loading was normalized for creatinine, i.e., a volume equivalent to 3 µg of creatinine was loaded). With respect to the size of proteins, tubular proteinuria is characterized by enhanced excretion of proteins the size of albumin or smaller. As shown in Fig. 3, this was precisely the case for Nhe3 +/- or Nhe3 -/- animals. In Nhe3 +/- animals, excretion of proteins 24 kDa was enhanced, whereas for larger proteins there was no difference. In Nhe3 -/- animals, excretion of proteins 24 kDa was also enhanced. In this group, we observed in addition an increased excretion of larger proteins. Most likely, the 60- to 70-kDa band corresponds to albumin and the 48-kDa band could correspond to vitamin D-binding protein ( 5 ). Protein excretion of Nhe3 -/- animals, therefore, resembles the pattern observed in megalin-deficient mice or in CLC-5-deficient mice, which are both typical models of tubular proteinuria ( 7, 39 ). Enhanced excretion of larger proteins, typical for glomerular proteinuria, was not observed. Thus proximal tubular NHE3 deficiency leads to reduced protein reabsorption.
Fig. 3. SDS-PAGE analysis of urinary proteins in mice. Urine samples from 2 animals of each group were normalized for creatinine and separated on 12 or 15% gels. Proteins were visualized either by Coomassie blue or silver staining.
The data presented in this study show for the first time that proximal tubular NHE3 supports protein endocytosis not only in cultured cells but also in vivo. Given the fact that the Na + - and H + -transporting function as well as the regulation of NHE3 are very comparable in vivo and in cultured cells, we think that the mechanisms linking NHE3 and endocytosis in cultured cells ( 14 - 16 ) are also suitable to explain our in vivo findings. Enhanced tubular protein reabsorption, for example, after glomerular injury, is one of the major factors determining the development of tubulointerstitial injury ( 3 ). Therefore, inhibition of excessive protein reabsorption would be a rational therapeutic strategy to prevent such damage. In line with this, cell culture data suggest that prevention of apical protein uptake by inhibition of NHE3 reduces the activation of inflammatory and fibrotic events ( 40 ). Our data presented in the present study provide a rational basis for evaluating the abovementioned strategy in vivo.
GRANTS
The work was supported in part by the Wilhelm-Sander-Stiftung (grant to M. Gekle) and by National Institutes of Health Grant DK-50594 (to G. E. Shull).
ACKNOWLEDGMENTS
We thank Dr. S. Silbernagl for stimulating discussions and Dr. H. Koepsell for support with handling of the mice.
【参考文献】
Biemesderfer D, Nagy T, DeGray B, and Aronson PS. Specific association of megalin and the Na + /H + exchanger isoform NHE3 in the proximal tubule. J Biol Chem 274: 17518-17524, 1999.
Brooks HL, Sorensen AM, Terris J, Schultheis PJ, Lorenz JN, Shull GE, and Knepper MA. Profiling of renal tubule Na + transporter abundances in NHE3 and NCC null mice using targeted proteomics. J Physiol 530: 359-366, 2001.
Burton C and Harris KPG. The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis 27: 765-775, 1996.
Camargo MSF, Sumpio BE, and Maack T. Renal hydrolysis of absorbed protein: influence of load and lysosomal pH. Am J Physiol Renal Fluid Electrolyte Physiol 247: F656-F664, 1984.
Christensen EI and Birn H. Megalin and cubilin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol 3: 256-266, 2002.
Christensen EI, Birn H, Verroust P, and Moestrup SK. Membrane receptors for endocytosis in the renal proximal tubule. Int Rev Cytol 180: 237-284, 1998.
Christensen EI, Devuyst O, Dom G, Nielsen R, Van Der Smissen P, Verroust P, Leruth M, Guggino WB, and Courtoy PJ. Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules. Proc Natl Acad Sci USA 100: 8472-8477, 2003.
Christensen EI and Willnow TE. Essential role of megalin in renal proximal tubule for vitamin homeostasis. J Am Soc Nephrol 10: 2224-2236, 1999.
Cui S, Verroust PJ, Moestrup SK, and Christensen EI. Megalin/gp330 mediates uptake of albumin in renal proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 271: F900-F907, 1996.
D'Amico G and Bazzi C. Pathophysiology of proteinuria. Kidney Int 63: 809-825, 2003.
D'Souza S, Garcia-Cabado A, Yu F, Teter K, Lukacs G, Skorecki K, Moore HP, Orlowski J, and Grinstein S. The epithelial sodium-hydrogen antiporter Na + /H + exchanger 3 accumulates and is functional in recycling endosomes. J Biol Chem 273: 2035-2043, 1998.
Dixon R and Brunskill NJ. Activation of mitogen pathways by albumin in kidney proximal tubule epithelial cells: implications for proteinuric states. J Am Soc Nephrol 10: 1487-1497, 1999.
Eddy AA and Giachelli CM. Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria. Kidney Int 47: 1546-1557, 1995.
Gekle M, Drumm K, Mildenberger S, Freudinger R, Ganer B, and Silbernagl S. Inhibition of Na + -H + exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum. J Physiol 520: 709-721, 1999.
Gekle M, Drumm K, Serrano OK, Mildenberger S, Freudinger R, Ganer B, Jansen HW, and Christensen EI. Na + /H + -exchange-3 serves as a molecular tool for cAMP-mediated regulation of receptor-mediated endocytosis. Am J Physiol Renal Physiol 283: F549-F558, 2002.
Gekle M, Freudinger R, and Mildenberger S. Inhibition of Na + -H + exchanger-3 interferes with apical receptor-mediated endocytosis via vesicle fusion. J Physiol 531.1: 619-629, 2001.
Gekle M, Mildenberger S, Freudinger R, Schwerdt G, and Silbernagl S. Albumin endocytosis in OK cells: dependence on actin and microtubules, regulation by protein kinases. Am J Physiol Renal Physiol 272: F668-F677, 1997.
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.
Gekle M, Mildenberger S, Freudinger R, and Silbernagl S. Functional characterization of albumin binding to the apical membrane of OK cells. Am J Physiol Renal Fluid Electrolyte Physiol 271: F286-F291, 1996.
Gekle M and Silbernagl S. The role of the proximal tubule in ochratoxin A nephrotoxicity in vivo: toxodynamic and toxokinetic aspects. Renal Physiol Biochem 17: 40-49, 1994.
Gruenberg J and Maxfield FR. Membrane transport in the endocytic pathway. Curr Opin Cell Biol 7: 552-563, 1995.
Guijarro C and Egido J. Transcription factor- B (NF- B) and renal disease. Kidney Int 59: 415-424, 2001.
Kapus A, Grinstein S, Wasan S, Kandasamy R, and Orlowski J. Functional characterization of three isoforms of the Na + /H + exchanger stably expressed in Chinese hamster ovary cells. J Biol Chem 269: 23544-23552, 1994.
Krug AW, Schuster C, Gassner B, Freudinger R, Mildenberger S, Troppmair J, and Gekle M. Human EGF receptor 1 (HER1) expression renders CHO cells sensitive to alternative aldosterone signaling. J Biol Chem 277: 45892-45897, 2002.
Lorenz JN, Schultheis PJ, Traynor T, Shull GE, and Schnermann J. Micropuncture analysis of single-nephron function in NHE3-deficient mice. Am J Physiol Renal Physiol 277: F447-F453, 1999.
Marshansky V and Vinay P. Proton gradient formation in early endosomes from proximal tubules. Biochim Biophys Acta 1284: 171-180, 1996.
Mellman I, Fuchs R, and Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem 55: 663-700, 1986.
Meyer MH, Meyer RAJ, Gray RW, and Irwin RL. Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography. Anal Biochem 144: 285-290, 1985.
Mukherjee S, Ghosh RN, and Maxfield FR. Endocytosis. Physiol Rev 77: 759-803, 1997.
Nakajima H, Takenaka M, Kaimori JY, Nagasawa Y, Kosugi A, Kawamoto S, Imai E, Hori M, and Okubo K. Gene expression profile of renal proximal tubules regulated by proteinuria. Kidney Int 61: 1577-1587, 2002.
Noel J and Pouysségur J. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na + /H + exchanger isoforms. Am J Physiol Cell Physiol 268: C283-C296, 1995.
Orlando RA, Rader K, Authier F, Yamazaki H, Posner BI, Bergeron JJM, and Farquhar MG. Megalin is an endocytic receptor for insulin. J Am Soc Nephrol 9: 1759-1766, 1998.
Rybak SL, Lanni F, and Murphy RF. Theoretical considerations on the role of membrane potential in the regulation of endosomal pH. Biophys J 73: 674-687, 1997.
Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, and Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na + /H + exchanger. Nat Genet 19: 282-285, 1998.
Schwark JR, Jansen HW, Lang HJ, Krick W, Burckhardt G, and Hropot M. S3226, a novel inhibitor of Na + /H + exchanger subtype 3 in various cell types. Pflügers Arch 436: 797-800, 1998.
Sonnenberg H and Deetjen P. Methode zur Durchströmung einzelner Nephronabschnitte. Pflügers Arch 278: 669-674, 1964.
Storrie B and Desjardins M. The biogenesis of lysosomes: is it a kiss and run, continuous fusion and fission process? Bioessays 18: 895-903, 1996.
Wang Y, Rangan GK, Tay YC, and Harris DCH. Induction of monocyte chemoattractant protein-1 by albumin is mediated by nuclear factor B in proximal tubule cells. J Am Soc Nephrol 10: 1204-1213, 1999.
Willnow TE, Hilpert J, Armstrong SA, Rohlmann A, Hammer RE, Burns DK, and Herz J. Defective forebrain development in mice lacking pg30/megalin. Proc Natl Acad Sci USA 93: 8460-8464, 1996.
Wohlfarth V, Drumm K, Mildenberger S, Freudinger R, and Gekle M. Protein uptake disturbs collagen homeostasis in proximal tubule-derived cells. Kidney Int Suppl 84: 103-109, 2003.
Woo AL, Noonan WT, Schultheis PJ, Neumann JC, Manning PA, Lorenz JN, and Shull GE. Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect. Am J Physiol Renal Physiol 284: F1190-F1198, 2003.
作者单位：1 Physiologisches Institut, University of Würzburg, 97070 Würzburg; 3 Physiologisches Institut, Universität Duisburg-Essen, 45122 Essen, Germany; and 2 Department of Molecular Genetics, Biochemistry, and Microbiology, The University of Cincinnati College of Medicine, Cincinnati, Ohio