Regulation of the Proximal Tubular Sodium/Proton Exchanger NHE3 in Rats with Puromycin Aminonucleoside (PAN)-Induced Nephrotic Syndrome
Valérie Besse-Eschmann*,
Jelena Klisic,
Vera Nief,
Michel Le Hir*,
Brigitte Kaissling* and
Patrice M. Ambühl,
*Department of Anatomy and Department of Physiology, University of Zurich, Irchel, Switzerland; and Renal Division, University Hospital, Zurich, Switzerland.
Correspondence to Dr. Patrice M. Ambühl, Renal Division, University Hospital, Rämistrasse 100, CH 8091 Zürich, Switzerland. Phone: 41-1-255-3815; Fax: 41-1-255-4593; E-mail: patrice.ambuehl{at}dim.usz.ch
ABSTRACT. Excessive proteinuria due to loss of glomerular permselectivityin nephrotic syndrome can cause disturbances in renal salt andwater handling with edema formation. Apart from oncotic andhydrostatic mechanisms associated with hypoalbuminemia, primaryderangements in renal tubular sodium transport may contributeto the pathogenesis of nephrotic edema. Whereas there is evidencefor an increase of cortical collecting duct sodium reabsorptionin nephrotic rats, it remains controversial whether proximaltubule sodium transport may also be activated in this condition.The regulation of the cortical Na/H exchanger NHE3, the mainpathway for Na reabsorption in the proximal tubule (PT), wasinvestigated in rats with puromycin aminonucleoside (PAN)-inducednephrotic syndrome. PAN rats developed reduced GFR, severe proteinuria,and sodium retention within 3 d. After 10 d, immunoblots ofbrush border vesicles revealed a decreased abundance of NHE3in nephrotic animals. However, the Na/H antiporter activityin the same vesicle preparations was not significantly altered.Antiporter activity normalized for NHE3 protein was increasedby 88% in nephrotic animals (P = 0.025). Immunohistochemistrywith the same polyclonal antibody as for immunoblots revealeda decrease of NHE3 abundance in PT. In contrast, immunoreactivityfor the monoclonal antibody 2B9, which specifically recognizesthe non-megalin-associated, transport-competent pool of NHE3,was higher in PAN-treated rats than in controls. In conclusion,increased sodium reabsorption might be associated with a shiftof NHE3 from an inactive pool to an active pool, thus contributingto sodium retention in a state of proteinuria.
The nephrotic syndrome (NS) is a common manifestation of renaldisease and is characterized by urinary protein loss due toglomerular damage and renal salt and water retention (1). Underregular conditions, permselectivity of intact glomerular structuresensures retention of most of the serum proteins in the vascularspace (2). Even under normal conditions, up to 5 g of proteinper day may be filtered by the glomeruli, which are then reabsorbedby the renal tubule (3,4). In glomerular diseases, two pathophysiologicallyimportant events occur. First, substantial amounts of protein(mainly albumin) are filtered through the damaged glomeruliand are lost into the urine. Second, increasing quantities ofthe filtered protein are reabsorbed by the renal tubule (5).Besides being a hallmark of glomerular disease, proteinuriahas been shown to be an independent factor that induces andmaintains renal damage (6). The current understanding is thatprotein reabsorption by the proximal tubule triggers inflammationand scarring by induction of several cytokines and growth factors(for example TGF) (7). The reabsorption of albumin is achievedpredominantly by endocytosis. Several recent studies have suggestedan interrelation of transcellular albumin transport by endocytosisand acidification of lysosomes (8,9) through endosomal Na/Hexchange (10,11). The nephrotic syndrome (NS) is also accompaniedby various degrees of salt and water retention and representsa major clinical problem in the management of patients withnephrosis (12,13). One proposed mechanism is systemic interstitialvolume sequestration due to hypoalbuminemia and secondary Na+retention. In addition, primary salt retention per se may contributesubstantially to systemic volume expansion in NS. The nephroticstate has been associated with activation of the Na/K-ATPasein the collecting duct (14,15). The effect of nephrotic syndromeon proximal tubule Na+ transporters has not been explored. Thebulk of sodium reabsorption in the proximal tubule is mediatedby the apical sodium/proton exchanger NHE3 (16). Given the potentialrole of proximal tubular sodium/proton exchange in both tubularprotein reabsorption and salt retention, we investigated theregulation of the cortical brush border membrane Na/H exchangerNHE3 in rats with nephrotic syndrome induced by puromycin aminonucleoside (PAN).
Materials and Supplies
All chemicals were obtained from Sigma Chemical Co. (St. Louis,MO) unless stated otherwise.
Animals
Male Sprague-Dawley rats (average 200 g) were injected witheither puromycin aminonucleoside (n = 9; PAN 150 mg/kg bodyweight intraperitoneally) or an identical volume of vehicle(H2O; n = 9). Animals were then placed separately into metaboliccages and allowed free access to distilled water. Control andPAN rats were pair-fed standard rat chow (No 9331 25 W10; EberleNafag AG, Gossau SG, Switzerland). Daily 24-h urine collectionswere performed in all animals. Urinary sodium concentration,as well as serum and urinary creatinine concentrations, weremeasured with an AVL984 electrode electrolyte analyzer (AVLMedical Instruments, Schaffhausen, Switzerland). Urine proteinconcentration was determined using the Bradford assay on a MerckVitalab Eclipse filter photometer. Creatinine clearance wascalculated from the urinary creatinine excreted over the last24 h before sacrifice and the serum creatinine concentrationfrom venous blood taken at sacrifice. Ten days after injectionof PAN or vehicle, rats were anesthetized with 0.25 ml/kg Dormitor(Pfizer, Sandwich, Kent, UK) (1 mg/ml medetomidini hydrochloridum)and 0.25 ml/kg Hypnorm (Janssen, New Brunswick, NJ) (0.315 mg/mlfentanyl citrate), and kidneys were excised, weighed, and placedin ice-cold phosphate-buffered saline (PBS).
Cortical Brush Border Membrane Vesicle Preparation
Renal cortical apical membrane vesicles were prepared by Mg2+aggregation, as described previously (17). Dissected kidneycortex was homogenized in membrane buffer (300 mM mannitol,18 mM HEPES, 5 mM EGTA, 100 µg/ml PMSF; pH 7.50) at 4°Cwith a Brinkmann Polytron (Brinkmann Instruments, Westbury,NY). MgCl2 was added to the homogenate to obtain a final concentrationof 15 mM, and the homogenate was pelleted at 4°C by centrifugationat 2500 x g for 30 min (Polytron; Kinematica GmbH, Kriens, Switzerland).Supernatant was transferred, an additional MgCl2 precipitationwas performed, and membranes were pelleted from the final supernatantat 48,000 x g at 4°C for 30 min (RC 5c Plus; DuPont Company,Sorvall Products, Wilmington, DE). Enrichment of brush bordermembrane vesicles was not affected by PAN treatment, as evidencedby comparable ratios of BBM to total cortical homogenate leucineaminopeptidase activities in control and PAN animals (x16 ±3 versus x13 ± 2, respectively, P = NS).
Immunoblot
Cortical brush border membrane pellets from the above preparationwere resuspended in membrane buffer (see above), and proteincontent was assessed according to Bradford. Thirty microgramsof protein were diluted 1:5 in 5x sodium dodecyl sulfate (SDS)loading buffer (1 mM Tris · HCl, pH 6.8, 1% SDS, 10%glycerol, 1% [vol:vol] 2-mercapttoethanol), boiled for 10 min,size fractionated by SDS-polyacrylamide gel electrophoresison 7.5% gels, and electrophoretically transferred to nitrocellulose.After blocking with 5% nonfat milk and 0.05% Tween 20 in PBSfor 1 h, blots were probed in the same buffer for 1 h with apolyclonal anti-rat NHE3 antibody directed against amino acids809 to 822 of the COOH-terminal cytoplasmic domain of rat NHE3(kindly provided by Dr. Orson W. Moe, University of Texas SouthwesternMedical Center, Dallas, TX) at a dilution of 1:10,000. Blotswere washed in 0.05% Tween 20 in PBS one time for 15 min andtwo times for 5 min, incubated with a 1:10,000 dilution of peroxidase-labeledsheep anti-rabbit IgG in 5% nonfat milk and 0.05% Tween 20 inPBS for 1 h, washed as above, and then visualized by enhancedchemiluminescence (Nitro-Block II, Applied Biosystems, FosterCity, CA; CDP-Star detection reagent, Amersham, Piscataway,NJ). NHE3 protein abundance was quantitated by densitometry(BioCapt software version 72.02 s for Windows, Vilbert Lourmat,France; Scion Image Beta 3b 1998, Scion Corporation, Frederick,MD). Signal was measured as an integrated volume with correctionfor a defined background.
Na+/H+ Antiporter Assay
Na+-H+ antiporter activity was determined by the acridine orangemethod as described by Moe et al. (18). Cortical brush bordermembrane vesicles were used as prepared for western blotting,with the exception that after the final precipitation step at48,000 x g, the pellet was resuspended in 140 mM NMDG gluconateand 5 mM MES at pH 5.5. The acid-loaded vesicles were then addedto a solution containing 120 mM NMDG-gluconate, 20 mM HEPESat pH 7.50, and 6 µM acridine orange (Molecular Probes,Eugene, OR). Fluorescence signal was followed in a spectrofluorometer(ex = 493 nm, ex = 530 nm; Shimadzu 5000, Japan). The protongradient-driven trapping of acridine orange in the vesiclesprovoked fluorescence quenching. Na+/H+ antiporter activitywas then assayed as the rate of increase in fluorescence (acridineorange efflux) occurring in response to Na+ addition to theextravesicular space. Specific activity of the Na+/H+ antiporterwas expressed as the slope of the initial Na+-dependent fluorescenceincrease divided by the initial quench.
Tissue Preparation for Light Microscopy and Immunohistochemistry
An additional set of animals (n = 3 for both PAN and control)undergoing identical treatment as described above (except forthe following modifications) was analyzed by light microscopyand immunohistochemistry. Nine days after injection of PAN orvehicle, rats were anesthetized with an intraperitoneal injectionof thiopental (0.5 mg/kg Pentothal; Abbott, Abbott Park, IL),and kidneys were fixed by vascular perfusion via the abdominalaorta as described previously (19). The fixative consisted of3% paraformaldehyde and 0.05% picric acid in 0.06 M cacodylatebuffer (pH 7.4; containing 3 mM MgCl2 and adjusted to 300 mOsmwith sucrose) and 10% hydroxyethyl starch (HAES, Fresenius,Switzerland). After fixation, the left renal artery was clampedand the fixative in the right kidney was washed out by perfusionwith 0.1 M cacodylate buffer. Both kidneys were then removed.
For light microscopy, the left unwashed kidney was cut in coronalslices and immersed for at least 24 h in the 3% paraformaldehydesolution, to which 0.1% glutardialdehyde (Fluka Chemie AG, Buchs,Switzerland) was added. Thereafter, the tissue was postfixedin 1% OsO4 and embedded in epoxy resin. Then, 1-µm-thicksections were cut from the epoxy resin-embedded tissue and stainedwith azure II-methylene blue. Coverslips were applied with DPXmounting medium (Agar Scientific, Stansted, Essex, UK).
For immunofluorescence, coronal slices of the right kidney weremounted on cork disks, frozen in liquid propane cooled by liquidN2 and stored at -80°C until use. Four-micrometer-thickcryosections were placed on chromalum/gelatin-coated slides.Sections were pretreated with 1% SDS in PBS for 4 min. Afterrinsing with PBS, they were covered with 10% normal goat serumin PBS containing 1% bovine serum albumin (PBS/BSA) for 10 min.Sections were then incubated overnight at 4°C with a mouseanti-rabbit NHE3 monoclonal antibody directed against a MaltoseBinding Protein fusion protein containing C-terminal 131 aminoacids of rabbit NHE3 (clone 2B9; Chemicon International, Temecula,CA) diluted 1:50, with a polyclonal anti-rat NHE3 antibody (seeImmunoblot section) diluted 1:500 in PBS/BSA or with a polyclonalanti-ecto-5'-nucleotidase antibody (19) 1:5000 for stainingthe brush border (without SDS-pretreatment in this case). Sectionswere then rinsed three times with PBS and covered for 1 h atroom temperature with FITC-conjugated goat anti-mouse IgG andIgM (Jackson Immunoresearch Laboratories, West Grove, PA) diluted1:50 and Cy3-conjugated goat anti-rabbit IgG (Jackson) diluted1:1000 in PBS/BSA together with normal rat serum 1:100. Finally,the sections were rinsed with PBS, coverslips were applied withDAKO-Glycergel (Dakopatts, Glostrup, Denmark) containing 2,5%1,4-diazabicyclo-(2.2.2)-octane (DABCO; Sigma) as a fading retardant,and the sections were examined by epifluorescence microscopy.The same protocol was followed for double-labeling, but thetwo primary antibodies as well as the two second antibodieswere mixed.
Renal Sodium and Protein Excretion; Creatinine Clearance
As shown in Figure 1, PAN-treated rats developed severe proteinuriaon day 4 and beyond, whereas renal protein excretion was minimalin control animals. Similarly, PAN rats showed a significantdecrease in urinary sodium excretion compared with control animalswithin 3 d after administration of the drug (Figure 2). A newsteady state in sodium balance was achieved on day 9 in PANanimals, with a urinary sodium excretion no longer statisticallydifferent from rats not treated with puromycin. Most of thePAN rats also exhibited ascites on the day of sacrifice. Takentogether, administration of puromycin resulted in the clinicalequivalent of nephrotic syndrome with proteinuria as well assalt and volume retention. Creatinine clearance was significantlylower in nephrotic versus control rats (0.67 ± 0.09 versus1.65 ± 0.15 ml/min, respectively; P = 0.0074).
Figure 1. 24-h urinary protein excretion. Protein excretion was determined from 24-h urine collections of control and puromycin aminonucleoside (PAN)-treated rats. Experimental rats developed severe proteinuria 4 d after administration of PAN, which persisted throughout the whole study period. Note the logarithmical scale of the y-axis. *P < 0.001.
Figure 2. 24-h urinary sodium excretion. Sodium excretion was determined from 24-h urine collections of control and PAN-treated rats. Whereas control rats were in sodium balance, as revealed by constant natriuresis over the whole study period, PAN rats retained sodium from day 3 through 8, as evidenced by almost undetectable sodium excretion during this period. However, natriuresis in PAN animals returned back to control levels at day 9. *P < 0.001.
Light Microscopy
The overview of control kidneys (Figure 3a) shows intact cortexand outer stripe of medulla with open tubules. In contrast,the kidneys of rats treated with PAN exhibit an irregular patternwith many partially collapsed tubules (Figure 3b). Moreover,many dilated proximal tubules, especially in the medullary rays,can be detected. At higher magnification, the proximal tubulesof control rats (Figure 4a) manifest with an intact brush border.In PAN rats (Figure 4b), a widespread shortening of the brushborder takes place in proximal tubules. The brush border alsoundergoes focal destruction, especially in S2 segments. Theproximal tubular cells display some variability in size, withmany cells being smaller than in control animals, but some cellsalso with an increase in size.
Figure 3. Light microscopic overview of cortex. 1-µm-thick epon sections. (a) control kidney; (b) PAN-treated animal showing some collapsed tubules, whereas many tubules are dilated in the cortical medullary rays.
Figure 4. Light microscopic view of S2-segment of proximal tubule. 1-µm-thick epon sections. The profiles are from the sections shown in Figure 1. (a) control; (b) PAN-treated rat with partial disruption of the apical brush border membrane.
Immunofluorescence
Two different antibodies were used for detection of NHE3 inthis study. With the monoclonal antibody 2B9, immunoreactivitywas homogeneously increased in PAN-treated animals (Figure 5).In contrast, with the polyclonal antibody there was an overalldecrease in PAN-treated animals. The levels of immunofluorescencewere similar to controls in some tubules, whereas they werevery low in other tubules (Figure 6). The decrease of immunoreactivitywith the polyclonal antibody may be related to the structuralalteration of the brush border described above. Indeed, theimmunoreactivity for two unrelated proteins of the luminal membrane,ecto-5'-nucleotidase and NaPi-IIa, decreased also (not shown).
Figure 5. NHE3 immunoreactivity with the monoclonal antibody 2B9 in the cortex. Cryosections were labeled with the 2B9 monoclonal antibody. In comparison with control animals (a), the immunofluorescence signal of the monoclonal antibody was increased in treated animals (b). A, arcuate artery.
Figure 6. NHE3 immunoreactivity with the polyclonal antibody in the cortex. Cryosections were labeled with a polyclonal antibody against NHE3. In comparison with control animals (a), the immunofluorescence signal obtained with the polyclonal antibody was decreased in treated animals (b). A, arcuate artery.
In both control and PAN-treated animals, the intracellular distributionof NHE3 immunoreactivity was strikingly different with the twoantibodies (Figure 7). NHE3 is mainly localized at the baseof the brush border in the so-called intermicrovillar compartment(20–22), which the polyclonal antibody labeled almostexclusively in the present study (Figure 7). In contrast, stronglabeling was detected with antibody 2B9 up to the tip of themicrovilli, as described previously (20). With 2B9 the intracellulardistribution pattern of immunoreactivity was somewhat alteredby PAN treatment. Whereas in control animals the intermicrovillarcompartment showed a distinctly higher immunofluorescence thanthe brush border, there was little difference between the twocompartments in PAN-treated animals (Figure 7). Although theoverall immunoreactivity with 2B9 was stronger in treated animals,its pattern often appeared irregular within tubular profilesat high magnification. This probably reflects the focal damagein the brush border, which has been observed also by light microscopy(see above). Indeed, the same irregular pattern was seen whenadjacent sections were double-labeled with 2B9 and with an antibodyagainst the brush border enzyme ecto-5'-nucleotidase (Figure 8).
Figure 7. NHE3 immunoreactivity with monoclonal and polyclonal antibody at high magnification. Cryosections were double-labeled with monoclonal and polyclonal anti-NHE3 antibodies. Two cross-sections of S1 segments of the proximal tubule in a control (panels a through c) and a treated rat (panels d through f) are shown. The two cryosections were processed together. In panels a and d, the immunofluorescence signal for monoclonal anti-NHE3 is shown; in panels b and e, the immunofluorescence signal for polyclonal anti-NHE3 antibody is shown. In panels c and f, the data from both channels are superimposed, with the signal obtained with monoclonal antibody shown in red and polyclonal antibody shown in green. With the monoclonal antibody 2B9, the intermicrovillar membrane compartment was distinctly visible in controls only. The immunoreactivity in the brush border appeared slightly higher in the tubules of PAN-treated rats. With the polyclonal antibody, immunoreactivity was mostly restricted to the intermicrovillar compartment and was markedly lower in the tubules of treated animals.
Figure 8. Colocalization of immunoreactivities with the anti-NHE3 monoclonal antibody and the brush border enzyme ecto-5'-nucleotidase. A profile of proximal tubule in a treated animal is shown. The cryostat section was double-labeled with a polyclonal antibody against the ecto-5'-nucleotidase (panel a; green in panel c) and with 2B9 (panel b; red in panel c). The damaged brush border shows short and discontinuous microvilli. The immunoreactivities with the two antibodies are congruent.
Na/H Exchange Activity and NHE3 Protein Abundance of Renal Cortical Brush Border Membrane Vesicles
Na/H exchange activity in cortical brush border membrane (BBM)vesicles was not significantly different between PAN and controlrats (Figure 9A). However, NHE3 immunoblot showed that NHE3protein abundance (per unit of total BBM vesicle protein) wasdecreased by 38% in animals that had been treated with puromycin(P = 0.003; Figure 9, B and C). This can be explained mainlyby the partial disruption of the cortical brush border fromPAN toxicity. To estimate specific Na/H exchange activity ofintact tubules, activity measurements were normalized to corticalbrush border NHE3 protein abundance, as Na/H exchange activityand NHE3 protein abundance were always determined from the samebrush border vesicle preparation. Specific Na/H exchange activitywas significantly increased in PAN rats by 88% versus controlanimals (P = 0.032; Figure 9D). As an internal control, Westernimmunoblot experiments were performed using an antibody directedagainst the rat sodium/phosphate cotransporter NaPi-2 (kindlyprovided by Dr. Jürg Biber, Institute of Physiology, Universityof Zurich-Irchel, Zurich, Switzerland). Unlike for NHE3, norelevant changes in NaPi-2 cortical BBM protein abundance couldbe detected between control and PAN treated animals (data notshown).
Figure 9. Proximal tubular Na/H antiporter activity and NHE3 protein abundance. Panel A depicts results of Na/H antiporter activity as determined from changes in intravesicular pH of cortical brush border membrane vesicles by acridine orange. No apparent difference exists in unadjusted measurements between control (white bar) and PAN-treated animals (black bar) regarding Na/H exchange activity. (B) Typical Western immunoblot from the same vesicle preparations as used for activity measurements showing substantially reduced NHE3 protein abundance in PAN rats compared with controls. (C) Quantification of NHE3 protein abundance in control and PAN-treated animals as determined by densitometry from Western immunoblot chemifluorescence signals. (D) Proximal tubular brush border Na/H exchange activity normalized for the amount of NHE3 protein abundance depicted in panels B and C. The adjusted Na/H exchange activity was increased by 88% in nephrotic rats (PAN) versus normal controls.
It is still a matter of debate whether nephrosis is a stateof underfill due to hypoalbuminemia or the result of primaryrenal salt and water retention from an activated renin-angiotensin-aldosteronesystem. Although our study was not designed to resolve thisquestion, the findings point to a novel mechanism for primaryrenal sodium retention in a model of nephrosis in the rat. PANin a dose of 150 mg/kg body wt resulted in salt retention within3 d of administration (Figure 1), preceding overt proteinuriaby 1 d (Figure 2). Besides a clear reduction in urinary sodiumexcretion, PAN rats also exhibited volume retention as evidencedby the development of ascites. Current evidence suggests thatthe collecting duct is one of the primary sites of salt retentionin nephrotic syndrome (23–25). The Na/K-ATPase seems tobe the major regulator of sodium reabsorption in this tubularsegment in rats with proteinuria (14, 15). However, other studieshave also shown increased sodium reabsorption to occur in theproximal tubule (26). Varying levels of intravascular volumestatus and GFR at time of study may account for the diversefindings with regard to the principal site of sodium reabsorptionin nephrotic subjects. As the bulk of sodium absorption occursin the proximal tubule, it seems reasonable to postulate thatthis segment contributes at least in part to salt and volumeretention in the nephrotic syndrome.
The combined functional and immunohistochemical data of ourstudy point toward an increase in activity of the Na/H exchangerin proximal tubules in PAN-induced nephrotic syndrome. Na/Hexchange activity normalized to NHE3 antigen was increased by88% in nephrotic versus control rats. As other BBM proteins,like the cortical sodium/phosphate cotransporter NaPi-2 andthe ecto-5'-nucleotidase, are not affected in PAN-treated animals,we postulate a specific effect of proteinuria on proximal tubularNHE3. Furthermore, our immunohistochemical data along with recentstudies by Biemesderfer et al. (20) provide the basis for apossible explanation how NHE3 activity may be regulated in thenephrotic syndrome. They demonstrated that NHE3 occurs in twodifferent pools. The majority of NHE3 is found as a 21 S, megalin-associated,inactive form in the intermicrovillar microdomain at the baseof the apical plasma membrane, whereas the remainder is presentmainly in a 9.6 S active form present in brush border microvilliunrelated to megalin. There are two separate lines of evidencein the present study that suggest that the relative distributionof NHE3 is shifted toward the megalin-free, active pool in nephroticanimals. First, in BBM vesicles, the Na/H exchange activityper unit of NHE3 protein increased in PAN-treated rats. Second,in tissue sections, the abundance of NHE3 detected with thepolyclonal antibody decreased, whereas increased immunoreactivitycould be noted with the monoclonal antibody 2B9, which is specificfor the megalin-free, active form of NHE3. Megalin is a receptorfor filtered proteins; increased protein filtration might thereforerepresent the link between PAN treatment and the postulateddissociation of NHE3 from the complexes with megalin. To substantiatethis proposed link between proteinuria and activation of theNa/H exchanger, it will be necessary to examine further modelsof proteinuria. However, regulation of NHE3 activity in opossumkidney (OKP) cells via trafficking of NHE3 protein between themicrovillar plasma membrane and a subapical compartment hasbeen shown previously by various stimuli such as acid (22),osmolarity (27), endothelin-1 (28), parathyroid hormone (29),and dopamine (30). Finally, in a preliminary report, we havedemonstrated that albumin increases Na/H exchange activity andapical cell surface NHE3 antigen in OKP cells (31).
The immediate signal for Na/H exchanger stimulation could beactivation of the renin-angiotensin system. Indeed, 10-11 Mangiotensin II activates Na/H exchange as shown in culturedproximal tubule cells. However, the literature on changes inlocal and systemic angiotensin II concentrations in PAN nephrosisis controversial. Whereas some investigators found increasedangiotensin II levels in nephrotic animals, others did not observesuch changes. PAN-treated rats had a decreased whole-animalGFR, as revealed by the significantly lower creatinine clearancecompared to control animals. Given the reduced total kidneyGFR, the "normal" total cortical BBM NHE3 activity suggestsa heightened level of proximal tubule reabsorption reflectinga reset level of glomerulotubular balance. The second implicationof our findings relates to proteinuria. Albumin enhances Na/Hexchange activity in OKP cells, as shown in a preliminary report(31). NHE3 participates in tubular albumin uptake (10,11); theincrease in NHE3-specific activity may therefore be in responseto the increased albumin load presented to the proximal tubule.
In conclusion, in a model of nephrotic syndrome, the abundanceof NHE3, its specific activity, and its subcellular localizationwere altered, suggesting a novel mechanism of control of Na/Hexchange in the proximal tubule in vivo.
Acknowledgments
PMA was supported by a grant from the Swiss National ScienceFoundation (31–54957.98) and the Hermann Klaus Foundation.JK was supported by the Novartis Science Foundation.
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Received for publication March 25, 2002.
Accepted for publication June 7, 2002.
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[Abstract][Full Text][PDF]
D. G. Fuster, I. A. Bobulescu, J. Zhang, J. Wade, and O. W. Moe Characterization of the regulation of renal Na+/H+ exchanger NHE3 by insulin
Am J Physiol Renal Physiol,
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[Abstract][Full Text][PDF]
S. de Seigneux, S. W. Kim, S. C. Hemmingsen, J. Frokiaer, and S. Nielsen Increased expression but not targeting of ENaC in adrenalectomized rats with PAN-induced nephrotic syndrome
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July 1, 2006;
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[Abstract][Full Text][PDF]
B. Sampaio-Maia, M. Moreira-Rodrigues, P. Serrao, and M. Pestana Blunted renal dopaminergic system activity in puromycin aminonucleoside-induced nephrotic syndrome
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314 - 323.
[Abstract][Full Text][PDF]
L. E. Yang, A. B. Maunsbach, P. K. K. Leong, and A. A. McDonough Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes
Am J Physiol Renal Physiol,
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F896 - F906.
[Abstract][Full Text][PDF]
S. W. Kim, W. Wang, J. Nielsen, J. Praetorius, T.-H. Kwon, M. A. Knepper, J. Frokiaer, and S. Nielsen Increased expression and apical targeting of renal ENaC subunits in puromycin aminonucleoside-induced nephrotic syndrome in rats
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May 1, 2004;
286(5):
F922 - F935.
[Abstract][Full Text][PDF]
J. Klisic, J. Zhang, V. Nief, L. Reyes, O. W. Moe, and P. M. Ambuhl Albumin Regulates the Na+/H+ Exchanger 3 in OKP Cells
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[Abstract][Full Text][PDF]
L. L. Hamm and V. Batuman Edema in the Nephrotic Syndrome: New Aspect of an Old Enigma
J. Am. Soc. Nephrol.,
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[Full Text][PDF]
E. M. Lee, C. A. Pollock, K. Drumm, J. A. Barden, and P. Poronnik Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells
Am J Physiol Renal Physiol,
October 1, 2003;
285(4):
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[Abstract][Full Text][PDF]
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Abstract
Introduction
) (7). The reabsorption of albumin is achieved predominantly by endocytosis. Several recent studies have suggested an interrelation of transcellular albumin transport by endocytosis and acidification of lysosomes (8,9) through endosomal Na/H exchange (10,11). The nephrotic syndrome (NS) is also accompanied by various degrees of salt and water retention and represents a major clinical problem in the management of patients with nephrosis (12,13). One proposed mechanism is systemic interstitial volume sequestration due to hypoalbuminemia and secondary Na+ retention. In addition, primary salt retention per se may contribute substantially to systemic volume expansion in NS. The nephrotic state has been associated with activation of the Na/K-ATPase in the collecting duct (14,15). The effect of nephrotic syndrome on proximal tubule Na+ transporters has not been explored. The bulk of sodium reabsorption in the proximal tubule is mediated by the apical sodium/proton exchanger NHE3 (16). Given the potential role of proximal tubular sodium/proton exchange in both tubular protein reabsorption and salt retention, we investigated the regulation of the cortical brush border membrane Na/H exchanger NHE3 in rats with nephrotic syndrome induced by puromycin amino nucleoside (PAN). 
ex = 493 nm, 
