1Department of Medicine, University of Sydney, Renal Research Group, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065; 4School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072; 3Department of Anatomy and Histology, University of Sydney, New South Wales 2006, Australia; and 2Physiologisches Institut, University of Würzburg, 97070 Würzburg, Germany
Submitted 30 December 2002 ; accepted in final form 2 June 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
proteinuria; sodium-hydrogen exchange; sodium retention
Under normal conditions, the kidneys filter 180 liters of filtrate and
reabsorb 1.7 kg of NaCl per day
(21), a function principally
performed by the Na+/H+ exchanger (NHE) isoform 3 (NHE3)
in the luminal membrane (3).
Several lines of experimental evidence suggest that increases in NHE3 activity
are linked to hypertension. Elevated levels of NHE3 protein and activity have
been observed in freshly isolated tubular cells and isolated intact tubules
from spontaneously hypertensive rats
(22). In NHE3 knockout mice,
systolic and arterial blood pressures are reduced, suggesting a key role for
NHE3 in maintaining these parameters
(27), whereas there is an
increase in NHE3 activity in puromycin aminonucleoside-induced nephrotic rats
that may contribute to the increased Na+ retention in these animals
(4). Significantly, a study in
hypertensive patients revealed that proximal tubule Na+
reabsorption was an independent determinant of the blood pressure response to
salt-induced hypertension (8).
It is important to keep in mind that although animal models of diabetes may
develop kidney disease that displays features in common with human diabetic
nephropathy, no single animal model develops renal changes identical to those
observed in humans (37). As a
result, it is critical to determine the response of cells of human proximal
tubular origin to exposure to conditions that mimic the proteinuric state.
In addition to Na+ reabsorption, it is estimated that several grams of albumin enter the proximal tubules on a daily basis, yet the urinary excretion of albumin is normally <50 mg/day; this suggests constitutive reabsorption of albumin (28). This reabsorption occurs in the proximal tubule via receptor-mediated endocytosis involving the scavenger receptor megalin (10). Albumin itself has been shown to exert a number of effects on the proximal tubule. Exposure of opossum kidney (OK) cells to pathophysiological concentrations of albumin has been shown to stimulate cellular proliferation (12) and also to impair albumin endocytosis by reducing the number of binding sites (Vmax) at the plasma membrane (19), whereas exposure to albumin has been shown to induce apoptosis in LLC-PK1 cells (16).
NHE3 has been recently demonstrated to play a critical role in
receptor-mediated albumin uptake in cell cultures. Studies in OK cells have
shown that failure to acidify the early endosome
(18) or inhibition of NHE3
with amiloride analogs significantly reduces albumin uptake
(17), an observation that is
supported by the finding that albumin uptake is abolished in NHE3-deficient OK
cells (20). Thus changes in
the regulation of endosomal pH may play a significant role in tubular
dysfunction (29). In rabbit
renal cortical membrane fractions, a substantial fraction of the cellular pool
of NHE3 has been shown to be associated with the scavenger receptor megalin
via interaction with its COOH-terminal tail
(6), and NHE3 in the
megalin-associated pools in the rabbit brush border was inactive
(5). These data indicate a dual
role for NHE3 in Na+ reabsorption and albumin uptake. In contrast
to the other proximal tubule NHE isoforms 1 and 2, NHE3 is known to exist
primarily in endosomes, with only 15% of the total cellular pool of NHE3
located in the plasma membrane
(2,
7). Rapid alterations in the
activity of NHE3 are accomplished by changes in the rates of deployment of
NHE3 from the endosomes or retrieval of NHE3 from the plasma membrane
(13). Furthermore, recent
evidence shows that NHE3 exists in lipid rafts in rabbit ileal brush borders
and that stimulation with epidermal growth factor (EGF), which increases NHE3
activity, results in a preferential increase in the amount of NHE3 in the
lipid raft fraction (26).
From these data, it is clear that NHE3 exists in functionally distinct pools at the plasma membrane, for example, pools associated with Na+ reabsorption and others involved in receptor-mediated albumin endocytosis. Thus there may be different retrieval/insertion mechanisms to and from these pools, such that the rates of insertion of NHE3 into the plasma membrane may occur independently of the rate of the NHE3 internalization in conjunction with receptor-mediated albumin endocytosis. This raises the intriguing possibility that increased tubular albumin may have two separate actions: 1) it may reduce the capacity of the albumin uptake pathway, thereby increasing levels of protein in the urine; and 2) if the rates of insertion of NHE3 into the different pools are not directly linked to the rate of receptor-mediated endocytosis, levels of functional NHE3 may be increased, resulting in increased Na+ retention by the proximal tubule. Such a model may provide an explanation for the link between hypertension and proteinuria in diabetic nephropathy. The aims of the present study were, therefore, to determine the effects of albumin on the expression, distribution, and activity of NHE3 in human proximal tubule cells (PTC) and to correlate these effects with changes in cellular growth.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental protocol. All experiments were carried out on passage 2 PTC. Cells were made quiescent by incubation in DMEM-F-12 containing 5 µg/ml human transferrin and 5 mM D-glucose without growth factors for 48 h. The cells were then cultured for a further 48 h in medium containing 5 mM D-glucose (control) or 5 mM D-glucose containing 0.1 or 1.0 mg/ml delipidated bovine serum albumin (Sigma Chemical).
Growth studies. Experiments to measure cell number and cell protein were performed in parallel, with the results obtained for total protein being adjusted to the total cell number in each of the treatments. Cell numbers in response to each treatment were determined by manual cell counts on trypsinized cells using a hemocytometer. The total protein content of cells was determined as a marker of cellular hypertrophy. Cells were solubilized with 0.2 M NaOH, and the protein was measured using a protein assay (Bio-Rad, Hercules, CA).
22Na+ uptake. 22Na+ uptake into cells was measured on the basis of the method of Rindler et al. (35). PTC were grown to confluence in 48-well culture plates, quiesced for 48 h, and then exposed to 0.1 or 1.0 mg/ml albumin or control conditions for 48 h. Cells were washed twice in HEPES-buffered saline (in mmol/l: 136 NaCl, 5.4 KCl, 1.2 CaCl2, 0.8 MgCl2,10 acidic-HEPES, and 5 glucose, pH 7.4) and incubated with Na+-free solution (HEPES-buffered saline with NaCl replaced by N-methyl-D-glucamine) for 15 min to deplete intracellular Na+. All uptake solutions contained the corresponding amounts of albumin. The cells were preincubated with 100 µM ouabain with or without the NHE blocker ethyliso-propylamiloride (EIPA, 100 µM) in Na+-free medium for a further 30 min. The Na+ solution was replaced with the uptake solution containing 22Na+ tracer (1 µCi/ml; New England Nuclear Geneworks, Boston, MA) in glucose-free HEPES-buffered saline for 20 min. At the end of the 22Na+ uptake period, the cells were washed rapidly three times with ice-cold 0.1 M MgCl2 and solubilized in 0.1 M NaOH. Cell lysate was mixed with scintillation fluid and counted in a beta scintillation counter (LKB Wallac, Turku, Finland). Parallel cell counts were performed, and the total 22Na+ uptakes were adjusted to cell number in each treatment and expressed as a percentage of the control values.
Cell cycle analysis. PTC were grown to confluence in six-well
plates and exposed to 0.1 or 1.0 mg/ml albumin or control conditions for 48 h.
The cells were then trypsinized, placed in 1.5-ml tubes, washed in 0.5 ml of
phosphate-buffered saline (PBS), centrifuged (1,000 rpm, 10 min, 4°C), and
fixed in 70% (vol/vol) ethanol at 20°C for 3 h. Cells were
washed in PBS and permeabilized in 0.5 ml of PBS with 0.1% Triton X-100 on ice
for 30 min. Cells were then centrifuged, and the pellet was resuspended in 0.5
ml of fluorochrome solution [propidium iodide (50 µg/ml), RNase (1 mg/ml),
and Triton X-100 (0.1% vol/vol)] in PBS and incubated for 1 h at 4°C. Cell
cycle analysis was performed using the FACSVan-tage SE flow cytometry system
(Becton Dickinson, San Diego, CA). The propidium iodide fluorescence of
individual nuclei and the forward and side scatter were measured using
identical instrument settings with
20,000 events.
Competitive RT-PCR. Competitive RT-PCR was performed to determine the changes in NHE3 expression level induced by exposure to albumin. PTC were grown to confluence in six-well plates and exposed to 0.1 mg/ml albumin for 48 h before RNA extraction. Total RNA was extracted using TRIzol reagent (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. RNA was reverse transcribed using the Superscript II reverse transcriptase kit (GIBCO BRL).
Competitive RT-PCR was performed using primers specific to the
COOH-terminal tail of human NHE3 (GenBank accession no. U28043
[GenBank]
). The primer
sequences were 5'-GTTCTTCACCGTCATCTTCCA (sense, bp 13451365) and
5'-CTGAGAGAAAATGTCAGCGCT (antisense, bp 17691789), and the
competitor primer sequence was
5'-CTGAGAGAAAATGTCAGCGCTGCCATCAGCTACGTGGCC (bp 16371655). These
primers produced products of 445 and 311 bp, respectively. Products were
sequenced and confirmed to be human NHE3. Competitive RT-PCR against
-actin was used as a control. Primers were designed against the human
-actin gene (GenBank accession no. M10277
[GenBank]
). The primer sequences were
5'-CATGTACGTTGCTATCCAG (sense, bp 20582076) and
5'-CGCAACTAAGTCATAGTCC (antisense, bp 30033021), and the
competitor primer sequence was 5'-CGCAACTAAGTCATAGTCCATGGAGCCGCCGATCCAC
(bp 28892906). These primers produced products of 964 and 849 bp,
respectively.
PCR were performed on cDNA using the sense and competitor primers, and the
products were gel purified and quantitated. For the PCR, the competitor cDNA
was used at 0.2, 0.5, 1.5, 4.6, and 13.7 fM. The reactions were for 35 cycles
with an annealing temperature of 60°C using the Expand High-Fidelity PCR
System (Roche, Mannheim, Germany). The products were run on a 2% agarose gel
stained with ethidium bromide and photographed. The photograph was then
scanned into a computer, and the relative intensities of the individual bands
were quantitated using NIH Image software (version 1.60). To normalize the
data to the levels of the housekeeping gene -actin, the ratios of the
concentration of competitor primer for NHE3 to the concentration of the
-actin primers at the equivalence point were determined. The equivalence
point is defined as the point at which the ratio of the intensity of the bands
for the competitor to native cDNA is unity, that is, the point at which the
concentration of the competitor is equivalent to the concentration of the
message for the target gene.
Polyclonal antibody to the extracellular domain of human NHE3. The peptide sequence of human NHE3 (GenBank accession no. NP_004165 [GenBank] ) was screened for potential extracellular epitopes. The polypeptide GGVEVEPGGAHGESGGF was selected. This peptide corresponds to amino acids 2642 of the predicted human sequence, a region that is within the predicted first extracellular loop. The SignalP algorithm was used to predict the position of any putative signal peptide cleavage site in the NH2-terminal region of human NHE3 (31). The peptide was synthesized, and an NH2-terminal cysteine was added to the epitope for conjugation via diphtheria toxin using maleimidocaproyl-N-hydroxy-succinimide (Chiron Mimotopes, Clayton, Victoria, Australia). Polyclonal antibodies were raised in rabbits, and the immune serum IgG was affinity purified.
Confocal immunofluorescence. Confocal microscopy was performed on PTC grown on Cell-Tak (BD Biosciences, Bedford, MA)-coated coverslips as follows. PTC were fixed with 4% paraformaldehyde in PBS for 2 min. Where required, cells were permeabilized with 0.1% DMSO in 2% normal horse serum, 0.1% Triton X-100, and 0.1% bovine serum albumin in PBS for 2 min. Permeabilized and nonpermeabilized cells were blocked with 20% normal horse serum in PBS for 20 min. The cells were then incubated with the anti-NHE3 antiserum (1:100) for 2 h at room temperature, washed, and incubated with a Cy3 anti-rabbit antibody (1:200; Jackson Immunochemicals) for a further 45 min. Slides were sealed with mounting medium (Dako, Carpenteria, CA) and visualized under a Leica TCS NT laser confocal microscope (Leica, Solms, Germany) with excitation at 488 nm and emission at 570 nm.
To demonstrate the specificity of the polyclonal anti-NHE3 antibody, 80 µM epitope peptide was preincubated with the antibody for 10 min to block the binding of the antibody-binding sites. Surface NHE3 distribution was determined on PTC quiesced in 5 mM glucose medium for 48 h and then exposed to 0.1 or 1.0 mg/ml albumin for 48 h. Cells were nonpermeabilized to determine surface levels of NHE3 or permeabilized to determine total NHE3 in the same apical plane used to determine the surface levels. Images were processed with Adobe Photoshop (version 5.02), and pixel densities at the apical pole of the cells were quantitated using NIH Image software (version 1.60).
Statistical analysis. Experiments were performed at least in triplicate on a minimum of four different cell culture preparations. Unless otherwise stated, results are expressed as percentage of control values (cells grown in the absence of albumin for the experimental period). Statistical comparisons between groups were made by analysis of variance or paired t-tests where appropriate. Analyses were performed using the software package Statview (version 4.5, Abacus Concepts, Berkley, CA). P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Apoptosis. Fluorescein-activated cell sorter analysis revealed that, under control conditions, 0.77 ± 0.16% of cells were in the pre-G1 peak, which represents apoptotic cells. No significant changes were observed in the numbers of cells in the pre-G1 peak after exposure to 0.1 or 1.0 mg/ml albumin for 48 h: 0.85 ± 0.37 and 0.71 ± 0.25%, respectively. These data indicated that exposure to albumin did not increase the levels of apoptosis of PTC.
22Na+ uptake. Total 22Na+ uptake was significantly increased in PTC after exposure to albumin for 48 h at 0.1 and 1.0 mg/ml: 119.1 ± 6.3% (n = 13, P = 0.0005) and 115.6 ± 5.3% (n = 11, P < 0.006) of control levels, respectively (Fig. 2). Incubation of the control cells with 100 µM EIPA reduced 22Na+ uptake to 42.4 ± 2.8% (n = 13) of baseline levels (Fig. 2). Similarly, incubation of the albumin-treated cells with EIPA reduced 22Na+ uptake to the level observed in control cells: 38.6 ± 4.3% (n = 13) in 0.1 mg/ml albumin and 47.9 ± 3.7% (n = 11) in 1.0 mg/ml albumin (Fig. 2). Thus the increase in total 22Na+ uptake was abolished by treatment with EIPA, and these data show that albumin exposure resulted in significant increases in the EIPA-sensitive component of 22Na+ uptake to 131.0 ± 6.3% (P = 0.0001) for 0.1 mg/ml albumin and 124.7 ± 8.6% (P < 0.005) for 1.0 mg/ml albumin compared with control cells not exposed to albumin (100%). Importantly, 1.0 µM EIPA had no effect on 22Na+ uptake (data not shown), indicating that this component of 22Na+ uptake was not occurring via NHE isoform 1 (32). Furthermore, no effects were observed on 22Na+ uptake in cells exposed to lower concentrations of albumin (0.001 or 0.01 mg/ml) for 48 h (data not shown).
|
Competitive RT-PCR. Competitive RT-PCR was performed to determine
whether the increases in 22Na+ uptake observed in
response to exposure to 0.1 mg/ml albumin were paralleled by an increase in
NHE3 mRNA expression levels. A representative gel of the products of the
competitive RT-PCR is shown in Fig.
3A, and logarithmic plots of the ratio of the target to
competitor bands against competitor concentration for NHE3 and the
housekeeping gene -actin are shown in
Fig. 3B. There is a
small but significant shift in the equivalence point for NHE3, whereas no
change is observed with
-actin. The NHE3 ratios were standardized to the
-actin ratios to account for any changes in total mRNA. Overall, there
was a significant increase in the levels of mRNA for NHE3 to 161.4 ±
15.1% (n = 6, P < 0.005) relative to the total mRNA pool
(as reflected by
-actin) in PTC exposed to albumin for 48 h
(Fig. 3C). These data
confirm that exposure of PTC to 0.1 mg/ml albumin, a concentration that
increases NHE3 activity, also results in a parallel increase in NHE3 message
levels.
|
Confocal immunofluorescence. Surface labeling was performed on nonpermeabilized and permeabilized PTC. When a confocal x-y scan was taken through the upper (subapical) part of the cells, nonpermeabilized cells exhibited a punctate distribution of labeling, principally at the cell periphery (Fig. 4A), whereas permeabilized cells also exhibited significant levels of intracellular staining (Fig. 4B). z-Axis scans on nonpermeabilized and permeabilized cells revealed that NHE3 was primarily localized to the apical domain of the cells (Fig. 4, C and D). These staining patterns are consistent with the known distribution of NHE3 in other cell types (2, 9, 33). To confirm the antibody specificity, the antiserum was preincubated with the antigenic peptide before the cells were labeled. This resulted in a dramatic decrease in the fluorescent signal (Fig. 4, E and F), indicating that the antibody staining was indeed specific for the epitope on NHE3. A similar decrease in fluorescence was also observed with a lower peptide concentration (10 µM; data not shown).
|
After exposure to albumin for 48 h, there was a significant increase in the levels of cell surface NHE3 (Fig. 5, A and B). Similarly, when x-y scans were performed on permeabilized cells viewed through the same optical plane, albumin also induced an increase in total NHE3 (Fig. 5, C and D). Measurements of relative pixel intensities in individual cells revealed a significant increase in the level of fluorescence intensity with both concentrations of albumin. In cells exposed to 0.1 mg/ml albumin, the level of fluorescence at the apical surface was 115.4 ± 2.7% (n = 20, P < 0.0001) compared with control levels (Fig. 6A). A similar increase in fluorescence (118.1 ± 3.2%, n = 17, P < 0.0001 compared with control) was observed in cells exposed to 1.0 mg/ml albumin (Fig. 6A). A similar analysis of pixel density at the apical pole was performed on permeabilized cells to determine the amounts of NHE3 on the cell surface relative to the intracellular pool in the immediate vicinity of the apical membrane. In response to albumin, there was an increase in total NHE3 at the apical membrane increasing to 118.7 ± 2.4% (n = 19, P < 0.0001) and 109.8 ± 2.7% (n = 20, P < 0.02), respectively (Fig. 6B). When the pixel intensities for the nonpermeabilized cells were standardized to the permeabilized cells, these data revealed that 76.1 ± 2.1% of the total apical NHE3 pool was located at the cell surface (Fig. 6C). Significantly, although the total amount of NHE3 at the cell surface increased, the proportion of NHE3 at the cell surface relative to the total subapical pool did not change with albumin exposure: 74.0 ± 1.7% and 81.9 ± 2.2% of total NHE3 for 0.1 and 1.0 mg/ml albumin, respectively (Fig. 6C). These data suggest that the increase in NHE3 activity we observed in response to prolonged exposure to albumin resulted from an increase in total NHE3 that translated into a parallel increase in the plasma membrane, rather than altered rates of insertion from submembrane stores.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our demonstration that albumin at 1.0 mg/ml resulted in a pronounced proliferative effect is consistent with findings in OK cells, where exposure to albumin increased cell number and total thymidine uptake, with a maximal effect at 1.0 mg/ml albumin (12). The fact that we observed no effects on cell growth at the lower concentration of albumin (0.1 mg/ml) may simply reflect a difference in the cell types studied. These data show that delipidated albumin alone, at a concentration in the high pathophysiological range and in the absence of growth factors, is able to induce cell proliferation. It has been shown that, after induction of experimental diabetes in a rat model, nephromegaly is preceded by an initial hyper-plastic phase over the first few days (23). Consequently, this initial hyperplasia may, in part, be accounted for by the proliferative effect of elevated albumin.
Albumin alone (at >0.5 mg/ml) has been shown to protect cultured murine
PTC from apoptosis by a mechanism involving the scavenging of reactive oxygen
species (24). In the present
study, we observed no significant changes in the levels of apoptosis in the
presence and absence of albumin; thus our data are consistent with a
renoprotective effect of albumin. These findings contrast with those reported
in LLC-PK1 cells, where exposure to albumin induced apoptosis;
however, in these studies, much higher concentrations of albumin were used
(>5 mg/ml) (16). An in vivo
study in protein-overload rats showed an increase in the number of
proliferating cells as determined by in situ hybridization for histone mRNAs.
However, this increase was counteracted by an even greater increase in the
number of apoptotic cells, with tubular atrophy being the net result
(36). Furthermore, it has been
shown in LLC-PK1 cells
(42), OK cells
(14), and primary cultures of
rat PTC (38) that exposure to
pathophysiological levels of albumin results in an activation of NF-B
that results in enhanced cytokine/chemokine production. Thus the increases in
PTC number that we observe in culture may result in vivo in the increased
cytokine/chemokine production that underlies the inflammatory phase of
tubulointerstitial pathogenesis
(41).
NHE3 has been shown to exist primarily in subapical endosomal pools and a juxtanuclear compartment in OK cells (2) and AP-1 cells transfected with NHE3 (15). It has been demonstrated that as little as 15% of the total cellular NHE3 is present in the plasma membrane (1, 2, 7). To directly monitor changes in levels of NHE3 at the cell surface, we developed an antibody to a predicted extracellular epitope in the first extracellular loop of human NHE3 on the basis of the model of NHE3 with 12 transmembrane loops and intracellular NH2 and COOH termini. The epitope recognized by our antibody corresponds to amino acids 2642 of the published human NHE3 sequence (GenBank accession no. NP_004165 [GenBank] ). A recent topological analysis of rabbit NHE3, however, showed that the first 30 amino acids of NHE3 form a signal peptide that is cleaved during processing, resulting in an extracellular NH2 terminus (40). We found a predicted signal peptide cleavage site between Gly27 and Val28 of the human sequence comparable to the predicted cleavage site in rabbit NHE3 between Gly29 and Ala30 (40). Thus our antibody recognizes the first 16 amino acids of the NH2 terminus of human NHE3 after signal peptide cleavage, and the binding of our antibody to the surface of nonpermeabilized PTC confirms that this region of human NHE3 is extracellular.
Surface labeling of PTC revealed a punctate distribution of NHE3 similar to
that observed in OK cells (2,
39). In permeabilized PTC,
there was considerably more NHE3 in the cytosol, and this intracellular NHE3
appeared to be associated largely with endosomal compartments in the subapical
region of the PTC (Fig. 4).
Therefore, these data obtained from confocal sections taken through the cell
body are in agreement with the overall cellular distribution of NHE3 in other
cell types, e.g., PS120 and OK cells
(2,
15), where only 1015%
of the total cellular NHE3 pool was reported to be inserted at the cell
surface (1,
2,
7). Interestingly, our data,
derived from confocal sections taken in the plane of the apical cell surface
on permeabilized and nonpermeabilized cells (Figs.
5 and
6), suggest that a significant
proportion (70%) of the total NHE3 in the immediate direct vicinity of
the cell membrane is present at the cell surface. However, because of the
difficulties in precise quantitation of relative intensities due to
overlapping stores of NHE3 visualized under permeabilized and nonpermeabilized
conditions, it is possible that this method may overestimate the proportion of
total NHE3 at the cell surface. Nevertheless, our data support the study in OK
cells showing that NHE3 exists in different functional domains and is present
in large complexes at the apical surface of OK cells, with the intracellular
pools acting as reservoirs for membrane recruitable NHE3
(2). It has been reported that
NHE3 exists as an active oligomer in the microvillar domain and as an inactive
megalin-associated form in intermicrovillar domains
(5) and that NHE3 levels
increase preferentially in lipid rafts after stimulation with EGF
(26). Thus the increase in
surface staining for NHE3 and increased NHE3 activity that we observed after
exposure to albumin may reflect an enhanced association with the plasma
membrane domains involved in mediating Na+ reabsorption. This
model, on the basis of the present data, is consistent with a recent finding
in nephrotic rats, where the increase in NHE3 activity was associated with a
shift from the inactive megalin-associated pool to the active pool in the
brush border of proteinuric rats
(4), potentially contributing
to increased Na+ retention. Our findings are also in agreement with
a report that albumin increased NHE3 activity and immunoreactivity in OK
cells, although higher concentrations of albumin (
5 mg/ml) were required
to elicit a response (30).
The increase in NHE3 expression and activity suggests that exposure to albumin increases the Na+ reabsorptive capacity of the human proximal tubule. Significantly, albumin induced increases in Na+ reabsorption at concentrations below which it exerted its proliferative effect. High concentrations of albumin are reported to enhance the proliferation of PTC (11); however, the differential effects we observed at lower concentrations indicate a specific role of albumin in regulating Na+ reabsorption.
In summary, the present study demonstrates the concentration-dependent uptake of albumin by primary cultures of human PTC and shows that albumin uptake is associated with an increase in the activity of NHE3. A sustained 1020% increase in the transcription and activity of NHE3 in response to elevated albumin may lead to a significant increase in Na+ retention, contributing to the development of hypertension, whereas a concomitant reduction in the tubular absorption of albumin in the presence of elevated albumin would result in the increased excretion of albumin manifest as proteinuria. Thus the data in the present study present a possible mechanism to explain the link between reduced albumin uptake and increased Na+ retention as observed in diabetic nephropathy.
![]() |
DISCLOSURES |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|