1 Department of Cell Biology, In chronic renal failure (CRF), reduction in renal mass leads
to an increase in the filtration rates of the remaining
nephrons and increased excretion of sodium per nephron. To address the mechanisms involved in the increased sodium excretion, we determined the total kidney levels and the densities per nephron of the major renal NaCl transporters in rats with experimental CRF. Two weeks after
5/6 nephrectomy (reducing the total number of nephrons to ~24 ± 8%), the rats were azotemic and displayed increased Na excretion. Semiquantitative immunoblotting revealed significant reduction in the
total kidney levels of the proximal tubule Na transporters NHE-3 (48%
of control), NaPi-II (13%), and Na-K-ATPase (30%). However, the
densities per nephron of NHE-3, NaPi-II, and Na-K-ATPase were not
significantly altered in remnant kidneys, despite the extensive
hypertrophy of remaining nephrons. Immunocytochemistry confirmed the
reduction in NHE-3 and Na-K-ATPase labeling densities in the proximal
tubule. In contrast, there was no significant reduction in the total
kidney levels of the thick ascending limb and distal convoluted tubule
NaCl transporters BSC-1 and TSC, respectively. This corresponded to a
3.6 and 2.5-fold increase in densities per nephron, respectively
(confirmed by immunocytochemistry). In conclusion, in this rat CRF
model: 1) increased fractional sodium excretion is associated with altered expression of proximal tubule Na transporter expression (NHE-3, NaPi-II, and Na-K-ATPase), consistent with glomerulotubular imbalance in the face of increased single-nephron glomerular filtration rate; and
2) compensatory increases in BSC-1
and TSC expression per nephron occur in distal segments.
fractional excretion of sodium; renal hypertrophy; glomerulotubular
imbalance
AS THE POPULATION of surviving nephrons is reduced in
chronic renal failure (CRF), the remaining tissue undergoes hypertrophy and the function is dramatically altered. It is well documented that
when total renal mass is reduced, single-nephron glomerular filtration
rate (SNGFR) in the remaining nephrons markedly increases (19). The
elevated filtration rate leads to an increased reabsorptive burden on
the residual nephrons. This together with the compensatory renal
hypertrophy is associated with marked alterations in tubular reabsorptive capacities of sodium and water (8, 14, 20, 45).
In CRF, there is an adaptive increase in sodium excretion by each
residually functioning nephron (21, 44, 46). In this way, sodium
balance can be maintained despite a diminishing GFR when intake of salt
and water is unaltered in CRF. The mechanisms responsible for the
increased fractional sodium excretion and marked natriuresis per
nephron in remnant kidney have been investigated (8, 14, 21, 45, 49,
53). However, the tubular sites for the markedly altered renal
transport of sodium in CRF have not been clearly elucidated, and the
results, especially concerning the proximal tubule, have been
conflicting (4, 8, 21, 32, 45, 53). Several investigators have
suggested that increased excretion of sodium per nephron in CRF was
achieved through a reduction in proximal tubular reabsorption (32, 45),
whereas others reported no changes in the proximal tubule (21). Since the type 3 Na/H exchanger (NHE-3) is a major sodium transport pathway
in the renal proximal tubule (3), we speculate that the altered levels
of apically expressed NHE-3 (2, 6) and basolaterally
expressed Na-K-ATPase in the kidney tubules (23, 25) in response to the
marked increase in SNGFR may account for the marked changes in proximal
tubule sodium and hence water reabsorption in remnant kidney.
Furthermore, in renal proximal tubules, transport of phosphate
(Pi) through the proximal apical membrane is largely performed by the type II
Na-Pi cotransporter (NaPi-II) (5,
38). Thus we speculate that NaPi-II may also be dysregulated together
with other proximal tubule transporters.
Another characteristic urinary manifestation of CRF is that the urinary
concentrating ability decreases in response to a reduction of
functioning renal mass (13, 30, 47). In contrast, the ability to dilute
urine has been demonstrated to be well maintained long after impairment
of the concentrating ability has taken place (28). Concentration of the
urine requires establishment and maintenance of a hypertonic medullary
interstitium. The loop of Henle generates a high osmolality in renal
medulla by driving the countercurrent multiplication, which is
dependent on the NaCl absorption by the thick ascending limb (TAL)
(29). Furthermore, urinary dilution is also mediated by NaCl absorption
in the TAL and distal convoluted tubule (DCT) (29). The apically
expressed Na-K-2Cl cotransporter (rat type 1 bumetanide-sensitive
cotransporter: BSC-1 or NKCC2) (10, 15, 24, 26, 39, 56) and type 3 Na/H
exchanger (NHE-3) (2, 3, 6), as well as basolaterally expressed
Na-K-ATPase, are key components responsible for sodium reabsorption by
the TAL.
In the rat model of CRF induced by 5/6 nephrectomy, delivery of sodium
and water to the TAL and DCT has been demonstrated to be significantly
increased (8). This is caused by an increase in filtered load and a
decrease in the fractional reabsorption of water and sodium in proximal
tubule (8). Thus, in the remaining functioning nephrons where single
nephron filtered load of fluid and solute is very high, we hypothesize
that changes in the expression of NaCl transporters may play a critical
role in the altered sodium handling and the urinary
concentration and diluting ability in CRF. Interestingly,
adriamycin-induced nephrotic syndrome in rats has been demonstrated to
be associated with a marked decrease in the abundance of Na-K-2Cl
cotransporter (BSC-1), NHE-3, and Na-K-ATPase, in association with a
marked decrease in urinary concentrating ability and derangement of
urinary sodium excretion (12). Thus it can be hypothesized that changes
in the expression of NaCl transporters may play a critical role in the
development of altered sodium handling in experimental CRF.
Furthermore, a change in the expression of these NaCl transporters may
play a role in the impairment of the urinary concentrating ability
seen in response to a reduction of functioning renal mass.
In the present study, we performed semiquantitative immunoblotting and
immunocytochemistry with the following purposes:
1) to examine whether there are
changes in the total kidney levels or densities per nephron of major
proximal tubule sodium transporters and major distal tubule sodium
transporters in rats with CRF induced by 5/6 nephrectomy; and
2) to examine whether these changes
are associated with changes in the urinary sodium excretion in rats with CRF.
Experimental animals. The studies were
performed on 54 adult Munich-Wistar rats initially weighing 265 ± 8 g (Møllegard Breeding Centre). The rats were maintained on a
standard rodent diet (Altromin, Lage, Germany) with free access to water.
Induction of CRF by surgical reduction of renal
mass. Experimental CRF was induced by excision of about
two-thirds of the left kidney and right total nephrectomy, using the
so-called excision remnant kidney model (18). The protocols used in
this study are depicted in Fig. 1.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Diagram of the study design. Protocol
1: chronic renal failure (CRF) induced in rats by 2/3
nephrectomy followed by contralateral nephrectomy
(n = 17). Protocol
2: sham-operated rats matching
protocol 1 (n = 11). Protocol
3: CRF rats treated with
1-deamino-[8-D-arginine]vasopressin
(DDAVP) using implantable osmotic minipumps for 7 days
(n = 6). Protocol
4: sham-operated rats treated with normal saline using
implantable osmotic minipumps for 7 days
(n = 6). Protocol
5: normal rats with excision of both poles of left
kidney (2/3 nephrectomy) (n = 6).
Protocol 6: kidneys of rats with CRF
(n = 4, protocol identical to the one
described in protocol 1) and of
sham-operated rats (n = 4, protocol
identical to protocol 2) were
perfusion fixed for immunocytochemistry (protocol
6 is not indicated in diagram). In CRF groups,
two-thirds of the left kidney (the upper and lower poles) was excised,
and 1 wk later, the right total kidney was removed. Rats were
maintained in metabolic cages at the days marked with asterisk,
allowing monitoring of urine excretion rates. Urine volume, osmolality,
creatinine, and sodium and potassium concentrations were measured.
Plasma was collected at the time of right nephrectomy and at the time
of death for measurement of sodium and potassium concentration, plasma
urea nitrogen, creatinine, and osmolality.
The rats were anesthetized with halothane (Christian Friis, Copenhagen, Denmark) and during surgery, the rats were placed on a heated table to maintain rectal temperature at 37-38°C. The left kidney was exposed through left flank incision and gently dissected free from the adrenal gland, and approximately two-thirds of the left kidney including the upper and lower pole was excised (consistently ~50% of the kidney mass was removed; see RESULTS section). One week later, these rats were again anesthetized with halothane, and the right kidney was removed through right flank incision after dissecting it free from the adrenal gland. The wound was closed with 4-0 Mersilene and metal clamps. Immediately after each surgical procedure, buprenorphium 0.2 mg/kg sc (Temgesic, Reckitt and Colman) was injected to relieve pain for 1 day, and during this time rats were allowed to recover from anesthesia and surgery in cages with free access to water and standard rat chow.
As a control group, rats were subjected to sham operations identical to those used for CRF rats except that kidneys or poles of kidney were not removed. Sham-operated rats were monitored for 2 wk in parallel with 5/6 nephrectomized rats. All rats were killed under light halothane anesthesia, and kidneys were rapidly removed and processed for membrane fractionation and immunoblotting at the same day.
We have chosen 2-wk followup after induction of 5/6 nephrectomy to 1) produce severe chronic renal insufficiency, 2) to allow significant hypertrophy of the remnant kidney, and 3) to minimize interstitial fibrosis. The functional data revealed marked chronic renal insufficiency, and the light microscopical evaluation confirmed a very mild fibrosis (see RESULTS).
Clearance studies. The rats were maintained in the metabolic cages, allowing quantitative urine collections and measurements of water intake (Fig. 1). Urine volume, osmolality, creatinine, and sodium and potassium concentration were measured. Plasma was collected from tail vein at the time of right nephrectomy, and from abdominal aorta at the time of death for measurement of sodium and potassium concentration, creatinine, and osmolality.
Experimental protocol. The following protocols were followed (Fig. 1). For protocol 1, CRF was induced in rats by excision remnant kidney model (n = 17). For protocol 2, rats were subjected to sham operation (n = 11). For protocol 3, CRF rats were treated with 1-deamino-[8-D-arginine]vasopressin (DDAVP, Sigma V1005) using implantable osmotic minipumps (model 2002; Alzet, Palo Alto, CA) for 7 days (n = 6). For protocol 4, sham-operated rats were treated with 0.9% NaCl solution using implantable osmotic minipumps for 7 days (n = 6). For protocol 5, in normal rats, both poles of left kidney were excised (n = 6). For protocol 6, for immunocytochemistry, kidneys of rats with CRF (n = 4, protocol identical to protocol 1) and of sham-operated rats (n = 4, protocol identical to protocol 2) were perfusion fixed (see below).
Implantation of osmotic minipumps. To examine whether the changes in expression of NaCl transporters in response to CRF are altered by long-term exogenous DDAVP treatment, osmotic minipumps were implanted in CRF (n = 6) and sham-operated (n = 6) rats (protocols 5 and 6). For implantation, minipumps were filled with DDAVP in a carrier solution containing 5% dextrose and 0.05% acetic acid for CRF rats or 0.9% NaCl solution for sham-operated rats. The pumps were equilibrated with normal saline for 4 h before insertion (35). Rats were anesthetized with inhalation of halothane, and the minipumps were inserted into the subcutaneous tissue under the skin of the back. Under these conditions, the pumps delivered 0.1 µg/h (0.5 µl/h) of DDAVP to CRF rats or 0.5 µl/h of 0.9% NaCl solution to sham-operated rats for 7 days.
Induction of chronic metabolic acidosis. To examine whether there are changes in the expression of NHE-3 levels in response to chronic metabolic acidosis (CMA) per se, Munich-Wistar rats were treated orally with 0.28 M NH4Cl in the drinking tap water for 2 wk ad libitum (n = 8) (1, 34). Control rats were received tap water ad libitum (n = 6).
Membrane fractionation for immunoblotting. The kidneys were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) using an Ultra-Turrax T8 homogenizer (IKA Labortechnik), at maximum speed for 10 s and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles (36). Gel samples (Laemmli sample buffer containing 2% SDS) were made of this pellet.
Primary antibodies. For
semiquantitative immunoblotting and immunocytochemistry, we used
previously characterized monoclonal and polyclonal antibodies
summarized in the following. 1) For NHE-3 (L546), an affinity-purified polyclonal antibody to NHE-3 was
previously characterized (12). 2)
For NaPi-II (L696), we prepared an affinity-purified polyclonal
antibody to NaPi-II using a synthetic peptide corresponding to the
final 24 amino acids of the COOH-terminal tail. A similar antibody was
previously characterized by Biber et al. (5).
3) For Na-K-ATPase, a monoclonal
antibody against the 1-subunit of Na-K-ATPase was previously
characterized (25). 4) For BSC-1
(L320), an affinity-purified polyclonal antibody to the apical Na-K-2Cl
cotransporter of the TAL was characterized by Kim et al. (26).
5) For the thiazide-sensitive Na-Cl
cotransporter, TSC (L573), an affinity-purified polyclonal antibody to
the apical Na-Cl cotransporter of the DCT was characterized by Kim et
al. (27). 6) For Tamm-Horsfall
protein, an affinity-purified polyclonal antibody to Tamm-Horsfall
protein has been characterized (22).
Electrophoresis and immunoblotting. Samples of membrane fractions from total kidney were run on 6-16% gradient polyacrylamide minigels (Bio-Rad Mini Protean II) for Na-K-ATPase, BSC-1, TSC, or 12% polyacrylamide minigels for NHE-3, NaPi-II, and Tamm-Horsfall protein. For each gel, an identical gel was run in parallel and subjected to Coomassie staining to assure identical loading (48). The other gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with affinity-purified primary antibodies (see above). The labeling was visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P447 or P448, diluted 1:3,000; DAKO, Glostrup, Denmark) using an enhanced chemiluminescence system (ECL, Amersham International).
Quantitation of total kidney levels of NaCl transporters and Tamm-Horsfall protein. ECL films with bands within the linear range were scanned (36) using an AGFA scanner (ARCUS II) and Corel Photopaint Software to control the scanner. The labeling density was determined of blots where samples from CRF kidneys were run on each gel with samples from control kidneys from sham-operated animals (36). The labeling density was corrected by densitometry of Coomassie-stained gels. The total kidney levels of NaCl transporters in the experimental animals were calculated as a fraction of the mean sham control value for that gel after correction for the fraction which was loaded of the total kidney mass. The loading fraction of total kidney mass was calculated by using the loading volumes of the gel samples to get identical densities of the Coomassie-stained lanes and by correction for body weight and kidney weight as follows: Loading fraction of total kidney mass = (loading volume of gel sample to get identical Coomassie staining)/(total volume of entire gel sample) × (the volume fraction that the entire gel sample constitute out of total kidney homogenate used for preparation of gel sample) × (total kidney weight per 100 g body wt corresponding to the left remnant kidney in CRF and the left plus right kidneys in sham-operated rats)/(numbers of kidney being 1 in CRF and 2 in sham-operated rats).1
Quantitation of densities per nephron of NaCl transporters and Tamm-Horsfall protein. To estimate the changes in the expression of NaCl transporters per single nephron to the increased SNGFR in remnant kidneys, total kidney levels of NaCl transporters and Tamm-Horsfall protein were divided by the fraction of the total kidney mass that remained immediately after 5/6 nephrectomy. Since the reduction in the number of nephrons in the remaining kidney should be roughly proportional to the initial reduction in renal mass, this provides an estimation of the changes in final protein expression per nephron of NaCl transporters in remnant kidney. Since there is no way to determine exactly the weight of the remaining tissue after polectomy, we used an additional protocol to estimate this. The fraction of the total kidney mass remaining immediately after 5/6 nephrectomy in protocol 1 (rats with CRF) was estimated from protocol 5 (normal rats with left 2/3 nephrectomy). This appeared to be a reasonable approach, since the weight of the surgically removed renal tissue from the left kidney in protocol 5 was nearly identical to that removed in rats from protocol 1 (0.176 ± 0.008 vs. 0.172 ± 0.008 g/100 g body wt in protocols 5 and 1, respectively).2
Statistical analyses. Values were presented in the text as means ± SE. Comparisons between groups were made by unpaired t-test. P < 0.05 was considered significant.
Preparation of tissue for immunocytochemistry. The kidneys from four CRF rats and four sham-operated rats were fixed by retrograde perfusion via the aorta with periodate-lysine-paraformaldehyde (PLP: 0.01 M NaIO4, 0.075 M lysine, and 2% paraformaldehyde, in 0.0375 M Na2HPO4 buffer, pH 6.2). In a separate experiment, 0.1% glutaraldehyde was added. Tissue blocks prepared from the cortex, outer stripe of outer medulla, inner stripe of outer medulla, and inner medulla were infiltrated for 30 min with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid nitrogen (40). For light microscopy, the frozen tissue blocks were cryosectioned (0.8-1 µm, Reichert Ultracut S Cryoultramicrotome), sections were incubated with primary antibodies, and the labeling was visualized with HRP-conjugated secondary antibodies (P447 or P448, 1:100; DAKO), followed by incubation with diaminobenzidine. For fluorescence microscopy, the label was visualized using goat-anti-rabbit IgG (Z0421, 1:50; DAKO) and FITC-conjugated rabbit-anti-goat antibody (F250, 1:50, DAKO).
Freeze-substitution of kidney tissue.
The frozen samples were freeze-substituted (model AFS; Reichert,
Vienna, Austria) (30, 39). Briefly, the samples were
sequentially equilibrated over 3 days in methanol containing 0.5%
uranyl acetate at temperatures gradually increasing from 80 to
70°C, and then rinsed in pure methanol for 24 h while
increasing the temperature from
70 to
45°C. At
45°C, the samples were infiltrated with Lowicryl HM20 and
methanol 1:1, 2:1, and, finally, pure Lowicryl HM20 before ultraviolet
polymerization for 2 days at
45°C and 2 days at 0°C. For
electron microscopy, immunolabeling was performed on ultrathin Lowicryl
HM20 sections (60-80 nm), which were incubated overnight at
4°C with primary antibodies diluted in PBS with 0.1% BSA or 0.1%
skimmed milk. The labeling was visualized with goat-anti-rabbit IgG
conjugated to 10-nm colloidal gold particles (GAR.EM10; BioCell Research Laboratories, Cardiff, UK) diluted 1:50 in PBS with 0.1% BSA.
The sections were stained with uranyl acetate and lead citrate before
examination in Philips CM100 or Philips 208 electron microscopes.
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RESULTS |
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Five-sixths nephrectomy is associated with significant
renal insufficiency and renal hypertrophy. As shown in
Table 1, the levels of plasma urea nitrogen
in CRF rats (24 ± 4 mmol/l, n = 17) were significantly higher (P < 0.05) than in sham-operated rats (6 ± 0.5 mmol/l,
n = 11). Furthermore, the levels of
plasma creatinine in CRF rats (136 ± 15 µmol/l) were
significantly higher (P < 0.05) than
in sham-operated rats (34 ± 1 µmol/l). Consistent with
this, the creatinine clearance was significantly reduced in rats with
CRF: 0.21 ± 0.03 in CRF vs. 0.81 ± 0.05 ml · min1 · 100 g body wt
1 in sham-operated
rats (P < 0.05), consistent with
chronic renal insufficiency.
|
Another known feature following 5/6 nephrectomy is renal hypertrophy of the remnant kidney (19). The weight of kidneys where both poles have been removed (i.e., corresponding to the initial 2/3 left nephrectomy) is only 0.18 ± 0.01 g/100 g body wt (protocol 5). Two weeks after induction of 5/6 nephrectomy, remnant kidneys in CRF rats were significantly hypertrophied and weighed 0.41 ± 0.02 g/100 g body wt compared with 0.31 ± 0.02 g/100 g body wt of left kidney of sham-operated rats (P < 0.05). Despite the hypertrophy, the total renal mass of the remnant kidney was only 65 ± 3% of total renal mass in sham-operated rats (P < 0.05).
Light microscopical examination revealed extensive hypertrophy of all the nephron and collecting duct segments with the marked dilatation. Very mild interstitial fibrosis with few fibroblasts was only observed in the inner medulla but not in cortex and outer medulla of remnant kidneys at the light and electron microscopic level (not shown).
Quantity of sodium excreted in urine increases
progressively as the creatinine clearance is reduced in rats with
CRF. As shown in Fig. 2,
the urinary sodium excretion rate increased markedly after induction of
CRF established by 5/6 nephrectomy compared with sham-operated rats
[0.50 ± 0.03 in rats with CRF
(n = 17) vs. 0.39 ± 0.02 µmol · min1 · 100 g body wt
1 in sham-operated
controls (n = 11),
P < 0.05]. Fractional
excretion of sodium (FENa) after
2 wk of induction of 5/6 nephrectomy was also significantly increased
(2.7 ± 0.6% in CRF rats vs. 0.4 ± 0.1% in sham controls,
P < 0.05; Table 1). Furthermore, the
fractional excretion of potassium
(FEK) was much higher in rats
with CRF (132 ± 9% in CRF vs. 39 ± 2% in sham controls,
P < 0.05; Table 1). As shown in Fig.
3, A and
B, the fractional excretion of sodium
and potassium significantly increased progressively as the creatinine
clearance was reduced in experimental rats, suggesting the quantity of
sodium and potassium excreted per nephron progressively increases as
the renal functions were reduced during development of CRF.
|
|
Urine production is increased and urinary
concentrating ability is decreased in rats with CRF.
During the period of acclimation, daily urine production averaged 40 ± 2 µl · min1 · kg
1
in rats with CRF (n = 17). As reported
earlier (30), this rose to 111 ± 3 µl · min
1 · kg
1
after induction of 5/6 nephrectomy (P < 0.05). There was a parallel increase in water intake, from 94 ± 4 to 155 ± 5 µl · min
1 · kg
1
per day (P < 0.05). In contrast,
urine production and urine osmolalities were unchanged in sham-operated
rats (n = 11). Thus the urine output
of a single remnant kidney was approximately four times that of one
kidney of sham control rats. This marked increase in urine output was
associated with significant impairment of urinary concentrating ability
in CRF rats: urine osmolality was 494 ± 16 in CRF vs. 1,326 ± 56 mosmol/kgH2O in sham-operated
rats (P < 0.05). Consistent with
changes in urine output and urine osmolalities, the urine-to-plasma
osmolality ratio
[(U/P)osm] was 1.6 ± 0.1 in CRF vs. 4.6 ± 0.4 in sham-operated controls, and the
solute-free water reabsorption
(TcH2O)
was 65 ± 12 in CRF vs. 141 ± 13 µl · min
1 · kg
1
in sham-operated rats, respectively (P < 0.05, Table 1).
Total kidney levels of NHE-3 are decreased in rats
with CRF. In renal proximal tubules, apical Na/H
exchange mediates most of the transcellular NaCl transport and
two-thirds of the transcellular NaHCO3 (43), and NHE-3 plays a
major role in this (3). The affinity-purified anti-NHE-3 antibody
recognized an ~87-kDa band in membrane preparations from the whole
kidney, consistent with studies from other investigators (2, 6, 12). As
shown in Fig. 4, densitometric analysis of
all samples from CRF and sham-operated rats revealed a marked decrease
in total kidney NHE-3 levels to 48 ± 14% in rats with CRF
(n = 13) of levels in sham-operated
rats (100 ± 19%, n = 11, P < 0.05; Table
2).3
The density of NHE-3 per nephron in remnant kidney was estimated to 199 ± 58% of levels in sham-operated rats (100 ± 19%,
P > 0.05, Table 3).
|
|
|
CMA often accompanies CRF, and rats with 5/6 nephrectomy are often acidotic (33). To examine whether acidosis per se would affect NHE-3 levels, we have investigated this directly using an additional protocol where rats were treated with NH4Cl for 2 wk to induce CMA (1, 34). Chronic acid loading produced reduced plasma HCO3 levels in rats with CMA, consistent with CMA (18.7 ± 1 in CMA vs. 30.5 ± 0.4 mmol/l in controls, P < 0.05). Compared with controls, densitometric analysis of all samples from CMA and control rats revealed a significant increase in NHE-3 levels in rats with CMA to 143 ± 12% (n = 8) of levels in control rats (100 ± 12%, n = 6, P < 0.05, not shown), consistent with previous observations (1). Thus acidosis per se appears not to be the cause of the reduced expression of NHE-3 in CRF.
Total kidney levels of NaPi-II are decreased in rats
with CRF. In renal proximal tubules, transport of
Pi through the proximal apical
membrane is largely performed by the NaPi-II cotransporter. To confirm
whether apical proximal tubule NaCl transporter expression other than
NHE-3 can also be altered in the remnant kidneys, we examined the
changes in density and total kidney levels of NaPi-II. The
affinity-purified anti-NaPi-II antibody recognized an ~85-kDa main
band in membrane preparations from the whole kidney, consistent with
previous studies (5). As shown in Fig. 5, densitometric analysis of 85-kDa bands in all samples from CRF and sham-operated rats
revealed a marked decrease in total kidney NaPi-II levels in rats with
CRF to 13 ± 4% (n = 13) of levels
in sham-operated rats (100 ± 6%,
n = 11, P < 0.05; Table 2). The density of
NaPi-II per nephron in remnant kidney was estimated to 56 ± 20% of
levels in sham-operated rats (100 ± 10%,
P > 0.05; Table 3).
|
Total kidney levels of Na-K-ATPase are decreased in
rats with CRF. The renal tubule is rich in Na-K-ATPase,
an important transport protein capable of coupling hydrolysis of ATP to
the active translocation of sodium and potassium across the cell
membrane and secondary active transport of other solutes. Figure
6 shows an immunoblot of Na-K-ATPase, using membrane
preparations from the whole kidney of rats with CRF and sham-operated
rats. The 1-isoform-specific monoclonal antibody recognized a band
migrating at ~96 kDa (25). Densitometric analysis of all samples from
CRF and sham-operated rats revealed a marked decrease in total kidney
Na-K-ATPase levels in CRF rats to 30 ± 9%
(n = 13) of levels in sham-operated
rats (100 ± 8%, n = 11, P < 0.05; Table 2). Density per
nephron in remnant kidney was estimated to 124 ± 37% in rats with
CRF of levels in sham-operated rats (100 ± 8%,
P > 0.05; Table 3).
|
Total kidney levels of two major absorptive Na-K-Cl
cotransporters (BSC-1 in the TAL and TSC in the DCT) are not decreased in rats with CRF. Figure 7,
A and
C, shows immunoblots of the BSC-1 and
TSC, using membrane preparations from whole kidney of rats with CRF and
sham-operated rats. Affinity-purified anti-BSC-1 antibody recognized a
broad band of molecular mass 146-176 kDa centered at ~161 kDa,
consistent with previous observations (10, 26, 39). Affinity-purified
anti-TSC antibody recognized a broad band centered at ~165 kDa (27).
In contrast to the significant reductions in total kidney levels of
NHE-3, NaPi-II, and Na-K-ATPase in rats with CRF, densitometric
analysis revealed unchanged levels of total kidney BSC-1 in rats with
CRF corresponding to 88 ± 15% of levels in sham-operated rats (100 ± 21%; Fig. 7B and Table 2) and
TSC corresponding to 61 ± 15% of levels in sham-operated rats (100 ± 18%; Fig. 7D and Table 2)
Furthermore, significant increases in densities per nephron of BSC-1
(365 ± 62% of sham levels, P < 0.05) and TSC (253 ± 62%, P < 0.05) were estimated (Table 3), consistent with the observed absence of
a reduction in total kidney levels despite reduction in renal mass.
|
Total kidney levels of Tamm-Horsfall protein is
decreased in rats with CRF. We examined the abundance
of Tamm-Horsfall protein, a glycosyl-phosphatidylinositol-linked
protein expressed predominantly in the apical plasma membrane of TAL
cells. This was performed as a control to assess whether changes in the
BSC-1 expression are accompanied by changes in other TAL proteins. In
contrast to the significant increase in density of BSC-1 in the TAL
cells of rats with CRF as well as unchanged total kidney levels, there was a marked decrease in total kidney Tamm-Horsfall protein levels in
rats with CRF: 34 ± 8% in rats with CRF
(n = 13, P < 0.05; Fig. 8)
compared with 100 ± 7% in sham-operated controls
(n = 11; Table 2). Density per nephron
in remnant kidney was estimated to 141 ± 33% in rats with CRF of
levels in sham-operated rats (100 ± 7%; Table
3).
|
Total kidney levels of BSC-1 and Tamm-Horsfall
protein: modification of CRF response by long-term administration of
DDAVP. We examined whether changes in densities or
total kidney levels of NaCl transporters and Tamm-Horsfall protein seen
in CRF rats are modified in response to long-term exogenous DDAVP
administration (Table 4). A densitometric
analysis revealed that the total kidney BSC-1 levels in DDAVP-treated
rats with CRF (n = 6, 0.1 µg/h sc for 7 days) increased to 202 ± 38% of levels in sham-operated rats
(Fig. 9), a substantially higher level of
BSC-1 expression than seen in response to CRF without DDAVP infusion.
The upregulation appeared to be rather specific, since total kidney
Tamm-Horsfall protein levels remained significantly decreased despite
DDAVP treatment, corresponding to 21 ± 4% of sham levels (100 ± 11%, P < 0.05; Table 4),
comparable to the levels seen without DDAVP infusion.
|
|
Immunocytochemical analysis of NaCl transporter
expression. In sham-operated rats, anti-NHE-3 antibody
labeled the apical plasma membrane domains of S1 and S2
segments of proximal convoluted tubule with a weaker staining of the
late S2 and S3 segments of proximal tubule (Fig.
10A,
arrows), as previously described (2). The labeling was exclusively
confined to the apical domains and intermicrovillar cleft of the
proximal tubule cells (arrows), whereas the apical brush border
(arrowhead) and basolateral plasma membranes were consistently
unlabeled (Fig. 10A). Furthermore, an intense labeling was also seen of the apical plasma membrane domains
of medullary TALs (MTALs) in sham-operated rats (not shown). In
contrast, immunocytochemistry showed that the labeling of NHE-3 in the
hypertrophied proximal tubules in the remnant kidneys (Fig. 10B, arrows) as well as of MTAL (not
shown) were much weaker. The NHE-3 labeling patterns were confirmed by
immunoelectron microscopy (not shown).
|
The proximal tubules exhibited strong Na-K-ATPase labeling of
basolateral plasma membranes (Fig. 10,
C and
D). No or little labeling was
detected in the thin descending limb of the loop of Henle (not shown),
whereas significant basolateral labeling was observed of collecting
duct principal cells, and very intense basolateral labeling was present
in the TAL cells in the inner stripe of the outer medulla. This strong
labeling also extended into the cortical portions of the TAL and of DCT
(not shown). This is consistent with previous evidence (25). In rats
with CRF, the labeling of the proximal tubule was much weaker (Fig. 10D), consistent with a significant
decrease in total kidney levels determined by immunoblotting. In
contrast, no major differences in the labeling density were seen in the
MTAL cells from rats with CRF compared with sham-operated rats (Fig.
11,
E and
F). This together with the observed
increase in TSC in the DCT (Fig.
11B) and of BSC-1 in the TAL (Fig.
11D) suggests that the NaCl
transporters in these segments are not decreased, despite a significant
reduction of renal mass in rats with CRF.
|
The TSC is expressed in the DCT and is responsible for a large fraction of the net sodium and chloride reabsorption in the distal portion of the mammalian renal tubule (9, 11, 27, 41, 50). In sham-operated rats, immunocytochemistry confirmed that TSC immunolabeling was seen in DCT in the apical and subapical regions (Fig. 11A). Importantly, a strong labeling of TSC was noted in DCT cells in rats with CRF (Fig. 11B), which was confirmed by electron microscopy (not shown). This is consistent with the increased TSC density determined by immunoblotting (Fig. 7C).
In sham-operated rats, an intense BSC-1 labeling was seen in apical and subapical domains of MTAL (Fig. 11C) and cortical TAL cells (not shown) as previously shown (10, 24, 26, 39, 56). Rats with CRF also exhibited strong labeling of TAL cells (Fig. 11D), consistent with the increased BSC-1 density observed by immunoblotting (Fig. 7A).
The microscopy also confirmed that the cells of proximal tubules (Fig. 10), descending thin limbs, MTAL, DCT (Fig. 11), and collecting ducts all underwent a marked hypertrophy with increased tubular diameters in rats with CRF.
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DISCUSSION |
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Consistent with previous evidence, rats with CRF induced by 5/6 nephrectomy had 1) extensive hypertrophy and dilatation of remaining nephrons, 2) significant azotemia, 3) significant increases in urinary sodium excretion, and 4) marked increase in urine output with an impairment in urinary concentrating ability. In association with this, we report that there is a significant decrease in total kidney levels of the major proximal tubule sodium transporter NHE-3, NaPi-II, and Na-K-ATPase in rats with CRF. Immunocytochemistry confirmed that NHE-3 and Na-K-ATPase levels in the proximal tubule are reduced. Consistent with this, densities per nephron of NHE-3, NaPi-II, and Na-K-ATPase did not increase proportionately to the extensive nephron hypertrophy, associated with the hyperfiltration in remnant nephrons. This is likely to play a significant role for the increased sodium excretion in CRF. In contrast, we report that total kidney levels of the TAL and DCT sodium transporters BSC-1 and TSC are not decreased in rats with CRF. Consistent with this, the densities per nephron of both BSC-1 and TSC are significantly increased, as also demonstrated by immunocytochemistry. Also, there was no decrease in Na-K-ATPase labeling in TAL and DCT. Thus expression of sodium transporters in the loop of Henle and DCT is not decreased in remnant kidneys, indicating that the altered tubular handling of sodium in CRF may be caused primarily by changes in proximal tubule NaCl transporter expression levels and that there appears to be a compensatory increase in NaCl transporter expression in the distal nephron.
The proximal tubule is the site of reabsorption of approximately two-thirds of the NaCl that enters the tubular fluid by glomerular filtration (3) and therefore is the main energy-expending compartment of kidney. Thus it is evident that structural and functional adaptation of the proximal tubule in response to increased SNGFR and tubular fluid flow rate, which are known to occur in remnant kidney, may play a critical role in the changes of reabsorption and excretion of sodium and water. It has been demonstrated that the absolute sodium reabsorption in the proximal tubule is increased in parallel with the rise in SNGFR in the remnant kidney (8, 14, 49), whereas the fraction of the filtered sodium and water reabsorbed in the proximal tubule was significantly reduced (8, 32, 45, 55).
In the "remnant kidney model," SNGFR increases uniformly to values that are two- to threefold greater than normal (8, 52). However, if the fraction of the filtered sodium load reclaimed by the proximal tubule is significantly decreased as suggested by many investigators (8, 32, 45, 55), then it appears that changes in sodium excretion are modulated by proximal nephron segments. Indeed, we have demonstrated a significant decrease in total kidney NHE-3, NaPi-II, and Na-K-ATPase levels associated with reduced labeling density of NHE-3 and Na-K-ATPase in the proximal tubule in rats with CRF. Moreover, estimated densities of NHE-3 and Na-K-ATPase per nephron in remnant kidneys revealed a modest increase, but this was not significant. The hypertrophy, manifested by an increase in tubular diameter and length of the proximal tubule in remnant kidneys, appears to offer an explanation for this modest increase in densities per nephron of NHE-3 and Na-K-ATPase. This may provide an explanation for the previously observed increase in absolute sodium reabsorption in the proximal tubule from CRF animals (14, 49). However, at the same time, the inability to increase the densities per nephron of these NaCl transporters proportionately to the increased SNGFR and tubular fluid flow rate may provide an explanation for 1) the previously observed reduced fractional reabsorption of sodium in proximal tubule, 2) the glomerulotubular imbalance, and 3) the increased urinary excretion of sodium in CRF.
The NaPi-II cotransporters are also expressed in the proximal tubule and contribute to the sodium and phosphate reabsorption in this segment. Especially, NaPi-II is known to be the rate-limiting and physiologically regulated step in proximal tubular phosphate reabsorption (5). CRF is well known to be associated with elevated serum parathyroid hormone (PTH) levels and changes in mineral ion homeostasis including secondary hyperparathyroidism (37). Since PTH is a potent inhibitor of proximal tubular NaPi-II by inducing the retrieval from the brush-border membrane and the lysosomal degradation (38), we speculate that the decreased density and total kidney levels of NaPi-II in rats with CRF in the present study may be at least partly attributed to the increased levels of serum PTH.
The delivery of sodium and water to the distal end of the proximal tubule of superficial nephrons and to the bend of Henle's loop of juxtamedullary nephrons is known to be increased in remnant kidneys due to an increase in the filtered load and a decrease in the fractional reabsorption of sodium and water in the proximal tubule (8). This condition may be similar to acute and chronic loading with isotonic saline in normal rats. A large amount of evidence indicates that isotonic saline loading is associated with an elevation of SNGFR rate as well as a decrease in fractional proximal NaCl reabsorption (54). Furthermore, Landwehr et al. (31) demonstrated that the increase in delivery to the loop of Henle is associated with a marked increase in NaCl absorption by the loop of Henle, presumably due in part to an increase in TAL NaCl absorption (31). Consistent with this, Ecelbarger et al. (10) demonstrated that chronic oral saline loading in rats markedly increased BSC-1 abundance in the TAL of rats. Our data showing increased densities of BSC-1 in CRF rats is also consistent with this view. Moreover, it was observed that long-term oral NaCl loading results in an increase in the Na-K-ATPase activity of the MTAL of rats (51). This is also consistent with our observation that the Na-K-ATPase labeling density of the TAL cells of remnant kidney was similar to the sham-operated controls, which contrasts the significant reduction in the labeling density of the proximal tubule. This also supports the view that a marked increase in the delivery of NaCl and water to the loop of Henle may partly upregulate several NaCl transporters across the TAL and DCT. Thus we speculate that the increase in density of BSC-1 seen following 2 wk after induction of 5/6 nephrectomy could at least partly be a consequence of a prolonged increase in the NaCl load delivered to the TAL partly due to both hyperfiltration and altered expression in the proximal sodium transporters in remnant kidneys. Other factors may also play an important role, including potential increases in plasma vasopressin levels, which have previously been found to be associated with CRF induced by surgical reduction in kidney mass (7). Consistent with this, we demonstrated that DDAVP treatment for 7 days of rats with CRF resulted in a greater increase in BSC-1 levels than seen in response to CRF alone, indicating that DDAVP can induce an increase in BSC-1 expression in CRF rats as demonstrated previously in normal rats (26). The TSC is expressed in the DCT of the kidney (41) and is responsible for a large fraction of the net sodium and chloride reabsorption that occurs in the distal portion of the mammalian renal tubule (9, 11, 42, 50). Micropuncture studies of superficial nephron segments of the rat have demonstrated that the distal tubule of the rat is responsible for reabsorption of 6-8% of the filtered load of sodium, whereas the collecting duct system normally reabsorbs 1% or less of the filtered load (16). Recently, it was demonstrated that the DCT is an important site of action of mineralocorticoids, and that aldosterone upregulates the expression of TSC significantly (27, 50). Since plasma aldosterone levels are known to be elevated significantly (17), this is likely to play a role in increasing TSC expression in 5/6 nephrectomized rats.
In summary, the reduction in total kidney levels of NHE-3, NaPi-II, and Na-K-ATPase with altered expression per nephron of these transporters in response to the increased SNGFR and tubular fluid flow rate may provide cellular and molecular mechanisms for the increase in urinary sodium excretion in remnant nephrons. In contrast, there are compensatory increases in BSC-1 and TSC densities and maintained Na-K-ATPase expression in TAL and DCT. This indicates a compensatory increase, which may be partly due to increased blood vasopressin and aldosterone levels and increased delivery of NaCl to the distal nephron in CRF. Additional studies will be required to determine whether NaCl transporters elsewhere along the renal tubule of remaining nephrons in CRF are regulated or altered.
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ACKNOWLEDGEMENTS |
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We thank Annette Blak Rasmussen, Zhila Nikrozi, Mette Vistisen, Inger-Merete Paulsen, and Gitte Christensen for expert technical assistance.
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FOOTNOTES |
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Support for this study was provided by the Karen Elise Jensen Foundation, Novo Nordic Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, the University of Aarhus, and the intramural budget of the National Heart, Lung, and Blood Institute.
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. §1734 solely to indicate this fact.
1 Sample calculation: 1) Loading volume of gel sample which gave identical Coomassie staining compared with other chronic renal failure (CRF) and sham samples: 2.0 µl. 2) Total volume of entire gel sample: 333 µl. 3) The volume fraction that the entire gel sample constitute out of total kidney homogenate used for preparation of gel sample: 18.75%. 4) Total kidney weight per 100 g body wt: 0.48 g. 5) Number of kidneys: 1 (CRF). This loading corresponds to (2.0/333) × 18.75% × 1 = 0.113% of total kidney mass.
2 Sample calculation of density per nephron in Na-K-ATPase. 1) Total kidney levels of Na-K-ATPase in rats with CRF: 30 ± 9% of sham levels. 2) Estimated fraction of total kidney mass which was remained immediately after 5/6 nephrectomy: 24.1%. 3) Density per nephron in remnant kidney 2 wk after induction of CRF was calculated by dividing the value obtained in 1) by that obtained in 2). This correspond to 124 ± 37% compared with sham-operated control rats.
3 These values were obtained after correction for the fraction of total kidney mass loaded and after densitometry of Coomassie-stained gels to correct for minor changes in loading.
Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
Received 9 November 1998; accepted in final form 12 April 1999.
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