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Late-Onset Downregulation of Napi-2 in Experimental Fanconi Syndrome
Late-Onset Downregulation of Napi-2 in Experimental Fanconi Syndrome
IPNA 2001
E X P E R I M E N TA L S T U D I E S / O R I G I N A L A R T I C L E
Introduction
Inorganic phosphate (Pi) is the most-abundant intracellular anion and is essential for normal cellular metabolism
and energy production. Regulation of serum Pi within a
narrow range is dependent on effective transcellular
transport systems. The renal tubules are a major determinant of Pi homeostasis, and are subject to hormonal and
dietary modulation [1]. The proximal tubule is the major
site of Pi reabsorption, where unidirectional Pi transport
involves uptake from the lumen across brush border
membrane (BBM) and efflux at the basolateral membrane [2]. BBM Pi uptake is the rate-limiting step in
renal Pi reabsorption, thereby being a primary site for
regulation. This regulation is mediated by apical Na-Pi
cotransporters and basolateral Na-K-ATPase that maintain a gradient for intracellular Na influx. Pi efflux
across the basolateral membrane is probably passive and
may involve anion exchange [3].
There are three types of Na-Pi cotransporters. Type I
and type II are expressed mainly in the kidney, while
type III is ubiquitous and may serve for housekeeping
Na-Pi cotransport. Type II is the most abundant and may
account for 85% of Pi reabsorption. Type II Na-Pi
cotransporter mRNA is expressed exclusively in the
proximal convoluted tubule, and its immunoreactive
protein is localized to the BBM [4, 5]. The dominant role
of Na-Pi 2 is demonstrated in murine gene-knockout
models of NaPi-2, where massive phosphaturia occurs
along with reduced BBM Na-Pi cotransport [6].
Na-Pi II is the major target for parathyroid hormone
(PTH) regulation. PTH induces endocytosis and retrieval
of NaPi-2 from the BBM and lysosomal degradation of
the protein. Therefore, recovery of NaPi-2 after the effect
of PTH requires de novo synthesis of the protein [4, 5].
Acute alterations in dietary Pi intake also inversely
affect NaPi-2 post-transcriptionally by recruiting pre-existing NaPi-2 protein to the apical membrane, or its retrieval and degradation, respectively [4, 5].
Maleic acid (MA) produces an experimental Fanconi
syndrome (FS) characterized by rapid bicarbonaturia,
413
Results
Serum and urine biochemical data are shown in Tables 1
and 2. These studies demonstrate the characteristics of FS,
namely phosphaturia, metabolic acidosis, and kaliuresis.
Random urine spot tests with Albustix were qualitatively
positive for glycosuria in rats given MA, and were negative in the control group. The administration of MA was
associated with an immediate onset of polyuria. While
mean urine output was 56 ml/24 h for control rats, animals administered MA produced 2530 ml of urine/day.
By 4 h following MA, the urine output was already of the
same magnitude as that of control rats over a 24-h period.
Phosphaturia was documented as early as 90 min following MA injection. Despite subsequent hypophosphatemia, phosphaturia was evident for at least 24 h after the
initial insult. As shown in Table 1, hypophosphatemia is
most pronounced after 90 min and 24 h, while after 4 h a
trend towards a better Pi conservation is observed. Hence,
we observed two phases of Pi excretion. The first phase
demonstrated an acute onset of phosphaturia occurring
414
Table 1 Serum biochemical
tests after maleic acid (MA)
injection
Control (n=4)
90 min (n=5)
4 h (n=6)
24 h (n=5)
Control (n=4)
90 min (n=5)
4 h (n=6)
24 h (n=5)
Sodium
(mEq/l)
Potassium
(mEq/l)
Phosphate
(mEq/l)
Calcium
(mEq/l)
Bicarbonate
(mEq/l)
145.52
140.44
1423
141.52
3.70.4
2.80.2
3.350.4
2.50.2*
2.90.2
1.710.1**
2.190.2*
1.580.1**
5.550.3
5.450.3
5.420.2
5.230.2
240.8
19.50.5**
18.20.8**
15.40.7**
Pi clearance
(ml/min)
Creatinine clearance
(ml/min)
Fractional excretion
of Pi
0.0450.002
0.9250.04*
0.460.02*
0.540.02*
1.450.03
1.260.05
1.460.04
1.20.07
3%0.06
73.4%2*
31%1*
45%1*
Fig. 2a, b NaPi-2 brush border membrane (BBM) protein in experimental Fanconi syndrome. a A representative Western blot. b A
bar graph of mean results of at least four rats (n is given in Table 3)
Control
90 min after MA
4 h after MA
24 h after MA
NaPi-2 mRNA
NaPi-2 protein
35.26.9 (n=4)
34.05.8 (n=5)
16.62.9 (n=5)*
9.82.9 (n=5)**
1.350.17 (n=9)
1.630.2 (n=4)
1.360.49 (n=4)
0.270.04 (n=5)***
415
Discussion
In kidneys of MA-treated rats, evidence of injury is
observed immediately after MA administration, which
progresses to extensive necrosis of the proximal tubules
by 24 h, while the loops of Henle and distal convoluted
tubules remain intact [9]. A disorder in Na gradientdependent solute uptake of renal BBM may be one of the
underlying mechanisms of experimental FS [10]. However, other studies suggested that FS induced by maleate
is not caused by an inhibition of BBM Na+-dependent
transport systems, but that protein phosphorylation may
play an important role in the molecular defect involved
in FS [11].
In the model used in this study, a rapid generalized
proximal injury induced by MA was evident by the rapid
onset of kaliuresis, metabolic acidosis, phosphaturia, and
glycosuria. MA administration to experimental animals
induces a rapid, reversible, complex dysfunction of
the renal tubule resembling FS. A defect in the proximal
tubule Na+-K+-ATPase appears to play a role in MAinduced FS [12]. Na-K-ATPase is the biochemical correlate of active cellular transmembrane sodium transport.
The transient early phosphaturia, independent of NaPi-2
protein abundance, may reflect the partial recovery of
Na-K pump activity and amino acid oxidation, well
known to occur after MA administration [12].
Another feature of the early, diffuse, and reversible
generalized proximal tubular injury may be the intracellular Pi depletion. This Pi depletion may affect membrane-bound, insoluble Pi in the proximal tubule, which
may interfere with proximal tubular transport [6]. While
Na+/H+ antiporter may induce only bicarbonaturia, extensive energy depletion may cause a generalized leak.
Pi infusion attenuates MA-induced bicarbonaturia [8,
13], while metabolic acidosis induced by MA worsens
renal tubular leak as intravenous bicarbonate infusion,
which corrects the metabolic acidosis, reduces the fractional excretion of Pi [13].
The time-scale of these effects of MA are of unique
significance. Our results suggest two phases of phosphaturia: a rapid phosphaturia, independent of NaPi-2, and a
late-onset inhibition of NaPi-2-dependent proximal tubular Pi reabsorption. Experimental FS may exhibit a complete inhibition of Na-dependent phosphate uptake by renal BBM vesicles, decreased ATP production by the renal tubule, and a reversible effect on renal mitochondria
[14]. The abnormal appearance of the mitochondria, with
compressed crystal membranes and flocculent densities,
is one of the most-impressive findings in MA-associated
FS [9]. Therefore, the early phase of phosphaturia may
416
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