Neurotrophin-3 inhibits HCO
absorption via
a cAMP-dependent pathway in renal thick ascending limb
David W.
Good and
Thampi
George
Departments of Medicine and Physiology and Biophysics, University
of Texas Medical Branch, Galveston, Texas 77555
 |
ABSTRACT |
Neurotrophins are expressed in the adult kidney, but their
significance is unclear. We showed previously that nerve growth factor
(NGF) inhibits HCO
absorption in the rat medullary
thick ascending limb (MTAL) via an extracellular signal-regulated
kinase (ERK)-dependent pathway. Here we examined whether other
neurotrophic factors affect MTAL HCO
absorption.
Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor had no effect. In contrast, neurotrophin-3 (NT-3,
0.7 nM) inhibited HCO
absorption by 40%
(half-maximal inhibition at ~0.4 nM). Inhibition by NT-3 was additive
to inhibition by NGF. Inhibitors of ERK activation that block
inhibition by NGF had no effect on inhibition by NT-3. In contrast,
8-bromo-cAMP or forskolin pretreatment blocked inhibition by NT-3 but
not NGF. Inhibition by NT-3 was also blocked by the specific protein
kinase A (PKA) inhibitor myristoylated PKI(14-22) amide and by vasopressin, which inhibits HCO
absorption via cAMP. Inhibitors of phosphatidylinositol 3-kinase or
protein kinase C did not affect NT-3-induced inhibition, but inhibition
by NT-3 was eliminated by genistein, consistent with involvement of a
receptor tyrosine kinase. These results demonstrate that NT-3 inhibits
HCO
absorption via a cAMP- and PKA-dependent
pathway. NT-3 and NGF regulate MTAL ion transport through different
signal transduction mechanisms. These studies establish a direct role
for NT-3 in regulation of renal tubule transport and identify the MTAL
as an important target for neurotrophins, which may be involved in the
control of renal acid excretion.
neurotrophic factors; kidney; Trk receptors; nerve growth factor; vasopressin
 |
INTRODUCTION |
NEUROTROPHIC FACTORS ARE
ESSENTIAL for the development and maintenance of the nervous
system (34, 50). The neurotrophin family of neurotrophic
factors includes nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5. These
factors play a major role in regulating the growth, survival, and
differentiation of neuronal cells (8, 34, 50).
Neurotrophins mediate these cellular responses through binding to two
distinct types of receptors: the Trk family of receptor tyrosine
kinases, which exhibit high selectivity for neurotrophin binding, and
the p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis
factor receptor family that binds all neurotrophins with similar
affinity (8, 14, 29, 50). In addition to their prominent
role in regulating neuronal processes, neurotrophins are expressed in a
variety of nonneural tissues including pancreas, liver, heart, spleen,
and epithelial organs such as salivary gland, kidney, and
gastrointestinal tract (4, 5, 8, 12, 26, 35, 36, 44, 45, 47, 49,
59). Neurotrophins are located in parenchymal cells and
epithelial cells in these organ systems, suggesting a nonneurotrophic
role. At present, however, the function of neurotrophins in peripheral tissues is poorly understood.
Neurotrophins and their receptors are expressed in the adult kidney
(5, 12, 26, 35, 36, 44, 45, 49, 59), but their
significance is unclear. The medullary thick ascending limb (MTAL) of
the mammalian kidney performs a number of important transport
functions, including reabsorption of NaCl that is essential for the
maintenance of sodium balance and the excretion of a dilute or
concentrated urine (37). The MTAL also participates in the regulation of acid-base balance by reabsorbing a sizable fraction of
the HCO
filtered at the glomerulus (16). Recently, we demonstrated that NGF regulates
transepithelial HCO
absorption in the MTAL of the rat through complex mechanisms. Specifically, NGF inhibits
HCO
absorption under basal conditions but stimulates
HCO
absorption in the presence of arginine
vasopressin (AVP) (19). These transport effects are
mediated through different signal transduction pathways: the
NGF-induced inhibition of HCO
absorption is mediated
through activation of extracellular signal-regulated kinase (ERK)
(53), whereas the AVP-dependent stimulation of HCO
absorption is mediated through activation of
protein kinase C (PKC) (19). In addition, NGF inhibits
HCO
absorption through a unique mechanism involving
inhibition of basolateral membrane Na+/H+
exchange activity, an effect opposite to the virtually universal stimulation of Na+/H+ exchange by growth
factors in other cells (54). These studies provided the
first evidence that neurotrophins directly regulate the function of
renal tubules and identified a possible role for NGF in the control of
urinary acidification (19).
The purpose of the present study was to determine whether neurotrophic
factors other than NGF influence HCO
absorption in
the MTAL and to identify signaling pathways involved in
neurotrophin-induced transport regulation. We examined the effects of
BDNF and NT-3, members of the neurotrophin gene family closely related
to NGF (8, 34), and glial cell line-derived neurotrophic
factor (GDNF), a member of the transforming growth factor-
superfamily that is essential for kidney development (1, 39, 40,
46). We demonstrate that NT-3 inhibits HCO
absorption via a cAMP- and protein kinase A (PKA)-dependent pathway that is distinct from the signaling pathways involved in regulation of
HCO
absorption by NGF. In contrast, BDNF and GDNF
have no effect on HCO
absorption.
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METHODS |
MTALs from male Sprague-Dawley rats (50-100 g body wt;
Taconic, Germantown, NY) were isolated and perfused in vitro as
described previously (15, 17, 19). In brief, the tubules
were dissected from the inner stripe of the outer medulla at 10°C in
control bath solution (see below), transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipettes for perfusion at 37°C. In all experiments, the lumen and
bath solutions contained (in mM) 146 Na+, 4 K+,
122 Cl
, 25 HCO
, 2.0 Ca2+,
1.5 Mg2+, 2.0 phosphate, 1.2 SO
, 1.0 citrate, 2.0 lactate, and 5.5 glucose (osmolality = 290 mosmol/kgH2O). Bath solutions also contained 0.2% fatty
acid-free bovine serum albumin. In one series of experiments,
hypertonic solutions were prepared by the addition of 75 mM NaCl to the
above control solution (final osmolality = 425 mosmol/kgH2O). All solutions were equilibrated with 95%
O2-5% CO2 and were pH 7.45 at 37°C. Bath
solutions were delivered to the perfusion chamber via a continuously
flowing exchange system (15). Experimental agents were
added to the bath solution as described in RESULTS. NT-3
(human recombinant) and GDNF (rat recombinant) were purchased from
Sigma; BDNF (human recombinant) was purchased from Promega; the
cell-permeant PKA inhibitor myristoylated PKI(14-22)
amide was purchased from Calbiochem. Solutions containing NGF and other
experimental agents were prepared as described previously (15,
17-19, 21).
The protocol for study of transepithelial HCO
absorption was as described previously (15, 19). The
tubules were equilibrated for 20-30 min at 37°C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per
unit tubule length) was adjusted to 1.5-2.0
nl · min
1 · mm
1. Two or
three 10-min tubule fluid samples were then collected for each period
(initial, experimental, and recovery). The tubules were allowed to
reequilibrate for 5-15 min after an experimental agent was added
to or removed from the bath solution. The absolute rate of
HCO
absorption
(pmol · min
1 · mm
1) was
calculated from the luminal flow rate and the difference between total
CO2 concentrations measured in perfused and collected fluids (15, 19). An average HCO
absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of
an experiment (initial and recovery periods), the values were averaged.
Single tubule values are presented in Figs 1-9. Mean values ± SE (n = number of tubules) are presented
in the text. Differences between means were evaluated using the
Student's t-test for paired data, with P < 0.05 considered statistically significant.

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Fig. 1.
Neurotrophin-3 (NT-3) inhibits HCO
absorption in the medullary thick ascending limb (MTAL).
A: the absolute rate of HCO
absorption (JHCO ) was measured
under control conditions and after addition of 0.7 nM NT-3 to
the bath solution. Data points are average values for single
tubules. Lines connect paired measurements made in the same tubule.
P value is for paired t-test. Mean values are
given in the text. B: effect of different NT-3
concentrations (added to the bath) on HCO
absorption. Data show the NT-3-induced inhibition of
HCO absorption, expressed as % of control transport
rate measured in the same tubule. Bars are means ± SE for 4 experiments in each group. *P < 0.005, NT-3 vs.
control.
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Fig. 2.
Effect of NT-3 (0.7 nM added to the bath) on
HCO absorption in hyperosmotic solutions
(Hyper). Hyperosmolality was produced by addition of 75 mM NaCl
to the lumen and bath. JHCO , data
points, lines, and P value as in Fig. 1A.
Mean values are given in the text.
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Fig. 3.
Effect of NT-3 (0.7 nM added to the bath) on
HCO absorption in the presence of nerve growth
factor (NGF). NGF (0.7 nM) was present in the bath throughout the
experiments. JHCO , data points, lines,
and P value as in Fig. 1A. Mean values are given
in the text.
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Fig. 4.
Effect of NT-3 (0.7 nM added to the bath) on
HCO absorption in the presence of U-0126 and
PD-98059, inhibitors of extracellular signal-regulated kinase (ERK)
activation. U-0126 (15 µM) or PD-98059 (15 µM) was present in the
bath throughout the experiments.
JHCO , data points, lines, and
P value as in Fig. 1A. Mean values are given in
the text.
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Fig. 5.
Effect of NT-3 (0.7 nM added to the bath) on
HCO absorption in the presence of the
phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002.
Tubules were bathed with 100 nM wortmannin or 20 µM LY-294002
throughout the experiments. JHCO , data
points, lines, and P value as in Fig. 1A. Mean
values are given in the text.
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Fig. 6.
Effect of NT-3 (0.7 nM added to the bath) on
HCO absorption in the presence of the protein kinase
C inhibitors staurosporine and chelerythrine chloride. Tubules were
bathed with 10 7 M staurosporine or
10 7 M chelerythrine chloride throughout the experiments.
JHCO , data points, lines, and
P value as in Fig. 1A. Mean values are given in
the text.
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Fig. 7.
Effects of NT-3 (0.7 nM added to the bath) on
HCO absorption in the presence of arginine
vasopressin (AVP; A), 8-bromo-cAMP (8-BrcAMP) or forskolin
(B), and the protein kinase A inhibitor myristoylated
PKI(14-22) amide (C). AVP
(10 10 M), 8-BrcAMP (10 4 M), forskolin
(10 6 M), or PKI(14-22) (360 nM) was
present in the bath throughout the experiments.
JHCO , data points, lines, and
P values as in Fig. 1A. NS, not significant. Mean
values are given in the text.
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Fig. 8.
Effect of NT-3 (0.7 nM added to the bath) on
HCO absorption in the presence of genistein.
Genistein (70 µM; Gen) was present in the bath throughout the
experiments. JHCO , data points, lines,
and P value as in Fig. 1A. Mean values are given
in the text.
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Fig. 9.
Effects of brain-derived neurotrophic factor (BDNF) and
glial cell line-derived neurotrophic factor (GDNF) on
HCO absorption in the MTAL. BDNF (0.7 nM;
A) and GDNF (1.6 nM; B) were added to the bath
solution in the absence ( ) or presence
( ) of 10 10 M bath AVP.
JHCO , data points, lines, and
P values as in Fig. 1A.
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RESULTS |
Effects of NT-3 on HCO
Absorption
NT-3 inhibits HCO
absorption.
Addition of 0.7 nM NT-3 to the bath decreased HCO
absorption by 36%, from 12.2 ± 0.5 to 7.8 ± 0.7 pmol · min
1 · mm
1
(P < 0.001; Fig.
1A). The inhibition was
observed within 15 min of exposure to NT-3, was sustained for at least
60 min, and was reversible.
The effect of NT-3 concentration on HCO
absorption
is shown in Fig. 1B. Inhibition of HCO
absorption was observed with bath addition of 0.35 nM NT-3. A further
increase in inhibition was observed with higher NT-3 concentrations, up
to 3.5 nM. The range of NT-3 concentrations that decreases HCO
absorption in the MTAL is in close agreement
with the effective concentrations of NT-3 that induce tyrosine
phosphorylation of the TrkC receptor (Refs. 27,
51, 57; see DISCUSSION) and is in
line with estimates of whole kidney NT-3 level in the Sprague-Dawley
rat (31). A NT-3 concentration of 0.7 nM was used for the
remainder of the experiments based on these considerations and to
permit comparison with our previous results (19, 53, 54)
on the transport effects of NGF, which was studied at the same concentration.
Hyperosmolality does not prevent inhibition by NT-3.
Hyperosmolality inhibits HCO
absorption in the
MTAL (17) and prevents inhibition by NGF
(19). We therefore tested the interaction between
hyperosmolality and NT-3. Hyperosmolality was produced by the addition
of 75 mM NaCl to the lumen and bath solutions (17).
Addition of 0.7 nM NT-3 to the bath decreased HCO
absorption from 6.7 ± 0.9 to 2.7 ± 0.5 pmol · min
1 · mm
1 in the
hyperosmotic solutions (P < 0.025; Fig.
2). Thus the inhibition of
HCO
absorption by NT-3 occurs independently of
inhibition by hyperosmolality.
NT-3 inhibits HCO
absorption in presence of NGF.
The finding that hyperosmolality blocks inhibition of
HCO
absorption by NGF but not by NT-3 suggests that
NGF and NT-3 inhibit HCO
absorption through
different mechanisms. To test this hypothesis further, we examined the
interaction between NT-3 and NGF directly. In MTAL bathed with 0.7 nM
NGF, addition of 0.7 nM NT-3 to the bath decreased
HCO
absorption by 36%, from 8.6 ± 1.2 to
5.5 ± 1.2 pmol · min
1 · mm
1
(P < 0.001; Fig. 3).
Thus the inhibitory effects of NT-3 and NGF are additive, which
suggests that these factors regulate HCO
absorption
through different signaling pathways.
Signal Transduction by NT-3
Prominent signaling pathways that are activated through
neurotrophin receptors and that influence HCO
absorption in the MTAL include Ras/ERK, phosphatidylinositol 3-kinase (PI3-K), and PKC (14, 29, 48, 57). We therefore examined the importance of these and other pathways in mediating inhibition by NT-3.
Role of ERK.
Inhibition of HCO
absorption by NGF is mediated
through activation of the ERK signaling pathway (53). To
determine whether ERK is involved in inhibition by NT-3, we examined
the effects of U-0126 and PD-98059, selective inhibitors of the
mitogen-activated protein kinase kinase MEK1/2, the direct activator of
ERK (2, 13). These inhibitors block activation of ERK by
osmotic stress and NGF in the MTAL (52, 53) and eliminate
ERK-dependent inhibition of HCO
absorption by NGF
(53). In tubules bathed with 15 µM U-0126 or 15 µM
PD-98059, addition of 0.7 nM NT-3 to the bath decreased HCO
absorption by 40%, from 13.0 ± 0.9 to 7.7 ± 0.9 pmol · min
1 · mm
1
(P < 0.001; Fig. 4).
Thus the inhibition by NT-3 is not mediated through activation of ERK.
Role of PI3-K.
PI3-K mediates certain biological responses induced by neurotrophins
(30, 48) and influences apical
Na+/H+ exchange activity and
HCO
absorption in the MTAL (21). The
role of PI3-K in mediating inhibition of HCO
absorption by NT-3 was investigated using wortmannin and LY-294002,
selective inhibitors of PI3-K that block PI3-K activation in the MTAL
(21). In MTAL bathed with 100 nM wortmannin or 20 µM
LY-294002, addition of 0.7 nM NT-3 to the bath decreased
HCO
absorption by 35%, from 13.0 ± 0.7 to
8.4 ± 0.9 pmol · min
1 · mm
1
(P < 0.001; Fig. 5).
Thus inhibition by NT-3 does not involve PI3-K.
Role of PKC.
Neurotrophin signaling may involve stimulation of phospholipase C
(PLC)-
, leading to activation of PKC (48, 57). To
determine whether PKC is involved in the inhibition of
HCO
absorption by NT-3, we examined the effects of
staurosporine and chelerythrine chloride, PKC inhibitors that
selectively abolish PKC-dependent regulation of HCO
absorption in the MTAL (17-19). In tubules bathed
with 10
7 M staurosporine or 10
7 M
chelerythrine chloride, addition of 0.7 nM NT-3 to the bath decreased
HCO
absorption by 37%, from 12.3 ± 0.6 to
7.7 ± 1.0 pmol · min
1 · mm
1
(P < 0.001; Fig. 6).
Thus inhibition of PKC does not prevent inhibition of
HCO
absorption by NT-3.
Role of cAMP.
cAMP inhibits HCO
absorption in the MTAL
(15). Three protocols were used to determine whether cAMP
is involved in mediating inhibition by NT-3. In the first protocol, we
examined the interaction between NT-3 and AVP, which inhibits
HCO
absorption by increasing cell cAMP
(15). In MTAL bathed with 10
10 M AVP (a
maximal inhibitory concentration), addition of 0.7 nM NT-3 to the bath
had no effect on HCO
absorption [7.9 ± 0.9 (AVP) vs. 7.9 ± 1.0 (AVP + NT-3)
pmol · min
1 · mm
1;
n = 4, P = not significant (NS); Fig.
7A]. Thus inhibition by AVP
prevents inhibition by NT-3. These results suggest that AVP and NT-3
inhibit HCO
absorption via a common mechanism.
To examine further the role of cAMP, a second protocol was
carried out in which MTAL were bathed with 8-bromo-cAMP (8-BrcAMP) or
forskolin, agents that induce maximal cAMP-dependent inhibition of
HCO
absorption (15). In the presence of
10
4 M 8-BrcAMP or 10
6 M forskolin, addition
of 0.7 nM NT-3 to the bath had no effect on HCO
absorption [8.0 ± 0.9 (agent) vs. 8.1 ± 0.9 (agent + NT-3) pmol · min
1 · mm
1;
n = 4, P = NS; Fig. 7B].
These results demonstrate that NT-3 inhibits HCO
absorption via a cAMP-dependent pathway. 8-BrcAMP and forskolin do not
prevent inhibition of HCO
absorption by NGF
(19). Thus these findings support the conclusion that NT-3
and NGF inhibit HCO
absorption through different
signal transduction mechanisms.
To extend these results further, a third protocol was performed to
examine the effect of the specific PKA inhibitor myristoylated PKI(14-22) amide (24). In MTAL bathed
with 360 nM PKI peptide, addition of 0.7 nM NT-3 to the bath had no
effect on HCO
absorption {13.3 ± 0.7 [PKI(14-22)] vs. 13.6 ± 0.8 [PKI(14-22) + NT-3] pmol · min
1 · mm
1;
n = 4, P = NS; Fig. 7C}.
PKI(14-22) also blocked inhibition of
HCO
absorption by forskolin (data not shown),
indicating that the peptide inhibits regulation of HCO
absorption through PKA. These data indicate that
the inhibition by NT-3 is dependent on PKA activity. Together, our
results support the conclusion that NT-3 inhibits
HCO
absorption by increasing intracellular cAMP and
stimulating PKA.
The experiments in Fig. 7A show that the transport effect of
NT-3 is prevented by pretreatment with a maximal dose of AVP (10
10 M) (15). To determine whether NT-3 and
AVP can interact physiologically to regulate HCO
absorption, further experiments were carried out using a submaximal AVP
concentration of 2 × 10
12 M, which inhibits
HCO
absorption by half of the maximum amount induced
by 10
10 M AVP (15, 20). In MTAL bathed with
2 × 10
12 M AVP, addition of 0.7 nM NT-3 to the bath
decreased HCO
absorption from 10.3 ± 0.8 to
7.3 ± 0.7 pmol · min
1 · mm
1
(n = 4; P < 0.001). Bath addition of
2 × 10
12 M AVP alone decreased
HCO
absorption by 22 ± 2% (n = 4; P < 0.005), confirming previous results
(20). These data demonstrate that NT-3 inhibits
HCO
absorption in the presence of a physiological
concentration of AVP, supporting the physiological relevance of
NT-3-induced transport regulation.
Genistein blocks inhibition by NT-3.
Neurotrophins trigger cell signals and regulate cellular responses
through binding to specific receptor tyrosine kinases (Trks) (8,
50). To test for the involvement of tyrosine kinase pathways in
the inhibition by NT-3, we examined the effect of the protein tyrosine
kinase inhibitor genistein. Genistein selectively inhibits tyrosine
kinase-dependent regulation of HCO
absorption in the
MTAL (17, 19) and blocks neurotrophin-induced signaling
via tyrosine phosphorylation in neuronal cells (41). In
MTAL bathed with 70 µM genistein, addition of 0.7 nM NT-3 to the bath
had no effect on HCO
absorption [13.5 ± 0.7 (genistein) vs. 13.4 ± 0.8 (genistein + NT-3)
pmol · min
1 · mm
1;
n = 4, P = NS; Fig.
8]. These results are not due to a
direct action of genistein on the cAMP-PKA system because genistein
does not prevent cAMP-mediated regulation of HCO
absorption by other stimuli (17). These findings support a
role for tyrosine kinase pathways in mediating the inhibition of
HCO
absorption by NT-3 and are consistent with the
involvement of a receptor tyrosine kinase.
Effects of BDNF and GDNF on HCO
Absorption
To investigate the range of neurotrophic factors that influences
MTAL function, we examined the effects of BDNF and GDNF. BDNF is a
member of the neurotrophin gene family that includes NGF and NT-3
(8, 34). GDNF is a member of the transforming growth
factor-
superfamily (1). Both factors were tested in the absence and presence of AVP in view of the effect of AVP to unmask
stimulation of HCO
absorption by NGF
(19). The results in Fig. 9
show that addition of either 0.7 nM BDNF or 1.6 nM GDNF to the bath had
no effect on HCO
absorption.
 |
DISCUSSION |
Neurotrophins and their receptors are present in the kidney and
other epithelial organs, but their significance for epithelial cell
function is not understood. In the present study, we demonstrate that
NT-3 inhibits HCO
absorption in the MTAL via a cAMP-
and PKA-dependent signaling pathway. We also show that NT-3 and NGF
regulate HCO
absorption independently, through
different signal transduction mechanisms. In contrast,
HCO
absorption is not affected by GDNF, the
prototypical member of the GDNF family of neurotrophic factors that is
essential for kidney development (1, 39, 40, 46). We also
observed no effect of BDNF, indicating that the regulatory response of
the MTAL is limited to specific members within the neurotrophin family.
These studies provide the first evidence that NT-3 regulates the
function of renal tubules and establish a direct role for NT-3 in the
regulation of epithelial ion transport. They also identify the MTAL as
an important target within the mammalian nephron for neurotrophins, which may be involved in the control of urinary acidification.
NT-3 mRNA is highly expressed in the adult kidney (36,
44), and NT-3 protein has been localized by immunohistochemistry to tubule epithelial cells in the renal cortex and medulla (26, 59). Our findings in the MTAL show that the high levels of NT-3 in the kidney correlate directly with a physiologically relevant, nonneurotrophic role in the regulation of renal tubule transport. The
precise nephron segments that express NT-3 have not been fully identified; however, the collecting ducts appear to be a site of NT-3
expression (26, 59). The location of NT-3 in collecting ducts in the outer medulla could provide a source for its biological action on the surrounding MTALs. In studies in which neurotrophins were
expressed in a renal epithelial cell line (Madin-Darby canine kidney,
MDCK), NT-3 was secreted preferentially across the basolateral membrane
(25). Basolateral secretion by medullary collecting duct
cells in vivo could provide a source of interstitial NT-3 for its
direct action in the MTAL. In this way, NT-3 may represent a previously
unrecognized factor that mediates communication between, and
coordinates the function of, the thick ascending limbs and collecting
ducts in the outer medulla and medullary rays. Further work is needed
to explore this hypothesis. The possibility that NT-3 may be produced
and secreted by the MTAL and act as an autocrine factor also remains to
be determined. Whether NT-3 acts on nephron segments other than the
MTAL is unknown.
The biological effects of neurotrophins are mediated through binding to
two types of cell-surface receptors: the Trk family of receptor
tyrosine kinases, which bind neurotrophins with high selectivity, and
p75NTR, which lacks intrinsic tyrosine kinase activity and binds all
neurotrophins with similar affinity (8, 14, 50). Although
the neurotrophin receptors present in the MTAL have not been
identified, some preliminary insights can be gained from our
experiments. The finding that BDNF does not affect HCO
absorption suggests that the transport effects
of NT-3 and NGF may be mediated through Trk receptors rather than
through p75NTR, because BDNF can effectively bind and activate p75NTR
(9, 34, 42, 43). In addition, the transport effects of
NT-3 and NGF are fully additive and are mediated through different
signaling pathways, consistent with these ligands acting through
different membrane receptors (see below). A role for tyrosine kinase
receptors is supported further by the observation that the transport
effects of NT-3 and NGF are blocked by tyrosine kinase inhibitors.
Three members of the Trk gene family have been identified: TrkA, which
preferentially binds NGF; TrkB, which binds BDNF and NT-4/5; and TrkC,
which binds NT-3 (8, 27, 50). [Trk receptors have some
capacity to bind neurotrophins other than their primary ligands;
however, the physiological significance of this cross-reactivity is
unclear (8, 27, 50).] Both TrkA and TrkC receptors are
expressed in the kidney and have been localized to renal tubules
(26, 35, 49). Thus the simplest explanation for our
results is that the separate actions of NT-3 and NGF on
HCO
absorption are mediated through the selective
binding of NGF to TrkA and NT-3 to TrkC. Confirmation will require
direct evidence for the expression of TrkA and TrkC in the MTAL. Also,
our studies do not exclude a possible role for p75NTR in mediating
NT-3- and/or NGF-induced signaling and transport regulation because
p75NTR has been shown to interact with and modify the function of Trk
receptors in other systems (8, 9, 14).
Neurotrophin signaling through Trk receptors involves the activation of
several parallel pathways, predominantly Shc/Ras/ERK, PI3-K, and
PLC-
/PKC (14, 29, 48, 57). In the MTAL, these pathways
(particularly ERK and PKC) play essential roles in mediating regulation
of HCO
absorption by NGF (19, 53). In
contrast, we found no role for ERK, PI3-K, or PKC in inhibition of
HCO
absorption by NT-3. Instead, the inhibition by
NT-3 was mediated through a cAMP-dependent signaling pathway. This
conclusion is supported by several observations: 1)
inhibition by NT-3 is blocked by 8-BrcAMP and forskolin, an agent that
elevates cAMP; 2) the specific PKA inhibitor
PKI(14-22) eliminates NT-3-induced inhibition; and
3) a maximal dose of vasopressin, which inhibits
HCO
absorption by increasing cAMP (15),
blocks inhibition by NT-3. Neurotrophin treatment leads to increased
levels of intracellular cAMP in some neuronal systems (10, 11,
23, 28, 33, 56, 58), possibly by increasing the activity of
Ca2+-calmodulin-dependent adenylyl cyclases
(10). In addition, NT-3 has been reported to induce rapid
stimulation of PKA activity in neural growth cones (58).
Together, these findings support the conclusion that NT-3 inhibits
HCO
absorption in the MTAL by elevating cAMP and
increasing PKA activity. We also found that NT-3 inhibits
HCO
absorption in the presence of a submaximal AVP
concentration, which suggests that NT-3 and AVP regulate
HCO
absorption through additive effects on the
cAMP-PKA pathway. Further work will be necessary to provide direct
evidence for NT-3-induced stimulation of the cAMP-PKA pathway and to
identify upstream signaling events leading to cAMP-mediated regulation.
NT-3 inhibits HCO
absorption in the MTAL by an
amount quantitatively similar to that observed with NGF
(19). Our results show, however, that NT-3 and NGF inhibit
HCO
absorption through different pathways: NT-3
inhibits via a cAMP-dependent pathway, whereas NGF inhibits via an
ERK-dependent pathway (53). This conclusion is supported
by several lines of evidence: 1) inhibition by NT-3 is
additive to inhibition by NGF; 2) 8-BrcAMP and forskolin
block inhibition by NT-3 but not by NGF (19);
3) inhibitors of ERK activation block inhibition by NGF
(53) but not by NT-3; and 4) hyperosmolality
prevents inhibition by NGF (19) but not by NT-3. The
latter finding is consistent with NT-3 acting via a cAMP-dependent
pathway because hyperosmolality does not prevent cAMP-mediated
transport inhibition (16, 17). In addition, the additivity
of the effects of NT-3 and NGF cannot be attributed to the use of a
submaximal NGF concentration because inhibition of
HCO
absorption by NGF is maximal at a concentration
of 0.7 nM (D. Good, unpublished observations). It is likely that
activation of the different signaling pathways by NT-3 and NGF leads to
inhibition of HCO
absorption through different
transport mechanisms. Absorption of HCO
in the MTAL
is mediated by the apical membrane Na+/H+
exchanger NHE3 (3, 6, 16, 55). Activation of the cAMP-PKA pathway is coupled directly to inhibition of NHE3 activity (7, 15, 16, 38). It is likely, therefore, that NT-3 inhibits HCO
absorption by acting via cAMP to inhibit NHE3.
In contrast, NGF has no direct effect on apical Na+/H+ exchange (NHE3) activity but instead
acts via ERK to inhibit basolateral Na+/H+
exchange activity (53, 54). The inhibition of basolateral Na+/H+ exchange results secondarily in
inhibition of apical Na+/H+ exchange due to
cross talk between the exchangers (54). We propose,
therefore, that the effects of NGF and NT-3 to inhibit HCO
absorption are additive because they act via
different signaling pathways to inhibit different transporters: NGF
acts via ERK to inhibit primarily basolateral Na+/H+ exchange activity (53, 54),
and NT-3 acts via cAMP to inhibit primarily apical
Na+/H+ exchange activity. Confirmation of the
latter mechanism will require further direct studies of the effects of
NT-3 on the apical Na+/H+ exchanger. Our
results establish, however, that NT-3 and NGF inhibit
HCO
absorption through functionally separate
pathways and are capable of mediating distinct physiological responses
in MTAL cells through the activation of different signal transduction mechanisms.
Although our studies identify a direct role for NT-3 in the
regulation of renal epithelial transport, the significance of neurotrophins for kidney function remains to be determined. Our results
show that NT-3 inhibits HCO
absorption over a
concentration range that agrees closely with the dose response for
NT-3-induced tyrosine phosphorylation of the TrkC receptor
(EC50
0.2-0.4 nM; Refs. 27,
51, 57), suggesting that NT-3 regulates MTAL
transport at physiologically relevant concentrations. In addition, the
inhibition of HCO
absorption by NT-3 in the MTAL is
comparable in magnitude to that observed with other regulatory factors
such as angiotensin II, chronic metabolic acidosis and alkalosis,
vasopressin, and aldosterone (15, 16, 22). This finding,
coupled with the fact that NGF also influences
Na+/H+ exchange activity and
HCO
absorption in the MTAL (19, 54),
suggests that neurotrophins may play a role in the regulation of renal
acid excretion. It is also possible, in view of their biological roles
in nerve cells, that neurotrophins are involved in mediating growth,
differentiation, survival, and/or repair of MTAL cells. Whether
neurotrophins are survival factors for renal epithelial cells or
participate in the renal response to injury is presently unknown.
Finally, an intriguing possibility arises from the differing
interactions of NT-3 and NGF with vasopressin. Vasopressin plays an
important role in the regulation of multiple renal transport processes,
most notably H2O absorption but also NaCl absorption,
K+ secretion, urea absorption, and H+
secretion/HCO
absorption (16, 37). Our
studies of HCO
absorption in the MTAL show that NT-3
mimics the transport and signaling effects of AVP, whereas NGF reverses
the transport effect of AVP (19). It is possible,
therefore, that NT-3 and NGF may function as locally produced factors
in the kidney that reproduce or modulate the physiological actions of
vasopressin. Further work is needed to test this hypothesis and to
determine the role of neurotrophins in acid-base regulation and other
critical renal functions. Understanding the biological actions of
neurotrophins on renal tubule function and survival will be important
in evaluating the possible renal effects that may result from the
systemic use of neurotrophins in treatment of neurological disease
(4, 32, 47).
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-38217.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. W. Good, 4.200 John Sealy Annex 0562, Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0562 (E-mail:
dgood{at}UTMB.edu).
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.
Received 18 April 2001; accepted in final form 20 July 2001.
 |
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