Neurotrophin-3 inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neurotrophins are expressed in the adult kidney, but their significance is unclear. We showed previously that nerve growth factor (NGF) inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> filtered at the glomerulus (16). Recently, we demonstrated that NGF regulates transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL of the rat through complex mechanisms. Specifically, NGF inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption under basal conditions but stimulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is mediated through activation of extracellular signal-regulated kinase (ERK) (53), whereas the AVP-dependent stimulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is mediated through activation of protein kinase C (PKC) (19). In addition, NGF inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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-beta superfamily that is essential for kidney development (1, 39, 40, 46). We demonstrate that NT-3 inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via a cAMP- and protein kinase A (PKA)-dependent pathway that is distinct from the signaling pathways involved in regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NGF. In contrast, BDNF and GDNF have no effect on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP>, 2.0 Ca2+, 1.5 Mg2+, 2.0 phosphate, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Neurotrophin-3 (NT-3) inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the medullary thick ascending limb (MTAL). A: the absolute rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. Data show the NT-3-induced inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in hyperosmotic solutions (Hyper). Hyperosmolality was produced by addition of 75 mM NaCl to the lumen and bath. JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P value as in Fig. 1A. Mean values are given in the text.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the presence of nerve growth factor (NGF). NGF (0.7 nM) was present in the bath throughout the experiments. JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P value as in Fig. 1A. Mean values are given in the text.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P value as in Fig. 1A. Mean values are given in the text.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P value as in Fig. 1A. Mean values are given in the text.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P value as in Fig. 1A. Mean values are given in the text.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P values as in Fig. 1A. NS, not significant. Mean values are given in the text.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of NT-3 (0.7 nM added to the bath) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the presence of genistein. Genistein (70 µM; Gen) was present in the bath throughout the experiments. JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P value as in Fig. 1A. Mean values are given in the text.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 9.   Effects of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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 (open circle ) of 10-10 M bath AVP. JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, data points, lines, and P values as in Fig. 1A.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of NT-3 on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> Absorption

NT-3 inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. Addition of 0.7 nM NT-3 to the bath decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is shown in Fig. 1B. Inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NT-3 occurs independently of inhibition by hyperosmolality.

NT-3 inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>absorption in presence of NGF. The finding that hyperosmolality blocks inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NGF but not by NT-3 suggests that NGF and NT-3 inhibit HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption through different signaling pathways.

Signal Transduction by NT-3

Prominent signaling pathways that are activated through neurotrophin receptors and that influence HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL (21). The role of PI3-K in mediating inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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)-gamma , leading to activation of PKC (48, 57). To determine whether PKC is involved in the inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NT-3, we examined the effects of staurosporine and chelerythrine chloride, PKC inhibitors that selectively abolish PKC-dependent regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NT-3.

Role of cAMP. cAMP inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via a cAMP-dependent pathway. 8-BrcAMP and forskolin do not prevent inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NGF (19). Thus these findings support the conclusion that NT-3 and NGF inhibit HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by forskolin (data not shown), indicating that the peptide inhibits regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption, further experiments were carried out using a submaximal AVP concentration of 2 × 10-12 M, which inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by 22 ± 2% (n = 4; P < 0.005), confirming previous results (20). These data demonstrate that NT-3 inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by other stimuli (17). These findings support a role for tyrosine kinase pathways in mediating the inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NT-3 and are consistent with the involvement of a receptor tyrosine kinase.

Effects of BDNF and GDNF on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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-beta 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL via a cAMP- and PKA-dependent signaling pathway. We also show that NT-3 and NGF regulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption independently, through different signal transduction mechanisms. In contrast, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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-gamma /PKC (14, 29, 48, 57). In the MTAL, these pathways (particularly ERK and PKC) play essential roles in mediating regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by NGF (19, 53). In contrast, we found no role for ERK, PI3-K, or PKC in inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL by elevating cAMP and increasing PKA activity. We also found that NT-3 inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the presence of a submaximal AVP concentration, which suggests that NT-3 and AVP regulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption through different transport mechanisms. Absorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption over a concentration range that agrees closely with the dose response for NT-3-induced tyrosine phosphorylation of the TrkC receptor (EC50 congruent  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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> 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<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (16, 37). Our studies of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Airaksinen, MS, Titievsky A, and Saarma M. GDNF family neurotrophic factor signaling: four masters, one servant? Mol Cell Neurosci 13: 313-325, 1999[ISI][Medline].

2.   Alessi, DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489-27494, 1995[Abstract/Free Full Text].

3.   Amemiya, M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, and Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney Int 48: 1206-1215, 1995[ISI][Medline].

4.   Apfel, SC. Neurotrophic factors in peripheral neuropathies: therapeutic implications. Brain Pathol 9: 393-413, 1999[ISI][Medline].

5.   Berkemeier, LR, Winslow JW, Kaplan DR, Nikolics K, Goeddel DV, and Rosenthal A. Neurotrophin-5: a novel neurotrophic factor that activates trk and trkB. Neuron 7: 857-866, 1991[ISI][Medline].

6.   Biemesderfer, D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997[Abstract/Free Full Text].

7.   Borensztein, P, Juvin P, Vernimmen C, Poggioli J, Paillard M, and Bichara M. cAMP-dependent control of Na+/H+ antiport by AVP, PTH, and PGE2 in rat medullary thick ascending limb cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F354-F364, 1993[Abstract/Free Full Text].

8.   Bothwell, M. Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci 18: 223-253, 1995[ISI][Medline].

9.   Bothwell, M. p75NTR: a receptor after all. Science 272: 506-507, 1996[ISI][Medline].

10.   Boulanger, L, and Poo M. Gating of BDNF-induced synaptic potentiation by cAMP. Science 284: 1982-1984, 1999[Abstract/Free Full Text].

11.   Cai, D, Shen Y, De Bellard M, Tang S, and Filbin MT. Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron 22: 89-101, 1999[ISI][Medline].

12.   Ernfors, P, Ibanez CF, Ebendal T, Olson L, and Persson H. Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain. Proc Natl Acad Sci USA 87: 5454-5458, 1990[Abstract].

13.   Favata, MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 18623-18632, 1998[Abstract/Free Full Text].

14.   Friedman, WJ, and Greene LA. Neurotrophin signaling via Trks and p75. Exp Cell Res 253: 131-142, 1999[ISI][Medline].

15.   Good, DW. Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb. J Clin Invest 85: 1006-1013, 1990[ISI][Medline].

16.   Good, DW. The thick ascending limb as a site of renal bicarbonate reabsorption. Semin Nephrol 13: 225-235, 1993[ISI][Medline].

17.   Good, DW. Hyperosmolality inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in rat medullary thick ascending limb via a protein tyrosine kinase-dependent pathway. J Biol Chem 270: 9883-9889, 1995[Abstract/Free Full Text].

18.   Good, DW. PGE2 reverses AVP inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in rat MTAL by activation of protein kinase C. Am J Physiol Renal Fluid Electrolyte Physiol 270: F978-F985, 1996[Abstract/Free Full Text].

19.   Good, DW. Nerve growth factor regulates HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in thick ascending limb: modifying effects of vasopressin. Am J Physiol Cell Physiol 274: C931-C939, 1998[Abstract/Free Full Text].

20.   Good, DW, Caflisch CR, and George T. Prostaglandin E2 regulation of ion transport is absent in medullary thick ascending limbs from spontaneously hypertensive rats. Am J Physiol Renal Fluid Electrolyte Physiol 269: F47-F54, 1995[Abstract/Free Full Text].

21.   Good, DW, Di Mari J, and Watts BA, III. Hyposmolality stimulates Na+/H+ exchange and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in renal thick ascending limb via PI3-kinase. Am J Physiol Cell Physiol 279: C1443-C1454, 2000[Abstract/Free Full Text].

22.   Good, DW, George T, and Wang DW. Angiotensin II inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via a cytochrome P450-dependent signaling pathway in rat medullary thick ascending limb. Am J Physiol Renal Physiol 276: F726-F736, 1999[Abstract/Free Full Text].

23.   Halegoua, S, Armstrong RC, and Kremer NE. Dissecting the mode of action of a neuronal growth factor. Curr Top Microbiol Immunol 165: 119-170, 1991[ISI][Medline].

24.   Harris, TE, Persaud SJ, and Jones PM. Pseudosubstrate inhibition of cyclic AMP-dependent protein kinase in intact pancreatic islets: effects on cyclic AMP-dependent and glucose-dependent insulin secretion. Biochem Biophys Res Commun 232: 648-651, 1997[ISI][Medline].

25.   Heymach, JV, Jr, Krüttgen A, Suter U, and Shooter EM. The regulated secretion and vectorial targeting of neurotrophins in neuroendocrine and epithelial cells. J Biol Chem 271: 25430-25437, 1996[Abstract/Free Full Text].

26.   Huber, LJ, Hempstead B, and Donovan MJ. Neurotrophin and neurotrophin receptors in human fetal kidney. Dev Biol 179: 369-381, 1996[ISI][Medline].

27.   Ip, NY, Stitt TN, Tapley P, Klein R, Glass DJ, Fandl J, Greene LA, Barbacid M, and Yancopoulos GD. Similarities and differences in the way neurotrophins interact with the Trk receptors in neuronal and nonneuronal cells. Neuron 10: 137-149, 1993[ISI][Medline].

28.   Kalman, D, Wong B, Horvai AE, Cline MJ, and O'Lague PH. Nerve growth factor acts through cAMP-dependent protein kinase to increase the number of sodium channels in PC12 cells. Neuron 2: 355-366, 1990.

29.   Kaplan, DR, and Miller FD. Signal transduction by the neurotrophin receptors. Curr Opin Cell Biol 9: 213-221, 1997[ISI][Medline].

30.   Kaplan, DR, and Miller FD. Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 10: 381-391, 2000[ISI][Medline].

31.   Katoh-Semba, R, Kaisho Y, Shintani A, Nogahama M, and Kato K. Tissue distribution and immunocytochemical localization of neurotrophin-3 in the brain and peripheral tissues of rats. J Neurochem 66: 330-337, 1996[ISI][Medline].

32.   Kernie, SG, and Parada LF. The molecular basis for understanding neurotrophins and their relevance to neurologic disease. Arch Neurol 57: 654-657, 2000[Free Full Text].

33.   Knipper, M, Beck A, Rylett J, and Breer H. Neurotrophin induced cAMP and IP3 responses in PC12 cells. FEBS Lett 324: 147-152, 1993[ISI][Medline].

34.   Lewin, GR, and Barde YA. Physiology of the neurotrophins. Annu Rev Neurosci 19: 289-317, 1996[ISI][Medline].

35.   Lomen-Hoerth, C, and Shooter EM. Widespread neurotrophin receptor expression in the immune system and other nonneural rat tissues. J Neurochem 64: 1780-1789, 1995[ISI][Medline].

36.   Maisonpierre, PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM, and Yancopoulos GD. Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247: 1446-1451, 1990[ISI][Medline].

37.   Masilamani, S, Knepper MA, and Burg MB. Urine concentration and dilution. In: The Kidney, , edited by Brenner BM.. Philadelphia, PA: Saunders, 2000, vol. I, p. 595-635.

38.   Moe, OW. Acute regulation of proximal tubule apical membrane Na/H exchanger NHE-3: role of phosphorylation, protein trafficking, and regulatory factors. J Am Soc Nephrol 10: 2412-2425, 1999[Free Full Text].

39.   Moore, MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, and Rosenthal A. Renal and neuronal abnormalities in mice lacking GDNF. Nature 382: 76-79, 1996[ISI][Medline].

40.   Pichel, JG, Shen L, Sheng HZ, Granholm A-C, Drago J, Grinberg A, Lee EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, and Westphal H. Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382: 73-76, 1996[ISI][Medline].

41.   Qiu, M-S, and Green SH. NGF and EGF rapidly activate p21ras in PC12 cells by distinct, convergent pathways involving tyrosine phosphorylation. Neuron 7: 937-946, 1991[ISI][Medline].

42.   Rodriguez-Tebar, A, Dechant G, and Barde YA. Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron 4: 487-492, 1990[ISI][Medline].

43.   Rodriguez-Tebar, A, Dechant G, Götz R, and Barde YA. Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor. EMBO J 11: 917-922, 1992[Abstract].

44.   Rosenthal, A, Goeddel DV, Nguyen T, Lewis M, Shih A, Laramee GR, Nikolics K, and Winslow JW. Primary structure and biological activity of a novel human neurotrophic factor. Neuron 4: 767-773, 1990[ISI][Medline].

45.   Salido, EC, Barajas L, Lechago J, Laborde NP, and Fisher DA. Immunocytochemical localization of nerve growth factor in mouse kidney. J Neurosci Res 16: 457-465, 1986[ISI][Medline].

46.   Sanchez, MP, Silos-Santiago I, Frisen J, He B, Lira SA, and Barbacid M. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382: 70-73, 1996[ISI][Medline].

47.   Saragovi, HU, and Gehring K. Development of pharmacological agents for targeting neurotrophins and their receptors. Trends Pharmacol Sci 21: 93-98, 2000[ISI][Medline].

48.   Segal, RA, and Greenberg ME. Intracellular signaling pathways activated by neurotrophic factors. Annu Rev Neurosci 19: 463-489, 1996[ISI][Medline].

49.   Shibayama, E, and Koizumi H. Cellular localization of the Trk neurotrophin receptor family in human non-neural tissues. Am J Pathol 148: 1807-1818, 1996[Abstract].

50.   Snider, WD. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 77: 627-638, 1994[ISI][Medline].

51.   Tsoulfas, P, Soppet D, Escandon E, Tessarollo L, Mendoza-Ramirez J-L, Rosenthal A, Nikolics K, and Parada LF. The rat trkC locus encodes multiple neurogenic receptors that exhibit differential response to neurotrophin-3 in PC12 cells. Neuron 10: 975-990, 1993[ISI][Medline].

52.   Watts, BA, III, Di Mari JF, Davis RJ, and Good DW. Hypertonicity activates MAP kinases and inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via distinct pathways in thick ascending limb. Am J Physiol Renal Physiol 275: F478-F486, 1998[Abstract/Free Full Text].

53.   Watts, BA, III, and Good DW. Extracellular signal-regulated kinase mediates inhibition of Na+/H+ exchange and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by nerve growth factor in medullary thick ascending limb (Abstract). J Am Soc Nephrol 10: 11A, 1999.

54.   Watts, BA, III, George T, and Good DW. Nerve growth factor inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange. J Biol Chem 274: 7841-7847, 1999[Abstract/Free Full Text].

55.   Watts, BA, III, and Good DW. Hyposmolality stimulates apical membrane Na+/H+ exchange and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in renal thick ascending limb. J Clin Invest 104: 1593-1602, 1999[Abstract/Free Full Text].

56.   Yao, H, York RD, Misra-Press A, Carr DW, and Stork PJS The cyclic adenosine monophosphate-dependent protein kinase is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor. J Biol Chem 273: 8240-8247, 1998[Abstract/Free Full Text].

57.   Yuen, EC, and Mobley WC. Early BDNF, NT-3, and NT-4 signaling events. Exp Neurol 159: 297-308, 1999[ISI][Medline].

58.   Zhang, HL, Singer RH, and Bassell GJ. Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones. J Cell Biol 147: 59-70, 1999[Abstract/Free Full Text].

59.   Zhou, X-F, and Rush RA. Localization of neurotrophin-3-like immunoreactivity in peripheral tissues of the rat. Brain Res 621: 189-199, 1993[ISI][Medline].


Am J Physiol Cell Physiol 281(6):C1804-C1811
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Good, D. W.
Articles by George, T.
Articles citing this Article
PubMed
PubMed Citation
Articles by Good, D. W.
Articles by George, T.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online