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Am J Physiol Renal Physiol 293: F1468-F1475, 2007. First published August 22, 2007; doi:10.1152/ajprenal.00246.2007
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Characterization of a putative intrarenal serotonergic system

Jie Xu, Bing Yao, Xiaofeng Fan, Melissa M. Langworthy, Ming-Zhi Zhang, and Raymond C. Harris

Division of Nephrology, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 29 May 2007 ; accepted in final form 21 August 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Serotonin [5-hydroxytryptamine (5HT)] acts through multiple G protein-coupled 5-HT receptors, and its activity is also regulated by the 5-HT transporter. The current studies report the expression and localization of the 5-HT receptors and transporter in the kidney. In addition, the enzymatic pathway mediating 5-HT synthesis is present in renal cortex, especially in the proximal tubules and glomerular epithelial cells and mesangial cells. Expression of the 5-HT receptors and 5-HT transporter was detected by RT-PCR in cell lines of these cell types. In cultured proximal tubule cells and podocytes, 5-HT activated ERK1/2 and increased the expression of connective tissue growth factor and transforming growth factor-beta, two key mediators of extracellular matrix accumulation. Immunohistochemistry and real-time RT-PCR studies also indicated that 5-HT stimulated expression of vascular endothelial growth factor in podocytes in vitro and in vivo. Therefore, these results indicate the presence of an integrated intrarenal serotonergic system and suggest a possible role for 5-HT as a mediator of renal fibrosis in the kidney.

serotonin transporter; serotonin receptor; connective tissue growth factor; transforming growth factor-beta; vascular endothelial growth factor

ALTHOUGH SEROTONIN (5-HT) has been intensively studied in the central neural system, there is abundant evidence that it can also serve as an extraneural hormone. 5-HT is synthesized from L-tryptophan by the sequential actions of L-tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC). 5-HT acts through multiple G protein-coupled serotonin receptors (5-HTR), and its activity is also regulated by the serotonin transporter (SERT), which transports 5-HT from the extracellular to intracellular space. Seven serotonin receptor subfamilies have been identified to date. Previous reports identified a number of serotonin receptor subtypes in the kidney or in cultured cells of renal origin, such as 5-HT1AR in human embryonic kidney 293 cells (8), 5-HT1DR in Madin-Darby canine kidney cells (17), and 5-HT2R in rat primary cultured mesangial cells (3). SERT expression is controlled by many factors, including cytokines and gene promoter polymorphisms (12). Previous reports demonstrated the existence of serotonin receptors and SERT in mesangial cells (14), and it has been reported that 5-HT can activate mesangial cell growth and may also participate in transcription cascades leading to glomerulosclerosis (3, 13).

In the current studies, we examined the expression and localization of TPH/AADC, the serotonin receptors and the serotonin transporter in the renal cortex by immunohistochemistry and in situ hybridization. We also demonstrated that 5-HT stimulates ERK activity through a 5-HT2AR-mediated signaling pathway and increases expression of profibrotic growth factors in cultured proximal tubule cells and podocytes.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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Cell culture. The cultured mouse proximal tubular epithelial cell line, MCT (7), derived from SJL mice, was maintained in DMEM containing 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Cultured immortalized mouse podocytes (IMP) (11) were maintained in RPMI 1640 medium with 100 U/ml penicillin, 100 µg/ml streptomycin, 1x insulin/transferrin/selenite (ITS; Sigma), and 10% fetal calf serum. IMP cells were grown on type I collagen-coated plates either at the permissive temperature of 33°C (in 5% CO2) to promote cell propagation as a proliferating phenotype with 10 U/ml {gamma}-interferon (Sigma) or at the nonpermissive temperature of 37°C (in 5% CO2) to allow the cells to differentiate (without {gamma}-interferon).

Animals. Male Sprague-Dawley rats (150–200 g) were treated with (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI; Sigma), a 5-HT2AR agonist (1 mg·kg–1·day–1 ip), for 3 days. Male C57/BL6J mice (3 mo old) were also used for immunohistochemistry, in situ hybridization, and Northern analysis. Generally, one kidney was fixed in buffered formalin (10%) and then embedded in paraffin for light microscopy. The other kidney was separated into cortex and medulla for protein and RNA isolation. All animal study protocols were reviewed and approved by the Vanderbilt University Institutional Animal Care and Use Committee. All experiments were conducted according to National Institutes of Health guidelines.

Isolation of primary proximal tubule epithelial cells. Proximal tubule epithelial cells were isolated using modification of the methods of Vinay et al. (19). Briefly, medullas and cortices from 3–5 male C57/BL6J mice were collected and minced in Krebs-Hensleit saline (KHS) buffer containing 115 mM NaCl, 24 mM NaHCO3, 10 mM HEPES, 5 mM glucose, 5 mM KCl, 2 mM NaH2PO4, 1.5 mM MgSO4, and 1 mM alanine. The above solution was then enriched with 0.15% (wt/vol) collagenase type I, 0.5% (wt/vol) bovine serum albumin, and 0.01% soybean trypsin inhibitor and incubated at 37°C for 1 h to digest the cortices. The suspension was strained through a 100-µm sieve, washed, and centrifuged at 600 rpm 3 times. The pellets were combined with 47% Percoll (Amersham) solution mixed with 2x KHS and centrifuged at 16,300 rpm for 30 min at 4°C. The lowest band enriched with proximal tubule segments was washed with KHS buffer three times and utilized for RNA isolation.

Northern analysis. The mRNA levels of different genes of interest in renal cortex and medulla from animals or renal cells (MCT or IMP) were quantified by Northern analysis. RNA was isolated using TriReagent (Molecular Research Center) and concentrations were quantitated by absorbance at 260 nm by BioRad spectrophotometer (BioRad). Twenty micrograms of total RNA were electrophoresed on 1% formaldehyde-agarose gels and transferred to nylon membranes (Scheicher and Schell). Blots were hybridized with the random prime-labeled (Megaprime DNA labeling system, Amersham) rat or mouse SERT. Every Northern analysis was repeated two or three times from independent RNA isolations.

Real-time RT-PCR analysis. The expression of SERT, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta), vascular endothelial growth factor (VEGF), and serotonin receptor subtypes was determined by real-time RT-PCR analysis with the iCycler IQ Real-Time PCR detection system using a SYBR Green kit (BioRad). cDNA synthesis was performed on 5 µg of total RNA using random hexamer and SuperScript RT (Invitrogen). Sequences of real-time PCR primer sets are listed in Table 1. The thermal cycling consisted of a denaturation step at 95°C for 5 min followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Quantitative values were calculated from the threshold PCR cycle numbers, which were derived from the exponential phase of each PCR reaction. The relative mRNA level in each sample was normalized for GAPDH mRNA content. Every experiment was repeated at least three times with different RNA isolations.


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  		Table 1. Real-time PCR primer sets

 
In situ hybridization. Digoxigenin (DIG)-labeled sense and antisense probes of mouse SERT were synthesized with the DIG RNA labeling kit (Roche) from linearized plasmids containing the mouse SERT fragment (600 bp in the coding region of SERT mRNA). After fixation in freshly prepared buffered formalin (10%), hemisected kidneys were cut into 5-µm sections and subjected to in situ hybridization. The DIG nucleic acid detection kit (Roche) was used to visualize hybridized probes according to the manufacturer's directions. Eosin was used for counter staining. The slides were examined via a microscope equipped with differential interference contrast (DIC) optics.

Immunohistochemistry. The animals were anesthetized with Nembutal (70 mg/kg ip), given heparin (1,000 U/kg ip) to minimize coagulation, and perfused with FPAS (3.7% formaldehyde, 10 mM sodium m-periodate, 40 mM phosphate buffer, and 1% acetic acid) through the aortic trunk cannulated via the left ventricle (23). After fixation, kidneys were dehydrated and paraffin-embedded. The slides were deparaffinized, rehydrated, and stained with TPH antibody (COVACE), VEGF-A antibody (R&D systems), and AADC antibody (Chemicon International) according to previous reports (22).

Western analysis. Cells were cultured in DMEM/F12 (MCT) or RPMI 1640 (IMP) medium without serum for 2 days. Growth-arrested cells were incubated with or without 5-HT at indicated doses and times. When selective inhibitors were applied, they were added 30 min before the application of 5-HT. Cellular protein was extracted in lysis buffer containing 100 mM Tris·HCl (pH 8.1), 1% Triton X-114, 10 mM ethylenediaminetetraacetic acid, 0.2 mM sodium orthovanadate, and a mixture of protease inhibitors (Boehringer, Mannheim, Germany). Protein concentration was determined by the Bradford method according to the instructions of the manufacturer (BioRad). Ten to twenty micrograms of total protein from each sample were subjected to 10–15% SDS-PAGE gels and then transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was probed with a phospho-specific ERK1/2 antibody (P-ERK) or a total ERK1/2 antibody (ERK, Santa Cruz Biotechnology) or VEGF-A antibody (R&D systems).

Statistical analysis. Statistical significance was evaluated with Student's t-test for two-group comparisons when indicated. A value of P < 0.05 (as indicated by * in figures) was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Expression of TPH, AADC, and SERT in the renal cortex. Immunohistohemical analysis indicated that two key enzymes in 5-HT biosynthesis, TPH (Fig. 1, A and B) and AADC (Fig. 1D), were present in mouse kidney cortex, predominantly in proximal tubules, but also in glomerular epithelial cells and mesangial cells. There was minimal expression of TPH (Fig. 1C) and AADC (data not shown) in the medulla.


Figure 1
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  		Fig. 1. Renal expression of L-tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC). TPH, the initial and rate-limiting enzyme in serotonin biosynthesis, was detected by immunohistochemistry in kidney cortex (A, x160; B, x400) but not medulla (C, x160). TPH was predominantly expressed in proximal tubules (PT), glomerular epithelial cells (GEC), and mesangial cells. A similar expression pattern in the renal cortex was observed for AADC, which is also involved in serotonin biosynthesis (D, x400).

 
A similar expression pattern was found for SERT in C57/BL6J mice by in situ hybridization. While no staining was observed with the sense SERT probe, strong positive SERT staining was clearly evident in proximal tubules, mesangial cells, and glomerular epithelial cells with the SERT anti-sense probe (Fig. 2A). Of interest, although immunoreactive TPH and AADC expression appeared to be stronger in parietal glomerular epithelial cells, SERT mRNA expression was detectable in both parietal and visceral glomerular epithelial cells. These results indicated the colocalization of serotonin synthetic machinery and the serotonin transporter. Northern analysis confirmed higher expression of SERT in mouse cortex than medulla. Furthermore, there was SERT expression in freshly isolated mouse proximal tubules (Fig. 2B). In nine separate experiments, real-time PCR analysis confirmed that SERT expression was significantly higher in mouse cortex than medulla (Fig. 2C). A similar expression pattern of SERT mRNA was found in rat kidney (data not shown).


Figure 2
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  		Fig. 2. Renal expression of SERT. In situ hybridization (B, x400) with a SERT anti-sense probe indicated that SERT was present primarily in the mouse kidney cortex, with positive staining in GEC, mesangial cells (M), podocyte (P), and PT. By comparison, no positive signal was detected with the SERT sense probe (A, x400). Northern blot analysis (B) demonstrated higher SERT expression in mouse kidney cortex than medulla and confirmed high expression in a PT fraction. Real-time RT-PCR confirmed that the SERT was predominantly expressed in the mouse kidney cortex (C). *P < 0.01.

 
Serotonin receptor and transporter expression in MCT cells. Since 5-HT and its transporter were both expressed in proximal tubule cells in vivo, we measured the mRNA expression levels for 5-HT1A/1B/1D/1FR and 5-HT2A/2B/2CR by real-time RT-PCR using the mouse proximal tubular epithelial cell line, MCT, as an in vitro correlate. Of these receptor subtypes, 5-HT2BR displayed the highest relative expression, while 5-HT1AR and 5-HT1BR had the lowest relative mRNA expression (Fig. 3). mRNA expression of SERT was also confirmed in MCT cells by RT-PCR (data not shown).


Figure 3
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  		Fig. 3. Real-time RT-PCR analysis of 5-hydroxytryptamine (5-HT) receptor mRNA expression in mouse proximal tubular epithelial (MCT) cells. Total RNA was isolated from MCT cells and expression of 7 subtypes of 5-HT receptor mRNA was evaluated by real-time RT-PCR analysis. Data are expressed as means ± SE (n = 3).

 
Serotonin stimulated phospho-ERK1/2 expression through 5-HT2AR in MCT cells. 5-HT stimulated ERK1/2 phosphorylation in MCT in a dose-dependent manner (Fig. 4A). Compared with basal pERK1/2 expression (control), 5 min of exposure of 5-HT (100 nM or higher) led to increased pERK1/2. The induction of ERK1/2 phosphorylation was rapid and transient (Fig. 4B). ERK1/2 activation reached a peak at 5 min and returned to basal levels by 30 min.


Figure 4
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  		Fig. 4. 5-HT stimulated phosphorylation activity of ERK1/2 in MCT. A: serotonin stimulated ERK1/2 phosphorylation activity in a dose-dependent manner. B: serotonin stimulation of ERK1/2 phosphorylation was rapid and transient. C: serotonin-induced phosphorylation of ERK1/2 was partially abolished by ketanserin (a 5-HT2AR antagonist). D: DOI, a 5-HT2A agonist stimulated ERK phosphorylation. Top: blots were performed using a phospho-specific ERK1/2 antibody with arrows indicating pERK. Bottom: blots were reprobed with an antibody that recognized total ERK1/2 with arrows indicating ERK.

 
Ketanserin (a 5-HT2AR antagonist) dose dependently attenuated the 5-HT-induced ERK1/2 activation (Fig. 4C). In contrast, SB 206553 (a 5-HT2B/2CR antagonist, 100 nM), WAY 100635 (a 5-HT1AR antagonist, 100 nM), and GR 127835 (a 5-HT1B/1DR antagonist, 100 nM) all failed to inhibit 5-HT-stimulated ERK1/2 activation (data not shown). In addition, DOI, a 5-HT2A receptor-selective agonist, stimulated ERK1/2 activation at concentrations of 10 and 100 nM (Fig. 4D). These results indicated predominant involvement of 5-HT2AR in stimulation of ERK1/2 activity in this cultured proximal tubule cell line.

Serotonin stimulated the expression of TGF-beta and CTGF in MCT cells. The effects of 5-HT on the expression of TGF-beta and CTGF mRNA were investigated by real-time RT-PCR. TGF-beta expression increased fivefold after 1-h treatment of 100 nM 5-HT, with a return to baseline by 6 h, and 5-HT-induced TGF-beta expression was completely blocked by a MAPK kinase pathway (MEK) inhibitor, PD-98059 (Fig. 5, A and B). Similar to 5-HT, DOI also stimulated TGF-beta expression (Fig. 5C). These results suggest that 5-HT may stimulate TGF-beta expression via MEK-ERK pathway through activation of 5-HT2AR.


Figure 5
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  		Fig. 5. Real-time PCR analysis of 5-HT stimulation of TGF-beta mRNA in MCT. A: time course of 5-HT stimulation of TGF-beta expression. B: 5-HT-induced TGF-beta expression was abolished by the MAPK kinase (MEK) inhibitor, PD-98059. C: DOI stimulated TGF-beta expression in a time-dependent manner. *P < 0.01, compared with control, n = 3.

 
Similar to stimulation of TGF-beta, 5-HT also stimulated CTGF mRNA expression in MCT cells. However, CTGF expression was not detectable at 1 h (data not shown) but increased sevenfold after 6 h of treatment (Fig. 6). Both the inhibitor of the TGF-beta type I receptor kinase activity, SB 431542 (9), and the inhibitor of MEK, PD-98059, inhibited 5-HT-stimulated CTGF expression. DOI also stimulated CTGF expression. These data suggest that both TGF-beta and the MEK-EKR pathway are involved in 5-HT-mediated CTGF expression.


Figure 6
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  		Fig. 6. Real-time PCR analysis of 5-HT stimulation of connective tissue growth factor (CTGF) expression in MCT. 5-HT-induced CTGF expression (6-h treatment) was abolished by the TGF-beta receptor kinase inhibitor, SB 431542, and by the MAPK kinase (MEK) inhibitor, PD-98059. DOI also stimulated TGF-beta expression. *P < 0.01, compared with control. {dagger}P < 0.01, compared with 5-HT treatment, n = 3.

 
Serotonin receptor and transporter expression in IMP cells. Since TPH, AADC, and SERT were expressed not only in proximal tubules but also in glomerular epithelial cells (Figs. 1 and 2), IMPs were employed to examine the expression of serotonin receptor subtypes by real-time RT-PCR. Unlike the expression pattern in MCTs, the most abundant 5-HT receptor subtypes in IMPs were 5-HT2BR and 5-HT1BR (Fig. 7). Differences in expression patterns were also observed between differentiated and undifferentiated (proliferating) IMPs, with higher expression in the differentiated cells (Fig. 7). Expression of SERT correlated with expression of 5-HT receptor and was also higher in differentiated IMP cells, as determined by both Northern analysis (Fig. 8A) and real-time RT-PCR (Fig. 8B).


Figure 7
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  		Fig. 7. 5-HT receptor expression pattern in cultured immortalized mouse podocyte (IMP) cells by real-time RT-PCR. Data are expressed as means ± SE, n = 4.

 

Figure 8
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  		Fig. 8. SERT expression in IMP cells. A, top: Northern analysis of the SERT mRNA expression in the proliferating and differentiated IMP cells. Bottom: same membrane was reprobed with 18S rRNA as a loading control. B: real-time RT-PCR of the SERT expression in the proliferating and differentiated IMP cells. *P < 0.01, compared with the proliferating IMP cells. Data are expressed as means ± SE, n = 3.

 
Serotonin stimulated the expression of CTGF and VEGF in IMP cells. To investigate potential roles of 5-HT in glomerular extracellular matrix (ECM) accumulation and/or renal injury, the effects of 5-HT on CTGF, TGF-beta, and VEGF expression were investigated in vitro and in vivo. Real-time RT-PCR indicated that in IMP cells, 5-HT stimulated mRNA expression of CTGF nearly threefold after 1 h of treatment, and CTGF expression remained elevated for at least 6 h compared with untreated controls (Fig. 9). No significant stimulation of TGF-beta expression was observed in this cell type (data not shown). In contrast, expression of VEGF increased nearly 10-fold after 1 h, which also persisted at least 6 h after 5-HT treatment (Fig. 10A). Conditioned medium from overnight treatment with 100 nM 5-HT showed higher VEGF expression than in control conditioned medium (Fig. 10B). In vivo studies in rats demonstrated localization of VEGF expression mainly in glomerular podocytes, and administration of DOI for 3 days led to higher VEGF expression compared with control littermates (Fig. 10C).


Figure 9
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  		Fig. 9. Real-time RT-PCR of the 5-HT stimulation of the CTGF expression in the differentiated IMP cells at different time points. *P < 0.01, compared with control. Data are expressed as means ± SE, n = 3.

 

Figure 10
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  		Fig. 10. 5-HT stimulation of the vascular endothelial growth factor (VEGF) expression in vitro and in vivo. A: real-time PCR analysis of VEGF expression in differentiated IMP cells at different time points. *P < 0.01, compared with the control. Data are expressed as means ± SE, n = 3. B: Western blot analysis of VEGF expression in differentiated IMP cells. C: VEGF expression in glomeruli from the control and DOI-treated rats. Arrows indicated glomerular epithelial cells (x400).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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These studies sought to determine whether components of the serotonin system are intrinsic to the mammalian kidney. While serotonergic innervation can be a source of ligand, we examined whether intrinsic renal cells also possess the capacity to produce 5-HT. To investigate this hypothesis, we examined TPH, the initial and rate-limiting enzyme, and AADC, the catalyzing enzyme, in serotonin synthesis. We detected TPH expression by immunohistochemistry mainly in the renal cortex, especially in proximal tubule cells and in the glomerulus in epithelial and mesangial cells. Similar to TPH, AADC was also found to be primarily localized to the proximal tubule (Fig. 1). The localization of TPH and AADC in the current studies agrees with previous studies of TPH and AADC activity that indicated the highest activities of both enzymes were found in the proximal tubule of rat and/or human kidneys (18). Serotonin activity is regulated not only by the 5-HT receptors, but also by the serotonin transporter. Unlike the presence of many subtypes of 5-HT receptors, only one serotonin transporter has been reported to date (5, 10). In addition, we also determined that the expression pattern of the serotonin transporter correlated with the TPH/AADC expression pattern in the cortex (Fig. 2). Together, these results suggest the presence of an intrarenal serotonergic system.

Previous studies revealed the existence of 5-HT1R and 5-HT2R in rat mesangial cells and functional activation (ERK phosphorylation and proliferation) through 5-HT and 5-HT2AR interaction (3, 4). Since our findings suggested colocalization of 5-HT production and SERT in proximal tubules and podocytes as well as mesangial cells, mouse proximal tubule cells (MCT) (20) and IMPs (11) were selected as experimental models in this study. 5-HT2A/2BR and 5-HT1DR were the dominant 5-HT receptor subtypes (Fig. 3) in MCTs. Of the 5-HT1R and 5-HT2R antagonists tested, only the 5-HT2AR antagonist, ketanserin, dose dependently attenuated the 5-HT-induced ERK1/2 activation, and DOI, a selective 5-HT2AR agonist, stimulated ERK1/2 activation (Fig. 4). Therefore, the 5-HT2ARs seem to be the major receptor subtype mediating the increased ERK1/2 activity in MCTs by serotonin. Since even 100 nM ketanserin did not completely inhibit ERK1/2 activation, other pathways may also mediate the 5-HT-pERK signaling, in addition to 5-HT2AR activation.

IMPs are conditionally immortalized podocyte cells, with an ability to proliferate at 33°C and to differentiate at 37°C (11). Compared with MCT cells, a distinct serotonin receptor expression pattern was found in IMP cells (Fig. 7). The 5-HT2BR and 5-HT1BR were the major receptor subtypes in differentiated IMP, with higher expression in differentiated IMP cells compared with proliferating IMP cells (Fig. 7). Unfortunately, since even low concentrations of the 5-HT receptor antagonists led to IMP detachment and apoptosis, we were not able to explore the possible 5-HT receptor involvement in 5-HT stimulation of ERK1/2 activity in IMP cells (data not shown).

5-HT2B receptors have been implicated in cardiac valve fibrosis (16). To investigate further a possible role for 5-HT in ECM accumulation and/or renal injury, real-time RT-PCR was employed to measure the expression levels of TGF-beta and CTGF. The profibrogenic role of TGF-beta in various fibrotic diseases is well established. More recently, CTGF has also been proposed as a key cytokine responsible for elevated deposition of ECM proteins (21). In MCT cells, 5-HT treatment led to increased expression of both TGF-beta and CTGF, which was blocked by inhibition of MEK activity (Figs. 5 and 6). In addition, 5-HT-induced CTGF expression was completed blocked by the TGF-beta receptor kinase activity inhibitor, SB 431542 (9), indicating that CTGF expression was TGF-beta dependent, as has been described in mesangial cells (6). In mesangial cells, 5-HT stimulated both TGF-beta and CTGF expression through activation of 5-HT2AR (4, 6). However, in mesangial cells, activation of ERK is necessary for 5-HT-induced TGF-beta expression, but not 5-HT-induced CTGF expression (4, 6). The different role of ERK activation in 5-HT-induced CTGF in mesangial cells and MCT cells will require further investigation.

In contrast, we could not detect any significant increase in TGF-beta expression by 5-HT in IMP cells (data not shown) nor any effect of the TGF-beta receptor kinase inhibitor, SB 431542, on CTGF expression. In these cells, 5-HT significantly stimulated CTGF expression within 1 h of 5-HT administration (Fig. 9), suggesting a TGF-beta-independent 5-HT-induced CTGF expression pathway in IMP cells. We speculate that these results may be due in part to the different 5-HT receptor expression pattern between MCT cells and IMP cells, but further studies will be required to test this hypothesis.

VEGF can regulate vascular permeability, endothelial cell migration, proliferation, and survival. In renal cortex, the glomerular filtration barrier consists of glomerular visceral epithelial cells (podocytes), fenestrated endothelial cells, and an intervening glomerular basement membrane. VEGF-A and other VEGF isoforms are expressed in both developing and mature podocytes, and reductions in VEGF levels produce dramatic consequences for glomerular development and health (2). In IMP cells, VEGF-A expression was stimulated by 5-HT treatment (Fig. 10, A and B). In rats, podocyte VEGF-A expression was stimulated by DOI. We realize that the specificity of DOI decreases with increasing concentrations. However, this result at least suggests that activation of the 5-HT2A receptor subtype increases podocyte VEGF expression in vivo. Since alterations of VEGF-A and CTGF expression are found in diabetic nephropathy associated with podocyte loss (1, 15), a possible role of 5-HT-stimulated VEGF-A/CTGF expression in treatment of diabetic nephrophathy is worthy of further investigation. Further studies will be required to elucidate physiological and/or pathophysiological roles for serotonin in the kidney.


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 ABSTRACT
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This work was supported by National Institutes of Health Grants DK-51265 and DK-62794 and funds from the Department of Veterans Affairs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Harris, Division of Nephrology, Vanderbilt Univ. School of Medicine, MCN C3121, 1161 21st St. S., Nashville, TN 37232 (e-mail: ray.harris{at}vanderbilt.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.


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  1. Baelde HJ, Eikmans M, Lappin DW, Doran PP, Hohenadel D, Brinkkoetter PT, van der Woude FJ, Waldherr R, Rabelink TJ, de Heer E, Bruijn JA. Reduction of VEGF-A and CTGF expression in diabetic nephropathy is associated with podocyte loss. Kidney Int 71: 637–645, 2007.[CrossRef][Web of Science][Medline]
  2. Eremina V, Quaggin SE. The role of VEGF-A in glomerular development and function. Curr Opin Nephrol Hypertens 13: 9–15, 2004.[Web of Science][Medline]
  3. Gooz M, Gooz P, Luttrell LM, Raymond JR. 5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells. J Biol Chem 281: 21004–21012, 2006.[Abstract/Free Full Text]
  4. Grewal JS, Mukhin YV, Garnovskaya MN, Raymond JR, Greene EL. Serotonin 5-HT2A receptor induces TGF-beta1 expression in mesangial cells via ERK: proliferative and fibrotic signals. Am J Physiol Renal Physiol 276: F922–F930, 1999.[Abstract/Free Full Text]
  5. Guignabert C, Izikki M, Tu LI, Li Z, Zadigue P, Barlier-Mur AM, Hanoun N, Rodman D, Hamon M, Adnot S, Eddahibi S. Transgenic mice overexpressing the 5-hydroxytryptamine transporter gene in smooth muscle develop pulmonary hypertension. Circ Res 98: 1323–1330, 2006.[Abstract/Free Full Text]
  6. Hahn A, Heusinger-Ribeiro J, Lanz T, Zenkel S, Goppelt-Struebe M. Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. J Biol Chem 275: 37429–37435, 2000.[Abstract/Free Full Text]
  7. Haverty TP, Kelly CJ, Hines WH, Amenta PS, Watanabe M, Harper RA, Kefalides NA, Neilson EG. Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis. J Cell Biol 107: 1359–1368, 1988.[Abstract/Free Full Text]
  8. Kushwaha N, Harwood SC, Wilson AM, Berger M, Tecott LH, Roth BL, Albert PR. Molecular determinants in the second intracellular loop of the 5-hydroxytryptamine-1A receptor for G-protein coupling. Mol Pharmacol 69: 1518–1526, 2006.[Abstract/Free Full Text]
  9. Laping NJ, Grygielko E, Mathur A, Butter S, Bomberger J, Tweed C, Martin W, Fornwald J, Lehr R, Harling J, Gaster L, Callahan JF, Olson BA. Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542. Mol Pharmacol 62: 58–64, 2002.[Abstract/Free Full Text]
  10. Levy RJ. Serotonin transporter mechanisms and cardiac disease. Circulation 113: 2–4, 2006.[Free Full Text]
  11. Mundel P, Reiser J, Zuniga Mejia Borja A, Pavenstadt H, Davidson GR, Kriz W, Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248–258, 1997.[CrossRef][Web of Science][Medline]
  12. Olson TP, Snyder EM, Frantz RP, Turner ST, Johnson BD. Repeat length polymorphism of the serotonin transporter gene influences pulmonary artery pressure in heart failure. Am Heart J 153: 426–432, 2007.[CrossRef][Web of Science][Medline]
  13. Parbtani A, Frampton G, Yewdall V, Kasai N, Cameron JS. Platelet and plasma serotonin in glomerulonephritis. III. The nephritis of systemic lupus erythematosus. Clin Nephrol 4: 164–172, 1980.
  14. Pizzinat N, Girolami JP, Parini A, Pecher C, Ordener C. Serotonin metabolism in rat mesangial cells: involvement of a serotonin transporter and monoamine oxidase A. Kidney Int 56: 1391–1399, 1999.[CrossRef][Web of Science][Medline]
  15. Roestenberg P, van Nieuwenhoven FA, Joles JA, Trischberger C, Martens PP, Oliver N, Aten J, Hoppener JW, Goldschmeding R. Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice. Am J Physiol Renal Physiol 290: F1344–F1354, 2006.[Abstract/Free Full Text]
  16. Rothman RB, Baumann MH, Savage JE, Rauser L, McBride A, Hufeisen SJ, Roth BL. Evidence for possible involvement of 5-HT(2B) receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications. Circulation 102: 2836–2841, 2000.[Abstract/Free Full Text]
  17. Schoeffter P, Bobirnac I. 5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. Naunyn Schmiedebergs Arch Pharmacol 352: 256–262, 1995.[Web of Science][Medline]
  18. Sole MJ, Madapallimattam A, Baines AD. An active pathway for serotonin synthesis by renal proximal tubules. Kidney Int 29: 689–694, 1986.[Web of Science][Medline]
  19. Vinay P, Gougoux A, Lemieux G. Isolation of a pure suspension of rat proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 241: F403–F411, 1981.[Abstract/Free Full Text]
  20. Wolf G, Kalluri R, Ziyadeh FN, Neilson EG, Stahl RA. Angiotensin II induces alpha3(IV) collagen expression in cultured murine proximal tubular cells. Proc Assoc Am Physicians 111: 357–364, 1999.[CrossRef][Web of Science][Medline]
  21. Yokoi H, Mukoyama M, Sugawara A, Mori K, Nagae T, Makino H, Suganami T, Yahata K, Fujinaga Y, Tanaka I, Nakao K. Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol 282: F933–F942, 2002.[Abstract/Free Full Text]
  22. Zhang MZ, Harris RC, McKanna JA. Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal glucocorticoids and mineralocorticoids. Proc Natl Acad Sci USA 96: 15280–15285, 1999.[Abstract/Free Full Text]
  23. Zhang MZ, Yao B, Cheng HF, Wang SW, Inagami T, Harris RC. Renal cortical cyclooxygenase 2 expression is differentially regulated by angiotensin II AT(1) and AT(2) receptors. Proc Natl Acad Sci USA 103: 16045–16050, 2006.[Abstract/Free Full Text]



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