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【摘要】 Interactions between transforming growth factor- (TGF- ) and nitric oxide (NO) are important in the pathophysiology of unilateral ureteral obstruction (UUO). Dopamine (DA) is a vasoactive renal mediator active at the D 1A receptor (D 1A R), which has not been studied in UUO; therefore, we examined the interactions among DA, TGF-, and NO in UUO. In vivo, UUO was carried out in rats with or without concurrent treatment with 1D11 , a monoclonal antibody to TGF-, for 14 days. In vitro, NRK-52E cells (normal rat kidney tubules) were treated with DA, and NO and TGF- release were examined. UUO resulted in a 70% decrease in the expression of renal D 1A R, confirmed by both Western blot analysis and immunohistochemistry. 1D11 treatment restored expression to 60% of control values. DA treatment decreased NRK-52E release of TGF- by 80%; conversely, DA significantly increased NO release from NRK-52E cells. These results suggest that DA modulates the release of cytokines, which are involved in the fibrotic and apoptotic sequelae of UUO, and that these effects are independent of DA's known vasoactive properties.
【关键词】 kidney obstructive uropathy apoptosis unilateral ureteral obstruction D A receptor
DOPAMINE ( DA ) IS A CATECHOLAMINE serving important roles both centrally and in the periphery ( 8, 13, 17 ). The central neurotransmitter role of DA in the regulation of motor function and behavior is well established. Historically, research on the dopaminergic system was dominated by the study of Parkinson's disease. More recently, DA has emerged as a peripheral hormone regulating renal sodium excretion, renal blood flow (RBF), and glomerular filtration rate (GFR) ( 1, 4, 17 ). DA exerts its biological effects through interaction with at least five known receptors ( 1, 17 ). The D 1A receptor (D 1A R) was recently localized to the renal vascular smooth muscle from the main renal artery to the afferent arterioles, in the proximal convoluted tubule, cortical collecting duct, and medullary thick ascending limbs ( 25, 26 ).
The dopaminergic system is targeted regularly by physicians. Low-dose DA is a common treatment to maintain RBF and GFR in clinical scenarios where renal perfusion is compromised. Due to its direct tubule suppression of sodium reabsorption, DA and D 1A R are now studied in the pathophysiology of hypertension ( 35 ). Radioligand study demonstrated that DA and DA agonists decrease angiotensin II (ANG II) binding and antagonize its stimulatory effects on renal sodium transport ( 28 ). In vitro treatment of proximal tubule cells with varying concentrations of DA was also shown to decrease mRNA levels of the ANG II receptor AT 1 ( 2 ). These tubule effects were reversed with blockade of D 1A R.
Our interests in the renal dopaminergic system as a paracrine modulator in the kidney stem from our previous studies on unilateral ureteral obstruction (UUO) in the rat. In the hours after UUO, documented changes in the kidney include a decline in GFR and in RBF ( 32 ). With continued obstruction, the kidney will develop marked interstitial edema, leukocytic infiltration, apoptosis, and interstitial fibrosis ( 10, 19 ). Various cytokines have been implicated in UUO, including those that promote fibrosis, apoptosis, and, perhaps, the resultant renal function decline, and those that protect the kidney from such damage. It has been previously shown that nitric oxide (NO) is antifibrotic and antiapoptotic in the UUO model and that NO synthase (NOS) levels are decreased in UUO ( 16, 23, 24 ). Transforming growth factor- (TGF- ), on the other hand, is upregulated in UUO and has been well established as a profibrotic, proapoptotic messenger in UUO ( 5, 18, 33 ). Additionally, TGF- has been shown to decrease NO production in tubule cells. Our laboratory, using an anti-TGF- antibody, and others, using antisense oligonucleotides, have shown that blockade of TGF- ameliorates tubular apoptosis, blunts interstitial fibrosis, and restores NOS levels ( 15, 22 ).
DA has been shown to release NO in endometrial tissue ( 31 ). Conversely, it was recently shown that NO can increase expression of D 1A R in proximal tubules ( 11 ). While there are several studies that examine the regulation of D 1A R expression in other systems (e.g., 13, 14, 17, 38 ), there are few that have examined such expression in UUO. Therefore, we examined the effect of UUO on D 1A R expression in vivo. Furthermore, because NO and TGF- can play opposing roles in the kidney in UUO, and NO and DA are known to interact, we examined the effect of DA on NO and TGF- release from a cultured proximal tubule cell line. In doing so, we attempted to link for the first time the actions of an intact dopaminergic system to known cytokines involved in UUO, NO and TGF-.
METHODS
In vivo UUO model. Sprague-Dawley rats underwent left unilateral ureteral ligation as described previously ( 18 ). Monoclonal antibody to TGF- (1D11 ) was generously provided (Genzyme) and was administered to three groups of rats at 0.5 mg/rat, 2 mg/rat, or 4 mg/rat, by intraperitoneal injection. To a separate group, an anti-verotoxin antibody (2 mg/rat) was administered as an isotype control. 1D11 was injected at days - 1, 0, 2, 4, 6, 8, 10, and 12 of UUO. Animals were euthanized at day 14, and both obstructed and unobstructed kidneys were harvested ( 22, 23 ). Mice with a targeted deletion of the inducible NOS gene (iNOS-/-; Jackson Labs) and their wild-type (WT) counterparts underwent 14-day UUO as described previously ( 12 ). 1D11 was administered daily at a dose of 0.2 mg/kg 1 day before UUO and daily for 14 days. Kidneys were harvested as above. Animal treatment adhered to approved institutional guidelines.
Western blot analysis for DA 1A R. Samples were prepared and Western blotting was carried out as previously described ( 22, 23 ). Seventy-five micrograms of protein, obtained from homogenized total kidney, were loaded per lane. The DA receptor is a typical G protein-coupled receptor and therefore traverses the membrane. Primary antibody, anti-D 1A R, specific for the third extracellular domain of the D 1A R (0.029 mg/ml, rabbit polyclonal, a generous gift from Dr. Robert Carey, University of Virginia), was diluted 2,000-fold and incubated for 1 h at room temperature. The antibodies obtained are specific for the D 1A R. After this, a 1-h incubation with goat-anti-rabbit-horseradish peroxidase (HRP)-conjugated secondary antibody was carried out. After being washed, streptavidin-HRP was applied to the membrane for 30 min, followed by washing and application of Opti-4-chloro-1 naphthol (HRP substrate) until development. The membrane was scanned, and the intensity of each band was quantitated by National Institutes of Health Image software (downloaded from the Internet). The D 1A R was detected as a band at 75 kDa. Intensity was expressed in arbitrary units. Western blot analysis was performed at n = 3-5/group. Intensities are presented as the average of the intensity for each group.
Immunohistochemistry for the DA 1A R. Wedges of hemisected obstructed and contralateral kidneys were prepared as previously described ( 22, 23 ). To retrieve antigen, slides were heated to 42°C for 12 min. Primary antibody, anti-D 1A R, was diluted 100-fold and incubated at room temperature for 1 h. For negative controls, the antibody was preabsorbed with peptide (GSEETQPFC, representing amino acids 299-307 of the D 1A R; see complete structure at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=protein&list_uids=118229&dopt=GenPept ). Biotinylated anti-rabbit secondary antibody (Vector) was incubated for 30 min. Sections were then incubated with avidin-biotin-peroxidase complex (Vector Labs) and developed with diaminobenzidene. After the slides were washed, counterstaining was done with 10% hematoxylin for 1-2 min.
Cell culture. NRK-52E cells, a clonal line established from normal rat kidney tubular epithelial cells ( 20 ), were obtained from the American Type Culture Collections (Rockville, MD). DMEM and FBS were obtained from GIBCO BRL (Grand Island, NY). Cells were seeded at a density of 5 x 10 5 cells/flask or 4 x 10 5 cells/well in a six-well plate and grown with DMEM plus 10% FBS. After attachment, media was replaced with 1% FBS alone or with respective treatment. Cells and media were collected at 9-, 16-, and 24-h intervals.
TGF- ELISA. Cells, at 4 x 10 5 cells/well, were grown in six-well plates until cell attachment was complete. Media was replaced with 1% FBS. DA (100 µM) was incubated for 9, 16, and 24 h. In addition, cells were incubated for 16 h with 5, 10, and 20 µM SCH-23390, a specific D 1A R antagonist, in combination with 100, 75, and 25 µM DA. Separately, 1 µM propranolol was incubated with 100 µM DA for 16 h. A cell count was performed just before media collection. Media of both control and treatment groups was collected and stored at -80°C until use. All samples were prepared and assayed using the Quantikine kit from R&D Systems (Minneapolis, MN). In brief, the samples were activated with 1 N HCl and neutralized with 1.2 N NaOH/0.5 M HEPES. The assay was completed using minimal light exposure. Samples were analyzed using a microplate reader set to 450 nm with correction set at 550 nm. Analysis was completed using dual-wavelength readings. A standard curve and all samples were run in duplicate. To ensure that DA would not affect the validity of the assay, we added 100 µM DA to a separate standard curve; no effect was noted. All experiments were repeated at n = 3-6.
Measurement of intracellular NO. NRK-52E cells were plated to six-well plates at a density of 4 x 10 5 cells/well and grown on 25-mm 2 glass coverslips. Cells were grown in media as described above. Cells were loaded with 20 µM membrane-permeant NO indicator diaminofluorescein-2-diacetate (DAF-2; Molecular Probes) for 1 h in the incubation medium. After the dye was loaded, the cells were rinsed with HEPES-buffered Na + -Ringer solution (pH 7.4). The dye used in this study is the same as that used by Matsuo ( 21 ), who studied NO release from human trabecular cells. The coverslip with the dye-loaded cells was then attached to the bottom of a flow-through superfusion chamber and mounted on the stage of an inverted epifluorescence microscope (Nikon Diaphot) interfaced to a Universal Imaging work station equipped with a frame-transfer type cooled charge-coupled device. The cells in the chamber were then superfused and maintained at 37°C as previously described ( 29 ). Cells were visualized with a Nikon CF Fluor oil-immersion objective ( x 40/1.3 numerical aperture). The cells were excited with 490-nm light from a 75-W xenon lamp with emission collected at 520 nm, at 15-s intervals for periods of about 10-15 min. The relative fluorescence intensity of the cells in the experimental field was analyzed singularly and independently from their neighbors. The relative change in intensity is expressed as a ratio of the difference between the average initial intensity (pre-DA) and peak intensity (post-DA exposure) divided by the initial intensity; n is the total number of cells studied.
Statistical analysis. Results are presented as means ± SE. ANOVA was used for analysis of Western blot analysis data, and Student's t -test was used for the in vitro experiments, with P < 0.05 being considered statistically significant.
RESULTS
D 1A R expression in UUO. We confirmed that there is a normal detectable level of D 1A R in the kidney ( Fig. 1 ). We sought to determine whether UUO would affect expression of D 1A R. Western blot analysis was carried out on kidney tissue samples collected as described above. As shown in Fig. 1, obstruction significantly decreased the expression of the D 1A R compared with the unobstructed control ( P < 0.01). The levels found in the obstructed kidney were decreased by 70% compared with that of the unobstructed control ( Table 1 ). To evaluate the influence that TGF- has on D 1A R expression in this model, we utilized an antibody to TGF-, 1D11 . Blockade of TGF- in a dose-dependent manner increased receptor expression ( Table 1 ). Statistical difference was achieved with the highest dose of 1D11 used ( P < 0.05). At this dose, the expression of D 1A R was increased more than twofold compared with the obstructed control kidney treated with control antibody.
Fig. 1. Expression of dopamine IA receptor (D 1A R) protein in contralateral and 14-day obstructed kidneys. Kidneys were obstructed in control rats or rats treated with 1D11 at doses of 0.5, 2, and 4 mg/rat as described in METHODS. Western blot analysis was carried out with an antibody specific for the D 1A R. Protein was identified at 75 kDa. Lane 1, contralateral kidney; lane 2, obstructed control kidney; lane 3, obstructed kidney treated with 0.5 mg 1D11 ; lane 4, obstructed kidney treated with 2 mg 1D11 ; lane 5, obstructed kidney treated with 4 mg 1D11 ; lane 6, additional contralateral kidney. Western blotting was performed with an n = 3-5/group.
Table 1. Dopamine 1A receptor quantification in rat kidney
Staining of tissue sections for the D 1A R enabled us to localize the receptor in both normal and obstructed kidneys. Receptor density was greatest in the proximal tubule cells of the medulla. Cell staining was homogenous in the control kidney ( Fig. 2 A ). Preincubation of antibody with peptide representing amino acids 299-307 of the D 1A R (see METHODS ) eliminated the staining ( Fig. 2 B ). Both inter- and intratubular staining was uniform throughout the slide. The obstructed kidney, however, lost such homogeneity. Rather, staining became heterogeneous, without a detectable pattern ( Fig. 2 C ). Most dramatic was the decreased staining found in dilated tubule cells in the obstructed groups.
Fig. 2. Immunohistochemistry for the D 1A R in rat kidneys. Kidneys were obstructed and harvested at 14 days of unilateral ureteral obstruction (UUO), and immunohistochemistry was carried out as described in METHODS. A : unobstructed kidney. B : unobstructed kidney+peptide. C : obstructed kidney. Magnification: x 200 ( n = 8 rats/group).
D 1A R expression in iNOS-/- mice. To examine the role of NO in the modulation of D 1A R, we utilized kidneys of iNOS knockout (KO) mice. WT and iNOS-/- mice were obstructed and treated with 1D11 or control antibody. Western blot analysis was performed on their kidneys. As shown in Fig. 3 and quantified in Table 2, there was no difference in D 1A R levels between the unobstructed WT group and the KO unobstructed kidneys. Furthermore, as was observed with rat kidneys, there was a significant decrease in receptor level in the obstructed kidney of both WT and KO mice, compared with the unobstructed control ( P < 0.01). When treated with 1D11 , kidneys of obstructed KO mice showed a decrease in D 1A R that was similar to that seen in untreated obstructed KO mice.
Fig. 3. Expression of D 1A R protein in control and 14-day obstructed kidneys in wild-type (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice. Kidneys were obstructed in WT or iNOS-/- mice and treated with 1D11 as described in METHODS. Western blot analysis was carried out with an antibody specific for the D 1A R. Protein was identified at 75 kDa. Lane 1, WT obstructed kidney; lane 2, WT contralateral kidney; lane 3, iNOS-/- 1D11 -treated obstructed kidney; lane 4, iNOS-/- 1D11 -treated contralateral kidney; lane 5, iNOS-/- control obstructed kidney; lane 6, iNOS-/- control contralateral kidney. Western blotting was performed with an n = 3-5/group.
Table 2. D 1A R Western blot quantification in iNOS-/- mice
Effect of DA on TGF- secretion in NRK-52E cells. We first confirmed via Western blot analysis that there is a detectable level of D 1A R in this cell line (data not shown). We treated NRK-52E cells for 9, 16, and 24 h with 100 µM DA as described. We found that these cells constitutively secreted TGF-. TGF- levels increased in the untreated control groups from 171.1 ± 18.5 pg/ml at 9 h to 353 ± 48.4 pg/ml at 24 h ( P < 0.02). As illustrated in Fig. 4, treatment of proximal tubule cells with 100 µM DA significantly decreased the secreted levels of TGF- found in the media. At 24 h, in the control group TGF- levels reached 353 ± 48.4 pg/ml. In the DA-treated group, TGF- levels at 24 h reached 72.3 ± 3.4 pg/ml, an inhibition of TGF- secretion by DA of 80% ( P < 0.02). Additionally, TGF- secretion in each treated group was significantly blunted by DA compared with its matched timed control ( P < 0.02). At 16 h, we show that DA can exert its effects in a dose-dependent manner. Compared with control at 16 h, 100 µM DA inhibited TGF- secretion by 75% ( P < 0.01), 75 µM DA inhibited TGF- secretion by 49% ( P < 0.01), and 25 µM DA inhibited TGF- secretion by 12% ( P < 0.01). Incubation of 1 µM propranolol with 100 µM DA blunted its effect noted at 16 h by 11% ( P < 0.01). Cell counts were unchanged compared with pretreatment values. Incubation of 5, 10, and 20 µM SCH-23390 with 100, 75, or 25 µM DA consistently resulted in significant cell death. Each concentration of inhibitor was incubated with each concentration of DA. Cell loss was 50% in all groups. TGF- concentration could not be accurately assessed in these groups.
Fig. 4. Transforming growth factor- (TGF- ) expression in dopamine-treated NRK-52E cells. Cells were treated with or without 100 µM dopamine for 9, 16, and 24 h, and TGF- secretion was quantified by ELISA.
Effect of DA on NO synthesis in NRK-52E cells. To ascertain whether DA can release NO from NRK-52E cells, DAF-2-loaded cells were acutely exposed to either 10 or 100 µM DA. If exposure to DA induces synthesis of NO in the cells, then the fluorescence of DAF-2 should increase. Figure 5 A is a representative trace of the relative fluorescence intensity of a DAF-2-loaded cell in the field of view. DA (100 µM) was added (arrow). As shown in the trace, the fluorescence increased in this cell 5 min after exposure to DA. NO production was spontaneous and varied over time points from cell to cell. Figure 5 B compares the response of the DAF-2-loaded cells to 10 and 100 µM DA. As shown in the graph, there was no increase in the relative DAF-2 fluorescence when 10 µM DA was used; however, 100 µM DA resulted in an increase in the DAF-2 fluorescence {-0.09 ± 0.01 [ intensity (peak - initial)/initial], n = 147 cells at 10 µM vs. 0.09 ± 0.01, n = 173 cells at 100 µM DA}, indicating that DA releases NO from NRK-52E cells. There was a significant increase ( P < 0.05) in the relative fluorescence of the cells treated with 100 µM DA compared with the pretreated fluorescence intensity (56,937 ± 1,360 arbitrary fluorescence units post-DA treatment vs. 52,202 arbitrary fluorescence units ± 1,065 pretreatment, n = 173). Figure 5 C is an example of the changes in fluorescence intensity, shown in pseudocolor, of images obtained in the same field of DAF-2-loaded NRK-52E cells before and after exposure to 100 µM DA. The blue pseudocolor represents the lowest fluorescence intensity, and red represents the highest fluorescence intensity. Both images were analyzed and are represented at the same settings. The relative fluorescence increased in the majority of the cells in this field with 7% increase in the DAF-2 fluorescence intensity.
Fig. 5. A : an increase in fluorescent intensity indicates nitric oxide (NO) release from NRK-52E cells. A representative trace of the relative fluorescence intensity of a diaminofluorescein-2-diacetate (DAF-2)-loaded cell is shown. Dopamine (DA; 100 µM) was added (arrow). Fluorescence increased in this cell subsequent to exposure to DA. B : comparison of NRK-52E cell fluorescence intensity in response to 10 and 100 µM DA. There was no increase in the relative DAF-2 fluorescence when 10 µM DA was used; however, 100 µM DA resulted in an increase in the DAF-2 fluorescence {-0.09 ± 0.01 SE, [ intensity (peak - initial)/initial], n = 147 cells at 10 µM vs. 0.09 ± 0.01, n = 173 cells at 100 µM DA}. C : DAF-2-loaded NRK-52E cells before and after exposure to 100 µM DA. Both images represent the same field of view. Blue pseudocolor represents the lowest fluorescence intensity, and red represents the highest fluorescence intensity. This representative field captures the increase in NO production by NRK-52E cells in response to 100 µM DA.
DISCUSSION
DA exerts its biological effect through interaction with several receptors; prominent among them is the D 1A R. In the kidney, it has been established using in vivo models that activation of the D 1A R causes direct inhibition of the Na + -K + -ATPase, with resultant natriuresis ( 13, 14 ). Acute blockade of the D 1A R with a receptor antagonist (SCH-23390) induces significant decreases in urinary flow rate and sodium excretion ( 6 ). In an in vivo model of obstruction, we found a decreased expression pattern of D 1A R after 14 days of UUO. Treatment of obstructed kidneys with the TGF- antagonist 1D11 blunted the decrease in receptor level. In vitro, DA increased NO release from NRK-52E cells and decreased the release of TGF-. These results suggest novel roles for DA in UUO, which may be unrelated to its previously demonstrated hemodynamic or tubular effects.
Renal interstitial fibrosis and tubular atrophy are principal histopathological prognostic indicators in UUO ( 10 ). Several candidate mediators of fibrosis have been identified, including TGF- and ANG II. It has been shown by many authors that when UUO develops, the obstructed kidney shows an increase in TGF-, with resultant tubular apoptosis and interstitial fibrosis ( 5, 7, 16, 22 ). Blockade of TGF- with monoclonal antibody, 1D11 , or antisense oligonucleotides blunts apoptosis and fibrosis ( 15, 22 ). Angiotensin-converting enzyme (ACE) inhibitors, AT 1 receptor antagonism, and reduction of the endogenous renin-angiotensin system via genomic manipulation of the angiotensinogen gene have all been shown to mitigate renal tubular and interstitial injury due to UUO ( 7, 16 ). Furthermore, blockade of AT 1 receptors has been shown to decrease TGF- expression in UUO in vivo ( 19 ).
Interestingly, expression of the AT 1 receptor in renal proximal tubule cells has been shown to be decreased with the addition of DA. DA (10 µM) administered to proximal tubule cells for 4 h decreased basal AT 1 levels by 67% and decreased 125 I-ANG II binding by 41% ( 2 ). In the present experiments, we showed that DA treatment of NRK-52E cells for 24 h blunted TGF- secretion by nearly 80% of the control value. Because 10 µM DA decreased AT 1 receptor expression in vitro, and in vivo blockade of AT 1 is associated with decreased TGF- expression, DA may be working through AT 1 receptors to exert its effects in the present experiment.
We attempted to block the effects of DA with a specific DA receptor blocker, SCH-23390. Unfortunately, in our experiment the combination of DA and SCH-23390 resulted in significant cell death. We used several doses of DA and SCH-23390 with the same results. We are unable to explain this result at this time, because SCH-23390 by itself had no effect.
DA at high doses may bind to other adrenergic receptors. To ensure that the observed effect was not due to nonspecific adrenergic binding, we utilized a nonspecific -blocker. We found that 1 µM propranolol incubated with 100 µM DA blunted DA's inhibitory effect by 11% ( P < 0.01). While this effect is statistically significant in our model, others have shown that similar doses of propranolol are much more effective at adrenergic blockade. Propranolol (2 µM) was able to block the apoptotic effect seen with norepinephrine (NE) treatment in a myocyte cell line by nearly 100% ( 3 ). In a perfused rat heart model, 1 µM propranolol completely blocked the arrhythmia-potentiating effect of NE ( 37 ). That propranolol only modulated TGF- secretion by 11% suggests that our findings with high-dose DA are not dependent on adrenergic receptors. It will be interesting to further elucidate the signal transduction by which DA and TGF- interact. Nevertheless, we identified for the first time a direct renal interaction between TGF- and the dopaminergic system. Although an interaction between TGF- and DA has been suggested in studies of Parkinson's patients ( 27 ), there has been no previous evidence of a direct effect of DA on TGF- synthesis. Furthermore, DA could modulate fibrotic events in UUO through its effects on TGF-.
In contrast, reciprocal interactions between DA and NO have been well documented. It has been shown that dopaminergic stimulation will upregulate neuronal NOS in a dose-dependent manner, most likely through D 1 receptors ( 34 ). Tseng et al. ( 31 ) reported that NO release is modulated by DA in endometrial tissue. It is suggested that D 1A R sensitization may be regulated by cAMP ( 1, 17 ). NO is known to induce cGMP levels. It was recently shown that NO can increase the expression of D 1A R in renal proximal tubule cells. Under normal cell culture conditions, the addition of L -arginine and other NO precursors to renal proximal tubule cells was shown to increase mRNA levels of D 1A R ( 11 ).
We found that DA induced NO production in renal proximal tubule cells. Using DAF-2, a dye which fluoresces in the presence of NO, and a real-time observation system, we followed cells treated with DA over a 900-s time course. We were able to monitor individual proximal tubule cells, both before and subsequent to DA stimulation. There was a significant increase ( P < 0.05) in the relative fluorescence of the cells treated with 100 µM DA compared with the pretreated fluorescence intensity. Our findings with DAF-2 are similar to those found in the study by Matsuo ( 21 ) examining trabecular cell NO synthesis, in which only four cells were monitored.
In contrast to TGF-, NO appears to be protective in the obstructed kidney. NO generation has been shown to ameliorate tubulointerstitial fibrosis in the case of UUO. Animals fed an arginine-supplemented diet were found to have increased levels of NO and an associated decrease in fibrosis compared with untreated animals ( 22 ). Using iNOS-/- animals, Hochberg et al. ( 12 ) were able to demonstrate that with UUO, WT mice kidneys had significantly less interstitial fibrosis and a smaller interstitial volume compared with the iNOS-/- kidneys. Additionally, after a 7-day obstruction, kidneys of iNOS-/- mice were found to have significantly more apoptotic cells than the obstructed kidneys in WT mice ( 23 ). As NO has been shown to upregulate the renal D 1A R, and we have shown that DA can release NO from NRK-52E, DA may be involved in a positive loop that supports the antifibrotic and antiapoptotic effects of NO.
Zeng et al. ( 37a ) showed that D 1A R expression is decreased in spontaneously hypertensive rats (SHR). Others have suggested that in SHR the inability of D 1A R agonists to produce natriuresis may include a defect in receptor G protein coupling, thereby impairing the transduction of the dopaminergic signal ( 13, 17 ). We have demonstrated a decline in D 1A R levels associated with a 14-day UUO. This was found in both rats and mice, suggesting that the decline is not a species-specific phenomenon. The decline in receptor expression was confirmed both by Western blot analysis and immunohistochemisty. Using Western blotting, the decrease at 14 days of UUO was 70%. However, there was not only a change in the amount of receptor but also in its distribution. In the unobstructed kidney, there is uniform D 1A R distribution. Such homogeneity is clearly lost with UUO. There is also clearly less staining in UUO compared with control. Change is not as dramatic using immunohistochemistry, as was determined using Western blotting because staining only illustrates membrane-bound receptors, whereas Western blotting represents both membrane-bound and vesicular receptors, i.e., total protein.
By using 1D11 and gene-deletion strategies, we also addressed the in vivo regulation of D 1A R. In rats treated with 1D11 , an antibody to TGF-, the decline in D 1A R in UUO was reversed. With high-dose 1D11 treatment, receptor levels were restored to 60% of normal. This suggests that TGF- may regulate the expression of D 1A R. In contrast, the iNOS does not appear to be involved in regulating D 1A R expression. We noted similar receptor expression levels in corresponding groups of mice in both the KO and WT mice. Similarly, the decreased expression of DA receptor in UUO was found in both KO and WT mice. However, iNOS may be important for the effects of TGF- on D 1A R expression, because the decrease in D 1A R was similar in both treated and untreated KO mice.
The findings in this report suggest that modulation of the dopaminergic system, with its interactions between NO and TGF-, could be protective to the kidney in UUO. The cellular mechanisms involved in the modulation of these systems remain to be elucidated. It is possible that TGF- increases D 1A R degradation or slows its production within the cell. Nonetheless, the increased TGF- found in UUO could be responsible for the decreased expression of the D 1A R. In addition, the interruption of an intact dopaminergic system in UUO may be one of the many factors that contribute to altered levels of NO and TGF-. This interruption in the DA pathway may contribute to the changes in RBF and GFR seen in UUO. These results suggest a role for DA in UUO and new pathways to be investigated.
Conclusion. In addition to its known hemodynamic effects and interaction with renal tubules, DA may be important in the pathological changes associated with UUO. UUO was found to result in decreased expression of the D 1A R. Furthermore, DA was shown to inhibit TGF- release and activate NO synthesis in renal proximal tubule cells. Taken together, these results suggest novel biological effects of DA in the kidney, which may be important in UUO and other renal fibrotic processes. DA and its interaction with the D 1A R may be new targets for therapeutic intervention in these diseases.
GRANTS
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58355 (D. Felsen) and New York Academy of Medicine, Edwin Beer Fellowship (D. Poppas).
ACKNOWLEDGMENTS
We thank Dr. Robert Carey, University of Virginia, for generously donating an antibody to the dopamine receptor and the Genzyme Corporation for generously donating 1D11 .
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作者单位：1 The Institute for Pediatric Urology, Department of Urology, New York Presbyterian Children‘s Hospital-Weill Cornell Medical College, and 2 Department of Physiology, Weill Medical College of Cornell University, New York, New York 10021