Dopamine-1 Receptor Coupling Defect in Renal Proximal Tubule Cells in Hypertension
Abstract—The ability of the dopamine-1 (D1)-like receptor to stimulate adenylyl cyclase (AC) and phospholipase C (PLC), inhibit sodium transport in the renal proximal tubule (RPT), and produce natriuresis is attenuated in several rat models of hypertension. Since the inhibitory effect of D1-like receptors on RPT sodium transport is also reduced in some patients with essential hypertension, we measured D1-like receptor coupling to AC and PLC in cultures of human RPT cells from normotensive (NT) and hypertensive (HT) subjects. Basal cAMP concentrations were the same in NT (n=6) and HT (n=4). However, the D1-like receptor agonist fenoldopam increased cAMP production to a greater extent in NT (maximum response=67±1%) than in HT (maximum response=17±5%), with a potency ratio of 105. Dopamine also increased cAMP production to a greater extent in NT (32±3%) than in HT (14±3%). The fenoldopam-mediated increase in cAMP production was blocked by SCH23390 (a D1-like receptor antagonist) and by antisense D1 oligonucleotides in both HT and NT, indicating action at the D1 receptor. The stimulatory effects of forskolin and parathyroid hormone–related protein of cAMP accumulation were not statistically different in NT and HT, indicating receptor specificity and an intact G-protein/AC pathway. The fenoldopam-stimulated PLC activity was not impaired in HT, and the primary sequence and expression of the D1 receptor were the same in NT and HT. However, D1 receptor serine phosphorylation in the basal state was greater in HT than in NT and was not responsive to fenoldopam stimulation in HT. These studies demonstrate the expression of D1 receptors in human RPT cells in culture. The uncoupling of the D1 receptor in both rats (previously described) and humans (described here) suggests that this mechanism may be involved in the pathogenesis of hypertension; the uncoupling may be due to ligand-independent phosphorylation of the D1 receptor in hypertension.
Dopamine produced by renal proximal tubules (RPT) has been shown to be a paracrine regulator of sodium transport in humans and in animals during sodium-replete conditions.1 During states of positive sodium balance, endogenous renal dopamine facilitates sodium excretion caused by a decrease in proximal as well as distal ion and water transport.1 2 3 4 5 The natriuretic effect of exogenous and endogenous renal dopamine (and dopamine-1 [D1]–like receptor agonists) is impaired in 2 animal models of hypertension.1 In the spontaneously hypertensive rat (SHR), the impaired natriuretic effect of dopamine and D1-like receptor agonists is associated with a decreased ability to inhibit Na+-H+exchanger1 3 6 and Na+,K+-ATPase activity in RPT.3 7 8 The decreased ability of dopamine and D1-like receptor agonists to inhibit these transporters has been related to a defective dopaminergic stimulation of second messenger production by adenylyl cyclase (AC), phospholipase C (PLC), and phospholipase A2.6 7 8 9 10 These phenotypes may be manifestations of a defective gene important in controlling blood pressure, since the defective dopaminergic regulation of RPT transport and sodium excretion cosegregates with hypertension in rats, and disruption of one of the D1-like receptor genes (D1A receptor) in mice produces hypertension.6
A defective regulation of renal proximal tubule sodium transport by D1-like receptors is also present in human essential hypertension.11 12 We hypothesized that the coupling between a D1-like receptor and the G protein/effector enzyme complex may also be defective in some patients with essential hypertension, similar to that seen in animal models of genetic hypertension. Therefore, we compared the effect of dopamine and fenoldopam on AC and PLC activity in human RPT in culture.
Two D1-like receptors have been cloned in mammals, the D1 and D5 receptors, also known as D1A and D1B, respectively, in rodents.1 Since the D1A receptor seems to be important in the regulation of blood pressure in mice,6 we studied the expression of the D1 receptor in the kidneys and RPT in culture from hypertensive (HT) and normotensive (NT) subjects. We also studied the expression and function of parathyroid hormone–related protein (PTHrP), since there is no impairment of the parathyroid hormone (PTH)/AC cascade in the 2 animal models of genetic hypertension that have an uncoupling of renal D1-like receptors from their effector enzyme complex.1 9
Source of Kidney Tissue
Histologically normal sections of fresh human kidneys from patients (NT subjects: n=6; mean age, 65 years; 3 men, 3 women; HT subjects: n=4; mean age, 65 years; 3 men, 1 woman) who had unilateral nephrectomy due to renal carcinoma or trauma were grown in culture. The hospital and outpatient records of the subjects were reviewed and classified into those with either normal blood pressure (n=6) or essential hypertension (n=4). All patients signed a consent form agreeing that the tissues taken from them become the property of the Department of Pathology and such tissues can be used for study. All studies were approved by the Institutional Review Board of the University of Virginia Center for the Health Sciences.
Human RPT (hRPT) cells were grown in serum-free medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium supplemented with selenium (5 ng/mL), insulin (5 μg/mL), transferrin (5 μg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL).13 hRPT cells (5×105 cells per well in 24 well plastic plates coated with 0.075% type I collagen), passage 6 and 7 (except when indicated), were incubated at 37°C in 95% O2/5% CO2 for 48 to 72 hours. Electron microscopic studies showed characteristics consistent with RPT origin.13 The cells express Na+-H+ exchanger, isoform 3 (NHE-3) and PTH receptors (NHE-3 and PTH receptors are present in proximal tubules; NHE-3 is not present in distal tubules) as well as a brush border marker enzyme, γ-glutamyltranspeptidase.
Determination of cAMP Accumulation
cAMP accumulation was determined in the presence of 1 mmol/L 3-isobutyl-1-methylxanthine in Dulbecco’s PBS.9 The cells were incubated at 37°C for 30 minutes with or without drugs: dopamine and the D1-like receptor agonist fenoldopam, the D1-like receptor antagonist SCH23390 (Research Biochemicals International), PTHrP, amino acids 1 to 34 (Bachem), and forskolin (Sigma Chemical Co).
Determination of PLC
Cytosol and membranes from hRPT were assayed for PLC activity as utilized in our laboratory with [3H] phosphatidylinositol-4,5-bisphosphate used as substrate.14 The reaction proceeded for 15 minutes at 37°C and was terminated by adding a stop solution containing 100 μL of 1% bovine serum albumin and 500 μL of 10% trichloroacetic acid.
Light Microscopic Immunohistochemistry
Antibodies were raised against synthetic peptide sequences: rabbit polyclonal antibody human D1 receptor GSGETQPFC (amino acids 299 to 307)15 and murine monoclonal antibody PTH/PTHrP receptor RRCDRNGSWELVPGHNRTWANYSE (amino acids 146 to 169).16 The polyclonal antibodies were immunoglobulin (IgG) or affinity purified (Research Genetics).
The cells and tissue sections were immersion-fixed in HISTOCHOICE for 10 minutes (hRPT) or 4 to 5 hours (kidney tissue) at room temperature and cryoprotected overnight at 4°C in PBS containing 30% sucrose, and immunohistochemistry was performed.17 The tissue sections, 5 to 10 μm thick, cut from paraffin blocks, and hRPTcells were incubated for 24 hours at 4°C with one of the following, diluted 1:500 in PBS: IgG- or affinity-purified D1 receptor polyclonal antibody or monoclonal anti-PTH/PTHrP receptor antibody, preimmune sera, IgG- or affinity-purified D1 or PTH/PTHrP receptor antibodies preadsorbed against their respective immunizing peptides. Immunostaining and visualization were detected with an avidin-biotin immunoperoxidase kit (Vectastain ABC kit, Vector Laboratories) and diaminobenzidine (Sigma Fast DAB Tablets, Sigma). The cells and tissue sections were lightly counterstained with hematoxylin.
The coincident location of the D1 and the PTH/PTHrP receptors was determined in HISTOCHOICE-fixed hRPT (20 to 30 minutes). The hRPT were exposed overnight at 4°C with primary antibodies (D1 at 1:500 and PTH/PTHrP at 1:200) in PBS blocking buffer (0.1 mol/L PBS, pH 7.4, 1% BSA, 1% nonfat dry milk, and 0.05% Triton X-100). Subsequent incubations were at room temperature. The secondary antibodies (biotinylated horse anti-mouse IgG antibody, 1:200, Vector Laboratories) were also diluted with PBS blocking buffer. Fluorescein-conjugated affinity-purified goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc) was applied at 1:100 for 30 minutes, followed by 0.1 mol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, and 0.5% Dupont Blocking Reagent for 30 minutes, streptavidin–horseradish peroxidase for 30 minutes, and tetramethylrhodamine tyramide in amplification diluent (1:50) for 10 minutes. Any remaining horseradish peroxidase activity was deactivated with 1% H2O2 in PBS (pH 7.0) for 15 minutes. Thereafter, the slides were incubated with antifluorescein–horseradish peroxidase (NEN Life Science Products) for 30 minutes and followed by a 10-minute incubation with 1:50 dilution of fluorescein tyramide in ×1 amplification diluent. Immunostaining was detected with the Tyramide Signal Amplification kit (NEN Life Science Products) followed by immunofluorescence visualization.
Immunoblotting and Immunoprecipitation
The tissues or cells were lysed with ice-cold lysis buffer (PBS with 1% NP40, 0.5% Na cholate, 0.1% SDS, 1 mmol/L PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mmol/L sodium vanadate). The lysates were incubated with D1 antibody for 1 hour followed with protein-A agarose for 12 hours with rocking at 4°C. The proteins separated by SDS–polyacrylamide gel electrophoresis were electrophoretically transferred onto nitrocellulose membranes. The transblot sheets were blocked with 5% to 10% nonfat dry milk in 10 mmol/L Tris-HCl, pH 7.5 and 0.1% Tween-20 and incubated with diluted affinity-purified polyclonal antiphosphoserine antibody (Zymed Laboratory)18 for 1 hour at room temperature or overnight at 4°C. The transblots were washed with buffer and then incubated with peroxidase-conjugated affinity-purified donkey anti-rabbit IgG for 1 hour at room temperature. The immunoblots, visualized with ECL System (Amersham), were quantified by densitometry.14
Determination of D1-like Receptor Specificity
We determined the involvement of the D1 receptor using antisense phosphorothioate/propyne-modified oligonucleotides purified by high-performance liquid chromatography (Genosys). Sense (5 nmol/L) (5′ ATG AGG ACT CTG AAC ACC 3′) and antisense (5 nmol/L) 3′ GGT GTT CAG AGT CCT CAT 5′) oligonucleotides were incubated with cultures of hRPT for 4 to 16 hours, washed, and reincubated for another 4 to 48 hours (total incubation time of 20 to 48 hours). The ability of antisense but not sense oligonucleotides to prevent expression of the D1 receptor was verified by Western blotting.
The sequences of the cDNA from the reverse transcriptase–polymerase chain reaction products of RNA from hRPT in culture were determined by the Sanger dideoxy chain termination method with Sequenase (US Biochemical).
Data are reported as mean±SE. Within-group differences were determined by ANOVA for repeated measures and Scheffé’s test. Differences between HT and NT were determined by t test.
Enzyme Activity Studies
Basal cAMP accumulation in hRPT was not statistically different between NT (n=6) and HT (n=4) subjects. The D1-like receptor agonist fenoldopam increased cAMP accumulation in hRPT to a greater extent in NT than in HT patients (Figure 1⇓). The maximum response was 67±1% in NT and 17±5% in HT, with a potency ratio of 105. Dopamine (10−7 mol/L) also increased cAMP accumulation to a greater extent in NT (32±3%, 8 experiments, 3 subjects, passages 6 to 10) than in HT (14±3%, 7 experiments, 2 subjects, passages 6 to 9). The differential effect of dopamine and fenoldopam on cAMP accumulation in hRPT from NT and HT subjects was not affected by the number of passages from 5 to 11 (data not shown). The effect was exerted at the D1-like receptor since the D1-like receptor antagonist SCH23390 significantly diminished the stimulatory effect of fenoldopam (Figure 2⇓) and dopamine (data not shown) on cAMP accumulation in hRPT cells from both NT and HT.
The differential effect of dopamine and fenoldopam on cAMP accumulation in hRPT from NT and HT was not due to intrinsic differences in AC enzyme because forskolin (100 nmol/L), which stimulates AC directly, increased cAMP accumulation to the same extent in NT and HT (Figure 3⇓). There was receptor specificity of the differential effect of dopamine and fenoldopam on cAMP accumulation in hRPT from NT and HT since PTHrP (10−11 to 10−6 mol/L) increased cAMP accumulation to the same extent in NT and HT (Figure 3⇓).
Previous studies in rodents have shown that the D1-like receptor is linked to both AC and PLC activation.1 7 10 The ability of D1-like receptor agonists to stimulate AC and PLC activity is attenuated in rat models of genetic hypertension.1 6 7 8 9 10 However, in hRPT, fenoldopam (5 μmol/L) stimulated PLC activity to a greater extent in HT than in NT at 1 minute (Figure 4⇓). Since the cells were studied contemporaneously, the preservation of the PLC, forskolin, and PTHrP response but not the cAMP response in HT may be taken to indicate that a selective dedifferentiation of HT cells did not occur with multiple passaging.
Light Microscopic Immunohistochemistry
Kidney Section Studies
Immunohistochemical staining for the D1 receptor was found in proximal and distal tubules and cortical collecting ducts but was absent in glomeruli, juxtaglomerular apparatuses, small blood vessels, and medulla.17 No staining was observed in consecutive sections processed with either the antibody preadsorbed against the immunizing peptide or the preimmune serum.17 As with the D1 receptor, the PTH/PTHrP receptor was present in both proximal and distal tubules in HT and NT (not shown). No staining was observed in consecutive sections processed with the antibody preadsorbed against the immunizing peptide (not shown).
RPT Cells in Culture
There were no differences in the immunostaining pattern of the D1 and PTH/PTHrP receptors between hRPT cells from NT and HT (not shown). No staining was observed in consecutive sections processed with either the antibodies preadsorbed against the immunizing peptide or preimmune serum (for the D1 receptor) (not shown).
Immunofluorescence of D1 and PTH/PTHrP Receptors in hRPT
The presence of D1 and PTH/PTHrP receptors in hRPT was corroborated by the immunofluorescence studies; there were also no differences in the immunofluorescence pattern of the D1 and PTH/PTHrP receptors in hRPT cells from NT and HT (Figures 5⇓). No staining was observed with either the antibodies preadsorbed against the immunizing peptide or preimmune serum (for the D1 receptor) (not shown).
D1 Receptor Sequence, Immunoblotting, and D1-like Receptor Specificity
There were no differences in the primary sequence (coding and noncoding region) of the D1 receptor gene (generated by reverse transcriptase–polymerase chain reaction of RNA from hRPT) between NT and HT (data not shown). Western blot analysis revealed that bands of ≈55 to 60 kDa, 70 to 90 kDa, and 113 kDa were detected by the D1 antibody in renal cortex and hRPT from NT and HT; no bands were observed in preimmune serum (data not shown). There were no differences in the density of immunostaining between NT (51.6±2.1% area, n=4) and HT (48.4±2.1%, n=5) at either the 55- to 60-kDa or 70- to 90-kDa band. Antisense but not sense oligonucleotides inhibited translation of the D1 receptor and completely blocked the ability of fenoldopam to stimulate cAMP accumulation in HT and nearly so in NT (Figure 6⇓). Thus, the D1-like receptor involved in the fenoldopam-stimulated cAMP accumulation is due mainly to the D1 receptor subtype.
D1 Receptor Phosphorylation
The uncoupling of the D1-like receptor from its G protein/AC complex is similar but distinct from homologous desensitization that is due to phosphorylation of the receptor after ligand occupation.1 19 We hypothesized that the D1 receptor in RPT in hypertension may be already phosphorylated even in the basal state and is therefore essentially desensitized. Since the D1 receptor is phosphorylated at its serine residues,19 we compared the serine phosphorylation of the receptor with and without 1 μmol/L fenoldopam. As shown in Figure 7⇓, the quantity of serine-phosphorylated D1 receptor is greater in HT than in NT at basal conditions. Moreover, fenoldopam increased the phosphorylation of the D1 receptor in NT but not in HT subjects. These data are in accord with the hypothesis that the decreased ability of fenoldopam to stimulate cAMP accumulation in HT subjects is associated with increased phosphorylation of the D1 receptor in the basal state.
These studies show a selective defect in the D1-like receptor signal transduction pathway involving cAMP in hRPT in culture from human subjects with essential hypertension. The impaired action of D1-like receptors on cAMP accumulation is not due to an aberrant AC enzyme or an epiphenomenon, since forskolin and PTHrP stimulated cAMP accumulation to the same extent in both groups. Because the human proximal tubule cells were studied after several passages, the possible confounding effect of dedifferentiation needs to be considered. However, the preservation of the responses to forskolin and PTHrP makes it unlikely that selective dedifferentiation of the hRPT cells from HT subjects caused the differential response to the D1-like receptor. Moreover, basal and PTHrP-stimulated cAMP levels were also similar to those reported by others using microdissected and cultured human proximal tubules.20 21
The decreased ability of D1-like receptors to stimulate cAMP production in hRPT from subjects with essential hypertension is similar to observations in RPT and arterioles in animal models of genetic hypertension.1 8 9 22 Because the D1-like receptor–mediated natriuresis is, in part, caused by cAMP, we have suggested that the impaired natriuretic effect of D1-like receptors in the SHR is in part due to decreased D1-mediated stimulation of cytoplasmic second messengers.1 The preservation of the natriuretic effect of the D1-like agonist fenoldopam in essential hypertension may be due to an intact D1-like response at more distal sites along the nephron (eg, cortical collecting duct).12 The ability of guanine nucleotides to stimulate AC activity in the RPT is also not impaired in the SHR, suggesting that the defect is probably located proximal to G proteins, presumably at a D1-like receptor.1 9 There is organ specificity of the defect since fenoldopam stimulated AC activity in the striatum of the brain to the same extent in SHR and its normotensive control.1 There is receptor specificity since PTH stimulated AC activity to the same extent in the RPT of these rat strains, an observation similar to that noted in the present study.1 9 There is, however, one appreciable difference. In the SHR, the ability of dopamine and D1-like agonists to stimulate PLC and phospholipase A2 activity in RPT is also impaired.7 10 In the present study, D1-like activation of PLC may be actually greater in HT than in NT subjects. The significance of the apparently greater increase in PLC activity after D1-like receptor stimulation in HT than in NT remains to be determined.
The 2 D1 receptor subtypes (D1 and D5 subtypes) cloned in mammals are expressed in RPT.1 There is no difference in the amount or distribution (data not shown) of D1 or D5 receptors in the kidney of HT and NT subjects. The apparent uncoupling of a D1-like receptor from AC is also not due to decreased receptor density, similar to studies in genetically hypertensive rats.1 9 The ability of antisense D1 receptor oligonucleotide to completely block the stimulatory effect of fenoldopam on cAMP accumulation in HT, and nearly so in NT, indicates that D1 receptor function predominates over D5 receptor function. Moreover, we have reported that disruption of the D1A receptor in mice increases blood pressure and produces diastolic hypertension.6 Although a defect of the D5 receptor or its regulation may be involved in hypertension, this dopamine receptor subtype has not been linked to PLC activation.1 It is also unlikely that a novel D1-like receptor linked to PLC is involved in hypertension because D1-like receptor–mediated PLC activation is not impaired in hRPT from HT subjects.
We have not found a mutation of the D1 receptor in our patients with essential hypertension; we have not found a mutation in the D1A or D1B receptor in genetically hypertensive rats, either.23 We have suggested that the uncoupling of D1-like receptor from its effector complex in RPT is akin to homologous desensitization.1 However, the desensitization is ligand independent since it occurs in cultures of RPT that are not exposed to dopamine. (RPT cannot synthesize dopamine in the absence of L-DOPA.1 ) The present studies show that the renal proximal tubular D1 receptor is “hyper”-serine-phosphorylated in HT compared with NT and does not respond to fenoldopam stimulation, similar to the cAMP accumulation studies. Since G protein–coupled receptor–related kinases (GRKs) are involved in the phosphorylation of receptors, resulting in their desensitization, it is of interest that GRK activity and GRK2 expression are increased in lymphocytes of patients with essential hypertension.24 GRK2 is involved in the phosphorylation of several G protein–coupled receptors, including D1 receptors.25 Increased activity of GRK2 could be responsible for the desensitization of the D1-like receptor. However, increased GRK2 activity does not explain the apparent importance of the kidney in the pathogenesis of essential hypertension, since GRK2 is expressed in many organs other than the kidney. Because GRK2 is involved in the desensitization of PTH receptor,25 an increase in GRK2 activity should have resulted in the desensitization of the PTH receptor in hypertension. However, renal PTH action is not impaired in rats with genetic hypertension and in humans with essential hypertension.1 9 Thus, the desensitization of the D1 receptor in hypertension may be due to a kinase but not necessarily GRK2.
In summary, we have found that dopamine D1 and PTH/PTHrP receptors are expressed in RPT of human kidneys. Although the distribution and quantity of D1 receptor expression are similar in RPT from NT and HT subjects, the ability of dopamine and a D1-like receptor agonist to stimulate cAMP production is impaired in HT subjects. The impairment is not due to a defective AC enzyme akin to the ligand-independent desensitization reported in genetically hypertensive rats. The defect is also receptor specific because PTHrP responsiveness is intact. Although the relationship between the increased serine phosphorylation of the D1 receptor and ligand-independent uncoupling of a D1-like receptor remains to be determined, it is suggested that this uncoupling may be involved in the pathogenesis of essential hypertension.
This study was supported in part by grants from the National Institutes of Health (HL23081, DK39308, and DK44756).
Previously published in abstract form (J Am Soc Nephrol. 1997;8:307A and J Invest Med. 1997;45:277A).
- Received July 16, 1998.
- Revision received August 18, 1998.
- Accepted December 7, 1998.
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