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【摘要】 Dopamine via activation of renal D 1 -like receptors inhibits the activities of Na-K-ATPase and Na/H exchanger and subsequently increases sodium excretion. Decreased renal dopamine production and sodium excretion are associated with hyperglycemic conditions. We have earlier reported D 1 -like receptor-G protein uncoupling and reduced response to D 1 -like receptor activation in streptozotocin (STZ)-treated hyperglycemic rats (Marwaha A, Banday AA, and Lokhandwala MF. Am J Physiol Renal Physiol 286: F451-F457, 2004). The present study was designed to test the hypothesis that oxidative stress associated with hyperglycemia increases basal D 1 -like receptor serine phosphorylation via activation of the PKC-G protein receptor kinase (GRK) pathway, resulting in loss of D 1 -like receptor-G protein coupling and function. We observed that STZ-treated rats exhibited oxidative stress as evidenced by increased lipid peroxidation. Furthermore, PKC activity and expression of PKC- I- and - -isoforms were increased in STZ-treated rats. In addition, in STZ-treated rats there was increased GRK2 translocation to proximal tubular membrane and increased basal serine D 1 -like receptor phosphorylation. Supplementation with the antioxidant tempol lowered oxidative stress in STZ-treated rats, led to normalization of PKC activity, and prevented GRK2 translocation. Furthermore, tempol supplementation in STZ-treated rats restored D 1 -like receptor-G protein coupling and inhibition of Na-K-ATPase activity on D 1 -like receptor agonist stimulation. The functional consequence was the restoration of the natriuretic response to D 1 -like receptor activation. We conclude that oxidative stress associated with hyperglycemia causes an increase in activity and expression of PKC. This leads to translocation of GRK2, subsequent phosphorylation of the D 1 -like receptor, its uncoupling from G proteins and loss of responsiveness to agonist stimulation.
【关键词】 G protein receptor kinases protein kinase C antioxidant streptozotocin SKF
RENAL DOPAMINE PLAYS AN IMPORTANT role in maintaining sodium homeostasis, particularly during increases in sodium intake. In the renal proximal tubule, dopamine inhibits the activity of sodium transporters such as the sodium hydrogen exchanger 3 and Na-K-ATPase, thus leading to natriuresis and diuresis ( 6, 15, 24, 26, 28 ). Dopamine exerts its action via stimulation of the D 1 -like (D 1 and D 5 in humans; D 1A and D 1B in rodents) and the D 2 -like (D 2, D 3, and D 4 ) receptors, which belong to the family of G protein-coupled receptors. However, the natriuretic and diuretic response to dopamine is due to activation of D 1 -like receptors in proximal tubules ( 2, 21, 22, 24 ). Moreover, of the D 1 -like receptors expressed in renal proximal tubules, only D 1 /D 1A (and not D 5 /D 1B ) receptors contribute to inhibition of sodium and water reabsorption by dopamine ( 1 ). Interestingly, dopamine receptor signaling is impaired in conditions associated with sodium retention, such as hypertension and diabetes. Furthermore, in hypertension and diabetes, sodium retention coexists with increased oxidative stress ( 37, 38 ).
Hyperglycemia associated with diabetes plays an important role in the generation of reactive oxygen species (ROS), leading to increased oxidative stress. Numerous studies using experimental models of both immune and nonimmune glomerular injury demonstrate ROS to be primary mediators in the pathogenesis of these disorders and show that the kidney is, in fact, susceptible to oxidative stress ( 8, 13, 33, 40 ). Enhanced oxidative stress has been documented in all three compartments of the renal cortex, i.e., glomeruli ( 19 ), tubulointerstitium ( 14 ), and vasculature ( 37 ). Studies in animal models of diabetes reveal that some of these functional and morphological abnormalities can be prevented by antioxidants, which act by lowering ROS ( 12, 27, 30, 31, 42 ).
We have reported that there is impaired renal D 1 -like receptor-G protein coupling, expression, and function in streptozotocin (STZ)-treated hyperglycemic rats ( 29 ). STZ-treated hyperglycemic rats are in a state of increased oxidative stress, and reducing oxidative stress via antioxidant supplementation normalizes various renal parameters in these rats ( 37 ). It is possible that defective renal D 1 -like receptor function in states of hyperglycemia could be a consequence of increased oxidative stress associated with hyperglycemia.
In this study, we tested the hypothesis that the defective renal D 1 -like receptor function in STZ-induced hyperglycemic rats is caused by increased oxidative stress. Therefore, decreasing the oxidative stress with the antioxidant tempol should restore D 1 -like receptor-G protein coupling, expression, and function. In addition, we explored the mechanisms, which may be responsible for D 1A receptor dysfunction in proximal tubules of STZ-induced hyperglycemic rats. These include the determination of D 1A receptor expression, receptor-G protein coupling, serine phosphorylation of D 1A receptors, PKC and G protein-coupled receptor kinase (GRK) expression in proximal tubules of control, hyperglycemic, and tempol-supplemented control and hyperglycemic rats.
MATERIALS AND METHODS
Induction of hyperglycemia and tempol supplementation. All experimental protocols were reviewed and approved by the University of Houston Institutional Animal Care and Use Committee. Male Sprague-Dawley rats were divided into four groups: 1 ) control rats (C); 2 ) STZ-treated rats (S); 3 ) control rats supplemented with tempol (CT); and 4 ) STZ-treated rats supplemented with tempol (ST; n = 6-8 rats/group). The C and CT groups were given a single intraperitoneal injection of the vehicle (5 mM sodium citrate, pH 4.5). The S and ST groups were made hyperglycemic by a single intraperitoneal injection of STZ (55 mg/kg). Rats were provided either tempol supplementation (1 mM in tap water; CT and ST groups) or tap water (C and S groups). Tempol supplementation was started 1 wk before STZ injection and continued for 1 wk additionally. All the groups had free access to drinking water. The animals were kept in individual cages, and their water intake was measured on a daily basis. Furthermore, the rats were weighed on a weekly basis.
Biochemical marker of oxidative stress. The quantification of the oxidation products, thiobarbituric acid-reactive substances, namely, malondialdehyde, was determined after homogenization of kidney cortical slices as described previously ( 4 ).
Preparation of renal proximal tubular suspension. An in situ enzyme digestion procedure ( 11 ) was used to isolate renal proximal tubules from all four groups. The proximal tubular suspension was used for the Na-K-ATPase assay and membrane preparation for subsequent experiments. Protein was determined by the bicinchoninic acid method (Pierce, Rockford, IL) using bovine serum albumin as a standard.
Measurement of basal PKC activity. Basal PKC activity in the kidney homogenate was measured using a nonradioactive PKC kit following the protocol supplied by the manufacturer (Promega, Madison, WI).
Preparation of proximal tubular membranes. Proximal tubular membranes were prepared as described previously ( 29 ). Briefly, proximal tubular suspensions were homogenized in a buffer (10 mM Tris·HCl, 250 mM sucrose, 2 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail; pH 7.4) and centrifuged at 20,000 g for 25 min at 4°C. The upper fluffy layer of the pellet was resuspended in the homogenization buffer and was considered the membrane fraction. The supernatant was considered to be the cytosolic fraction.
Western blotting of PKC- I, PKC-, GRK2, and D 1A receptor. Proximal tubular membranes (15, 15, and 40 µg proteins for PKC- I, PKC-, and GRK2, respectively) or the whole cell lysate (4 and 40 µg proteins for D 1A receptor and GRK2, respectively) and cytosolic fraction (40 µg protein for GRK2) were resolved by SDS-PAGE. The resolved proteins were electrophoretically transblotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). After incubation with primary and secondary antibodies, the membranes were incubated with enhanced chemiluminescence reagent, and the bands were visualized on X-ray film. The bands were quantified by densitometric analysis using Scion Image Software provided by the National Institutes of Health.
Immunoprecipitation-immunoblotting of serine-phosphorylated D 1A receptors. A previously described method ( 3 ) was used for immunoprecipitation of D 1A receptors from the proximal tubular cell lysate with slight modification. The immunoprecipitated samples (20 µl) were resolved by 10% SDS-PAGE, and the proteins were electrotransferred onto an Immobilon P membrane. A specific phosphoserine antibody (1:200) was used to detect serine phosphorylation on D 1A receptors. Horseradish peroxidase-conjugated secondary antibody (goat anti-mouse; 1:3,000) was used to probe the phosphoserine antibody, and the bands were visualized with an enhanced chemiluminiscence reagent kit. These immunoprecipitated samples were used for immunoblotting of D 1A receptors. Band density for serine-phosphorylated D1A receptors was normalized by band density for D 1A receptors.
Measurement of [ 35 S]guanosine 5'-O-(3-thiotriphosphate) binding. A [ 35 S]guanosine 5'- O -(3-thiotriphosphate) (GTP S) binding assay was performed as described earlier ( 20 ). Nonspecific [ 35 S]GTP S binding was determined in the presence of 100 µM unlabeled GTP S.
Radioligand [ 3 H]SCH-23390 binding. Binding of a D 1 -receptor antagonist, [ 3 H]SCH-23390, to the proximal tubular membrane was performed as described previously ( 23 ). Nonspecific binding was defined using 10 µM unlabeled SCH-23390.
Na-K-ATPase activity assay in proximal tubules. Na-K-ATPase activity was measured as previously described ( 20 ). It was measured as the function of liberated inorganic phosphate (P i ) in triplicate, was calculated as the difference between the total and ouabain-insensitive ATPase activity, and is represented as the percentage of basal, where basal was normalized to 100%.
Surgical procedures and experimental protocol for renal function studies. Rats were anesthetized with Inactin (100 mg/kg ip). Surgical interventions were made as described previously ( 29 ). The effect of SKF-38393 on sodium and water excretion was determined in all four groups. The protocol consisted of a 45-min stabilization period after completion of surgery followed by five consecutive 30-min collection periods: C1, C2, D, R1, and R2 (see Fig. 7 ). During C1 and C2, saline alone was infused; during D, SKF-38393 (1 µg·kg -1 ·min -1 in saline) was infused; and during R1 and R2 (recovery), only saline was infused. Urine samples were collected throughout the 30-min periods, and blood samples were collected at the end of each period. Plasma was separated by centrifuging blood samples at 1,500 g for 15 min at 4°C. Urine and plasma creatinine and sodium were measured as described previously ( 29 ). Urine flow, urinary sodium excretion, and fractional excretion of sodium were calculated as described previously ( 29 ). Blood glucose was measured by a glucose analyzer (Accuchek Advantage, Roche). Plasma insulin was measured by radioimmunoassay using a rat insulin kit (RI-13k, Linco Research, St. Charles, MI).
Fig. 1. Basal activity and expression of protein kinase C (PKC) in control (C), STZ-treated (S), and tempol-supplemented control (CT) and STZ-treated (ST) rats. A : PKC activity was measured as the ability to phosphorylate fluorescent-tagged peptide substrate using a nonradioactive PKC kit (Promega). B and C : proximal tubular membranes (15-20 µg protein) were prepared from proximal tubules and used for Western blotting for PKC- I and - as described in MATERIALS AND METHODS. Top : representative Western blots. A single band at 80 kDa was observed. Bottom : densitometric values. Values are means ± SE; n = 3-4. * P < 0.05 compared with control rats using 1-way ANOVA followed by Newman-Keuls multiple comparison test.
Fig. 2. G protein receptor kinase (GRK)2 protein distribution in control and STZ-treated rats. A : proximal tubular membrane. B : whole cell lysate. C : cytosol of control and STZ-treated rats. Proximal tubular membranes, whole cell lysate, and cytosol (30-40 µg protein) were prepared from proximal tubules and used for Western blotting for GRK-2 protein as described in MATERIALS AND METHODS. Top : representative Western blots. A single band at 80 kDa was observed. Bottom : densitometric values. Values are means ± SE; n = 3. * P < 0.05 compared with control rats using paired Student's t -test.
Fig. 3. GRK2 expression in the proximal tubular membranes of control (C), STZ-treated (S), and tempol-supplemented control (CT) and STZ-treated (ST) rats. Proximal tubular membranes (30-40 µg protein) were prepared from proximal tubules and used for Western blotting for GRK2 protein as described in MATERIALS AND METHODS. Top : representative Western blot. A single band at 80 kDa was observed. Bottom : densitometric values. Values are means ± SE; n = 3. * P < 0.05 compared with control rats using 1-way ANOVA followed by Newman-Keuls multiple comparison test.
Fig. 4. Serine phosphorylation of dopamine D 1A receptors (D 1A R) in proximal tubules of control (C), STZ-treated (S), and tempol-supplemented control (CT) and STZ-treated (ST) rats. Proximal tubular cell lysate was used for immunoprecipitation of D 1A R. Immunoprecipitated samples were then used for immunoblotting of serine-phosphorylated D 1A R and total D 1A R. Top : representative immunoblots of serine-phosphorylated D 1A R and total D 1A R. Bottom : densitometric analysis of serine-phosphorylated D 1A R protein normalized to immunoprecipitated D 1A R protein density. Values are means ± SE; n = 4. * P < 0.05 compared with control rats using 1-way ANOVA followed by Newman-Keuls multiple comparison test.
Fig. 5. G protein coupling and basal D 1A R abundance in renal proximal tubular membranes of control (C), STZ-treated (S), and tempol-supplemented control (CT) and STZ-treated (ST) rats. A : proximal tubular membranes from all 4 groups were incubated with [ 35 S]guanosine 5'- O -(3-thiotriphosphate) (GTP S), unlabeled GTP S (for nonspecific), and SKF-38393 (10 -6 mol/l) at 30°C for 60 min. Values are expressed as percent stimulation compared with basal binding. B : total number of D 1A R on the proximal tubular membrane as determined by binding of 20 nM [ 3 H]SCH-23390, a D 1 -like-receptor antagonist, to the proximal tubular membrane. Unlabeled SCH-23390 (10 µmol/l) was used for determining nonspecific binding. Specific binding was calculated as the difference between total binding and nonspecific binding. Values are means ± SE; n = 4. * P < 0.05 compared with control rats using 1-way ANOVA and Newman-Keuls test. # P < 0.05 compared with STZ-treated rats using 1-way ANOVA and Newman-Keuls test.
Fig. 6. Effect of dopamine D 1 -like-receptor agonist SKF-38393 on Na-K-ATPase activity in the proximal tubules of control (C), STZ-treated (S), and tempol-supplemented control (CT) and STZ-treated (ST) rats. Proximal tubular suspensions from all the groups were incubated with or without SKF-38393 (10 -6 mol/l) at 37°C for 15 min. Ouabain-sensitive Na-K-ATPase activity was measured as described in MATERIALS AND METHODS. Values are means ± SE; n = 4. Basal Na-K-ATPase activity (nmol P i ·mg protein -1 ·min -1 ) in proximal tubules of STZ-treated rats was similar in vehicle- and tempol-supplemented control rats and tempol-supplemented STZ-treated rats (98.32 ± 18.04; 90.26 ± 13.12; 108.18 ± 10.13). Basal activity was significantly higher in STZ-treated rats (138.2 ± 11.8). * P < 0.05 vs. control basal within the same group using ANOVA and Newman-Keuls test. # P < 0.05 vs. STZ-treated rats using unpaired Student's t -test. ¶ P < 0.05 vs. control rats using ANOVA and Newman-Keuls test.
Fig. 7. D 1 -like receptor-mediated natriuresis in vehicle- and tempol-supplemented STZ-treated and control rats. A : urine flow (UF). B : urinary sodium excretion (U Na V). C : fractional excretion of sodium (FE Na ). D : glomerular filtration rate (GFR) before, during, and after 1 µg·kg -1 ·min -1 SKF-38393. C1 and C2 : basal values before drug administration; D, values during drug administration; R1 and R2 : values after drug infusion was terminated. All the time intervals ( C1, C2, D, R1, and R2 ) were 30 min. Urine and plasma samples were collected for each time interval and analyzed for sodium and creatinine. UF, U Na V, and FE Na were calculated as described in MATERIALS AND METHODS. Values are means ± SE; n = 6-8. * P < 0.05 vs. control values of the same group using ANOVA and Newman-Keuls test. # P < 0.05 vs. control rats using Student's unpaired t -test.
Materials. The following chemicals and materials were purchased from the source indicated: [ 3 H]SCH-23390, [ 35 S]GTP S (DuPont New England Nuclear, Boston, MA); rabbit anti-rat D 1A receptor polyclonal antibodies, horseradish peroxidase-conjugated anti-rabbit antibodies (Alpha Diagnostics, San Antonio, TX); anti-mouse phosphoserine antibodies (Calbiochem, San Diego, CA); rabbit anti-human PKC- I, polyclonal antibodies, mouse anti-horseradish peroxidase-conjugated anti-mouse antibodies, protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA); and protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). All other chemicals were purchased from Sigma, St. Louis, MO, and were of highest grade available.
Data analysis. Where applicable, data are presented as means ± SE. The P value of <0.05 was considered statistically significant. Statistical analysis was done using GraphPad Prism, version 3.02 (GraphPad Software, San Diego, CA).
RESULTS
Effect of tempol supplementation on lipid peroxidation, fasting blood glucose, plasma insulin, body weight, water intake and cardiovascular parameters in control and STZ-treated rats. There was a significant increase in lipid peroxidation levels in the renal cortical slices from STZ-treated rats. Tempol supplementation reduced the oxidative stress, as reflected in a decrease in lipid peroxidation in these animals ( Table 1 ). STZ-treated rats had significantly higher fasting blood glucose levels and significantly lower plasma insulin levels compared with control rats. Tempol supplementation did not cause any significant change in the fasting blood glucose or the plasma insulin levels of STZ-treated rats ( Table 1 ). There was a significant reduction in the plasma insulin levels in the tempol-supplemented control rats, indicating that tempol may improve insulin sensitivity ( Table 1 ). The water intake was significantly higher in STZ-treated rats compared with control rats. Tempol supplementation did not alter water intake in STZ-treated rats ( Table 1 ). Furthermore, tempol supplementation in STZ-treated rats did not prevent the weight loss associated with STZ treatment ( Table 1 ). There were no differences in mean blood pressure and heart rate between vehicle- and tempol-supplemented control and STZ-treated rats ( Table 1 ). Kidney weight was significantly increased in STZ-treated rats, indicating some degree of hypertrophy. In tempol-supplemented STZ-treated rats, kidney weight was not significantly different from that of control rats ( Table 1 ). Glomerular filtration rate tended to be lower in vehicle- and tempol-supplemented STZ-treated rats compared with control rats, but this was not significantly different.
Table 1. Effects of tempol supplementation on basal parameters
Effect of tempol supplementation on PKC activity and expression in proximal tubular membranes from control and STZ-treated rats. Basal PKC activity was 65% higher in the STZ-treated rats compared with control rats ( Fig. 1 A ). Tempol supplementation significantly reduced PKC activity in STZ-treated rats ( Fig. 1 A ). In STZ-treated rats, there were an approximately fourfold increase in the expression of PKC- I and an approximately twofold increase in the expression of PKC- compared with control rats. Tempol supplementation normalized the increased PKC- I and PKC- protein expression in proximal tubular membranes of STZ-treated rats ( Fig. 1, B and C ).
Effect of tempol supplementation on GRK2 expression in proximal tubular membranes of control and STZ-treated rats. There was a 70% increase in GRK2 immunoreactivity in proximal tubular membranes isolated from STZ-treated rats compared with that of control rats ( Fig. 2 A ). Moreover, there was a significant decrease in GRK2 immunoreactivity in the cytosolic fraction of STZ-treated rats compared with control ( Fig. 2 C ), indicating an increased translocation of GRK2 to the proximal tubular membranes in the STZ-treated rats. There was no change in GRK2 immunoreactivity in the whole cell lysate from STZ-treated rats ( Fig. 2 B ). Tempol supplementation significantly reduced GRK2 protein density in the proximal tubular membranes from STZ-treated rats ( Fig. 3 ).
Effect of tempol on serine phosphorylation of D 1A receptors in proximal tubules of control and STZ-treated rats. In proximal tubules of STZ-treated rats, basal serine phosphorylation of D 1A receptors was about twofold higher compared with that of control rats ( Fig. 4 ). Tempol supplementation in STZ-treated rats caused a significant reduction in the basal serine phosphorylation of D 1A receptors, similar to the levels seen in control rats ( Fig. 4 ).
Effect of tempol on G protein coupling of D 1A receptors and D 1 -like receptor expression in proximal tubular membranes in control and STZ-treated rats. SKF-38393 increased [ 35 S]GTP S binding in proximal tubular membranes from control rats but not in STZ-treated rats ( Fig. 5 A ). However, when these rats were supplemented with tempol, stimulation of [ 35 S]GTP S binding by SKF-38393 was observed in the proximal tubular membranes. There was no difference in the basal [ 35 S]GTP S binding in proximal tubular membranes from the rats in all the four groups.
To determine whether tempol supplementation normalizes the D 1 -like receptor numbers on proximal tubular membranes of STZ-treated rats, single-point radioligand binding with 20 nM [ 3 H]SCH-23390 was performed. In vehicle-supplemented STZ-treated rats, there was an 68% reduction in specific [ 3 H]SCH-23390 binding. Tempol supplementation in STZ-treated rats significantly increased the specific [ 3 H]SCH-23390 binding ( Fig. 5 B ). However, the specific [ 3 H]SCH-23390 binding in tempol-supplemented STZ-treated rats was still significantly less than that in vehicle- and tempol-supplemented control rats ( Fig. 5 B ).
Effect of tempol on SKF-38393 induced inhibition of Na- K-ATPase activity in renal proximal tubules of control and STZ-treated rats. SKF-38393 (10 -8 -10 -6 mol/l) caused inhibition of Na-K-ATPase activity in proximal tubules from all four groups. However, the ability of SKF-38393 to inhibit Na-K-ATPase activity was significantly diminished in the STZ-treated animals ( Fig. 6 ). The maximal inhibition of 12% was produced by 10 -6 mol/l SKF-38393 in the proximal tubules of STZ-treated rats compared with 37% inhibition in control rats. Tempol supplementation in STZ-treated rats restored SKF-38393-induced inhibition of Na-K-ATPase activity; a maximal inhibition of 22% was produced by 10 -6 mol/l SKF-38393 in the proximal tubules of tempol-supplemented STZ-treated rats. SKF-38393-mediated inhibition of Na-K-ATPase activity was not significantly different in vehicle- and tempol-supplemented control rats. Although tempol supplementation in STZ-treated rats significantly restored SKF-38393-mediated inhibition of Na-K-ATPase, it was still significantly less compared with vehicle- and tempol-supplemented control rats ( Fig. 6 ). Basal Na-K-ATPase activity (nmol P i ·mg protein -1 ·min -1 ) in proximal tubules of STZ-treated rats was similar in vehicle- and tempol-supplemented control rats and tempol-supplemented STZ-treated rats (98.32 ± 18.04; 90.26 ± 13.12; 108.18 ± 10.13). Basal activity was significantly higher in STZ-treated rats (138.2 ± 11.8 nmol P i ·mg protein -1 ·min -1 ).
Effect of tempol on SKF-38393-mediated natriuresis in control and STZ-treated rats. Intravenous administration of SKF-38393 (1 µg·kg -1 ·min -1 ) produced significant increases in urine flow, urinary sodium excretion, and fractional sodium excretion in control rats ( Fig. 7, B and C ). However, SKF-38393 did not produce natriuresis in STZ-treated rats, although a modest diuresis was observed ( Fig. 7 A ). The basal urine flow was significantly elevated in STZ-treated rats compared with control rats ( Fig. 7 A ). The natriuretic response to SKF-38393 administration was restored in tempol-supplemented STZ-treated rats ( Fig. 7, B and C ). Also, SKF-38393 produced similar increases in urine flow and sodium excretion in vehicle- and tempol-supplemented control rats ( Fig. 7, A - C ). There was a significant increase in fractional excretion of sodium after SKF-38393 administration in tempol-supplemented STZ-treated rats and control rats ( Fig. 7 C ). However, the extent of increase in fractional excretion of sodium was less than the extent of increase in urinary sodium excretion ( Fig. 7 B ), indicating that tempol supplementation increased the SKF-38393-mediated natriuresis via increases in filtration fraction along with tubular mechanisms. Glomerular filtration rate in tempol-supplemented control rats tended to be higher than in control rats however, this was not significantly different ( Fig. 7 D ).
DISCUSSION
We have reported earlier that D 1 -like receptor-mediated natriuresis is impaired in STZ-treated rats as a result of reduced D 1 -like receptor expression on the proximal tubular membrane and receptor-G protein uncoupling ( 29 ). In this study, we have examined the mechanisms responsible for the dysfunction of D 1 -like receptors in STZ-treated rats. In addition, we studied the role of oxidative stress in D 1 -like receptor dysfunction by examining the effect of tempol, a superoxide dismutase mimetic, in restoring dopamine D 1 -like receptor function in STZ-treated hyperglycemic rats.
Although a large body of data has accumulated to indicate that hyperglycemia produces oxidative stress in humans as well as animal models ( 39, 41, 44 ), it is unclear whether this phenomenon is responsible for impaired D 1 -like receptor function in STZ-treated rats. The results presented in our study provide the evidence for the involvement of oxidative stress in D 1 -like receptor dysfunction observed in STZ-treated rats, as the membrane-permeable free radical scavenger tempol ameliorated oxidative stress and normalized D 1 -like receptor coupling, expression, and function. Therefore, we propose the following model ( Fig. 8 ). Hyperglycemia increases oxidative stress, which leads to increase in the PKC activity and expression of PKC- I and -. This, in turn, causes increased translocation of GRK2 to the proximal tubular membrane in STZ-treated rats. GRK2 causes hyper-serine phosphorylation of D 1A receptors in proximal tubules of STZ-treated rats. These hyper-serine-phosphorylated D 1A receptors fail to couple to G proteins, which, in turn, leads to impaired D 1 -like agonist-mediated inhibition of Na-K-ATPase activity and natriuresis in STZ-treated rats. Tempol supplementation, by reducing oxidative stress, prevents these changes, thus normalizing D 1 -like receptor function.
Fig. 8. Diagrammatic representation of hyperglycemia-induced oxidative stress and the mechanisms leading to D 1 -like receptor-G protein uncoupling and loss of functional response to D 1 -like receptor activation.
It is reported that increased phosphorylation of serine residues in D 1A receptors is responsible for the attenuation of the natriuretic effects of dopamine in various animal models, including spontaneously hypertensive rats, obese Zucker rats, and old Fischer 344 rats ( 3, 9, 43, 46 ). Similarly, we found that in proximal tubules of STZ-treated rats, the basal serine phosphorylation of D 1A receptors is higher compared with control rats. The hyper-serine phosphorylation of D 1A receptors could be responsible for the uncoupling of the receptors from G proteins and the subsequent failure of the agonist to produce natriuresis in STZ-treated rats. In this regard, it is important to note that higher basal serine phosphorylation of the D 1 (analogous to rat D 1A ) receptor has been reported in the proximal tubular culture from essential hypertensive patients ( 16, 36 ). Therefore, it appears that an increase in the basal serine phosphorylation of D 1A receptors in hypertension ( 16, 46 ), aging ( 3 ), obesity ( 7, 43 ), and hyperglycemia (as found in this study) leads to its uncoupling from G proteins and loss of downstream signaling components in proximal tubules of the kidney.
The receptor phosphorylation and desensitization by GRKs can occur if the receptors are activated in the presence of an agonist or with overexpression of GRKs ( 17, 35 ). In the present study, we found increased translocation of GRK2 to the proximal tubular membranes in the absence of an agonist in STZ-treated rats. Increased translocation of GRK2 is particularly interesting in light of the observation that increased GRK expression caused agonist-independent serine phosphorylation of renal D 1A receptors in essential hypertension, obesity, and aging ( 3, 16, 43 ). Moreover, an increase in GRK4 activity and expression in proximal tubular cells from humans with essential hypertension has also been reported ( 36 ). This higher GRK4 activity and expression may lead, in turn, to hyperphosphorylation of D 1A receptors and subsequent uncoupling of the receptors from G proteins and desensitization ( 36 ).
One possible explanation for the increased translocation of GRK2 could be increased PKC activity and expression in the proximal tubules of STZ-treated rats. PKC has been shown to provide anchors for GRK2 on the plasma membrane and increase GRK2 activity ( 25, 45 ). We found that STZ-treated rats had significantly higher PKC activity. Furthermore, protein expression of PKC- I and - isoforms was significantly increased in the proximal tubular membranes of STZ-treated rats. Moreover, studies from our laboratory in other animal models associated with oxidative stress such as old Fischer 344 rats and obese Zucker rats show that basal PKC activity is elevated in these animals, which is responsible for an increase in GRK2 translocation to the plasma membrane ( 4, 7, 43 ).
Next, we wanted to determine whether reducing oxidative stress normalizes D 1 -like receptor function. We chose tempol for our study because it is a stable, metal-independent, low-molecular weight, cell-permeable superoxide dismutase. It has been shown to have beneficial effects in normalizing blood pressure in various models of hypertension as well as in restoring renal dysfunction in hypertension and improving endothelial dysfunction in STZ-induced diabetes ( 18, 32, 38 ).
Tempol supplementation significantly decreased the levels of lipid peroxidation in the kidney of STZ-treated rats and thus reduced the oxidative stress in STZ-treated rats. In this study, tempol supplementation did not alter the fasting blood glucose or plasma insulin levels in the STZ-treated rats. Tempol supplementation has been reported to improve insulin sensitivity in rat models of insulin resistance, namely, Ren-2 rats and obese Zucker rats ( 7, 10 ). In our study, tempol supplementation did not alter insulin sensitivity in STZ-treated rats, as these animals were incapable of producing insulin. Interestingly, tempol supplementation seemed to improve insulin sensitivity in control rats.
We found that STZ-treated rats had significantly higher kidney weight compared with control rats; tempol supplementation reduced kidney weight in STZ-treated rats. Results from our study are in agreement with other reports in which tempol supplementation in disease models associated with oxidative stress such as Dahl salt-sensitive rats reduced the degree of renal hypertrophy and improved creatinine clearance ( 18 ).
Tempol supplementation significantly reduced PKC activity and PKC- I and - expression in STZ-treated rats. This is in agreement with previous work from our laboratory in which reducing oxidative stress in old Fischer 344 rats by tempol led to normalization of PKC activity ( 5 ) and with other groups where lowering oxidative stress in hyperglycemia led to normalization of PKC activity ( 34 ). There was also a significant reduction in protein expression of PKC- I and -. We speculate that this normalization of PKC activity led to decreased translocation of GRK2 to proximal tubular membrane and normalization of the basal serine phosphorylation of D 1A receptor in tempol-supplemented STZ-treated rats.
Furthermore, D 1 -like receptor-G protein coupling was completely restored in tempol-supplemented STZ-treated rats. It is possible that reducing basal serine phosphorylation of the D 1A receptor led to the normalization of receptor-G protein coupling in the proximal tubules of STZ-treated rats. Hence reducing oxidative stress and thus normalizing receptor-G protein coupling contributed to the restoration of D 1 -like receptor-mediated inhibition of Na-K-ATPase activity and natriuretic response in tempol-supplemented STZ-treated rats.
In summary, we have established that hyperglycemia-induced oxidative stress leads to an increase in basal serine phosphorylation of the D 1A receptor resulting from increased membranous translocation of GRK2 in STZ-treated rats. We also found that there was increased PKC activity and expression in the proximal tubules of STZ-treated rats, contributing to increased GRK2 translocation in proximal tubules of STZ-treated rats. Therefore, oxidative stress causes dopamine D 1 -like receptor dysfunction in STZ-treated rats, as reducing the oxidative stress leads to restoration of dopamine D 1 -like receptor-G protein coupling and function.
GRANTS
This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58743.
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作者单位：Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, Texas