Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney
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糖尿病学杂志 2006年第6期
1 Division of Nephrology and Hypertension, Oregon Health and Science University, Portland, Oregon
2 Department of Medicine, Oregon Health and Science University, Portland, Oregon
3 Diabetes Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
4 Research Service, Portland VA Medical Center, Portland, Oregon
5 Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon
CAV-1, caveolin-1; eNOS, endothelial nitric oxide synthase; NOS, nitric oxide synthase
ABSTRACT
Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.
Experimental diabetes is associated with complex alterations in renal nitric oxide (NO) bioavailability and signaling. Studies exploring the diabetic renal NO system in different experimental settings have produced inconsistent and seemingly contradictory findings (1,2). For instance, in vitro studies suggest defective endothelium-dependent (i.e., endothelial NO synthase [eNOS]eCdependent) NO production or function in the diabetic kidney (3eC6). These studies contrast with observations from in vivo studies with NO synthase (NOS) inhibitors, which show substantial NO dependency of renal hemodynamics in hyperfiltering diabetic rats (7eC9).
To function as an endothelial NO-producing enzyme, eNOS (NOS III) requires a battery of cofactors, posttranslational modifications such as phosphorylation and dimerization, protein-protein interactions, and subcellular targeting (10). Phosphorylation of eNOS on Ser1177 by several serine/threonine kinases, such as Akt (protein kinase B) (11,12) or protein kinase A (13), in response to a variety of physiological stimuli is a critical control step for NO production by the enzyme. This process enhances the rate of electron flux from the reductase to the oxygenase domain of eNOS and reduces the calcium requirements for the enzyme, thus increasing NO synthesis (14). Homodimerization of eNOS is also crucial for NO production (15). Formation of a homodimer is crucial for NO production by eNOS (15,16), creating high-affinity binding sites for the NOS substrate L-arg and enabling electron transfer from the reductase domain of one NOS monomer to the oxygenase domain of the other (17).
Activation of eNOS is not only dependent on phosphorylation by upstream kinases and conformational changes but is also determined by its specific subcellular localization. A large pool of eNOS representing the activatable enzyme is localized within the plasma membrane and enriched in specialized structures called caveolae (18). Plasmalemmal caveolae are membrane invaginations that serve as domains for the sequestration and organization of a large number of molecules, including receptors and their downstream signaling effectors and modulators, enzymes, membrane transporters, structural molecules, and lipids (19,20).
Caveolin-1 (CAV-1) is the main structural component of caveolae in endothelial cells. It acts as a scaffolding protein and is involved in modulation of receptor signaling and function of caveolar enzymes (19,20). In unstimulated endothelial cells, eNOS is inhibited by its protein-protein interaction with CAV-1. The pathway of eNOS activation upon stimulation by agonist involves mobilization of intracellular Ca2+ and consequent interaction of calmodulin with eNOS. The eNOS/calmodulin interaction allows the release of eNOS from an inhibitory complex with CAV-1 (21,22). Therefore, alterations in CAV-1 abundance and eNOS interactions can impact eNOS function and consequently vascular function and modeling. A better understanding of the role of CAV-1 in mediating cellular functions in diabetes is needed for elucidation of NO pathophysiology in the diabetic kidney.
Although inactivation of NO by reactive oxygen species has been suggested as a major mechanism responsible for reduced bioavailability of eNOS-derived NO in diabetes (23), other factors related to direct changes in eNOS function and molecular integrity have also been suggested (24). In the present study, we explored the renal cortical expression of eNOS with respect to some of its functional determinants, including the cellular localization, phosphorylation status, and dimer/monomer formation, in normal and diabetic rats. Furthermore, we examined renal cortical expression and localization of the endogenous eNOS inhibitor CAV-1 and its colocalization with eNOS.
RESEARCH DESIGN AND METHODS
Studies were conducted in male Sprague-Dawley rats, with initial weights of 300 g. Rats were made diabetic with streptozotocin (65 mg/kg body wt i.p.; Sigma, St. Louis, MO). Diabetes was confirmed by measurement of tail blood glucose level using a reflectance meter (One Touch II; Lifescan, Milpetas, CA). Diabetic rats received daily evening injections of ultralente insulin (Iletin II; Eli Lilly, Indianapolis, IN) in doses individually adjusted to maintain blood glucose levels between 200 and 300 mg/dl. Moderately hyperglycemic rats at this stage exhibit characteristic renal hemodynamic changes sensitive to NOS inhibition (8,25,26). A diabetic subgroup (diabetic rats on intensive insulin treatment) received intensive insulin treatment (4 units ultralente insulin, twice daily) to achieve better metabolic control. Blood glucose levels were monitored at least weekly in all diabetic rats. Age-matched nondiabetic rats served as controls. All rats were fed standard rat chow (Rodent Laboratory Chow 5001; Ralston Purina, Richmond, IN) ad libitum. After 4 weeks of diabetes, the rats were anesthetized with methohexitone (Brevital; 50 mg/kg i.p.), and aortic blood was obtained for determination of blood glucose and glycosylated hemoglobin (HbA1c [A1C]), after which tissue was collected for protein expression studies. These studies were approved by the Portland Veterans Affairs Institutional Animal Care and Use Subcommittee.
Immunoblotting and immunohistochemistry
Immunoblotting.
The right kidneys were divided into cortical and medullary portions and snap frozen in liquid nitrogen. To obtain whole-cell homogenates, kidney cortex was homogenized in lysis buffer containing 50 mmol/l Tris, 150 mmol/l NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1.0% Triton-X 100 and protease inhibitors and centrifuged at 12,000g for 30 min at 4°C; the supernatant was saved at eC70°C. To obtain crude membrane (pellet) and cytosolic (soluble) fractions, cortical samples were homogenized in Tris-EDTA buffer (25 mmol/l Tris, 5 mmol/l EDTA, 40 e蘥/ml phenylmethylsulfonyl fluoride, 20 e蘥/ml leupeptin, and 20 e蘥/ml benzamidine) and centrifuged at 500 x g for 15 min at 4°C, and the resulting nuclei-free supernatant was centrifuged at 100,000 x g for 20 min at 4°C. Phosphatase inhibitors (1 mmol/l NaF, 1 mmol/l sodium vanadate, 5 nmol/l microcystin LR, 1 mmol/l sodium pyrophosphate, and 1 mmol/l p-nitro-phenylphosphate) were added to the lysis buffer when the samples were used for determination of phosphoprotein expression. Pellets were solubilized in Tris-EDTA buffer plus 1% deoxycholate. Total protein content in all fractions was determined by BCA analysis (Pierce).
Denatured proteins were separated through an SDS-polyacrylamide gel and transferred to polyvinylidine fluoride membranes (Bio-Rad). After blocking, membranes were incubated overnight with mouse anti-eNOS (Transduction; 1:800 for membrane fractions, 1:500 for cytosol), antieCphospho Ser1177 eNOS (Cell Signaling, Beverly, MA; 1:400), or rabbit antieCCAV-1 (1:800; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Immunodetection and visualization were accomplished with an enhanced chemiluminiscence as previously described (27). Resultant films (Kodak) were scanned using a flatbed scanner, and images were analyzed with NIH Image software. After detection of eNOS monomer and CAV-1, membranes were stripped in stripping buffer (Chemicon) for 15 min at room temperature, blocked, and reincubated for 1 h at room temperature with goat anti-actin antibody (1:800; Santa Cruz Biotechnology). This was followed by a 45-min incubation with antieCgoat-IgG secondary antibody conjugated with horseradish peroxidase (1:40,000; Pierce) and reaction with enhanced chemiluminiscence as above. For phospho-eNOS expression analysis, detection of phosphoprotein was followed by membrane stripping, detection of total eNOS expression, and then actin detection as described above.
To determine eNOS dimer expression by Western blotting, membrane and cytosolic samples were not denatured by heat. The samples were separated using "cold electrophoresis" (21) through an SDS-polyacrylamide gel at 4°C, with detection and visualization as above. All protein expression measurements were performed at least in triplicate.
Coimmunoprecipitation.
Membrane samples were immunoprecipitated with rabbit antieCCAV-1 antibody and protein A (2 e蘥; Santa Cruz Biotechnology) in a buffer containing 50 mmol/l Tris, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% NP-40, 0.25% sodium deoxycholate, 60 mmol/l octylglucoside, 40 e蘥/ml phenylmethylsulfonyl fluoride, 20 e蘥/ml leupeptin, and 20 e蘥/ml benzamidine. After washing in a buffer containing 25 mmol/l Tris, 150 mmol/l NaCl, 1% Triton X-100, and 5 mmol/l EDTA, the resulting complexes were separated through an SDS-polyacrylamide gel, and eNOS detection was performed as above. Samples incubated with nonimmune rabbit IgG instead of antieCCAV-1 antibody served as controls. All immunoblotting and immunoprecipitation measurements were performed at least in triplicate. Some membranes were stripped and analyzed for CAV-1 expression to validate coimmunoprecipitation.
Immunohistochemistry.
The left kidney was perfused with ice-cold PBS (30 ml), excised, and immersed in 10% formalin. The fixed kidneys were dehydrated through a graded series of ethanols, embedded in paraffin, and sectioned at 4 e蘭 thickness. The antibody described above (Transduction; Santa Cruz Biotechnology) was used for immunohistochemical detection of CAV-1 and confocal microscopy studies. Sections were deparaffinized in xylene, rehydrated through graded ethanols to water, and pretreated by steaming in 10% CITRA buffer (BioGenex, San Ramon, CA). After blocking, slides were incubated overnight at 4°C with primary antibody (1:200) or with the same concentration of nonimmune mouse IgG as a control. Endogenous peroxidase activity was blocked with 3% H2O2 solution in methanol. The primary antibody was localized using the Vectastain ABC-Elite peroxidase detection system (Vector Laboratories, Burlingame, CA). This was followed by reaction with diaminobenzidine as chromogen and counterstaining with hematoxylin (Sigma). Sections of each diabetic kidney were processed in parallel with the appropriate control tissue.
Immunoreactive eNOS and CAV-1 were colocalized using confocal microscopy. Paraffin sections were processed as above. After incubating with both primary antibodies, samples were washed three times in PBS for 10 min. The CAV-1 primary antibody was localized by immunofluorescent detection with a secondary Alexa Fluor-Green (488)eCtagged goat anti-rabbit antibody (1:200 dilution, 1-h incubation; Molecular Probes, Eugene, OR), and eNOS primary antibody was detected with a secondary Alexa Fluor red (568)eCtagged goat anti-mouse antibody (1:200 dilution, 1-h incubation; Molecular Probes). Samples were washed three times in PBS for 10 min to remove excess secondary antibody and then sealed by coverslip after application of SlowFade (Molecular Probes). Consecutive sections, 500 nm apart, were scanned alternating between 488- and 568-nm lasers with a Leica TCS SP confocal laser-scanning microscope. System settings were held constant for all imaging, and images were digitally captured. Colocalization was visualized by superimposing the green CAV-1 over the red eNOS using Adobe Photoshop (Adobe Systems, San Jose, CA), with the resultant yellow image representing the area of colocalization.
Statistical analysis.
Data are expressed as means ± SE. Analyses were performed by ANOVA followed by the Scheffee test, using Statview SE and Graphics software (Brainpower, Calabasas, CA). A P value <0.05 was viewed as statistically significant.
RESULTS
General characteristics of control and diabetic rats are shown in Table 1. Moderately hyperglycemic diabetic rats demonstrated lower weight gain, increases in the left kidney weight and kidney-to-body weight ratio, moderate hyperglycemia, and increased A1C levels compared with control rats (P < 0.001). In diabetic rats on intensive insulin treatment, kidney hypertrophy and elevation in levels of blood glucose and A1C were attenuated (P < 0.001 vs. moderately hyperglycemic rats), although blood glucose and A1C values remained higher than in control animals.
Renal cortical expression of eNOS in diabetes.
All groups of control and diabetic rats demonstrated similar expression of renal cortical eNOS monomer analyzed in whole-cell preparations (Fig. 1A). In contrast, eNOS protein in crude membrane fractions was reduced in cortical samples of moderately hyperglycemic diabetic rats compared with control rats and diabetic rats on intensive insulin treatment (Fig. 1B). In diabetic rats on intensive insulin treatment, membrane eNOS expression was similar to that in nondiabetic control rats. The cytosolic eNOS fraction was undetectable in most samples harvested from control rats (Fig. 1C). However, eNOS was present and abundant in all cytosolic samples from moderately hyperglycemic diabetic rats. Although the cytosolic eNOS was also detectable in diabetic rats on intensive insulin treatment, its abundance was attenuated by intensive insulin treatment (Fig. 1C).
In further experiments, we determined renal cortical phosphorylation status of eNOS, one of the crucial posttranslational modifications responsible for NO production by the enzyme. As shown in Fig. 2, moderately hyperglycemic diabetic rats demonstrated decreased phospho-Ser1177 eNOS expression in crude membrane fractions that was reversed by intensive insulin treatment. The differences between the groups in total eNOS membrane expression protein in these experiments were similar to those in the previous series. The ratio of phospho-Ser1177 eNOS/total eNOS, determined as another marker of eNOS activity, was not significantly altered in diabetic rats because of lower total eNOS expression.
Because dimer formation is critical for eNOS to functionally produce NO, we embarked on further studies to determine the proportion of eNOS existing as either dimer or monomer in the diabetic kidney (Fig. 3). In accordance with in vitro evidence (28), electrophoresis of membrane samples without prior heat denaturation revealed bands approximately twice the size of eNOS monomer (corresponding to 145-kDa bands), attributable to eNOS dimer. Moderately hyperglycemic diabetic rats demonstrated a significant reduction in the membrane-bound dimer-to-monomer ratio compared with control rats. In diabetic rats on intensive insulin treatment, the dimer-to-monomer ratio was not different from nondiabetic control rats.
Renal cortical expression of CAV-1 and CAV-1eCeNOS colocalization.
CAV-1 protein expression was abundant in membrane fractions in both control and diabetic rats. In renal cortical membrane preparations, CAV-1 expression significantly declined in moderately hyperglycemic diabetic rats compared with control rats (P < 0.01) (Fig. 4). Intensive insulin therapy restored CAV-1 expression to levels comparable with those in nondiabetic control rats (P < 0.01 vs. moderately hyperglycemic diabetic rats). In all groups of rats, CAV-1 was not detectable in cytosolic fractions (not shown). Immunohistochemical studies localized CAV-1 in the endothelia of renal vasculature and glomeruli. Positive staining was also localized in arteriolar vascular smooth muscle in both control and diabetic rats. Furthermore, immunoreactive CAV-1 was present in basolateral aspects of distal tubules (Fig. 5).
Further studies explored colocalization and protein-protein interactions of CAV-1 and eNOS in control and diabetic rats. Using confocal microscopy, eNOS and CAV-1 were colocalized in endothelial cells in both groups throughout the renal and glomerular vascular tree but not in tubules (Fig. 6). Both proteins were coimmunoprecipitated in membrane fractions in both control and diabetic rats.
DISCUSSION
These studies demonstrate complex alterations in renal cortical eNOS expression and posttranslational modifications important for enzymatic activity in moderately hyperglycemic diabetic rats. Moderately hyperglycemic diabetic rats demonstrated a decrease in expression of eNOS in crude membrane preparations. In contrast, eNOS expression in the cytosolic fraction, which was barely detectable in control rats, was increased in the setting of diabetes. These differences occurred despite similar eNOS expression in the whole-cell preparations. Phospho-Ser1177 eNOS was also decreased in moderately hyperglycemic diabetic rats compared with controls. In addition, the membrane-localized eNOS was observed in a higher proportion in the monomeric state with diabetes.
These eNOS alterations were associated with downregulation of the expression of CAV-1 in the diabetic kidney. CAV-1 and eNOS were colocalized in endothelial cells throughout the renal and glomerular vascular tree but not in tubules. Importantly, observed changes in eNOS expression, posttranslational modifications, and subcellular targeting, as well as CAV-1 expression, were reversed or attenuated by intensive insulin treatment.
Despite abundant evidence of impaired endothelium-dependent vasodilation and reduced NO renal bioavailability in renal and nonrenal vasculature in diabetes (3,4,6,29,30), previous studies have reported normal (31) or enhanced (26,32eC34) expression of eNOS in the diabetic kidney and, in some reports, even enhanced NO production in diabetic renal cortex (31). In accord with Ishii et al. (31), we found no differences between control and diabetic rats with respect to whole-cell eNOS expression. However, there were important differences in membrane-bound and cytosolic eNOS that could at least in part explain apparent disparities between eNOS renal expression, in vitro function, and endothelium-dependent vasodilation in diabetes.
Reduced eNOS expression in membrane fractions and the opposite trend in the cytosol confer implications for the functional status of the enzyme in the diabetic kidney. It has been postulated that eNOS membrane localization in caveolae is crucial for agonist-stimulated NO release (35eC37). Therefore, decreased expression of membrane-bound eNOS in the diabetic kidney suggests a reduction in the stimulatable enzyme pool that can be functionally coupled to specific agonists. This phenomenon may negatively impact endothelial function in the diabetic kidney. Therefore, it is possible that previously reported increases in eNOS expression in the diabetic renal cortex (26) could be in part attributable to the total cellular pool of enzyme and not just to that which is membrane localized.
In response to humoral and physical stimuli that enhance NO production by the enzyme, eNOS undergoes stimulatory phosphorylation on Ser1177 by several serine/threonine kinases, such as Akt (11,12) or protein kinase A (13). Therefore, eNOS phosphorylation status on Ser1177 is one of the crucial functional characteristics of the enzyme. eNOS phosphorylation has recently been the subject of substantial interest as one of the possible sites of diabetes-induced defects in endothelial NO generation. Recent studies have reported impairment of eNOS enzymatic function in hyperglycemic conditions in cultured endothelial cells (24,38) and in penile endothelia in diabetic rats (39) in association with O-linked N-acetylglucosamine modification of eNOS at the Akt phosphorylation site, resulting in reduced ability of the enzyme to be phosphorylated by Akt. These modifications are mediated by activation of the hexosamine pathway (24). Our present findings suggest that a similar process occurs in diabetic renal cortex. In addition, our data also suggest that a decrease in phospho-Ser1177 eNOS in moderately hyperglycemic diabetic rats may be due to reduced abundance of the enzyme in crude membrane fractions.
Previous reports on eNOS expression in diabetic renal cortex have not differentiated the membrane and cytosolic cellular partitions and posttranslational modifications, but Lee et al. (40) have recently addressed these issues in the renal medulla. The authors observed no differences in eNOS protein expression in membrane and cytosolic fractions, as well as in Ser1177 phosphorylation of eNOS between nondiabetic and diabetic rats, and no effects of intensive insulin treatment. However, there were differences in phosphorylation status of other residues, in particular in Thr495 inhibitory phosphorylation, resulting in enhanced eNOS activity in the diabetic renal medulla. It should be noted that the latter analysis is not readily comparable with our present data, because the eNOS phosphorylation was analyzed in whole-cell preparations; this could explain the disparate findings with respect to eNOS phosphorylation status in diabetes between their study and our observations.
Further experiments focused on eNOS dimer expression, another posttranslational eNOS modification important for NO generation (15eC17). Similar to the eNOS monomer expression, the eNOS dimer-to-monomer ratio was reduced in membrane fractions of diabetic kidneys. The reduction in membrane-bound dimer is likely to further reduce availability of the activatable enzyme in diabetic rats. Defects in eNOS cofactor function or availability may be responsible for impaired eNOS dimerization. A deficit in the cofactor tetrahydrobiopterin, which is essential for eNOS activity and seems to play a role in NOS dimer formation (41), has been implicated in the pathophysiology of diabetes-induced endothelial dysfunction (42,43). In the context of documented glycosylation of the Akt phosphorylation site responsible for reduced eNOS activation in hyperglycemia (24), we also cannot exclude the possibility that glycosylation of specific sites on the eNOS molecule may interfere with dimerization.
Considering the important roles of CAV-1 in the membrane targeting of eNOS and modulation of its enzymatic activity (21,44), changes in eNOS membrane and cytosolic expression in the diabetic kidney may also be related to CAV-1 abundance in the membrane. Theoretically, changes in CAV-1 could be a common denominator for most of the alterations of the eNOS molecule observed in the present studies. To address this issue, we determined CAV-1 expression in crude membrane and cytosolic fractions, and we document here a diabetes-dependent reduction in CAV-1 expression in crude membranes. CAV-1 was expressed and localized in endothelial cells of arteries, arterioles, and glomeruli. CAV-1 immunoreactivity was also apparent in the vascular smooth muscle cells of glomerular arterioles and in basolateral aspects of distal tubules. The distribution of CAV-1 in diabetic kidneys was similar to that in nondiabetic controls. Confocal microscopy studies found that eNOS and CAV-1 colocalized exclusively in endothelia throughout the renal vascular tree, both in control and diabetic animals. Furthermore, as in the extrarenal vasculature, coimmunoprecipitation studies demonstrated direct interaction of both proteins in control and diabetic renal cortex.
Studies have suggested that eNOS needs to be localized in caveolae to interact with proteins and signals for its activation (36,45). Thus, the lack of membrane CAV-1 could explain reductions of eNOS in crude membrane fractions and its rise in the cytosolic compartment. Furthermore, depletion of CAV-1 from caveolar fractions in endothelial cells has been associated with reduced agonist-induced eNOS phosphorylation (45,46). In support of the view that CAV-1 plays a role in diabetes-induced changes in eNOS targeting and phosphorylation are parallel shifts in membrane CAV-1, total eNOS, and phosphorylated eNOS expressions induced by moderate hyperglycemia and improved metabolic control with intensive insulin treatment. Of interest, a similar CAV-1 regulatory pattern as in the present studies (downregulation of its expression and its reversal with insulin treatment) has recently been reported in Schwann cells, implicating this process in diabetic neuropathy as well (47).
Most of our present findings support the abundant evidence demonstrating impaired NO generation and bioavailability in renal and extrarenal vasculature of diabetic animals (3eC6,30). However, considering the fact that eNOS and CAV-1 form an inhibitory complex, reduced CAV-1 expression may be interpreted as a factor favoring NOS enzymatic activity. This observation would be consistent with data suggesting enhanced NO dependency of renal hemodynamics in hyperfiltering diabetic rats and the role of NO in the pathogenesis of hyperfiltration (7eC9) and with those reports suggesting a role for eNOS-derived NO in this process (26,32,33). However, the latter reports relied on measurements of eNOS expression and constitutive renal NOS activity, and the role of eNOS could not be verified by measurement of renal hemodynamic responses to specific eNOS inhibitors. Nevertheless, we believe that the prevailing evidence in this study is compatible with an overall reduction in eNOS-derived NO bioavailability in the diabetic kidney. Supporting this interpretation are our previous renal hemodynamic studies implicating neuronal NOS as the major source of NO involved in the pathophysiology of hyperfiltration (25).
Intensive insulin treatment that achieved tighter metabolic control corrected or attenuated the changes seen in the moderately hyperglycemic diabetic rats. Expression of eNOS and CAV-1 in membrane fractions and the dimer-to-monomer ratio were similar in control rats and diabetic rats on intensive insulin treatment. Similarly, eNOS phosphorylation was also normalized in diabetic rats on intensive insulin treatment. This phenomenon and the previous report in Schwann cells (47) indicate that hyperglycemia and/or low plasma insulin levels are most likely major factors in this process. Moreover, reversal of impaired eNOS phosphorylation in diabetic rats on intensive insulin treatment could be attributable not only to improved glycemic control but even more closely to higher plasma insulin doses resulting in enhanced signaling via Akt kinase (11,12). On the other hand, incomplete reversal of some alterations as observed in diabetic rats on intensive insulin treatment may be attributable to the fact that intensive insulin treatment did not entirely normalize blood glucose levels and A1C.
The possible impact of reduced CAV-1 abundance per se in the diabetic kidney remains to be established. In addition to modulation of eNOS function, CAV-1, as a molecule involved in spatial organization of signaling molecules in caveolae, is a good candidate to play a role in processes implicated in the development of diabetic renal alterations. Caveolae are enriched in a wide array of receptors, growth and vasoactive factors (platelet-derived growth factor, epidermal growth factor, receptor for advanced glycosylation end products, bradykinin, and endothelin), their signaling molecules (diacylglycerol, protein kinase C, and mitogen-activated protein kinase), and enzymes (eNOS) that have been associated with development of diabetes complications.
In summary, we observed complex alterations in eNOS expression, cellular compartmentalization, phosphorylation, and homodimerization in the diabetic renal cortex. These changes were associated with reduced CAV-1 expression. Improvement of metabolic control with intensive insulin treatment corrected or attenuated the differences between control and diabetic rats, suggesting that these changes are related to hyperglycemia and/or insulin deficiency. These data support the view of decreased renal eNOS-derived NO bioavailability in diabetes.
ACKNOWLEDGMENTS
S.A. has received National Institutes of Health Grant DK-63231 and support from the VA Merit Review and the Juvenile Diabetes Research Foundation. S.M. has received support from the VA Merit Review. R.K. has received institutional grant MZO 00023001 of the Czech Ministry of Health Care, Czech Republic.
Parts of this study have been published in abstract form (48).
FOOTNOTES
DOI: 10.2337/db05-1595
The costs of publication of this article were defrayed in part by the payment of page charges. This 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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2 Department of Medicine, Oregon Health and Science University, Portland, Oregon
3 Diabetes Center, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
4 Research Service, Portland VA Medical Center, Portland, Oregon
5 Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon
CAV-1, caveolin-1; eNOS, endothelial nitric oxide synthase; NOS, nitric oxide synthase
ABSTRACT
Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.
Experimental diabetes is associated with complex alterations in renal nitric oxide (NO) bioavailability and signaling. Studies exploring the diabetic renal NO system in different experimental settings have produced inconsistent and seemingly contradictory findings (1,2). For instance, in vitro studies suggest defective endothelium-dependent (i.e., endothelial NO synthase [eNOS]eCdependent) NO production or function in the diabetic kidney (3eC6). These studies contrast with observations from in vivo studies with NO synthase (NOS) inhibitors, which show substantial NO dependency of renal hemodynamics in hyperfiltering diabetic rats (7eC9).
To function as an endothelial NO-producing enzyme, eNOS (NOS III) requires a battery of cofactors, posttranslational modifications such as phosphorylation and dimerization, protein-protein interactions, and subcellular targeting (10). Phosphorylation of eNOS on Ser1177 by several serine/threonine kinases, such as Akt (protein kinase B) (11,12) or protein kinase A (13), in response to a variety of physiological stimuli is a critical control step for NO production by the enzyme. This process enhances the rate of electron flux from the reductase to the oxygenase domain of eNOS and reduces the calcium requirements for the enzyme, thus increasing NO synthesis (14). Homodimerization of eNOS is also crucial for NO production (15). Formation of a homodimer is crucial for NO production by eNOS (15,16), creating high-affinity binding sites for the NOS substrate L-arg and enabling electron transfer from the reductase domain of one NOS monomer to the oxygenase domain of the other (17).
Activation of eNOS is not only dependent on phosphorylation by upstream kinases and conformational changes but is also determined by its specific subcellular localization. A large pool of eNOS representing the activatable enzyme is localized within the plasma membrane and enriched in specialized structures called caveolae (18). Plasmalemmal caveolae are membrane invaginations that serve as domains for the sequestration and organization of a large number of molecules, including receptors and their downstream signaling effectors and modulators, enzymes, membrane transporters, structural molecules, and lipids (19,20).
Caveolin-1 (CAV-1) is the main structural component of caveolae in endothelial cells. It acts as a scaffolding protein and is involved in modulation of receptor signaling and function of caveolar enzymes (19,20). In unstimulated endothelial cells, eNOS is inhibited by its protein-protein interaction with CAV-1. The pathway of eNOS activation upon stimulation by agonist involves mobilization of intracellular Ca2+ and consequent interaction of calmodulin with eNOS. The eNOS/calmodulin interaction allows the release of eNOS from an inhibitory complex with CAV-1 (21,22). Therefore, alterations in CAV-1 abundance and eNOS interactions can impact eNOS function and consequently vascular function and modeling. A better understanding of the role of CAV-1 in mediating cellular functions in diabetes is needed for elucidation of NO pathophysiology in the diabetic kidney.
Although inactivation of NO by reactive oxygen species has been suggested as a major mechanism responsible for reduced bioavailability of eNOS-derived NO in diabetes (23), other factors related to direct changes in eNOS function and molecular integrity have also been suggested (24). In the present study, we explored the renal cortical expression of eNOS with respect to some of its functional determinants, including the cellular localization, phosphorylation status, and dimer/monomer formation, in normal and diabetic rats. Furthermore, we examined renal cortical expression and localization of the endogenous eNOS inhibitor CAV-1 and its colocalization with eNOS.
RESEARCH DESIGN AND METHODS
Studies were conducted in male Sprague-Dawley rats, with initial weights of 300 g. Rats were made diabetic with streptozotocin (65 mg/kg body wt i.p.; Sigma, St. Louis, MO). Diabetes was confirmed by measurement of tail blood glucose level using a reflectance meter (One Touch II; Lifescan, Milpetas, CA). Diabetic rats received daily evening injections of ultralente insulin (Iletin II; Eli Lilly, Indianapolis, IN) in doses individually adjusted to maintain blood glucose levels between 200 and 300 mg/dl. Moderately hyperglycemic rats at this stage exhibit characteristic renal hemodynamic changes sensitive to NOS inhibition (8,25,26). A diabetic subgroup (diabetic rats on intensive insulin treatment) received intensive insulin treatment (4 units ultralente insulin, twice daily) to achieve better metabolic control. Blood glucose levels were monitored at least weekly in all diabetic rats. Age-matched nondiabetic rats served as controls. All rats were fed standard rat chow (Rodent Laboratory Chow 5001; Ralston Purina, Richmond, IN) ad libitum. After 4 weeks of diabetes, the rats were anesthetized with methohexitone (Brevital; 50 mg/kg i.p.), and aortic blood was obtained for determination of blood glucose and glycosylated hemoglobin (HbA1c [A1C]), after which tissue was collected for protein expression studies. These studies were approved by the Portland Veterans Affairs Institutional Animal Care and Use Subcommittee.
Immunoblotting and immunohistochemistry
Immunoblotting.
The right kidneys were divided into cortical and medullary portions and snap frozen in liquid nitrogen. To obtain whole-cell homogenates, kidney cortex was homogenized in lysis buffer containing 50 mmol/l Tris, 150 mmol/l NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1.0% Triton-X 100 and protease inhibitors and centrifuged at 12,000g for 30 min at 4°C; the supernatant was saved at eC70°C. To obtain crude membrane (pellet) and cytosolic (soluble) fractions, cortical samples were homogenized in Tris-EDTA buffer (25 mmol/l Tris, 5 mmol/l EDTA, 40 e蘥/ml phenylmethylsulfonyl fluoride, 20 e蘥/ml leupeptin, and 20 e蘥/ml benzamidine) and centrifuged at 500 x g for 15 min at 4°C, and the resulting nuclei-free supernatant was centrifuged at 100,000 x g for 20 min at 4°C. Phosphatase inhibitors (1 mmol/l NaF, 1 mmol/l sodium vanadate, 5 nmol/l microcystin LR, 1 mmol/l sodium pyrophosphate, and 1 mmol/l p-nitro-phenylphosphate) were added to the lysis buffer when the samples were used for determination of phosphoprotein expression. Pellets were solubilized in Tris-EDTA buffer plus 1% deoxycholate. Total protein content in all fractions was determined by BCA analysis (Pierce).
Denatured proteins were separated through an SDS-polyacrylamide gel and transferred to polyvinylidine fluoride membranes (Bio-Rad). After blocking, membranes were incubated overnight with mouse anti-eNOS (Transduction; 1:800 for membrane fractions, 1:500 for cytosol), antieCphospho Ser1177 eNOS (Cell Signaling, Beverly, MA; 1:400), or rabbit antieCCAV-1 (1:800; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Immunodetection and visualization were accomplished with an enhanced chemiluminiscence as previously described (27). Resultant films (Kodak) were scanned using a flatbed scanner, and images were analyzed with NIH Image software. After detection of eNOS monomer and CAV-1, membranes were stripped in stripping buffer (Chemicon) for 15 min at room temperature, blocked, and reincubated for 1 h at room temperature with goat anti-actin antibody (1:800; Santa Cruz Biotechnology). This was followed by a 45-min incubation with antieCgoat-IgG secondary antibody conjugated with horseradish peroxidase (1:40,000; Pierce) and reaction with enhanced chemiluminiscence as above. For phospho-eNOS expression analysis, detection of phosphoprotein was followed by membrane stripping, detection of total eNOS expression, and then actin detection as described above.
To determine eNOS dimer expression by Western blotting, membrane and cytosolic samples were not denatured by heat. The samples were separated using "cold electrophoresis" (21) through an SDS-polyacrylamide gel at 4°C, with detection and visualization as above. All protein expression measurements were performed at least in triplicate.
Coimmunoprecipitation.
Membrane samples were immunoprecipitated with rabbit antieCCAV-1 antibody and protein A (2 e蘥; Santa Cruz Biotechnology) in a buffer containing 50 mmol/l Tris, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% NP-40, 0.25% sodium deoxycholate, 60 mmol/l octylglucoside, 40 e蘥/ml phenylmethylsulfonyl fluoride, 20 e蘥/ml leupeptin, and 20 e蘥/ml benzamidine. After washing in a buffer containing 25 mmol/l Tris, 150 mmol/l NaCl, 1% Triton X-100, and 5 mmol/l EDTA, the resulting complexes were separated through an SDS-polyacrylamide gel, and eNOS detection was performed as above. Samples incubated with nonimmune rabbit IgG instead of antieCCAV-1 antibody served as controls. All immunoblotting and immunoprecipitation measurements were performed at least in triplicate. Some membranes were stripped and analyzed for CAV-1 expression to validate coimmunoprecipitation.
Immunohistochemistry.
The left kidney was perfused with ice-cold PBS (30 ml), excised, and immersed in 10% formalin. The fixed kidneys were dehydrated through a graded series of ethanols, embedded in paraffin, and sectioned at 4 e蘭 thickness. The antibody described above (Transduction; Santa Cruz Biotechnology) was used for immunohistochemical detection of CAV-1 and confocal microscopy studies. Sections were deparaffinized in xylene, rehydrated through graded ethanols to water, and pretreated by steaming in 10% CITRA buffer (BioGenex, San Ramon, CA). After blocking, slides were incubated overnight at 4°C with primary antibody (1:200) or with the same concentration of nonimmune mouse IgG as a control. Endogenous peroxidase activity was blocked with 3% H2O2 solution in methanol. The primary antibody was localized using the Vectastain ABC-Elite peroxidase detection system (Vector Laboratories, Burlingame, CA). This was followed by reaction with diaminobenzidine as chromogen and counterstaining with hematoxylin (Sigma). Sections of each diabetic kidney were processed in parallel with the appropriate control tissue.
Immunoreactive eNOS and CAV-1 were colocalized using confocal microscopy. Paraffin sections were processed as above. After incubating with both primary antibodies, samples were washed three times in PBS for 10 min. The CAV-1 primary antibody was localized by immunofluorescent detection with a secondary Alexa Fluor-Green (488)eCtagged goat anti-rabbit antibody (1:200 dilution, 1-h incubation; Molecular Probes, Eugene, OR), and eNOS primary antibody was detected with a secondary Alexa Fluor red (568)eCtagged goat anti-mouse antibody (1:200 dilution, 1-h incubation; Molecular Probes). Samples were washed three times in PBS for 10 min to remove excess secondary antibody and then sealed by coverslip after application of SlowFade (Molecular Probes). Consecutive sections, 500 nm apart, were scanned alternating between 488- and 568-nm lasers with a Leica TCS SP confocal laser-scanning microscope. System settings were held constant for all imaging, and images were digitally captured. Colocalization was visualized by superimposing the green CAV-1 over the red eNOS using Adobe Photoshop (Adobe Systems, San Jose, CA), with the resultant yellow image representing the area of colocalization.
Statistical analysis.
Data are expressed as means ± SE. Analyses were performed by ANOVA followed by the Scheffee test, using Statview SE and Graphics software (Brainpower, Calabasas, CA). A P value <0.05 was viewed as statistically significant.
RESULTS
General characteristics of control and diabetic rats are shown in Table 1. Moderately hyperglycemic diabetic rats demonstrated lower weight gain, increases in the left kidney weight and kidney-to-body weight ratio, moderate hyperglycemia, and increased A1C levels compared with control rats (P < 0.001). In diabetic rats on intensive insulin treatment, kidney hypertrophy and elevation in levels of blood glucose and A1C were attenuated (P < 0.001 vs. moderately hyperglycemic rats), although blood glucose and A1C values remained higher than in control animals.
Renal cortical expression of eNOS in diabetes.
All groups of control and diabetic rats demonstrated similar expression of renal cortical eNOS monomer analyzed in whole-cell preparations (Fig. 1A). In contrast, eNOS protein in crude membrane fractions was reduced in cortical samples of moderately hyperglycemic diabetic rats compared with control rats and diabetic rats on intensive insulin treatment (Fig. 1B). In diabetic rats on intensive insulin treatment, membrane eNOS expression was similar to that in nondiabetic control rats. The cytosolic eNOS fraction was undetectable in most samples harvested from control rats (Fig. 1C). However, eNOS was present and abundant in all cytosolic samples from moderately hyperglycemic diabetic rats. Although the cytosolic eNOS was also detectable in diabetic rats on intensive insulin treatment, its abundance was attenuated by intensive insulin treatment (Fig. 1C).
In further experiments, we determined renal cortical phosphorylation status of eNOS, one of the crucial posttranslational modifications responsible for NO production by the enzyme. As shown in Fig. 2, moderately hyperglycemic diabetic rats demonstrated decreased phospho-Ser1177 eNOS expression in crude membrane fractions that was reversed by intensive insulin treatment. The differences between the groups in total eNOS membrane expression protein in these experiments were similar to those in the previous series. The ratio of phospho-Ser1177 eNOS/total eNOS, determined as another marker of eNOS activity, was not significantly altered in diabetic rats because of lower total eNOS expression.
Because dimer formation is critical for eNOS to functionally produce NO, we embarked on further studies to determine the proportion of eNOS existing as either dimer or monomer in the diabetic kidney (Fig. 3). In accordance with in vitro evidence (28), electrophoresis of membrane samples without prior heat denaturation revealed bands approximately twice the size of eNOS monomer (corresponding to 145-kDa bands), attributable to eNOS dimer. Moderately hyperglycemic diabetic rats demonstrated a significant reduction in the membrane-bound dimer-to-monomer ratio compared with control rats. In diabetic rats on intensive insulin treatment, the dimer-to-monomer ratio was not different from nondiabetic control rats.
Renal cortical expression of CAV-1 and CAV-1eCeNOS colocalization.
CAV-1 protein expression was abundant in membrane fractions in both control and diabetic rats. In renal cortical membrane preparations, CAV-1 expression significantly declined in moderately hyperglycemic diabetic rats compared with control rats (P < 0.01) (Fig. 4). Intensive insulin therapy restored CAV-1 expression to levels comparable with those in nondiabetic control rats (P < 0.01 vs. moderately hyperglycemic diabetic rats). In all groups of rats, CAV-1 was not detectable in cytosolic fractions (not shown). Immunohistochemical studies localized CAV-1 in the endothelia of renal vasculature and glomeruli. Positive staining was also localized in arteriolar vascular smooth muscle in both control and diabetic rats. Furthermore, immunoreactive CAV-1 was present in basolateral aspects of distal tubules (Fig. 5).
Further studies explored colocalization and protein-protein interactions of CAV-1 and eNOS in control and diabetic rats. Using confocal microscopy, eNOS and CAV-1 were colocalized in endothelial cells in both groups throughout the renal and glomerular vascular tree but not in tubules (Fig. 6). Both proteins were coimmunoprecipitated in membrane fractions in both control and diabetic rats.
DISCUSSION
These studies demonstrate complex alterations in renal cortical eNOS expression and posttranslational modifications important for enzymatic activity in moderately hyperglycemic diabetic rats. Moderately hyperglycemic diabetic rats demonstrated a decrease in expression of eNOS in crude membrane preparations. In contrast, eNOS expression in the cytosolic fraction, which was barely detectable in control rats, was increased in the setting of diabetes. These differences occurred despite similar eNOS expression in the whole-cell preparations. Phospho-Ser1177 eNOS was also decreased in moderately hyperglycemic diabetic rats compared with controls. In addition, the membrane-localized eNOS was observed in a higher proportion in the monomeric state with diabetes.
These eNOS alterations were associated with downregulation of the expression of CAV-1 in the diabetic kidney. CAV-1 and eNOS were colocalized in endothelial cells throughout the renal and glomerular vascular tree but not in tubules. Importantly, observed changes in eNOS expression, posttranslational modifications, and subcellular targeting, as well as CAV-1 expression, were reversed or attenuated by intensive insulin treatment.
Despite abundant evidence of impaired endothelium-dependent vasodilation and reduced NO renal bioavailability in renal and nonrenal vasculature in diabetes (3,4,6,29,30), previous studies have reported normal (31) or enhanced (26,32eC34) expression of eNOS in the diabetic kidney and, in some reports, even enhanced NO production in diabetic renal cortex (31). In accord with Ishii et al. (31), we found no differences between control and diabetic rats with respect to whole-cell eNOS expression. However, there were important differences in membrane-bound and cytosolic eNOS that could at least in part explain apparent disparities between eNOS renal expression, in vitro function, and endothelium-dependent vasodilation in diabetes.
Reduced eNOS expression in membrane fractions and the opposite trend in the cytosol confer implications for the functional status of the enzyme in the diabetic kidney. It has been postulated that eNOS membrane localization in caveolae is crucial for agonist-stimulated NO release (35eC37). Therefore, decreased expression of membrane-bound eNOS in the diabetic kidney suggests a reduction in the stimulatable enzyme pool that can be functionally coupled to specific agonists. This phenomenon may negatively impact endothelial function in the diabetic kidney. Therefore, it is possible that previously reported increases in eNOS expression in the diabetic renal cortex (26) could be in part attributable to the total cellular pool of enzyme and not just to that which is membrane localized.
In response to humoral and physical stimuli that enhance NO production by the enzyme, eNOS undergoes stimulatory phosphorylation on Ser1177 by several serine/threonine kinases, such as Akt (11,12) or protein kinase A (13). Therefore, eNOS phosphorylation status on Ser1177 is one of the crucial functional characteristics of the enzyme. eNOS phosphorylation has recently been the subject of substantial interest as one of the possible sites of diabetes-induced defects in endothelial NO generation. Recent studies have reported impairment of eNOS enzymatic function in hyperglycemic conditions in cultured endothelial cells (24,38) and in penile endothelia in diabetic rats (39) in association with O-linked N-acetylglucosamine modification of eNOS at the Akt phosphorylation site, resulting in reduced ability of the enzyme to be phosphorylated by Akt. These modifications are mediated by activation of the hexosamine pathway (24). Our present findings suggest that a similar process occurs in diabetic renal cortex. In addition, our data also suggest that a decrease in phospho-Ser1177 eNOS in moderately hyperglycemic diabetic rats may be due to reduced abundance of the enzyme in crude membrane fractions.
Previous reports on eNOS expression in diabetic renal cortex have not differentiated the membrane and cytosolic cellular partitions and posttranslational modifications, but Lee et al. (40) have recently addressed these issues in the renal medulla. The authors observed no differences in eNOS protein expression in membrane and cytosolic fractions, as well as in Ser1177 phosphorylation of eNOS between nondiabetic and diabetic rats, and no effects of intensive insulin treatment. However, there were differences in phosphorylation status of other residues, in particular in Thr495 inhibitory phosphorylation, resulting in enhanced eNOS activity in the diabetic renal medulla. It should be noted that the latter analysis is not readily comparable with our present data, because the eNOS phosphorylation was analyzed in whole-cell preparations; this could explain the disparate findings with respect to eNOS phosphorylation status in diabetes between their study and our observations.
Further experiments focused on eNOS dimer expression, another posttranslational eNOS modification important for NO generation (15eC17). Similar to the eNOS monomer expression, the eNOS dimer-to-monomer ratio was reduced in membrane fractions of diabetic kidneys. The reduction in membrane-bound dimer is likely to further reduce availability of the activatable enzyme in diabetic rats. Defects in eNOS cofactor function or availability may be responsible for impaired eNOS dimerization. A deficit in the cofactor tetrahydrobiopterin, which is essential for eNOS activity and seems to play a role in NOS dimer formation (41), has been implicated in the pathophysiology of diabetes-induced endothelial dysfunction (42,43). In the context of documented glycosylation of the Akt phosphorylation site responsible for reduced eNOS activation in hyperglycemia (24), we also cannot exclude the possibility that glycosylation of specific sites on the eNOS molecule may interfere with dimerization.
Considering the important roles of CAV-1 in the membrane targeting of eNOS and modulation of its enzymatic activity (21,44), changes in eNOS membrane and cytosolic expression in the diabetic kidney may also be related to CAV-1 abundance in the membrane. Theoretically, changes in CAV-1 could be a common denominator for most of the alterations of the eNOS molecule observed in the present studies. To address this issue, we determined CAV-1 expression in crude membrane and cytosolic fractions, and we document here a diabetes-dependent reduction in CAV-1 expression in crude membranes. CAV-1 was expressed and localized in endothelial cells of arteries, arterioles, and glomeruli. CAV-1 immunoreactivity was also apparent in the vascular smooth muscle cells of glomerular arterioles and in basolateral aspects of distal tubules. The distribution of CAV-1 in diabetic kidneys was similar to that in nondiabetic controls. Confocal microscopy studies found that eNOS and CAV-1 colocalized exclusively in endothelia throughout the renal vascular tree, both in control and diabetic animals. Furthermore, as in the extrarenal vasculature, coimmunoprecipitation studies demonstrated direct interaction of both proteins in control and diabetic renal cortex.
Studies have suggested that eNOS needs to be localized in caveolae to interact with proteins and signals for its activation (36,45). Thus, the lack of membrane CAV-1 could explain reductions of eNOS in crude membrane fractions and its rise in the cytosolic compartment. Furthermore, depletion of CAV-1 from caveolar fractions in endothelial cells has been associated with reduced agonist-induced eNOS phosphorylation (45,46). In support of the view that CAV-1 plays a role in diabetes-induced changes in eNOS targeting and phosphorylation are parallel shifts in membrane CAV-1, total eNOS, and phosphorylated eNOS expressions induced by moderate hyperglycemia and improved metabolic control with intensive insulin treatment. Of interest, a similar CAV-1 regulatory pattern as in the present studies (downregulation of its expression and its reversal with insulin treatment) has recently been reported in Schwann cells, implicating this process in diabetic neuropathy as well (47).
Most of our present findings support the abundant evidence demonstrating impaired NO generation and bioavailability in renal and extrarenal vasculature of diabetic animals (3eC6,30). However, considering the fact that eNOS and CAV-1 form an inhibitory complex, reduced CAV-1 expression may be interpreted as a factor favoring NOS enzymatic activity. This observation would be consistent with data suggesting enhanced NO dependency of renal hemodynamics in hyperfiltering diabetic rats and the role of NO in the pathogenesis of hyperfiltration (7eC9) and with those reports suggesting a role for eNOS-derived NO in this process (26,32,33). However, the latter reports relied on measurements of eNOS expression and constitutive renal NOS activity, and the role of eNOS could not be verified by measurement of renal hemodynamic responses to specific eNOS inhibitors. Nevertheless, we believe that the prevailing evidence in this study is compatible with an overall reduction in eNOS-derived NO bioavailability in the diabetic kidney. Supporting this interpretation are our previous renal hemodynamic studies implicating neuronal NOS as the major source of NO involved in the pathophysiology of hyperfiltration (25).
Intensive insulin treatment that achieved tighter metabolic control corrected or attenuated the changes seen in the moderately hyperglycemic diabetic rats. Expression of eNOS and CAV-1 in membrane fractions and the dimer-to-monomer ratio were similar in control rats and diabetic rats on intensive insulin treatment. Similarly, eNOS phosphorylation was also normalized in diabetic rats on intensive insulin treatment. This phenomenon and the previous report in Schwann cells (47) indicate that hyperglycemia and/or low plasma insulin levels are most likely major factors in this process. Moreover, reversal of impaired eNOS phosphorylation in diabetic rats on intensive insulin treatment could be attributable not only to improved glycemic control but even more closely to higher plasma insulin doses resulting in enhanced signaling via Akt kinase (11,12). On the other hand, incomplete reversal of some alterations as observed in diabetic rats on intensive insulin treatment may be attributable to the fact that intensive insulin treatment did not entirely normalize blood glucose levels and A1C.
The possible impact of reduced CAV-1 abundance per se in the diabetic kidney remains to be established. In addition to modulation of eNOS function, CAV-1, as a molecule involved in spatial organization of signaling molecules in caveolae, is a good candidate to play a role in processes implicated in the development of diabetic renal alterations. Caveolae are enriched in a wide array of receptors, growth and vasoactive factors (platelet-derived growth factor, epidermal growth factor, receptor for advanced glycosylation end products, bradykinin, and endothelin), their signaling molecules (diacylglycerol, protein kinase C, and mitogen-activated protein kinase), and enzymes (eNOS) that have been associated with development of diabetes complications.
In summary, we observed complex alterations in eNOS expression, cellular compartmentalization, phosphorylation, and homodimerization in the diabetic renal cortex. These changes were associated with reduced CAV-1 expression. Improvement of metabolic control with intensive insulin treatment corrected or attenuated the differences between control and diabetic rats, suggesting that these changes are related to hyperglycemia and/or insulin deficiency. These data support the view of decreased renal eNOS-derived NO bioavailability in diabetes.
ACKNOWLEDGMENTS
S.A. has received National Institutes of Health Grant DK-63231 and support from the VA Merit Review and the Juvenile Diabetes Research Foundation. S.M. has received support from the VA Merit Review. R.K. has received institutional grant MZO 00023001 of the Czech Ministry of Health Care, Czech Republic.
Parts of this study have been published in abstract form (48).
FOOTNOTES
DOI: 10.2337/db05-1595
The costs of publication of this article were defrayed in part by the payment of page charges. This 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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