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1Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio;
2Department of Medicine, Chonnam National University Hospital, Gwangju, South Korea;
3Audie L. Murphy Memorial Hospital Division and 4Geriatric Research, Education, and Clinical Center, South Texas Veterans Health Care System, San Antonio, Texas
ABSTRACT
Podocytes or glomerular epithelial cells (GECs) are important targets of the diabetic microenvironment. Podocyte foot process effacement and widening, loss of GECs and hypertrophy are pathological features of this disease. ANG II and oxidative stress are key mediators of renal hypertrophy in diabetes. The cellular mechanisms responsible for GEC hypertrophy in diabetes are incompletely characterized. We investigated the effect of high glucose on protein synthesis and GEC hypertrophy. Exposure of GECs to high glucose dose dependently stimulated [3H]leucine incorporation, but not [3H]yhymidine incorporation. High glucose resulted in the activation of ERK1/2 and Akt/PKB. ERK1/2 pathway inhibitor or the dominant negative mutant of Akt/PKB inhibited high glucose-induced protein synthesis. High glucose elicited a rapid generation of reactive oxygen species (ROS). The stimulatory effect of high glucose on ROS production, ERK1/2, and Akt/PKB activation was prevented by the antioxidants catalase, diphenylene iodonium, and N-acetylcysteine. Exposure of the cells to hydrogen peroxide mimicked the effects of high glucose. In addition, ANG II resulted in the activation of ERK1/2 and Akt/PKB and GEC hypertrophy. Moreover, high glucose and ANG II exhibited additive effects on ERK1/2 and Akt/PKB activation as well as protein synthesis. These additive responses were abolished by treatment of the cells with the antioxidants. These data demonstrate that high glucose stimulates GEC hypertrophy through a ROS-dependent activation of ERK1/2 and Akt/PKB. Enhanced ROS generation accounts for the additive effects of high glucose and ANG II, suggesting that this signaling cascade contributes to GEC injury in diabetes.
high glucose; angiotensin II; podocytes; reactive oxygen species; protein synthesis; hypertrophy
PODOCYTES, OR VISCERAL GLOMERULAR epithelial cells (GECs), are highly specialized cells covering the exterior glomerular basement membrane surface of the glomerular capillary (4, 36). In addition to abnormalities in the glomerular endothelium and mesangium, recent data suggest that diabetic nephropathy is accompanied by changes in GECs (49). The diabetic milieu, represented by hyperglycemia, causes broadening and effacement of GEC foot processes associated with the loss of podocytes themselves (30, 49). Because GECs do not usually proliferate, GEC loss results in an increase in size due to hypertrophy and additional widening/effacement of the foot processes to cover the denuded glomerular basement membrane (32, 52). While this is primarily compensatory, GEC hypertrophy is likely maladaptive and contributes to glomerulosclerosis (23, 32, 52).
Components of the local renin-angiotensin systems (RAS) are upregulated in diabetes in proximal tubular epithelial cells, mesangial cells, and GECs (6, 49). GECs can generate ANG II and do express the AT1 receptor (47, 49, 54). High glucose concentration induces ANG II formation in GECs through upregulation of angiotensinogen expression (36, 49). Angiotensin-converting enzyme inhibition or AT1 receptor blockade attenuates GEC foot process broadening/effacement and hypertrophy and prevents loss of GEC in animal models of diabetes or in cells exposed to high glucose, supporting a role for ANG II in GEC injury in diabetes (21, 36, 49, 54).
A growing body of evidence indicates that oxidative stress is a major mechanism of cellular dysfunction in diabetes and contributes to diabetic complications, including diabetic nephropathy (5, 27, 28). It has been suggested that overproduction of reactive oxygen species (ROS) is the main initiating and progression factor involved in the development of diabetic nephropathy (5, 27, 28). Increased ROS generation induces GEC dysfunction in glomerular diseases such as Heymann nephritis (34, 36, 49). However, to our knowledge, no data related to the role of ROS in GEC injury in diabetes have been reported.
In this study, we explored the effects of high glucose and ANG II on oxidant generation and hypertrophy of GECs. We describe an essential role for oxidative stress in high glucose-induced GEC hypertrophy. We provide the first evidence that in these cells high glucose stimulates protein synthesis via a redox-dependent activation of the protein kinases ERK1/2 and Akt/PKB. Finally, our data show that high glucose and ANG II elicit additive responses to activate these signals and to induce GEC hypertrophy.
MATERIALS AND METHODS
Cell culture and transfections. Rat visceral GECs (podocytes) were isolated and characterized as described by Kreisberg et al. (26). These cells were used between passages 10 and 12. Cells were maintained in DMEM supplemented with antibiotic/antifungal solution and 10% fetal bovine serum. GECs were transiently transfected with plasmid DNA [15 μg of vector alone or hemagglutinin (HA)-Akt(K179M)] via electroporation (Gene pulser, Bio-Rad) as previously described (14). The HA epitope-tagged mammalian expression construct HA-Akt(K179M) was a generous gift of Dr. T. F. Franke (Harvard Institutes of Medicine, Boston, MA).
Immunoprecipitation and ERK1/2 and Akt/PKB activity assay. GECs were grown to near confluency in 60- or 100-mm dishes and serum-deprived for 48 h. All incubations were carried out in serum-free DMEM at 37°C for a specified duration. The cells were lysed in radioimmune precipitation buffer [20 mM Tris?HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1% NP-40] at 4°C for 30 min. The cell lysates were centrifuged at 10,000 g for 30 min at 4°C. Protein was determined in the cleared supernatant using the Bio-Rad method. Immunoprecipitation and ERK1/2 and Akt/PKB activity assays were performed as described (14, 16). Anti-ERK1/2 or Akt/PKB immunoprecipitates were used in an immune complex kinase assay using myelin basic protein as a substrate. For immunoprecipitation, rabbit anti-ERK1/2 (Santa Cruz Biotechnology) or sheep anti-Akt1/PKB (Upstate Biotechnology) was used. The bands corresponding to phosphorylated myelin basic protein were quantitated by densitometry and/or PhosphorImager analysis.
Immunoblotting. GEC lysates were prepared as described above for ERK1/2 and Akt/PKB activity assay. For immunoblotting, proteins (2550 μg) were separated using 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated with mouse polyclonal phosphospecific ERK (1:1,000), rabbit anti-ERK1/2 (1:2,000) or rabbit polyclonal anti-Akt1/PKB (1:1,000, Cell Signaling Technology), and the primary antibodies were detected using horseradish peroxidase-conjugated IgG (1:2,500 or 1:5,000). Bands were visualized by enhanced chemiluminescence. Densitometric analysis was performed using National Institutes of Health Image software.
Detection of intracellular ROS. The peroxide-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescin diacetate (Molecular Probes) was used to assess the generation of intracellular ROS as described previously (14, 44). This compound is converted by intracellular esterases to 2',7'-dichlorodihydrofluorescin, which is then oxidized by intracellular ROS to the highly fluorescent 2',7'-dichlorodihydrofluorescein (DCF). Cells were grown in 120- or 24-well plates and serum-deprived for 48 h. Immediately before the experiments, cells were washed with HBSS and loaded with 50 μM 2',7'-dichlorodihydrofluorescin diacetate dissolved in HBSS for 30 min at 37°C. They were then incubated for 30 min with different inhibitors (see RESULTS) followed by the addition of a selected agonist or vehicle for various time periods. Subsequently, DCF fluorescence was detected at excitation and emission wavelengths of 488 and 520 nm, respectively, and measured with a multiwell fluorescence plate reader (Wallac 1420 Victor2, PerkinElmer).
Measurement of DNA synthesis. DNA synthesis is measured as the incorporation of [3H]thymidine into TCA-insoluble material as described previously (14, 16). Briefly, confluent GECs were washed with phosphate-buffered saline and incubated in serum-free medium for 48 h. Agonists were then added for an additional 48 h before being pulsed with 1 μCi/ml [3H]thymidine for 4 h. The medium was removed, and the cells were washed twice with ice-cold 5% TCA to remove unincorporated [3H]thymidine. Cells were solubilized by adding 0.75 ml of 0.25 N NaOH and 0.1% SDS. A 0.5-ml volume of this cell lysate was neutralized and counted in a scintillation counter.
[3H]leucine incorporation. [3H]leucine incorporation was measured as previously shown (14, 16). GECs were grown is six-well dishes, made quiescent in serum-free medium for 48 or 72 h. GECs were then incubated with or without agonist for 48 h. Six hours before being harvested, GECs were pulsed with 2 μCi/ml [3H]leucine. At the end of this incubation period, cells were washed three times with phosphate-buffered saline and solubilized overnight with 1.5 ml of 0.1% SDS. The content of two wells was pooled and transferred into a tube containing 60 μl of 10% bovine serum albumin. Proteins were precipitated with 300 μl 20% TCA and left overnight at 4°C. Samples were then centrifuged at 2,000 g for 30 min at 4°C, the supernatant was discarded, and the pellet was resuspended in 0.5 N NaOH. Duplicate aliquots (0.5 ml) were removed and counted in a scintillation counter.
Statistical analysis. Results are expressed as means ± SE. Statistical significance was assessed by Student's unpaired t-test. Significance was determined at P < 0.05.
RESULTS
High glucose concentration induces protein synthesis and hypertrophy of GECs. To study GECs, hypertrophy was measured as the ratio of the incorporation of [3H]eucine, a measure of protein synthesis, to the incorporation of [3H]thymidine, a measure of DNA synthesis. Exposure of quiescent confluent GECs to various concentrations of glucose (530 mM) dose dependently stimulated [3H]leucine incorporation. The maximal effect occurred at 2530 mM and represented a 1.5- to 1.9-fold increase in [3H]leucine incorporation compared with cells treated with normal glucose concentration (5 mM) (Fig. 1A). Exposure of the cells to high glucose concentration (25 mM) had no significant effect on [3H]thymidine incorporation (Fig. 1B), suggesting that high glucose induces GEC hypertrophy. FCS (10% vol/vol), which served as a positive control, significantly stimulated GEC DNA synthesis. Figure 1C shows that no change in protein synthesis was observed in cells exposed to mannitol (20 mM) as an osmotic control.
Effects of high glucose on ERK1/2 and Akt/PKB activity in GECs. The MAPK family members ERK1/2 and the serine-threonine kinase Akt/PKB play a critical role in cell growth and hypertrophy in various cell types, including renal cells (11, 14, 16, 19, 20). We postulated that ERK1/2 and Akt/PKB activation contributes to glucose-induced protein synthesis and hypertrophy in GECs. To examine the effect of glucose on the activity of ERK1/2 and Akt/PKB, cultured GECs were incubated with either 25 mM glucose for different periods of time or with various concentrations of glucose (0.0011 μM) for 15 min. Anti-ERK1/2 or anti-Akt/PKB immunoprecipitates were used in an immune complex kinase assay using myelin basic protein as a substrate. As shown in Figs. 2, A and C, high glucose concentration caused a rapid increase in ERK1/2 and Akt/PKB activity in a time-dependent manner, an effect that peaked at 15 min and subsided by 180 min. Stimulation of GECs with different concentrations of glucose showed dose-dependent increase in ERK1/2 and Akt/PKB activity, with a threshold at 20 mM and a maximal effect occurring at 2530 mM (Fig. 1, B and D). Mannitol (20 mM) did not affect ERK1/2 or Akt/PKB activity (Fig. 2E). Glucose had no significant effect on ERK1/2 and Akt/PKB protein levels, as determined by immunoblotting (Figs. 1, AD, middle).
Role of ERK1/2 and Akt/PKB in high glucose-induced GEC hypertrophy. The above-mentioned studies suggest that high glucose is a strong activator for ERK1/2 and Akt/PKB; therefore, we postulated that these kinases might be involved in glucose-induced protein synthesis. Inhibition of the ERK1/2 pathway by treatment of the cells with MEK inhibitor PD-98059 abrogated high glucose-induced [3H]leucine incorporation (Fig. 3A). To assess the role of Akt/PKB in GEC protein synthesis, we tested the effect of HA-tagged kinase-inactive Akt/PKB with a point mutation in the ATP-binding site [HA-Akt(K179M)] on high glucose concentration-stimulated [3H]leucine incorporation. As shown in Fig. 3B, inhibition of Akt by transient transfection of the cells with HA-Akt(K179M) abolished glucose-induced [3H]leucine incorporation. Of note, the dose-responses of high glucose-induced [3H]leucine incorporation paralleled the dose-responses of high glucose-induced ERK1/2 and Akt/PKB activation. Taken together, these findings are consistent with a critical role for ERK1/2 and Akt/PKB in the hypertrophic response of GECs to high glucose.
Effect of high glucose on ROS production in GECs. High glucose has been reported to increase ROS production in vascular cells as well as in renal cells including tubular epithelial cells or mesangial cells but not in GECs (5, 25, 27, 28). To evaluate the role of high glucose concentration as a signal leading to oxidative stress in GECs, we examined their effect on the generation of ROS. The production of intracellular ROS by GECs in response to high glucose concentration was demonstrated with a fluorescence-based assay using peroxide-sensitive fluorophore DCF. Quantification of DCF fluorescence was performed with a multiwell fluorescence plate reader. Incubation of GECs with 25 mM glucose resulted in a rapid and time-dependent increase in DCF fluorescence; a significant increase was seen as early as 2.5 min after treatment (Fig. 4A). Catalase (300 U/ml) completely blocked the oxidative effect of high glucose, suggesting that intracellular hydrogen peroxide (H2O2) is primarily responsible for the fluorescence signal (Fig. 4B). Moreover, high glucose-induced ROS production was almost completely blocked by diphenylene iodonium (DPI; 10 μM), an inhibitor of flavoprotein-containing enzymes, such as the NAD(P)H oxidase systems, or by the antioxidant N-acetylcysteine (NAC; 20 mM), (Fig. 4B). Exposure of the cells to mannitol (20 mM) had no effect on ROS generation (Fig. 4C).
Collectively, these results demonstrate that high glucose elicits ROS production in GECs. Furthermore, it appears that the time courses of intracellular ROS generation in response to glucose are consistent with a potential role for these ROS in downstream signaling events and, in particular, regulation of redox-sensitive protein kinases.
Role of ROS in high glucose-induced ERK1/2 and Akt/PKB activation and in GEC hypertrophy. It has been reported that the activity of ERK1/2 and Akt/PKB is redox sensitive in various cells type including renal proximal tubular epithelial cells or mesangial cells (7, 14, 16, 19, 46). The correlation between the time courses of high glucose-induced ERK1/2 and Akt/PKB activation and the kinetics of oxidant production in response to glucose (compare Figs. 2, A and B, and 4A) indicates that ROS may mediate the effects of high glucose on ERK1/2 and Akt/PKB. To test this hypothesis, we studied the effect of H2O2 on ERK1/2 and Akt/KB activation in GECs. As shown in Fig. 4, D and E, 100 μM H2O2 induced robust activation of ERK1/2 and Akt/PKB. The kinetics were consistent with the time courses of ERK1/2 and Akt/PKB activation by high glucose. Next, we examined the effect of NAC and DPI on high glucose-induced ERK1/2 and Akt/PKB activation. As shown in Fig. 4, F and G, NAC (20 mM) and DPI (10 μM) significantly inhibited both high glucose-induced ERK1/2 and Akt/PKB activation. These data strongly suggest that high glucose concentration-induced ERK1/2 and Akt/PKB activation is mediated by ROS.
Treatment of GECs with H2O2 stimulates [3H]leucine incorporation (Fig. 4H), indicating that ROS induce protein synthesis. Exposure of GECs to 300 U/ml of catalase, 20 mM NAC or 5 μM DPI, concentrations that blocked the activation of ERK1/2 and Akt/PKB and ROS generation, inhibited the increase in [3H]leucine incorporation induced by high glucose (Fig. 4I). Moreover, these data provide additional evidence for the involvement of a ROS-dependent mechanism in the high glucose-induced ERK1/2 and Akt/PKB activation and implicate ROS as mediators of the signaling pathway activated by high glucose, leading to GEC hypertrophy via ERK1/2 and Akt/PKB.
Additive effect of high glucose and ANG II on the redox-, ERK1/2-, and Akt/PKB-dependent pathways modulating GEC hypertrophy. Under hyperglycemic conditions, the vasoactive peptide ANG II has been shown to contribute to the alteration of vascular and renal cell function that leads to hypertrophy and fibrosis, the two major manifestations of diabetic vasculopathy or nephropathy (6, 49, 52). Activation of ERK1/2 and Akt/PKB pathways plays a key role in ANG II-induced deleterious effects such as cell hypertrophy (14, 19, 28, 46). Furthermore, hyperglycemia enhances the signaling pathways triggered by ANG II in various cell types (1, 2, 33, 40, 45, 50). Whether high glucose concentrations and ANG II exert additive effects on the hypertrophic cascade involving ROS, ERK1/2, and Akt/PKB, however, has not been previously investigated in GECs. First, we evaluated the effect of treatment of GECs with ANG II on ERK1/2 and Akt/PKB activation. As shown in Fig. 5, A and B, ANG II (1 μM) caused an increase in ERK1/2 and Akt/KB activity in a time-dependent manner, an effect that peaked at 5 min and returned to basal levels within 180 min. ANG II had no significant effect on ERK1/2 or Akt/PKB levels as determined by immunoblotting. This indicates that, similar to what is observed with high glucose concentration, ANG II stimulates the two kinases in GECs. We next investigated whether an additive or a synergistic interaction exists between high glucose and ANG II and assessed its potential role in the regulation of the redox-, ERK1/2- and Akt/PKB-dependent hypertrophic cascade accounting for GEC hypertrophy. To evaluate the effect of high glucose and ANG II on ERK1/2 and Akt/PKB activity, GECs were incubated for 5 days in media containing either normal glucose (5 mM) or high glucose (25 mM) before exposure to ANG II (1 μM) for 5 min. As depicted in Fig. 6, A and B, high glucose alone or ANG II under normal glucose increased ERK1/2 and Akt/PKB activity. Importantly, high glucose significantly enhanced ANG II-induced ERK1/2 and Akt/PKB activation (Fig. 6, A and B), demonstrating that high glucose and ANG II displayed an additive response. Similarly, both high glucose and ANG II increased protein synthesis. Combined exposure of GECs to high glucose and ANG II caused an augmentation in [3H]leucine incorporation greater than that observed with high glucose or ANG II alone (Fig. 6C). These data indicate that high glucose and ANG II also act in an additive manner to induce protein synthesis. Finally, the redox dependence of this additive effect was tested. Figure 6D showed that treatment with catalase, DPI, or NAC totally abrogated the additive effect of high glucose and ANG II on ERK1/2 and Akt/PKB activity. Moreover, the induction of ERK1/2 and Akt/PKB activity by high glucose or ANG II alone is also mediated by ROS. Similarly, catalase and DPI abolished the additive action of high glucose and ANG II on protein synthesis as well as the increase in [3H]leucine incorporation due to high glucose or ANG II by themselves (Fig. 6E). This suggests that the additive response to high glucose concentration and ANG II is a redox-sensitive process and that augmented ROS generation may account for the amplified ERK1/2 and Akt/PKB activation and subsequent GEC protein synthesis and hypertrophy.
DISCUSSION
In this study, we provide evidence that exposure of GECs to high glucose activates MAPK ERK1/2 and the serine-threonine protein kinase Akt/PKB through generation of ROS. We also demonstrate that this redox-dependent activation of these signaling molecules mediates high glucose-induced GEC protein synthesis and cell hypertrophy. Furthermore, high glucose concentration and ANG II have additive effects on ERK1/2 and Akt/PKB activation and protein synthesis in GECs. The data indicate that oxidative stress contributes to podocyte injury in diabetes.
In a number of cells, high glucose promotes an increase in ERK1/2 and Akt/PKB activity (12, 18, 38, 41, 42). In GECs, we found that the increase in the activity of the two kinases was observed after a short (5180 min) as well as prolonged exposure (5 days) to high glucose. Prolonged exposure of GECs results in ERK1/2 activation (22). However, this is the first report of activation of the MAPK after short-term exposure to high glucose. It is also the first report of Akt/PKB activation by high glucose in these cells. Hence, activation of the ERK1/2 and Akt/PKB pathways represents a very proximal step in the intracellular signaling triggered by high glucose in GECs. Despite the demonstration that high glucose stimulates protein synthesis and hypertrophy in cultured GECs (22, 54) and that, in vivo, GEC hypertrophy contributes to glomerular enlargement in diabetic nephropathy (49), the mechanisms underlying these processes or the profile of growth of these cells in a diabetic milieu remains largely unknown. Understanding the events involved in GEC hypertrophy is important. Indeed, it is postulated that GECs do not replicate, most likely because of the constitutive overexpression of cell cycle-arresting proteins, which include cyclin-dependent kinase inhibitors such as p21Cip1 or p27Kip1 (36, 49). Therefore, GECs typically cope with injury in many instances by undergoing hypertrophy (23, 32, 52). Recent evidence suggests that GECs play a critical role in the early functional and structural changes of diabetic kidney disease. Hypertrophy of GECs with broadening and effacement of their foot processes, associated with a decrease in their number per glomerulus, occurs in type 1 and 2 diabetes (13, 31, 35, 41, 43, 44). Hypertrophy eventually leads to a degenerative process in GECs that accounts for the development of proteinuria and glomerulosclerosis in diabetes (8, 13, 20, 27, 29, 35, 43, 44). GEC hypertrophy occurs early in diabetes and accompanies glomerular enlargement (9, 20, 29). At this stage, broadening of the foot process occurs to cover the expanded basement membrane (9, 20, 29). The development of diabetic nephropathy in experimental animal models as well as in diabetic patients is associated with a decrease in the number of GECs (13, 35, 41, 43). Given that the GECs are unable to replicate, remaining GECs expand and broaden their foot processes to cover the denuded basement membrane (8, 13, 31, 35, 41, 43, 44). Moreover, Wiggins et al. (48), using a rat model of aging, provided evidence that there are recognizable stages of hypertrophy through which GEC pass en route to GEC loss and consequent glomerulosclerosis. These authors postulated that similar stages of GEC hypertrophy occur in association with glomerulomegaly caused by diabetes.
The present study provides evidence that ERK1/2 and Akt/PKB mediate high glucose-induced protein synthesis and establish a basis for the cellular mechanisms of the hypertrophic response to high glucose. It is known that Akt/PKB participates in the hypertrophy of renal cells, including mesangial cells and proximal tubular epithelial cells (14, 15, 39) as well as GECs, on stimulation with mechanical stretch (37). However, to the best of our knowledge, the kinase has not been identified as an intermediate in the signaling cascade linking high glucose to GEC hypertrophy. The involvement of the MEK/ERK1/2 pathway corroborates the report from Hoshi et al. (22) demonstrating that glucose-induced activation of ERK1/2 is responsible, at least partially, for GEC hypertrophy. Hyperglycemia and high glucose concentration induce several growth factors and upregulate their receptors in renal cells, which, in turn, result in growth and the production of extracellular matrix proteins (52). The vasoactive peptide ANG II is of obvious biological relevance as a major pathogenic mediator of diabetic nephropathy (52). In addition to its hemodynamic properties, ANG II exerts potent effects on several renal cell types to stimulate hypertrophy and synthesis of extracellular matrix proteins (6, 49, 52). Our present findings demonstrate for the first time that ANG II also elicits a robust activation of ERK1/2 and Akt/PKB in GECs. Interestingly, we found that high glucose and ANG II elicited additive responses on ERK1/2 and Akt/PKB activation and subsequent GEC hypertrophy. This supports the contention that high glucose and ANG II exert additive hypertrophic and prosclerotic effects in vascular and renal cells. For instance, high glucose and ANG II additively induce hypertrophy in renal tubular cells (50). Furthermore, additive effects of high glucose and ANG II on ERK1/2 and Janus-activated kinase/signal transducers and activators of transcription pathway activation are key mechanisms for the increased vascular smooth muscle cell or mesangial cell growth under high-glucose conditions (1, 2, 33, 40, 45).
The identification of ERK1/2 and Akt/PKB as mediators of GEC hypertrophy is consistent with the current hypothesis that high glucose- or ANG II-induced hypertrophy requires the cyclin-dependent kinase inhibitors p21Cip1 or p27Kip1 in several renal cells including podocytes. Indeed, it has been proposed that activation of ERK1/2 and Akt/PKB by ANG II mediates p27Kip1 upregulation, leading to hypertrophy of proximal tubular epithelial and mesangial cells (19, 56). Similarly, ERK1/2-dependent increased expression of p27Kip1 plays a key role in high glucose-induced mesangial cell hypertrophy (51). Moreover, blockade of the ANG II AT1 receptor lowers p27Kip1 expression in GEC exposed to high glucose (54). These observations suggest that a related signaling cascade exists in GECs.
There is now considerable evidence that ROS function as classic second messenger molecules (10). It has been reported that ERK1/2 and Akt/PKB are activated by oxidative stress in a variety of cell types including renal cells (7, 14, 16, 19, 46). Oxidative stress has been implicated in the development of diabetic complications (5, 27, 28). High glucose induces ROS generation in vascular cells and renal cells (5, 27, 28). However, the effect of high glucose on ROS in GEC has not been investigated. Exposure of GECs to high glucose elicits a rapid increase in intracellular ROS production. This early effect supports the contention that ROS mediate early signaling events in response to high glucose. Importantly, we show that ROS, and specifically H2O2, directly induce ERK1/2 and Akt/PKB activation, as well as protein synthesis. Moreover, the effect of high glucose on ERK1/2 and Akt/PKB is markedly reduced by the antioxidants catalase, NAC, and DPI. Finally, the same antioxidants nearly abrogated high glucose-induced protein synthesis. Collectively, these findings indicate that ROS are signaling molecules responsible for ERK1/2 and Akt/PKB activation initiated by high glucose and resulting in protein synthesis. Although oxidants derived from a mitochondrial source were shown to be predominant in certain cell types in response to high glucose (5), others have reported that activation of NAD(P)H oxidases play a pivotal role in high glucose-induced ROS generation (3, 24, 25, 27, 28). We find that the generation of intracellular ROS, the activation of ERK1/1 and Akt/PKB, as well as the stimulation of protein synthesis in response to high glucose are abolished by DPI, an inhibitor of flavin-containing oxidases, such as NAD(P)H oxidase. In addition, the kinetics of oxidant generation together with the fact that exogenously applied catalase inhibits DCF fluorescence indicate that ROS are rapidly released from GECs. This strongly suggests that ROS generation happens most likely through enhanced NAD(P)H oxidase activity. While the NAD(P)H oxidase components p47phox, p67phox, p22phox, and gp91phox/Nox2 are known to be expressed in GECs (17, 34), the precise structure and the mechanism of activation of the various oxidases responsible for generation of ROS in these cells remain to be determined.
Interestingly, the antioxidants catalase, NAC, and DPI prevented the additive effects of high glucose and ANG II on ERK1/2 and Akt/PKB activation and on protein synthesis. This strongly suggests that increased ROS generation and the consequent augmentation in ERK1/2 and Akt/PKB activity constitute the molecular basis of the additive response exhibited by high glucose and ANG II. Importantly, the action of high glucose or ANG II per se on the two kinases and on protein synthesis are redox dependent as well. Therefore, it is tempting to propose that high glucose and ANG II signaling may converge at the level of ROS, resulting in the enhancement of ANG II-stimulated ERK1/2 and Akt/PKB activation as well as GEC hypertrophy in response to high glucose. Such regulation has been reported in adipocytes, where high glucose enhances insulin-stimulated ROS generation and augments its signaling (53). In aortic endothelial cells, the effects of leptin on ROS production are also additives to those of high glucose (55).
Our results unveil the pivotal role of ERK1/2 and Akt/PKB activation through ROS by conditions mimicking hyperglycemia in GEC protein synthesis and hypertrophy. Moreover, this study provides evidence that, in conditions characterized by activation of the renin-angiotensin system such as diabetes, high glucose and ANG II may exert additive effects on GEC hypertrophy, thereby altering cell function (Fig. 7). ROS generation has been implicated in GEC dysfunction in inflammatory diseases of the kidney (34, 36, 49). Therefore, understanding the GEC response to oxidative stress and the identification of the source(s) of ROS in diabetes may have direct therapeutic implications.
GRANTS
Y. Gorin was supported through a regular Research Grant from the Juvenile Diabetes Research Foundation, a Scientist Development Grant from the American Heart Association, and a National Kidney Foundation Young Investigator Grant. This work was supported by a Veterans Affairs Research Enhancement Award Program (G. G. Choudhury and H. E. Abboud), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43988 and DK-33665 (H. E. Abboud), the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases George M. O'Brien Kidney Center through P50 DK-61597 (H. E. Abboud), and a regular Research Grant from the Juvenile Diabetes Research Foundation (G. G. Choudhury).
ACKNOWLEDGMENTS
We thank Sergio Garcia for help with cell culture.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Amiri F, Shaw S, Wang X, Tang J, Waller JL, Eaton DC, and Marrero MB. Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose. Kidney Int 61: 16051616, 2002.
Amiri F, Venema VJ, Wang X, Ju H, Venema RC, and Marrero MB. Hyperglycemia enhances angiotensin II-induced janus-activated kinase/STAT signaling in vascular smooth muscle cells. J Biol Chem 274: 3238232386, 1999.
Asaba K, Tojo A, Onozato ML, Goto A, Quinn MT, Fujita T, and Wilcox CS. Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int 67: 18901898, 2005.
Barisoni L and Kopp JB. Update in podocyte biology: putting one's best foot forward. Curr Opin Nephrol Hypertens 12: 251258, 2003.
Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes 54: 16151625, 2005.
Carey RM and Siragy HM. The intrarenal renin-angiotensin system and diabetic nephropathy. Trends Endocrinol Metab 14: 274281, 2003.
Chen K, Thomas SR, Albano A, Murphy MP, and Keaney JF Jr. Mitochondrial function is required for hydrogen peroxide-induced growth factor receptor transactivation and downstream signaling. J Biol Chem 279: 3507935086, 2004.
Coimbra TM, Janssen U, Grone HJ, Ostendorf T, Kunter U, Schmidt H, Brabant G, and Floege J. Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int 57: 167182, 2000.
Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, and Fioretto P. Is podocyte injury relevant in diabetic nephropathy Studies in patients with type 2 diabetes. Diabetes 52: 10311035, 2003.
Droge W. Free radicals in the physiological control of cell function. Physiol Rev 82: 4795, 2002.
Fujita H, Omori S, Ishikura K, Hida M, and Awazu M. ERK and p38 mediate high-glucose-induced hypertrophy and TGF- expression in renal tubular cells. Am J Physiol Renal Physiol 286: F120F126, 2004.
Fujita H, Omori S, Ishikura K, Hida M, and Awazu M. ERK and p38 mediate high-glucose-induced hypertrophy and TGF- expression in renal tubular cells. Am J Physiol Renal Physiol 286: F120F126, 2004.
Gassler N, Elger M, Kranzlin B, Kriz W, Gretz N, Hahnel B, Hosser H, and Hartmann I. Podocyte injury underlies the progression of focal segmental glomerulosclerosis in the fa/fa Zucker rat. Kidney Int 60: 106116, 2001.
Gorin Y, Kim NH, Feliers D, Bhandari B, Choudhury GG, and Abboud HE. Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase. FASEB J 15: 19091920, 2001.
Gorin Y, Ricono JM, Kim NH, Bhandari B, Choudhury GG, and Abboud HE. Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells. Am J Physiol Renal Physiol 285: F219F229, 2003.
Gorin Y, Ricono JM, Wagner B, Kim NH, Bhandari B, Choudhury GG, and Abboud HE. Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells. Biochem J 381: 231239, 2004.
Greiber S, Munzel T, Kastner S, Muller B, Schollmeyer P, and Pavenstadt H. NAD(P)H oxidase activity in cultured human podocytes: effects of adenosine triphosphate. Kidney Int 53: 654663, 1998.
Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, and Kikkawa R. Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. Diabetes 46: 847853, 1997.
Hannken T, Schroeder R, Zahner G, Stahl RA, and Wolf G. Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27Kip1: role in angiotensin II-mediated hypertrophy of proximal tubular cells. J Am Soc Nephrol 11: 13871397, 2000.
Hixon ML, Muro-Cacho C, Wagner MW, Obejero-Paz C, Millie E, Fujio Y, Kureishi Y, Hassold T, Walsh K, and Gualberto A. Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization. J Clin Invest 106: 10111020, 2000.
Hoffmann S, Podlich D, Hahnel B, Kriz W, and Gretz N. Angiotensin II type 1 receptor overexpression in podocytes induces glomerulosclerosis in transgenic rats. J Am Soc Nephrol 15: 14751487, 2004.
Hoshi S, Nomoto K, Kuromitsu J, Tomari S, and Nagata M. High glucose induced VEGF expression via PKC and ERK in glomerular podocytes. Biochem Biophys Res Commun 290: 177184, 2002.
Hoshi S, Shu Y, Yoshida F, Inagaki T, Sonoda J, Watanabe T, Nomoto K, and Nagata M. Podocyte injury promotes progressive nephropathy in zucker diabetic fatty rats. Lab Invest 82: 2535, 2002.
Hua H, Munk S, Goldberg H, Fantus IG, and Whiteside CI. High glucose-suppressed endothelin-1 Ca2+ signaling via NADPH oxidase and diacylglycerol-sensitive protein kinase C isozymes in mesangial cells. J Biol Chem 278: 3395133962, 2003.
Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Utsumi H, and Nawata H. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 49: 19391945, 2000.
Kreisberg JI, Hoover RL, and Karnovsky MJ. Isolation and characterization of rat glomerular epithelial cells in vitro. Kidney Int 14: 2130, 1978.
Lee HB, Yu MR, Yang Y, Jiang Z, and Ha H. Reactive oxygen species-regulated signaling pathways in diabetic nephropathy. J Am Soc Nephrol 14: S241S245, 2003.
Li JM and Shah AM. ROS generation by nonphagocytic NADPH oxidase: potential relevance in diabetic nephropathy. J Am Soc Nephrol 14: S221S226, 2003.
Marshall CB and Shankland SJ. Cell cycle and glomerular disease: a minireview. Nephron Exp Nephrol 102: e39e48, 2005.
Marshall SM. Recent advances in diabetic nephropathy. Clin Med 4: 277282, 2004.
Mifsud SA, Allen TJ, Bertram JF, Hulthen UL, Kelly DJ, Cooper ME, Wilkinson-Berka JL, and Gilbert RE. Podocyte foot process broadening in experimental diabetic nephropathy: amelioration with renin-angiotensin blockade. Diabetologia 44: 878882, 2001.
Mundel P and Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 13: 30053015, 2002.
Natarajan R, Scott S, Bai W, Yerneni KK, and Nadler J. Angiotensin II signaling in vascular smooth muscle cells under high glucose conditions. Hypertension 33: 378384, 1999.
Neale TJ, Ullrich R, Ojha P, Poczewski H, Verhoeven AJ, and Kerjaschki D. Reactive oxygen species and neutrophil respiratory burst cytochrome b558 are produced by kidney glomerular cells in passive Heymann nephritis. Proc Natl Acad Sci USA 90: 36453649, 1993.
Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, Coplon NS, Sun L, and Meyer TW. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 99: 342348, 1997.
Pavenstadt H, Kriz W, and Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev 83: 253307, 2003.
Petermann AT, Pippin J, Durvasula R, Pichler R, Hiromura K, Monkawa T, Couser WG, and Shankland SJ. Mechanical stretch induces podocyte hypertrophy in vitro. Kidney Int 67: 157166, 2005.
Sautin YY, Lu M, Gaugler A, Zhang L, and Gluck SL. Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells. Mol Cell Biol 25: 575589, 2005.
Senthil D, Choudhury GG, McLaurin C, and Kasinath BS. Vascular endothelial growth factor induces protein synthesis in renal epithelial cells: a potential role in diabetic nephropathy. Kidney Int 64: 468479, 2003.
Shaw S, Wang X, Redd H, Alexander GD, Isales CM, and Marrero MB. High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway. J Biol Chem 278: 3063430641, 2003.
Sheu ML, Ho FM, Chao KF, Kuo ML, and Liu SH. Activation of phosphoinositide 3-kinase in response to high glucose leads to regulation of reactive oxygen species-related nuclear factor-B activation and cyclooxygenase-2 expression in mesangial cells. Mol Pharmacol 66: 187196, 2004.
Sheu ML, Ho FM, Yang RS, Chao KF, Lin WW, Lin-Shiau SY, and Liu SH. High glucose induces human endothelial cell apoptosis through a phosphoinositide 3-kinase-regulated cyclooxygenase-2 pathway. Arterioscler Thromb Vasc Biol 25: 539545, 2005.
Steffes MW, Schmidt D, McCrery R, Basgen JM; International Diabetic Nephropathy Study Group. Glomerular cell number in normal subjects and in type 1 diabetic patients. Kidney Int 59: 21042113, 2001.
Thabut G, El-Benna J, Samb A, Corda S, Megret J, Leseche G, Vicaut E, Aubier M, and Boczkowski J. Tumor necrosis factor- increases airway smooth muscle oxidants production through a NADPH oxidase-like system to enhance myosin light chain phosphorylation and contractility. J Biol Chem 277: 2281422821, 2002.
Tsiani E, Lekas P, Fantus IG, Dlugosz J, Whiteside C. High glucose-enhanced activation of mesangial cell p38 MAPK by ET-1, ANG II, and platelet-derived growth factor. Am J Physiol Endocrinol Metab 282: E161E169, 2002.
Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, and Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274: 2269922704, 1999.
Wang L, Flannery PJ, and Spurney RF. Characterization of angiotensin II-receptor subtypes in podocytes. J Lab Clin Med 142: 313321, 2003.
Wiggins JE, Goyal M, Sanden SK, Wharram BL, Shedden KA, Misek DE, Kuick RD, and Wiggins RC. Podocyte hypertrophy, "adaptation," and "decompensation" associated with glomerular enlargement and glomerulosclerosis in the aging rat: prevention by calorie restriction. J Am Soc Nephrol 16: 29532966, 2005.
Wolf G and Ziyadeh FN. Molecular mechanisms of diabetic renal hypertrophy. Kidney Int 56: 393405, 1999.
Wolf G, Chen S, and Ziyadeh FN. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy. Diabetes 54: 16261634, 2005.
Wolf G, Neilson EG, Goldfarb S, and Ziyadeh FN. The influence of glucose concentration on angiotensin II-induced hypertrophy of proximal tubular cells in culture. Biochem Biophys Res Commun 176: 902909, 1991.
Wolf G, Reinking R, Zahner G, Stahl RA, and Shankland SJ. Erk 1,2 phosphorylates p27Kip1: Functional evidence for a role in high glucose-induced hypertrophy of mesangial cells. Diabetologia 46: 10901109, 2003.
Wu X, Zhu L, Zilbering A, Mahadev K, Motoshima H, Yao J, and Goldstein BJ. Hyperglycemia potentiates H2O2 production in adipocytes and enhances insulin signal transduction: potential role for oxidative inhibition of thiol-sensitive protein-tyrosine phosphatases. Antioxid Redox Signal 7: 526537, 2005.
Xu ZG, Yoo TH, Ryu DR, Cheon Park H, Ha SK, Han DS, Adler SG, Natarajan R, and Kang SW. Angiotensin II receptor blocker inhibits p27Kip1 expression in glucose-stimulated podocytes and in diabetic glomeruli. Kidney Int 67: 944952, 2005.
Yamagishi SI, Edelstein D, Du XL, Kaneda Y, Guzman M, and Brownlee M. Leptin induces mitochondrial superoxide production and monocyte chemoattractant protein-1 expression in aortic endothelial cells by increasing fatty acid oxidation via protein kinase A. J Biol Chem 276: 2509625100, 2001.
Zeng L, Xu H, Chew TL, Chisholm R, Sadeghi MM, Kanwar YS, and Danesh FR. Simvastatin modulates angiotensin II signaling pathway by preventing Rac1-mediated upregulation of p27. J Am Soc Nephrol 15: 17111720, 2004.