Hydrogen Sulfide Inhibits High Glucose-induced Matrix Protein Synthesis by Activating AMP-activated Protein Kinase in Renal Epithelial Cells*♦

Background: Whether hydrogen sulfide regulates protein synthesis is not known. Results: In kidney cells, hydrogen sulfide inhibited high glucose-induced synthesis of proteins including matrix proteins by activating AMP-activated protein kinase and inhibiting events in mRNA translation. Conclusion: Hydrogen sulfide reduces high glucose stimulation of matrix protein synthesis in renal cells. Significance: Hydrogen sulfide induction may inhibit kidney matrix protein accumulation in diabetes. Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H2S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mM) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H 2 S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase ␤, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase ␤ inhibitor, had the same effect. Renal cortical content of cystathionine ␤-synthase and cystathionine ␥-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kid-ney, and reduction in its generation may contribute to kidney injury in diabetes.
Biologically active gases such as nitric oxide and carbon monoxide modulate tissue function. Vessel wall relaxation and vasodilatation occur in mice lacking both endothelial nitricoxide synthase and cyclooxygenase, suggesting the presence of other endothelium-dependent relaxation factors (1). Among the endothelium-dependent relaxation factors, hydrogen sulfide and carbon monoxide have attracted attention. Hydrogen sulfide was proposed as a physiologically active neurotransmitter in the brain (2). Until recently, there was controversy whether endogenous hydrogen sulfide had any physiological significance. A remarkable study reported that when hydrogen sulfide generation was genetically suppressed by deleting cystathionine ␥-lyase, an enzyme that generates hydrogen sulfide, mice developed hypertension when compared with the wild type mice despite similar endothelial NOS expression (3). Administration of sodium hydrosulfide (NaHS), 3 a source of hydrogen sulfide, and not ammonia rescued the cystathionine ␥-lyase knock-out mice from hypertension (3), demonstrating that hydrogen sulfide regulates hemodynamics in mammals. Hydrogen sulfide is produced in the wall of blood vessels and causes relaxation of smooth muscle cells by opening the K-ATP channels, without recruiting cyclic GMP (4).
There is evidence that in addition to its hemodynamic effects, hydrogen sulfide can affect fundamental cellular responses to injury such as macrophage infiltration, apoptosis, and mitochondrial respiration in the heart (5,6). The role of hydrogen sulfide in renal physiology and disease is beginning to be explored. Biochemical analysis has shown that the kidney produces hydrogen sulfide catalyzed by three enzymes: cystathionine ␥-lyase, cystathionine ␤-synthase, and 3-mercap-topyruvate sulfotransferase (7). Hydrogen sulfide content is decreased in the renal parenchyma in rats with streptozotocininduced diabetes, and administration of NaHS inhibited increases in TGF␤1, reactive oxygen species, and type IV collagen in diabetic rats (8).
Studies in the heart and kidney have indicated that hydrogen sulfide inhibits phenotypes that depend on increase in protein synthesis. NaHS inhibited hypertension-induced cardiac fibrosis, arteriolar hypertrophy, and oxidative stress in the spontaneously hypertensive rat, inhibited increase in left ventricular wall thickness and collagen deposition in the pressure overload model of cardiac hypertrophy, and reduced the expression of type IV collagen in the diabetic rat kidney (8 -10). However, the mechanisms underlying hydrogen sulfide modulation of protein synthesis have not been explored. Both renal hypertrophy and accumulation of matrix proteins such as type IV collagen and laminin are cardinal manifestations of diabetic nephropathy (11). The goal of the present study was to investigate whether hydrogen sulfide modulates molecular events involved in cell hypertrophy and matrix synthesis induced by high glucose employing renal glomerular epithelial cells.

EXPERIMENTAL PROCEDURES
Cell Culture-Glomerular epithelial cells of the rat (GECs) were grown in DMEM containing 7% FBS, 5 mM glucose, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine as described previously (12). These cells express nephrin and podocin, proteins synthesized by the renal glomerular podocytes in vivo (supplemental Fig. S1). GECs were quiesced in serum-free medium for 24 h and then incubated with 5 mM glucose (normal), 30 mM glucose (high glucose), or mannitol (5 mM glucose ϩ 25 mM mannitol, osmotic control) with or without NaHS (50 -500 M, Sigma) for the indicated times.
Protein Synthesis Measurement-Protein synthesis was measured as described previously (13). Serum-starved cells were labeled with 10 Ci/ml [ 35 S]methionine for the terminal 2 h of incubation. Cells were washed in PBS and lysed in radioimmunoprecipitation assay buffer followed by centrifugation at 14,000 rpm for 20 min at 4°C. Cell protein content was measured with a Bio-Rad reagent using bovine serum albumin as standard (Bio-Rad). An equal amount of protein (30 g) was spotted onto the 3 MM filter paper (Whatman, Maidstone, UK). Filters were washed three times by boiling for 1 min in 10% trichloroacetic acid (TCA) containing 0.1 g/liter methionine before determining radioactivity.
Cell Hypertrophy Measurement-GECs were serum-starved for 24 h followed by incubation with high glucose with or without NaHS for 48 h. Cells were harvested by trypsinization with 0.05% trypsin/EDTA, and then an equal volume of GECs was separated into two tubes. Total cell number was calculated in one of the tubes using a hemocytometer. The cells in the other tube were lysed, and total protein concentration was determined as described above. Cellular hypertrophy was calculated as cell protein/unit cell number (13).
Transfection with siRNA-siRNA was transfected according to the manufacturer's protocol (Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, scrambled RNA (control) or pools of siRNA for LKB1 or calcium calmodulin-dependent protein kinase kinase ␤ (CaMKK␤) were diluted into siRNA transfection medium to a final concentration of 2-20 nM. Diluted siRNA was incubated with 6 l of siRNA transfection reagent for 30 min at room temperature. GECs were washed with PBS twice and then incubated with the siRNA transfection medium for 30 min. After 30 min, cells were incubated with the diluted scrambled RNA or siRNA for LKB1 or CaMKK␤ for 8 h, and then medium was changed to growth medium for 48 h. After 48 h, GECs were quiesced in serum-free medium for 24 h before performing the experiment.
Immunoblotting-Immunoblotting was performed as described previously (13)(14)(15). Equal amounts of cell lysate protein (2-20 g) and tissue homogenates (30 g) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with primary antibody overnight at 4°C. After extensive washing, the membrane was incubated with secondary antibody linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA). Proteins were visualized by using enhanced chemiluminescence reagent (Pierce-Thermo Scientific). The membrane was stripped in 0.5 M NaOH and reprobed with the indicated antibodies to assess loading. Band intensities from the immunoblots were quantified by densitometric analysis. All primary antibodies were from Cell Signaling (Danvers, MA) except for those against fibronectin, actin (Sigma), and cystathionine-␤-synthetase (Abgent, San Diego, CA) and cystathionine-␥-lyase (Abcam, Cambridge, MA).
Animal Study-Animal protocols were approved by the Institutional Animal Care and Use Committee. OVE26 mice (The Jackson Laboratory, Bar Harbor, ME) develop hyperglycemia and type 1 diabetes soon after birth (16,17); control NJ and OVE26 mice were studied at the age of 3 months. The C57BL/KsJ lepr Ϫ/Ϫ db/db mice, a model of type 2 diabetes, and its lean littermates (db/m) (The Jackson Laboratory) were maintained on regular laboratory chow. Blood glucose concentration was monitored for the emergence of diabetes, which is usually evident between 6 and 8 weeks of age. In the present study, lean littermate control and diabetic mice were studied in the early phase, after 2 weeks of onset of hyperglycemia. The db/db mice develop renal hypertrophy at 2 weeks following onset of diabetes (18,19). Mice were sacrificed at the end of the experimental period, and renal cortex was dissected out and processed for further analysis.
Immunohistochemical Studies-Kidney sections from 3month-old NJ controls and type 1 OVE26 diabetic mice were fixed in 10% formalin and embedded in paraffin. The slides were heated in a microwave oven in citrate buffer for 15 min, quenched in 3% hydrogen peroxide for 6 min, and washed in Tris-buffered saline. The slides were then blocked with Sniper blocking buffer (Biocare, Concord, CA) for 20 min and incubated with the rabbit polyclonal anti-cystathionine ␤-synthase (1:500; Abgent, San Diego, CA) overnight at 4°C in a humidified chamber. After rinsing, the slides were incubated with goat anti-rabbit polymer-horseradish peroxidase (HRP) (Biocare) for 20 min at room temperature. Immunoreactivity was visualized with 3-3Ј-diaminobenzidine (Biocare). Negative controls were performed by omitting the incubation with the primary antibody. Sections were photographed using Olympus AX70 (Melville, NY). Renal cortical lysates were employed in immunoblotting studies employing antibodies against cystathionine ␤-synthase and cystathionine ␥-lyase.
Statistical Analysis-Data were obtained from at least three independent experiments or animals and expressed as mean Ϯ S.E. Statistical comparisons between multiple groups were performed by ANOVA single-way analysis, and post hoc analysis was done using Student-Newman-Keuls multiple comparison test employing the GraphPad Prism 4 software. Statistical analyses between two groups were performed by the Student's t test. A p value of Ͻ 0.05 was considered statistically significant.

Hydrogen Sulfide Decreases High Glucose-induced Protein
Synthesis, Cellular Hypertrophy, and Extracellular Matrix Protein Accumulation-Increased protein synthesis contributes to hypertrophy of renal cells and to the accumulation of extracellular matrix proteins in the renal parenchyma in diabetes (11). We examined the effect of hydrogen sulfide on high glucose stimulation of protein synthesis in the GECs. High glucose increased de novo protein synthesis by 30% at 48 h (Fig. 1A, p Ͻ 0.01 by ANOVA); equimolar mannitol did not affect protein synthesis for up to 72 h (supplemental Fig. S2A). Co-incubation with NaHS reduced high glucose-stimulated protein synthesis, reaching significance at 250 M (Fig. 1A, p Ͻ 0.05); however, NaHS did not affect the rate of protein synthesis in cells incubated with 5 mM glucose. Similar inhibition of de novo protein synthesis was also seen with mouse podocytes (supplemental Fig. S2B), suggesting that hydrogen sulfide evokes similar responses in GECs from two distinct species. Cellular hypertrophy, measured as protein content per unit cell number, was increased by 25% by high glucose at 48 h (Fig. 1B, p Ͻ 0.001). NaHS significantly inhibited high glucose-stimulated cellular hypertrophy (Fig. 1B, p Ͻ 0.001), the cell content of protein reaching levels seen in cells incubated with 5 mM glucose; NaHS alone did not affect cellular protein content.
Of relevance to diabetic nephropathy, high glucose promotes synthesis of extracellular matrix proteins in the GECs (20). We examined whether NaHS inhibits high glucose-induced expression of laminin ␥1 and collagen IV ␣5 in the GECs. Laminin ␥1 and collagen IV ␣5 are parts of laminin trimer (laminin ␣5, ␤2, ␥1, LM-521) and type IV collagen trimer (␣3, ␣4, ␣5), respectively, deposited in the mature glomerular basement membrane following synthesis by the GECs in vivo (21). High glucose increased the expression of laminin ␥1 and collagen IV ␣5 at 48 h in the GECs (Fig. 1C, p Ͻ 0.001, Fig. 1D, p Ͻ 0.05); these changes were significantly reduced by co-incubation with NaHS ( Fig. 1C, p Ͻ 0.001, Fig. 1D, p Ͻ 0.05). Similar inhibition of high glucose-stimulation of laminin ␥1 synthesis was also observed in mouse podocytes and proximal tubular epithelial cells (supplemental Fig. S2, C and D); laminin ␥1 is a component of the basement membrane of tubules. We also examined whether preincubation with NaHS for 30 min followed by removal would still result in inhibition of matrix protein synthesis in the GECs. Thus, GECs were exposed to NaHS or the vehicle for 30 min in a medium containing either 5 mM glucose or 30 mM glucose. Then, the media were changed with fresh media without NaHS with the respective glucose concentra-tions; laminin ␥1 expression was examined 48 h later. Brief exposure to NaHS was sufficient to inhibit high glucose-stimulated laminin ␥1 expression 48 h later, suggesting that NaHS sets metabolic reactions in motion in a rapid manner that result FIGURE 1. Hydrogen sulfide inhibits high glucose-stimulated protein synthesis, cellular hypertrophy, and matrix protein expression. Cells were quiesced in serum-free medium for 24 h followed by treatment with 100 or 250 M NaHS with or without 30 mM glucose (high Glu) for 48 h. Control cells were grown in normal growth medium containing 5 mM glucose. A, de novo protein synthesis was measured by [ 35 S]methionine incorporation into TCAprecipitable protein. Composite data from four experiments are shown in the graph (**, p Ͻ 0.01 versus control, #, p Ͻ 0.05 versus Glu by ANOVA). B, cellular hypertrophy was calculated as total cellular protein per unit cell number. Following treatment of GECs with high glucose for 48 h with or without NaHS, cells were lifted by trypsinization and equally divided into two tubes. Total cell number was counted in one tube, and the other tube was used for total protein estimation. Composite data from four experiments are shown in the graph (***, p Ͻ 0.001 versus control, ###, p Ͻ 0.001 versus Glu by ANOVA). C and D, GECs were treated as mentioned above, and equal amounts of cell lysate protein were separated by SDS-PAGE and immunoblotted with laminin ␥1 (Lam␥1) and Col IV ␣5 antibodies. Loading was assessed by immunoblotting for actin. A representative blot and composite graph from three experiments are shown (*, p Ͻ 0.05; ***, p Ͻ 0.001 versus control, #, p Ͻ 0.05; ###, p Ͻ 0.001 versus Glu by ANOVA). E, cells were quiesced in serum-free medium for 24 h followed by treatment with vehicle or 250 M NaHS with 5 or 30 mM glucose (G) for 30 min. After 30 min, the media were changed with fresh media without NaHS with respective glucose concentrations. Following incubation for 48 h, equal amounts of cell lysate protein were separated by SDS-PAGE and immunoblotted with laminin ␥1 antibody. Loading was assessed by immunoblotting for actin. A representative blot and composite graph from three experiments are shown (***, p Ͻ 0.001 versus 5 mM glucose ϩ vehicle, ###, p Ͻ 0.001 versus 30 mM glucose ϩ vehicle by ANOVA).
in sustained inhibition of matrix proteins for a long period of time. Together, these data show that hydrogen sulfide inhibits high glucose-induced general protein synthesis, cellular hypertrophy, and extracellular matrix protein synthesis in the GECs. We initiated the investigation on the underlying mechanism.
Hydrogen Sulfide Regulates Translation Initiation and Elongation Phases by Inhibiting mTOR Pathway-Translation of mRNA is a rate-limiting step in gene expression culminating in protein synthesis (22). High glucose stimulation of protein synthesis and hypertrophy in the GECs involves induction of initiation and elongation phases of mRNA translation (12,13). Several important events in these phases of translation are under the control of mTOR complex 1 (23,24). Phosphorylation of 4E-BP1 and p70S6 kinase serves as a readout of mTOR complex 1 activation (22,25). Under normal conditions, the eukaryotic initiation factor 4E (eIF4E) is held inactive by its binding partner 4E-BP1. Phosphorylation of 4E-BP1 results in the release of eIF4E, permitting it to bind other eIFs to form the eIF4F complex and bind to the cap of the mRNA (26). Mitigation of high glucose-induced protein synthesis by NaHS in the GECs prompted us to test whether NaHS affects the initiation phase of mRNA translation.
High glucose increased 4E-BP1 phosphorylation at 30 and 60 min (p Ͻ 0.05) that was abolished by NaHS ( Fig. 2A, p Ͻ 0.05). Incubation of GECs with high glucose increased p70S6 kinase phosphorylation on Thr-389 (p Ͻ 0.01) that was significantly reduced by co-incubation with NaHS (Fig. 2B, p Ͻ 0.05). In addition to phosphorylating ribosomal proteins, p70S6 kinase regulates the activation of eIF4F during translation initiation. During the initiation phase, eIF4A, a DEAD box protein, forms eIF4F complex by associating with eIF4G and eIF4E (26). As a part of eIF4F, eIF4A functions as a helicase to resolve complexities in the 5Ј-untranslated region of the mRNA and assist the 40 S ribosomal subunit in the preinitiation complex in locating the AUG start codon (27). This action is facilitated by eIF4B and eIF4H (28). Programmed cell death protein 4 (PDCD4) binds eIF4A at its MA3 domains (29) and keeps it in an inactive complex in the resting cell. When protein synthesis is stimulated, PDCD4 is phosphorylated by p70S6 kinase and undergoes ubiquitination by the E3 ligase Skp-cullin-F-box (SCF)-␤transducin repeat-containing protein (␤TRCP) and proteasomal degradation (30). In the GECs, high glucose reduced the expression of PDCD4 (Fig. 2C, p Ͻ 0.05), which was inhibited by NaHS (Fig. 2C, p Ͻ 0.001); NaHS alone tended to augment the expression of PDCD4. Expression of dominant negative p70S6 kinase abolished the ability of high glucose to reduce PDCD4 content in the GECs (Fig. 2D). The abundance of PDCD4 in control cells was augmented by dominant negative p70S6 kinase, indicating that the basal expression of the protein was also regulated by the kinase. Rapamycin also abolished high glucose inhibition of PDCD4 expression, confirming the role of mTOR complex 1 (supplemental Fig. S3). Co-immunoprecipitation experiments showed that high glucose promoted dissociation of eIF4A from PDCD4 (p Ͻ 0.05) and increased its association with eIF4G (p Ͻ 0.05) (Fig. 2E); these data showed that eIF4F formation was stimulated by high glucose. NaHS abrogated these reactions and maintained eIF4A binding to PDCD4 (Fig.  2E, p Ͻ 0.05). Due to the greater availability of PDCD4 following NaHS treatment, there was a trend toward greater binding to eIF4A in control cells incubated with 5 mM glucose. The addition of MG-132 inhibited (p Ͻ 0.05) high glucose-induced reduction in PDCD4 expression (p Ͻ 0.05) (Fig. 2F), confirming that high glucose promoted PDCD4 degradation via the proteasomal pathway; MG-132 alone did not alter the PDCD4 expression.
Activated p70S6 kinase regulates important aspects of the elongation phase of mRNA translation. During elongation phase, movement of aminoacyl tRNA from the A site in the ribosome to the P site is facilitated by the eukaryotic elongation factor 2 (eEF2), which is active when dephosphorylated on Thr-56 (31). Phosphorylation of eEF2 on Thr-56 is under the control of eEF2 kinase, a calcium/calmodulin-dependent kinase III (32). p70S6 kinase phosphorylates eEF2 kinase on Ser-366 and inhibits its activity (33). High glucose increases Ser-366 phosphorylation of eEF2 kinase and decreases eEF2 phosphorylation on Thr-56, resulting in eEF2 activation in the GECs and proximal tubular epithelial cells (12,13,19). High glucose promoted Ser-366 phosphorylation of eEF2 kinase (p Ͻ 0.05) and dephosphorylation of Thr-56 on eEF2 (p Ͻ 0.05); NaHS inhibited these changes (Fig. 3A, p Ͻ 0.05, Fig. 3B, p Ͻ 0.05). Unlike high glucose, equimolar mannitol did not affect any of the aforementioned phosphorylation events (supplemental Fig. S4). These data demonstrate that hydrogen sulfide abrogates high glucose-stimulated initiation and elongation phases of mRNA translation by inhibiting mTOR complex 1 activity.
Next, we sought to identify the kinases upstream of mTOR complex 1 that may be regulated by hydrogen sulfide. In the GECs, AMP-activated protein kinase (AMPK) inhibits mTOR complex 1 activation induced by high glucose (12,13). Therefore, we explored whether AMPK is activated by hydrogen sulfide in inhibiting mTOR complex 1 in the high glucose-treated GECs.
Hydrogen Sulfide Activates AMPK by Inhibiting High Glucose-induced AMPK Dephosphorylation-AMPK is a heterotrimer consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥). Phosphorylation of Thr-172 of the ␣ subunit is essential for AMPK activity (34). NaHS significantly increased AMPK phosphorylation at 5 and 15 min in the GECs and returned to baseline by 30 min (Fig. 4A, p Ͻ 0.05). NaHS dose-dependently increased AMPK phosphorylation at 5 min, which reached significance at 250 and 500 M (Fig. 4B, p Ͻ 0.05, p Ͻ 0.01). NaHS also induced AMPK phosphorylation in mouse podocytes and proximal tubular epithelial cells with approximately the same temporal profile (supplemental Fig. 5, A and  B). We next examined whether NaHS affects high glucose-induced change in AMPK phosphorylation. High glucose reduced AMPK phosphorylation at 5-60 min (p Ͻ 0.05 or p Ͻ 0.01), but equimolar mannitol, serving as osmotic control, had no effect on AMPK phosphorylation (Fig. 4C). NaHS restored high glucose-induced reduction in AMPK phosphorylation to normal at 30 (p Ͻ 0.05) and 60 min (p Ͻ 0.01) (Fig. 4D).
Hydrogen Sulfide Inhibits High Glucose-stimulated Protein Synthesis through AMPK Activation-AMPK inhibits protein synthesis induced by high glucose in the GECs; stimulation of AMPK with 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) inhibits renal hypertrophy in diabetic rodents (13,35). We next explored the role of AMPK in NaHS regulation of high glucose-stimulated protein synthesis. We employed Compound C, a selective inhibitor of AMPK (35)(36)(37). Preincubation with Compound C abrogated the ability of NaHS to increase AMPK phosphorylation in high glucose-treated cells at 30 and 60 min (p Ͻ 0.05, p Ͻ 0.01 versus GluϩNaHS) (Fig. 5A) without affecting the basal phosphorylation status.
We next examined the role of AMPK in NaHS-mediated inhibition of high glucose-induced protein synthesis in the GECs. High glucose increased de novo protein synthesis (p Ͻ 0.001 versus control) that was significantly inhibited by NaHS (p Ͻ 0.01 versus glucose (Glu)) (Fig. 5B); Compound C abolished NaHS regulation of the effect of high glucose on protein synthesis (Fig. 5B, p Ͻ 0.05 versus GluϩNaHS). In a similar manner, Compound C blocked the reduction of laminin ␥1 content induced by NaHS in GECs treated with high glucose (Fig. 5C, p Ͻ 0.05 versus GluϩNaHS). Although incubation with Compound C alone did not affect basal protein synthesis, there was a trend toward increasing laminin ␥1 expression that did not reach statistical significance. These data show that AMPK activation is required for hydrogen sulfide mitigation of high glucose-induced protein synthesis and laminin ␥1 expression in the GECs.
Hydrogen Sulfide Inhibits High Glucose-induced mRNA Translation via Activation of AMPK-NaHS blocked signaling events necessary for high glucose-induced stimulation of mRNA translation (Fig. 2). We examined whether AMPK activation is required for NaHS-induced inhibition of mRNA translation in high glucose-treated cells. High glucose significantly increased phosphorylation of 4E-BP1 (Fig. 6A, p Ͻ 0.01) and p70S6 kinase (Fig. 6B, p Ͻ 0.05). High glucose promoted dephosphorylation of eEF2 at Thr-56 (Fig. 6C, p Ͻ 0.01); NaHS significantly reversed high glucose-induced changes in phosphorylation of 4E-BP1, p70S6 kinase, and eEF2, respectively (Fig. 6, A-C, p Ͻ 0.05-p Ͻ 0.001). Compound C significantly abrogated the effect of NaHS on high glucose-induced changes in phosphorylation of 4E-BP1, p70S6 kinase (Fig. 6A, p Ͻ 0.05,  Fig. 6B, p Ͻ 0.001 versus GluϩNaHS), and eEF2 (Fig. 6C, p Ͻ 0.01 versus GluϩNaHS). Compound C alone increased phosphorylation of 4E-BP1 (p Ͻ 0.01) and p70S6 kinase (p Ͻ 0.001) and decreased phosphorylation of eEF2 (p Ͻ 0.001) (Fig. 6, B  and C). Furthermore, Compound C abolished the effect of hydrogen sulfide on high glucose-induced reduction in PDCD4 expression (Fig. 6D). These data show that the ameliorative effect of NaHS on mRNA translation and protein synthesis induced by high glucose occurs through the activation of AMPK. Activated AMPK in turn blocks mTOR complex 1-reg-  ulated pathways of initiation and elongation phases of mRNA translation, in agreement with a previous study (13).
Role of CaMKK␤ in Hydrogen Sulfide Induction of AMPK Phosphorylation-LKB1 and CaMKK␤ are two major kinases for AMPK phosphorylation (38). LKB1 is a tumor suppressor, and several human tumor cell lines, including HeLa cells, lack LKB1 expression (39). To identify the kinase upstream of AMPK, we first examined the effect of NaHS on AMPK phosphorylation in HeLa cells. NaHS-stimulated AMPK phosphorylation in HeLa cells was abrogated by STO-609, a CaMKK␤ specific inhibitor (40) (supplemental Fig. S6A, p Ͻ 0.01). These data suggested that CaMKK␤ may be activated by NaHS to induce AMPK phosphorylation.
Incubation of GECs with NaHS led to increased AMPK phosphorylation (Fig. 7A, p Ͻ 0.05). STO-609 abolished NaHS induction of AMPK phosphorylation (p Ͻ 0.05 versus NaHS); composite data from several experiments showed that it did not affect basal AMPK phosphorylation. These data suggested that CaMKK␤ serves as the upstream kinase induced by NaHS to stimulate AMPK phosphorylation in the GECs. To further explore upstream kinases, we employed pools of specific siRNA against LKB1 and CaMKK␤. Immunoblotting with the respec-   FEBRUARY 10, 2012 • VOLUME 287 • NUMBER 7 tive antibodies showed that the GECs express both LKB1 and CaMKK␤ (Fig. 7B), the former being more abundant. Expression of LKB1 and CaMKK␤ proteins was significantly reduced in cells transfected with the respective siRNA but not in the cells transfected with control scrambled RNA (Fig. 7B, supplemental Fig. S6B). Treatment of GECs with NaHS increased AMPK phosphorylation in the control RNA-transfected cells (p Ͻ 0.01 versus control by ANOVA) and in LKB1 siRNAtransfected cells (p Ͻ 0.05 versus control by ANOVA). NaHS could not significantly increase AMPK phosphorylation in CaMKK␤ siRNA-transfected cells when compared with control RNA-transfected cells treated with NaHS (p Ͻ 0.001). In cells transfected with siRNA for both CaMKK␤ and LKB1, NaHS was not able to induce phosphorylation of AMPK (Fig.  7B, top panel), suggesting that CaMKK␤ accounts for most of the phosphorylation of AMPK following exposure to NaHS, with a possible additional contribution from LKB1.

H 2 S Inhibits High Glucose-induced Matrix Protein Synthesis
Kidney Cortex Expression of Cystathionine ␤-Synthase and Cystathionine ␥-Lyase Is Decreased in Diabetes-We examined the expression of cystathionine ␤-synthase and cystathionine ␥-lyase, the enzymes that generate hydrogen sulfide, in the kidney cortex from type 1 and type 2 diabetic mice. Immunoblotting showed that the expression of cystathionine ␤-synthase and cystathionine ␥-lyase was reduced in the kidney cortex of OVE26 mice with type 1 diabetes (Fig. 8A, p Ͻ 0.05 versus control) and in db/db mice with type 2 diabetes (Fig. 8B, p Ͻ 0.01 versus control). Immunoperoxidase staining of the kidney showed that normal mice expressed cystathionine ␤-synthase mainly in the tubules; the expression of the enzyme was significantly reduced in diabetic mice (Fig. 8C). Reduction in expression of cystathionine ␤-synthase and cystathionine ␥-lyase occurred at a time when renal hypertrophy and onset of matrix laminin and type IV collagen accumulation are evident in these models of diabetes (17)(18)(19)41). These data suggest that conditions for decreased synthesis of hydrogen sulfide exist in the kidney in both type 1 and type 2 diabetes that would facilitate increased protein synthesis required for renal hypertrophy and matrix protein accumulation.

DISCUSSION
Our data show that hydrogen sulfide inhibits high glucose induction of synthesis of proteins and matrix protein expression in the GEC. The underlying mechanism involves activation of AMPK by its upstream kinase CaMKK␤. By promoting activity of AMPK, hydrogen sulfide inhibits mTOR complex 1 and events in the initiation and elongation phases of translation that are under its control. Reduction of cystathionine ␤-synthase and cystathionine ␥-lyase, enzymes that generate hydrogen sulfide, in the kidneys of mice with type 1 and type 2 diabetes supports the notion that inhibition of hydrogen sulfide contributes to clinical expression of diabetic nephropathy (Fig. 9).
Hydrogen sulfide is generated by the action of cystathionine ␥-lyase and cystathionine ␤-synthase on L-cysteine in the presence of pyridoxal 5Ј-phosphate; 3-mercaptopyruvate sulfotransferase desulfurates mitochondrial 3-mercaptopyruvate to release hydrogen sulfide (42). Both cystathionine ␥-lyase and cystathionine ␤-synthase are expressed in the kidney (7,43). Following release, hydrogen sulfide can be stored in cells as bound sulfane sulfur that can be released by reducing conditions (44). Bound sulfane sulfur is the major determinant of physiological functions of hydrogen sulfide (45)(46)(47). Recent studies indicate that hydrogen sulfide has diverse effects on cell function including cell survival, hemodynamics, and inflammation (44); however, its role in protein synthesis has not been examined in detail.
Our data suggest that in the context of high glucose exposure, hydrogen sulfide serves as an inhibitor of protein synthesis. The prediction would be that hydrogen sulfide generation is reduced in the kidney in diabetes. Previous studies have shown that plasma hydrogen sulfide level is lower in human subjects with type 2 diabetes (48,49) and in end stage kidney disease patients on hemodialysis (50). Relevant to diabetic kidney disease, hydrogen sulfide levels in the kidney are reduced in a chemical model of type 1 diabetes in the rat (8). Our finding of reduction in the expression of cystathionine ␤-synthase and cystathionine ␥-lyase in the kidney suggests that synthesis of hydrogen sulfide would be reduced, coinciding with renal hypertrophy and matrix accumulation in mice with either type FIGURE 8. Expression of hydrogen sulfide-generating enzymes is reduced in the kidney in diabetes. A and B, expression of cystathionine ␤-synthase (CBS) and cystathionine ␥-lyase (CSE) is reduced in the renal cortex of mice with type 1 diabetes (A) or type 2 (B) diabetes. Equal amounts of tissue homogenate protein were immunoblotted with antibody against CBS or cystathionine ␥-lyase; loading was assessed by immunoblotting for actin (*, p Ͻ 0.05; **, p Ͻ 0.01 versus control by t test). C, immunoperoxidase staining of the kidney showed reduction in the expression of cystathionine ␤-synthase in type 1 diabetic mice (OVE26) when compared with non-diabetic NJ control mice.
1 or type 2 diabetes. Reduction in hydrogen sulfide would remove an endogenous break on pathways regulating protein synthesis in the kidney and facilitate high glucose to induce renal cell hypertrophy and augment expression of matrix proteins.
Kidney hypertrophy and accumulation of extracellular matrix proteins causing renal fibrosis are cardinal manifestations of diabetic kidney disease. Previous studies have linked renal hypertrophy, which appears early in the course of diabetes, with accumulation of matrix proteins that becomes evident later (51). Hyperglycemia induces renal hypertrophy and podocyte apoptosis in type 1 diabetic mice by inhibiting AMPK activity (35). Both hypertrophy and renal fibrosis require stimulation of protein synthesis. Previous studies showed that hydrogen sulfide reduced hypertrophy of intramyocardial arterioles and cardiac ventricular fibrosis (9), supporting the contention that it is an inhibitor of protein synthesis. There is consensus that the initiation phase of mRNA translation is a rate-limiting step in synthesis of proteins (22). Because both the initiation and the elongation phases of mRNA translation are partly under the control of mTOR complex 1, hydrogen sulfide modulation of high glucose induction of mTOR complex 1 activity was explored, using phosphorylation of p70S6 kinase and 4E-BP1 as a readout. High glucose increased the phosphorylation of these two mTOR complex 1 targets that was inhibited by hydrogen sulfide. Our data show that hydrogen sulfide averted high glucose induction of phosphorylation of 4E-BP1 and p70S6 kinase and degradation of PDCD4 induced by high glucose. This would reduce formation of eIF4F by locking up eIF4E and eIF4A in their respective inhibitory complexes with 4E-BP1 and PDCD4, respectively. Hydrogen sulfide showed a trend toward increased PDCD4 expression in cells incubated with 5 mM glucose; these data suggest that the basal expression of PDCD4 may be under the control of AMPK induced by hydrogen sulfide. Because p70S6 kinase 1 governs PDCD4 degradation, these data are consistent with suppression of mTORC1-p70S6 kinase 1 axis by AMPK induced by hydrogen sulfide. Because the increment in PDCD4 was greater than basal levels seen in cells incubated with 5 mM glucose, it is possible that there are additional mechanisms by which hydrogen sulfide regulates PDCD4 expression. In addition to the events in the initiation phase, p70S6 kinase also regulates the elongation phase of translation. In the current study, high glucose induced changes in phosphorylation of p70S6 kinase 1, eEF2 kinase, and eEF2, which would stimulate the elongation phase of translation; these changes were significantly inhibited by hydrogen sulfide. Thus, the inhibitory effect of hydrogen sulfide on protein synthesis is achieved by interference with key events in both the initiation and the elongation phases of mRNA translation. Because hydrogen sulfide did not affect basal protein synthesis, it appears to be effective as an inhibitor in the context of stimulation of protein synthesis by agents such as high glucose in the GEC.
High glucose recruits mTORC1 to promote protein synthesis and hypertrophy in kidney epithelial and mesangial cells (13,19,35,(52)(53)(54). One mechanism by which mTOR complex 1 is stimulated by high glucose in the GECs involves inhibition of AMPK phosphorylation and activity (12,13). Given the similarity between AMPK and hydrogen sulfide as inhibitors of protein synthesis in the context of high glucose exposure, possible mediation of the effect of hydrogen sulfide by AMPK was investigated. Hydrogen sulfide dose-dependently stimulated Thr-172 phosphorylation of the catalytic subunit of AMPK. High glucose-induced reduction in AMPK phosphorylation was restored to normal level by hydrogen sulfide. Reversal of the ameliorative effect of hydrogen sulfide on high glucose-induced stimulation of protein synthesis and inhibition of AMPK phosphorylation by Compound C, an AMPK-selective inhibitor, demonstrated that AMPK was the mediator of the effect of hydrogen sulfide on protein synthesis in cells exposed to high glucose. Thr-172 phosphorylation of the catalytic subunit of AMPK is catalyzed by LKB1 and CaMKK␤. Observations with STO-609 and siRNA showed that in the GEC, hydrogen sulfide stimulation of AMPK is mediated by CaMKK␤. The mechanism by which AMPK inhibits mTOR complex 1 involves the TSC1/TSC2-Rheb pathway (55); recently, AMPK has also been shown to inhibit mTOR complex 1 by phosphorylating raptor, independent of TSC2 (56). Further investigation is needed to study the mechanism by which CaMKK␤ is stimulated by hydrogen sulfide. Hydrogen sulfide induction of AMPK phosphorylation has also been described in the brain in a rodent model of cardiac arrest (57).
There are potential clinical implications for the use of agents that generate hydrogen sulfide. As mentioned above, NaHS ameliorated kidney injury in a rat model of chemically induced diabetes; however, the molecular mechanisms by which hydrogen sulfide exerted its protective effect were not explored (8). NaHS has been shown to attenuate hypertension in an experimental model of renal artery stenosis by reducing the mRNA and protein expression of renin (58). Reduction in ischemiareperfusion-induced injury to the kidney has been reported by augmenting cystathionine ␥-lyase expression in the kidney (59). Sen et al. (60,61) have also described ameliorative effects of hydrogen sulfide on the progression of kidney disease in rodents with hyperhomocysteinemia. Hydrogen sulfide affords protection to the heart against ischemia-reperfusion injury (5,62), and clinical trials evaluating its therapeutic role in amelioration of ischemic heart disease are underway (44). The mechanisms of cardiac protection may involve diverse pathways and include inhibition of macrophage infiltration of the myocardium, reduction in mitochondrial respiration and preservation of mitochondrial structure, and inhibition of apoptosis (5,61). These protective phenotypes were reproduced by myocardium-specific overexpression of cystathionine ␥-lyase (5). Other mechanisms contributing to hydrogen sulfide protection of myocardium include opening of the K-ATP channels in mitochondria and/or sarcolemma (63,64), induction of antioxidant genes by Nrf-2, and resistance to apoptosis by stimulation of survival pathways (65). Recruitment of specific pathways by hydrogen sulfide in diverse tissues is likely to be dependent on the context of injury. Future studies are needed to explore whether some of these pathways are also stimulated by hydrogen sulfide in the kidney in the setting of hyperglycemia-induced injury. The availability of agents that promote hydrogen sulfide release in vivo (42) makes it worthwhile to test it as a therapeutic modality in diabetic kidney disease.