Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD + levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD + , silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD + levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD + depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-β1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.

diabetic nephropathy (dn), one of the most serious and common microvascular complications of diabetes, is considered the leading cause of chronic renal failure and primary indication for dialysis and transplantation (11). The identification of new therapeutic targets and compounds that can combat this devastating disease is an urgent matter. Excess amounts of reactive oxygen species (ROS) in diabetes mellitus (DM) can cause increased expression of extracellular matrix (ECM) genes, with progression to fibrosis, proteinuria, and end-stage renal disease (11, 33). In DM, the link between higher levels of ROS and ECM accumulation is multifactorial and may include the reduction of sirtuin 1 [Sirt-1; silent information regulator 2 (Sir2)] activity (16, 27). The Sirt-1 protein is the founding member of a family of NAD+-dependent deacetylases, which are best known for their acknowledged link to longevity associated with caloric restriction (CR) (7, 12, 16, 29). Sirt-1 activity is regulated via the availability of its cosubstrate, NAD+ (18, 19). It has been shown that a higher level of ROS promotes activation of the NAD+-dependent DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), with subsequent NAD+ depletion and downregulation of Sirt-1 activity (37). Whether this mechanism of Sirt-1 reduction is operative in the diabetic kidney is unknown and deserves further investigation. Transforming growth factor-β1 (TGF-β1) has a critical role in ECM accumulation in DM (49). Some experimental DM studies have shown that a reduction in Sirt-1 is associated with increased ECM accumulation (30, 41). However, the way in which low Sirt-1 activity contributes to kidney ECM accumulation in DM remains to be determined.

Several investigators (24, 26, 30, 41, 44, 45) have shown that the renal expression/activity of Sirt-1 is diminished in different models of experimental DM, although contradictory results have also been reported (25). Similarly, it has been shown in human kidneys that the expression of Sirt-1, as assessed by immunohistochemistry or mRNA expression, was significantly reduced in patients with DN compared with normal subjects or patients with minimal changes disease (6). The mechanism involved in Sirt-1 reduction in diabetic conditions is not totally clear (27), but a reduction in the phosphorylation of AMP-activated protein kinase (AMPK) may play a role (24). It has been shown that maneuvers that could increase Sirt-1 activity may lead to kidney protection in DM (24, 26, 45, 47).

Several experimental and clinical studies have demonstrated that cocoa may be beneficial to human health under various conditions (22). It is generally accepted that the salutary effects of cocoa are related to its high polyphenol content (22). However, we were surprised by our observation that cocoa with low polyphenol (CL; 0.5% of polyphenol, Table 1 and Fig. 1, A–E) was as efficient as cocoa enriched with polyphenol (CH; 60% of polyphenol) (36) in reducing ECM accumulation and oxidative stress in the kidneys of diabetic rats. This observation prompted us to investigate which compound in CL could be responsible for that beneficial effect, using ultra-performance liquid chromatography (UPLC)-mass spectrometry techniques. Chromatography analysis of components within the CL and CH showed the presence of a common component in both CH and CL. Mass spectrometry analysis in CH and CL showed the presence of a structure with a molar mass of 181 g/mol in the common compound identified. Fragmentation analysis of the structure identified by mass spectrometry following ionization showed the product ion spectra of m/z 181, which corresponds to theobromine, the nonpolyphenol component. The concentration of theobromine in both CH and CL extracts was estimated at ∼8% (data not shown).

Table 1. Physical and metabolic parameters of experimental groups after 16 wk of diabetes Group Initial Body Weight, g Final Body Weight, g HbA1c, % Kidney Weight/Body Weight, ×100 Systolic Blood Pressure, mmHg SHR CT 283 ± 17 346 ± 27 5.3 ± 0.28 0.38 ± 0.03 186.1 ± 9.4 SHR DM 280 ± 9 224 ± 59* 13.1 ± 0.37* 0.53 ± 0.09§ 181.7 ± 18.8 SHR DM CL 259 ± 26 258 ± 59* 13.0 ± 0.30* 0.53 ± 0.10§ 184.7 ± 16.9

Fig. 1.Cocoa with low polyphenol (CL 0.5%) and theobromine in diabetic spontaneously hypertensive rats (SHR) reduce extracellular matrix accumulation, oxidative stress, albuminuria, and renal hypertrophy, respectively. A–D: photomicrographs (A and C) and quantification (B and D) of the glomerular periodic acid-Schiff (PAS; A)- and collagen IV (C)-stained areas from all groups. A and C: original magnification ×400 counterstained with hematoxylin. Scale bar = 50 μm. Values are means ± SE of 3 independent experiments, and images are representative of 6 rats/group. *P = 0.0001 vs. SHR control (CT). E: NADPH-dependent reactive oxygen species (ROS) generation in renal cortical homogenate and expressed as relative luminescence units (RLU)/mg protein. Values are means ± SE of 3 independent experiments for 6 rats/group. *P = 0.013 vs. CT. ±P = 0.016 vs. diabetic SHR (DM). F: albumin excretion rate (AER) in control (SHR CT), control treated with theobromine (TB; SHR CT TB), diabetic (SHR DM), and diabetic treated with theobromine (SHR DM TB) rats. Values are means ± variance of 3 independent experiments for 6 rats/group. *P = 0.025 vs SHR CT. G–J: photomicrographs (G and I) and quantification (H and J) of the glomerular PAS (G)- and collagen IV (I)-stained areas from all groups. *P < 0.001 vs SHR CT. G and I: original magnification ×400 counterstained with hematoxylin. Scale bar = 50 μm. Values are means ± SE of 3 independent experiments, and images are representative of 6 rats/group. *P < 0.005 vs. SHR CT.

Theobromine is a methylxanthine that has been implicated as a contributor to the positive effects of cocoa (1, 23). A recent randomized clinical trial demonstrated that theobromine is safe and enhances HDL cholesterol in healthy volunteers (35). Whether theobromine is renoprotective and has any effect on Sirt-1 activity in DN is unknown.

The aims of the present study were to determine the mechanism of Sirt-1 activity reduction under diabetic conditions, the contribution of a reduction in Sirt-1 to mediation of TGF-β1 signaling and ECM accumulation, and whether theobromine exhibits renoprotective effects through increasing Sirt-1 activity.

MATERIALS AND METHODS Reagents. All reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. Identification and quantification of polyphenols and nonpolyphenols in cocoa extract. The identification of the components of cocoa extract was assessed by HPLC (Waters, Milford, MA) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) (13, 14). Animals and study design. This study protocol was approved by the local committee for ethics in animal research (CEEA/IB/UNICAMP, protocol no. 1834-1). Male spontaneously hypertensive rats (SHR) supplied by Taconic (Germantown, NY) were used in this study. We chose to induce diabetes in SHR because they present a more progressive form of renal disease (8) and we have a vast amount experience with this model (4, 10, 33, 38), and because of the frequent association of diabetes with hypertension in human diabetic kidney disease. Diabetes was induced in 12-wk-old male SHR via a single intravenous injection of streptozotocin (STZ; 60 mg/kg in sodium citrate buffer, pH 4.5) via the tail vein. Control SHR received only the vehicle (citrate buffer). Rats with plasma glucose concentrations >15 mmol/l were considered diabetic in these experiments. Systolic blood pressure was obtained with tail-cuff plethysmography in nonanesthetized rats using a 229 blood pressure amplifier/pump (ITCC Life Science, Woodland Hills, CA). Forty-eight hours after the STZ injection, the diabetic rats were randomly assigned to receive no treatment or treatment with theobromine (5 mg·kg−1·day−1 diluted in drinking water). Although the 8% of theobromine in 24 mg/kg of CL that the SHR received corresponds to 2 mg/kg of theobromine, we chose to treat them with 5 mg/kg. We made this decision because in the recent study in humans, the dose of theobromine was roughly 14 mg/kg (35), and it has been estimated that acute oral toxicity in rats is achieved with a much higher dose (950 mg/kg) (42). Therefore, 5 mg/kg of theobromine to treat rats is still quite a low dose. During the study, the diabetic rats received 2 U of insulin (human insulin HI-0310; Lilly, Indianapolis, IN) three times per week, subcutaneously. After 12 wk of diabetes, the rats were euthanized by carbon dioxide (CO 2 ) asphyxiation. Anesthetic procedures were used in full, and all precautions were taken to ensure that the animals did not suffer unduly during and after the experimental procedure. The kidneys were snap-frozen at −80°C for future assays. Albumin excretion rate. The albumin excretion rate (AER) of a 24-h urine collection was determined with an ELISA kit (Nephrat II; Exocell, Philadelphia, PA) (38). Renal histopathology. Four-micrometer kidney sections were stained with periodic acid-Schiff (PAS), and matrix mesangial expansion, quantified by Leica Application Suite (LAS Image Analysis, Leica Microsystems, Buffalo Grove, IL), was derived from the assessment of 30 glomeruli from each rat. Immortalized human mesangial cell culture. Immortalized human mesangial cells (iHMCs) were kindly provided by Dr. Nestor Schor (Dept. of Medicine, Nephrology Div., Federal University of São Paulo, São Paulo, Brazil) and from Dr. Bernhard Banas (Nephrology Center, Medical Policlinic, Ludwig-Maximilian University of Munich, Munich, Germany) (2, 9). The concentrations of treatments used in the high-glucose (HG; 30 mM) medium or TGF-β1 (5 ng/ml) in all experiments were chosen after carrying out a thiazolyl blue tetrazolium bromide (MTT) assay (data not shown). Mannitol was used as an osmotic control for 30 mM d-glucose. Experimental conditions of iHMCs. iHMCs were kept without serum in normal glucose (NG; 5.5 mM) or HG or TGF-β1 for 24 h in the presence of 44 nM theobromine or 0.5% polyphenols (0.25% epicatechin and 0.25% catechin), AMPK blocker compound C (CC; 10 μM), PARP-1 activity inhibitor (PJ-34, 1 μM), Sirt-1 activity blocker (EX-527), NAD+ (300 μM), 100 nM small interfering RNA (siRNA) or scrambled (Scr) for Sirt-1, or 200 nM for NOX4. Theobromine (44 nM) was used as it corresponds to its 8% composition within 100 ng/ml of CL. Transient transfection with siRNAs. The siRNA duplexes and Src siRNA corresponding to human NOX4 and Sirt-1 were obtained from Invitrogen (Carlsbad, CA) and Santa Cruz Biotechnology (Danvers, MA), respectively. The transient transfection of siRNAs was carried out using Lipofectamine transfection reagent (Invitrogen) (5, 47). NADPH oxidase activity. NADPH oxidase activity was measured by the lucigenin-enhanced chemiluminescence method as previously described, with a few modifications (38). Western blot and immunoprecipitation analysis. The samples and Western blots were prepared as previously described (4, 10, 38). The following primary antibodies were used: goat anti-type IV collagen (1:500; Southern Biotech, Birmingham, AL), rabbit anti-NOX4 (1:500; Santa Cruz Biotechnology), mouse anti-nitrotyrosine (1:2,000; EMD Millipore, Billerica, MA), mouse anti-PARP-1 (1:1,000; Trevigen, Gaithersburg, MD), rabbit phosphorylated AMPK (Thr172; 1:1,000; Cell Signaling Technology, Danvers, MA), rabbit total AMPK (1:1,000; Cell Signaling Technology), goat anti-fibronectin (1:500; Calbiochem, La Jolla, CA), and rabbit anti-acetyl and anti-SMAD3 (1:500; Cell Signaling Technology). To verify the uniformity of the protein load and transfer efficiency across the test samples, the membranes were reprobed with actin (goat polyclonal anti-actin antibody, diluted 1:1,000; Santa Cruz Biotechnology). For immunoprecipitation analysis, 1,000 μg of total protein in both renal cortex homogenates or iHMC cell lysates were immunoprecipitated with rabbit anti-SMAD3 (Cell Signaling Technology) or rabbit anti-Sirt-1 (Santa Cruz Biotechnology) using protein A agarose beads (Santa Cruz Biotechnology). Precipitates were then analyzed by Western blotting with rabbit anti-lysine (Cell Signaling Technology) and reprobed with rabbit anti-SMAD3 (Cell Signaling Technology) or with rabbit anti-NOX4 (Santa Cruz Biotechnology) or rabbit phosphorylated AMPK (Thr172; Cell Signaling Technology). Immunohistochemistry. To detect ECM accumulation and oxidative stress-induced DNA base modification, immunohistochemistry was carried out for collagen IV and 8-hydroxy-2′-deoxyguanosine (8-OHdG; a DNA base-modified product), respectively (4, 38). The primary antibodies used were goat anti-type IV collagen (Southern Biotech) and 1:50 dilution for mouse monoclonal anti-8-OHdG antibody (N45.1; Japan Institute for the Control of Aging, Fukuroi, Shizuoka, Japan). Collagen IV and nuclear 8-OHdG intensity in both the glomerulus and tubulointerstitial area were quantified using the Leica Application Suite in 50 sequential high-power microscopic fields (×400). Double immunofluorescence for Sirt-1 and PARP. Kidney halves fixed in 4% paraformaldehyde and frozen in Tissue-Tek O.C.T. compound-embedding medium were autoclaved for 10 min in 120 mmHg. Then, the tissue was permeabilized with 0.3% Triton X-100, and the unspecific sites were blocked by 1% BSA+0.3% Triton X-100. The primary antibodies used were rabbit anti-Sirt-1 diluted 1:10 (Santa Cruz Biotechnology) and mouse anti-PARP-1 diluted 1:10 (Enzo Life Sciences, Farmingdale, NY). Incubation with specific secondary antibodies associated with fluorochromes Alexa 488 or Alexa 594 was performed. The images were observed under confocal microscopy (Leica TCS SP5 II), and the colocalization of stainings was quantified by Image J software. 2′,7′-Dichlorodihydrofluorescein diacetate measurement of ROS production. Intracellular ROS levels were measured by 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) as previously described (10). Sirt-1 activity quantification. Sirt-1 activity levels in the iHMC lysates (47) and kidney cortexes of the SHR (41) were quantified based on an enzymatic reaction by a fluorometric Sirt-1 assay kit (Fluor-de-Lys Kit; Enzo Life Sciences) using a fluorogenic peptide encompassing residues 379–382 of p53, acetylated on lysine 382. The acetylated lysine residue was coupled to an aminomethylcoumarin moiety. The peptide was deacetylated by Sirt-1, followed by the addition of a proteolytic developer that released the fluorescent aminomethylcoumarin. Briefly, kidney cortex extract or iHMC lysates were harvested in a RIPA buffer that was composed of 50 mM Tris·HCl (pH 7.6), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, and a protease inhibitor cocktail (Complete; Boehringer-Mannheim, Indianapolis, IN). iHMC lysates or kidney cortex extracts were collected after sonication and subsequent centrifugation at 12,000 g for 15 min at 4°C. A 25-μl enzyme preparation consisting of 10 μl of assay buffer provided in the kit plus 5 μl of Sirt-1 enzyme (0.2 U/μl) and 10 μl of iHMC lysate or kidney cortex extract was incubated with 25 μl containing 170 μM NAD+ and 100 μM p53 fluorogenic peptide for 45 min at 37°C followed by incubation in a developer for 15 min at 37°C. Relative fluorescence of the fluorophore generated was measured using a fluorescence plate reader (SynergyMx; Biotek) at excitation and emission wavelengths of 360 and 460 nm, respectively. The relative fluorescence values were corrected by the amount of protein in the cell lysate or kidney cortex extract. NAD+, NADH, and NAD+/NADH ratio quantification. NAD+, NADH, and NAD+/NADH ratio were quantified using an NAD+/NADH quantification colorimetric kit (BioVision, Milpitas, CA) (29). Measurement of AMP and ATP levels. The measurement of intracellular of AMP and ATP nucleotides was performed as previously described (21), with some modification. Samples of iHMCs were deproteinized in 10% TCA, mixed vigorously, and left on ice for 10 min. Homogenates were subsequently centrifuged (3,500 g for 10 min at 4°C), and a known volume of supernatant was collected for neutralization with a solution of 6 M KOH and 2 M K 2 CO 3 . The neutralized samples were centrifuged for 10 min at 3,500 g, and the resulting supernatants were analyzed. Protein content was determined in the TCA precipitated by using a Bradford assay. The chromatographic analysis was performed HPLC using an ACQUITY UPLC Photodiode Array (PDA, Waters). The analytic column (100 × 4·6-mm ID) was packed with 5 μm of ODS Hypersil (Thermo Scientific). Chromatographic conditions were as follows: buffer A consisted of 150 mM KH 2 PO 4 , containing 150 mM KCl, adjusted to pH 6.0 with KOH. Buffer B consisted of a 15% (vol/vol) solution of acetonitrile in buffer A. The composition of the mobile phase was controlled by a low-pressure gradient mixing device with a flow rate of 0·9 ml−1·min−1 at 20°C. The gradient curve was as follows: buffer B was increased linearly between the following time points: 0 min, 0% B; 3·5 min, 7% B; 5·5 min, 50% B, 7·0 min, 100% B; 7·1 min, 0% B with a reequilibration time of 5 min. Sample peaks were integrated and quantified using a Waters chromatography data system. Peaks were monitored by absorption at 254 nm. A second UV detector set at 216 nm was connected in series to monitor peak purity. Immunofluorescence for collagen type IV. Immunofluorescence staining was performed by incubating the fixed cells with collagen IV primary antibody (1:15; Southern Biotech) and rhodamine-conjugated secondary antibody for collagen IV (1:50; BD Transduction Labs, San Jose, CA). Images were then taken with an Olympus FluoView confocal microscope (Olympus of the Americas, Melville, NY). Statistical analysis. The results, expressed as means ± SE, except for albuminuria, which is expressed as geometric mean and confidence of interval, were analyzed with the Kruskal-Wallis test (for multiple groups) and Mann-Whitney U-test (for 2 groups). Comparisons between multiple groups were performed with a one-way analysis of variance (ANOVA) followed by the Bonferroni test, and t-tests were used for comparisons between two groups. A value of P < 0.05 was considered significant. The analyses were performed using StatView software (SAS Institute, Cary, NC).

DISCUSSION Thus far, the mechanism of Sirt-1 depletion in the diabetic kidney has not been assessed in great detail (27, 34). Here, we showed that under diabetic conditions, Sirt-1 is reduced as a consequence of AMPK downregulation followed by NOX4 and PARP-1 activation, with subsequent NAD+ depletion in the kidney. Following Sirt-1 reduction, kidney ECM accumulation increased due to SMAD3 acetylation induced by TGF-β1. We also showed for the first time that a single-dose level of theobromine led to inhibition of NOX4-induced PARP-1 activation, possibly by preventing AMPK inactivation via an increase in AMP/ATP ratio levels (15, 17). This effect led to an increase in NAD+ levels, and hence, Sirt-1 activation, which prevented SMAD3 acetylation protecting the diabetic kidney from ECM accumulation (Fig. 15). Of interest, we have recently demonstrated (43), by matrix-free nano-assisted laser desorption-ionization mass spectrometry imaging, that indeed theobromine accumulates in the kidney after oral administration of theobromine to rats (Drug Testing). This latter finding further supports the concept that theobromine may have a potential as a renal-protective drug. Fig. 15.Schematic representation of the proposed mechanism by which diabetes/HG leads to Sirt-1 downregulation and ECM accumulation and the inhibitory action of theobromine in this sequence of events. Download figureDownload PowerPoint

ECM accumulation is a hallmark of kidney disease in DM (49). Some studies have shown that in DM reduction in the kidney expression and activity of Sirt-1 are associated with ECM accumulation and an elevated expression of TGF-β1 (30, 41). However, these studies did not assess the mechanism by which Sirt-1 reduction leads to ECM accumulation. A possible explanation is the reduction of Sirt-1 activity on deacetylating SMAD2 and SMAD3 (31). Acetylation involves the transfer of the acetyl moiety from acetyl coenzyme A to the ε-amino groups of lysine residues (31, 48). SMAD3 can be acetylated at the Lys-378 in the MH2 domain by p300/CBP (31, 20), which regulates SMAD3 DNA binding activity and subsequently mediates TGF-β1-induced collagen synthesis in fibroblasts (3), tubular cells (31), and 293 and HepG2 cell lines (3), suggesting that the acetylation of SMAD3 might play an important role in TGF-β1-driven tissue fibrosis. Our results support these studies, showing that acetylation in SMAD3 is vital in mediating diabetes or HG-induced renal ECM accumulation, possibly due to the lack of deacetylating activity of Sirt-1. This is the first description of a Sirt-1/SMAD3 interaction in DN and may represent a common mechanism in the regulation of members of the SMAD family, as a previous study reported that Sirt-1 can deacetylate SMAD7, which prevents TGF-β1-induced apoptosis in cultured glomerular mesangial cells (28). Dietary restriction (29, 26, 44) and maneuvers via the use of plant-derived polyphenols such as resveratrol (24, 45–47) have been shown to ameliorate renal injury in experimental diabetes via an association with the regulation of Sirt-1 protein. In particular, CR or activation of Sirt-1, has been associated with alleviation of ECM accumulation in experimental type 2 diabetes (26) and a unilateral ureteral obstruction model (31). In vitro, Sirt-1 activation or overexpression has been shown to ameliorate ECM accumulation in both mesangial cells exposed to HG (30, 41) and tubular and fibroblast kidney cells treated with TGF-β1 (31). A few years ago, it was suggested that theobromine, a methylxanthine present in cocoa, could at least partly account for the salutary effects of cocoa in humans (23). More recently, a number of potential beneficial actions of theobromine have been described, including a capacity to enhance HDL cholesterol concentrations in healthy volunteers (35). Of interest theophylline, a methylxanthine derivative (a component of tea polyphenol), protects against intracellular NAD+ depletion via PARP-1 inhibition and thus activates Sirt-1 activity in the macrophages and lung cells of patients with chronic obstructive pulmonary disease (34). Interestingly, while no study has been conducted on the effect of theobromine on diabetic nephropathy, a PubMed search with the terms “theobromine and diabetic nephropathy” resulted in 51 articles, most of which were related to the use of pentoxifylline in diabetic nephropathy. The reason might be that pentoxifylline, like theobromine, is a xanthine derivative. Pentoxifylline is a commercially available drug that is mostly indicated in the treatment of intermittent claudication. To date, a few studies have demonstrated a reduction in albuminuria in diabetic patients treated with pentoxifylline, with almost no side effects, although its real contribution in treating DN needs to be tested in large, randomized clinical trials (40). In the current study, we observed a nonsignificant reduction in blood pressure by theobromine in diabetic rats, which might have contributed to the beneficial effects of theobromine. In addition, the theobromine treatment in the diabetic rats was initiated soon after the induction of diabetes and before the development of renal disease, which is a situation that is not likely to occur clinically. Finally, we did not assess whether the effect of theobromine on phosphodiesterase inhibition, interfering with adenosine 3′,5′-phosphate cAMP signaling, contributed to the observed protective effects of theobromine. However, it is generally accepted that theobromine is a weaker phosphodiesterase inhibitor compared with other methylxanthines such as caffeine (1,3,7-trimethylxanthine) and theophylline (1,3-dimethylxanthine) (32, 39). In conclusion, in this study we identified a novel regulatory mechanism for Sirt-1 reduction in the diabetic kidney that involves the PARP-1 activation effect of increased ROS production by NADPH-enhanced NOX4 via AMPK inactivation, leading to the depletion of NAD+. The consequent reduction in Sirt-1 activity impairs SMAD3 deacetylation with the development of kidney ECM accumulation. AMPK activation by theobromine is able to interrupt the sequence of events leading to Sirt-1 activation, and hence, protects the diabetic kidney.

GRANTS This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Grant 2008/57560-0 and 2012/22452-8 ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Grant 301797/2009-9 ). A. Papadimitriou received a postdoctoral scholarship from FAPESP.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS Author contributions: A.P., K.C.S., E.B.P., J.M.L.F., and J.B.L.F. provided conception and design of research; A.P., K.C.S., E.B.P., C.M.B. performed experiments; A.P., K.C.S., E.B.P., J.M.L.F., and J.B.L.F. analyzed data; A.P., K.C.S., E.B.P., J.M.L.F., and J.B.L.F. interpreted results of experiments; A.P., K.C.S., E.B.P. prepared figures; A.P., K.C.S., E.B.P. drafted manuscript; A.P., K.C.S., E.B.P., J.M.L.F., and J.B.L.F. edited and revised manuscript; A.P., K.C.S., E.B.P., C.M.B., J.M.L.F., and J.B.L.F. approved final version of manuscript.