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Probucol ameliorates renal injury in diabetic nephropathy by inhibiting the expression of the redox enzyme p66Shc

Aims Probucol is an anti-hyperlipidemic agent and a potent antioxidant drug that can delay progression of diabetic nephropathy (DN) and reverses renal oxidative stress in diabetic animal models; however, the mechanisms underlying these effects remain unclear. p66Shc is a newly recognized mediator of mitochondrial ROS production in renal cells under high-glucose (HG) ambience. We previously showed that p66Shc can serve as a biomarker for renal oxidative injury in DN patients and that p66Shc up-regulation is correlated with renal damage in vivo and in vitro. Here, we determined whether probucol ameliorates renal injury in DN by inhibiting p66Shc expression. Results We found that the expression of SIRT1, Ac-H3 and p66Shc in kidneys of DN patients was altered. Also, probucol reduced the levels of serum creatinine, urine protein and LDL-c and attenuated renal oxidative injury and fibrosis in STZ induced diabetic mice. In addition, probucol reversed p-AMPK, SIRT1, Ac-H3 and p66Shc expression. Correlation analyses showed that p66Shc expression was correlated with p-AMPK and Sirt1 expression and severity of renal injury. In vitro pretreatment of HK-2 cells with p-AMPK and SIRT1 siRNA negated the beneficial effects of probucol. Furthermore, we noted that probucol activates p-AMPK and Sirt1 and inhibits p66shc mRNA transcription by facilitating the binding of Sirt1 to the p66Shc promoter and modulation of Ac-H3 expression in HK-2 cells under HG ambience. Innovation and conclusion Our results suggest for the first time that probucol ameliorates renal damage in DN by epigenetically suppressing p66Shc expression via the AMPK-SIRT1-AcH3 pathway.


Introduction
Diabetic nephropathy (DN) is the most common complication of diabetes mellitus leading to end-stage renal disease, and it is associated with a high mortality rate [18]. The initial phases of the DN are characterized by glomerular and tubular epithelial hypertrophy, and thickening of glomerular and tubular basement membranes, which is followed by ensuing hyperfiltration, albuminuria, glomerulosclerosis and tubulo-interstitial fibrosis [12,18]. Recently, various investigators have demonstrated that increased reactive oxygen species (ROS) generation seem to be one of the main pathophysiological change induced by high-glucose (HG) ambience [20]. The increased ROS production results in extracellular matrix (ECM) accumulation and another notable pathological event, i.e., apoptosis. Adaptor protein p66Shc is a newly recognized mediator of mitochondrial ROS production [10,18]. Previously, our laboratory reported that the p66Shc is mainly expressed in the renal tubular epithelium, and to a limited extent in the glomerulus. This mediator can be phosphorylated by PKC-β and PKC-δ under HG conditions [34,35], and that results in oxidative damage secondary to mitochondrial ROS overproduction in DN. Other studies have shown that p66Shc mRNA transcription is regulated by NAD-dependent deacetylase sirtuin-1 (Sirt1) via epigenetic Acetyl-Histone H3 (Ac-H3) chromatin modification [50]. Furthermore, AMP-activated protein kinase (AMPK) acts as regulator of Sirt1 activity [4], which suggests that the AMPK/Sirt1/Ac-H3 signaling pathway plays a critical role in attenuating renal oxidative damage by modulating p66Shc expression in DN.
Probucol is a compound with two characteristic phenolic rings, and it has been used for years as an anti-hyperlipidemic agent in patients with DN [32]. Over the last several years many clinical studies have shown that probucol can reduce urinary protein excretion and prevent DN progression [8]. In addition, there are certain reports in the literature which indicate that probucol acts as a potent oxygen radical scavenger, and thus can effectively prevent oxidative stress-induced tissue damage [16,26]. Along these lines it has been demonstrated that probucol suppresses oxidative stress and attenuates oxidative injury in the renal podocytes of db/db mice by inhibiting NADPH oxidase 2 (Nox2) expression [49]. Conceivably, by virtue of probucol being an antioxidant it has been proposed to inhibit renal tubular epithelialmesenchymal transition (EMT) in mouse models of DN [6,31]. However, the molecular mechanism(s) underlying this phenomena are not precisely delineated. Interestingly, recent studies have suggested that succinobucol, a metabolically stable derivative of probucol, is a more efficient modulator of mitochondrial homeostasis and mitochondrial ROS production than its parent compound [5]. Additionally, probucol has been shown to suppress human glioma cell proliferation in vitro by inhibiting ROS production and activating AMPK phosphorylation [17].
Given the above findings, it is plausible that the AMPK/Sirt1/ p66Shc signaling pathway plays a critical role in DN development. In this study, we noted altered expression of Sirt1, Ac-H3 and p66Shc in renal biopsy tissue samples of DN patients. We analyzed the relationship between the expression of these signaling proteins and patient clinical characteristics. In addition, we assessed the efficacy and novel pharmacological mechanism of probucol in the alleviation of renal injury in STZ induced diabetic mice and demonstrated that probucol attenuates renal injury by inhibiting p66Shc expression via the AMPK/ Sirt1 signaling pathway.

Renal p66Shc, Sirt1 and Ac-H3 expression in DN patients
The clinical characteristics of the DN patients and non-diabetic renal disease (NDRD) patients, who served as controls in this study, are shown in Table 1. DN patients exhibited significantly increased blood glucose and hemoglobin A1c (HbA1C) levels, as well as increased 24 h urine protein excretion, compared with control subjects. There were no significant differences between DN patients and control subjects with respect to age, sex. Changes in glomerular and tubulo-interstitial morphology, namely, glomerulosclerosis and focal tubular atrophy and interstitial fibrosis, were illustrated by H & E, PASM, PAS and Masson's trichrome staining of biopsy tissues of DN patients. Mesangial region expansion and tubular atrophy were noted in H & E and PAS staining. PASM and Masson's trichrome staining demonstrated tubular atrophy and interstitial fibrosis in DN patients compared with control subjects (Fig. 1A). Immunohistochemical (IHC) staining demonstrated significantly increased p66Shc and acetylated histone3 (Ac-H3) expression in the glomeruli and renal tubules of DN patients compared with NDRD patients, as well as decreased Sirt1 expression in DN patients compared with NDRD patients (Fig. 1B). Quantitatively, the tubular interstitial damage noted in DN patients was more severe than that noted in NDRD patients (Fig. 1C). P66Shc staining intensity was increased by 50% in DN patients (Fig. 1D), while Sirt1 staining intensity was decreased bỹ 30%. In contrast, Ac-H3 staining was increased by~32% in DN patients (Fig. 1E). Scatter plot analysis demonstrated a positive correlation between p66Shc expression and tubulo-interstitial damage severity and AcH3 expression ( Fig. 1F and H) and a negative correlation between p66Shc expression and SIRT1 expression (Fig. 1G).

Effects of probucol on biochemical parameters, p66Shc mRNA expression and glomerular injury in diabetic mice
To determine the long-term effects of probucol in a diabetic mice model, specific functional characteristics and biochemical indicators were analyzed. As shown in Table 2, probucol administration for 20 weeks had no effect on body weight in STZ-induced diabetic mice compared with DN mice (Table 2 and Fig. 2A). No changes in blood glucose levels were observed (Fig. 2B), while a~7.5-fold increase in proteinuria was observed in STZ mice compared with control mice. This increase was modestly reversed by probucol treatment (1.06 ± 0.21 mg/24 h vs 1.48 ± 0.68 mg/24 h, P < 0.05, Fig. 2C). Likewise, changes in serum creatinine, blood urea nitrogen (BUN),total triglyceride (TG) and total cholesterol (TC) levels were noted to be increased in STZ mice compared with control mice (Table 2). In addition, STZ mice exhibited significantly increased levels of urinary H 2 O 2 and TBARS. These increases were reversed following probucol treatment. No changes were observed in DMSO-treated mice ( Fig. 2D and E). Furthermore, renal malondialdehyde (MDA) and LDL-c levels were significantly increased in STZ-induced diabetic mice. These increases were reversed by probucol treatment (Fig. 2F and G). Significantly enhanced p66Shc mRNA expression was observed in the kidneys of STZ-induced mice. This was reversed by probucol treatment (Fig. 2H). Correlation analysis revealed a significantly positive correlation between p66Shc mRNA expression and urinary H 2 O 2 (R = 0.743, p < 0.01) (Fig. 2I) and LDL-c levels (R = 0.733, p < 0.01) (Fig. 2J). Further investigation demonstrated that probucol treatment significantly attenuated these changes in the kidneys of diabetic mice, particularly in the mesangial matrix and glomerulosclerosis, compared to untreated STZ-induced diabetic mice. No significant changes were observed in DMSO-treated mice compared to STZ-induced diabetic mice (Fig. 2K). In addition, probucol treatment significantly decreased the kidney weight/body weight ratio compared to untreated STZ-induced diabetic mice. No changes were observed in DMSO-treated mice (Fig. 2L). Similar findings were noted with respect to glomerulosclerosis, as indicated by glomerular damage scores (Fig. 2M) and glomerular tuft areas (Fig. 2M). Glomerular damage scores were positively correlated with p66Shc mRNA expression (R = 0.6, p < 0.05) (Fig. 2O).

Effects of probucol on tubulo-interstitial damage and renal oxidative injury in diabetic mice
To determine the effects of probucol on renal tubulo-interstitial  . We also noted a strong correlation between p66Shc mRNA expression and oxidative injury severity (R = 0.887, p < 0.01) (Fig. 3H). Furthermore, p66Shc mRNA expression was strongly correlated with apoptosis (R = 0.736, p < 0.01) (Fig. 3I).
2.4. Probucol treatment regulates renal phospho-AMPK, SIRT1, Ac-H3 and p66Shc expression in diabetic mice Western blot analysis showed that p-AMPK expression was significantly reduced in the kidneys of STZ-induced diabetic mice compared to control mice ( Fig. 4A and C, line 2 vs line 1). Similar results were observed regarding Sirt1 protein expression ( Fig. 4B and D, line 2 vs line 1). In contrast, the levels of Ac-H3 and p66Shc protein expression were markedly increased in the kidneys of STZ-induced diabetic mice ( Fig. 4B middle panel). Following probucol administration for 20 weeks, all of these changes were reversed ( Fig. 4A and B, line 4 vs line 2). No changes were observed in β-actin expression. In addition, p66Shc protein level was higher in the kidneys of STZ-induced diabetic mice than in the kidneys of control mice and was decreased by probucol treatment (Fig. 4F). To elucidate the relationships among these molecules, correlation analyses were performed and demonstrated that p-AMPK expression was positively correlated with Sirt1 protein expression but was negatively correlated with Ac-H3 expression (Fig. 4G). Further analysis showed that p66Shc expression was positively correlated with Ac-H3 expression, while p66Shc expression was negatively correlated with Sirt1 expression (Fig. 4H). Further investigation demonstrated that the concentration of p-AMPK was notably decreased in the renal cortical tissues of STZ-induced diabetic mice compared to control mice (Fig. 4I, line 2 vs line 1) and that this decrease was partially reversed by probucol treatment (line 4 vs line 2). Similar results were observed regarding Sirt1 expression (Fig. 4J). To confirm the above results, IHC staining was performed on kidney tissues sections (Fig. 4K). Sirt1 (upper panel) and AcH3 (middle panel) were mainly expressed in the glomeruli and tubular nuclei, and renal Sirt1 expression was notably reduced in STZ-induced diabetic mice compared with control mice (Fig. 4K, b vs a), while Ac-H3 expression was significantly increased in these mice compared to control mice (Fig. 4K,f vs e). p66Shc was mainly expressed in the renal tubules, as well as in the glomerulus although to a limited extent. Renal p66hc expression was also increased in STZ-induced diabetic mice ( Fig. 4K lower panels). However, these changes were reversed by probucol treatment.

Probucol regulates high-glucose-induced p-AMPK, Sirt1, Ac-H3 and P66Shc expression in HK-2 cells
In vitro studies were performed with HK-2 cells, human proximal tubular cells. Significantly decreased time-dependent p-AMPK expression was observed, as determined by Western blotting, in HK-2 cells exposed to 30 mM D-glucose (HG) for 1-6 h ( Immunofluorescence studies showed that HG decreased Sirt1 expression in HK-2 cells ( Fig. 5G upper panels) but significantly increased p66Shc expression. These effects were reversed by probucol treatment. In addition, we also measured p-AMPK concentrations in HK-2 cells treated with probucol. As shown in Fig. 5H, decreased p-AMPK concentrations were observed in HK-2 cells exposed to HG conditions compared to HK-2 cells exposed to LG conditions. These change were partially reversed by probucol treatment. Similar changes in Sirt1 expression were observed in HK-2 cells subjected to HG and probucol treatment (Fig. 5I).

Probucol inhibits p66Shc expression through the AMPK/Sirt1 pathway
To determine whether the AMPK-Sirt1 pathway modulates p66Shc expression regulation by probucol, studies with various inhibitors or activators were performed. HK-2 cells were exposed to different concentrations of D-glucose. They were also subjected to individual concomitant treatments of probucol and Dorsomorphin (selective AMPK inhibitor), 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, AMPK activator) or EX-527 (selective SIRT1 inhibitor). Treatment with probucol significantly reduced p66Shc mRNA expression in HK-2 cells exposed to HG conditions (   (caption on next page) expression through the AMPK/Sirt1 pathway. Similar results were noted regarding cellular lipid peroxidation injury, as determined by malondialdehyde (MDA) measurements (Fig. 6B). Immunofluorescence showed that probucol treatment significantly ameliorated the decreases in p-AMPK and Sirt1 expression elicited by HG conditions (Fig. 6C upper and middle panels). However, these changes were almost restored to baseline in cells treated with Dorsomorphin or EX-527. AICAR treatment enhanced the effects of probucol, but not in the presence of EX-527. The results regarding Sirt1 contrasted with those regarding p66Shc expression (Fig. 6C middle panels), which were similar to those regarding p66Shc mRNA expression. In addition, probucol treatment reduced HG-stimulated ROS generation, and this effect was partially blocked partially by treatment with Dorsomorphin or Ex527, as shown by DHE staining (Fig. 6C, lower panels). These findings were also confirmed by Western blot analyses ( Fig. 6D-F). These data suggest that probucol inhibits p66Shc expression and ROS generation via the AMPK-Sirt1 pathway in HK-2 cells exposed to HG conditions.

Probucol negatively regulates p66Shc expression via epigenetic chromatin modification
It has been reported that Sirt1 activation by p-AMPK is dependent upon its binding to the p66Shc promoter region, which decreases Ac-H3 levels and ultimately results in p66Shc gene transcription downregulation. We examined the effects of probucol on Sirt1 and Ac-H3 expression levels in HK-2 cells that were exposed to HG and treated with probucol or AMPK siRNA. Significantly decreased Sirt1 expression was observed in HK-2 cells exposed to HG conditions or transfected with AMPK siRNA, as determined by Western blotting (Fig. 7A). Sirt1 expression was partially restored by probucol treatment. Contrasting results were observed regarding Ac-H3 protein expression following various treatments (Fig. 7B). We considered the possibility that probucol-mediated p66Shc transcription regulation may occur at the chromatin level because histone deacetylation can inhibit p66Shc gene transcription. To confirm this possibility, we performed a ChIP assay in HK-2 cells under different D-glucose conditions and focused on the 1641-bp region of the p66Shc promoter, which is upstream of the transcription start site. Within this fragment, only one region (−508 bp to −250 bp) from the material immunoprecipitated with the anit-Sirt1 antibody was amplified by real-time PCR (Fig. 7C). We then assessed the interactions among Sirt1, Ac-H3 and the −508-bp to −250-bp region of the p66Shc gene promoter in HK-2 cells. As shown in Fig. 7D, the antibody directed against Sirt1 immunoprecipitated the DNA fragments from HK-2 cells containing potential binding sites for the p66Shc promoter. The binding of Sirt1 to the promoter region was dramatically decreased in HK-2 cells under HG conditions and following AMPK siRNA transfection compared to LG ambience. Binding was partially restored following probucol or AICAR treatment (Fig. 7D). Contrasting results were observed regarding the binding of Ac-H3 to the p66Shc promoter region (Fig. 7E), suggesting that probucol-mediated p66Shc transcription inhibition may involve epigenetic chromatin modification modulated by a Sirt1-AcH3 complex.

Discussion
The data of this study indicate that probucol ameliorates renal injury in STZ-induced diabetic mice, and it seems to exert these effects by quelling oxidative stress and apoptosis in diabetic kidneys by repressing p66Shc, an adaptor protein linked to longevity. In addition, the findings suggest that probucol inhibits p66Shc expression via an AMPK-SIRT1-AcH3-dependent pathway. Renal biopsy tissue staining results indicate that renal injury and fibrosis are significantly increased in DN patients (Fig. 1A). Unfortunately, the mechanisms underlying these changes have not been clearly delineated, and no suitable drug that can reverse these changes has been identified. The studies carried out over the last 2 decades indicate that DN is characterized by increased oxidative stress caused by excessive generation of reactive oxygen species (ROS) that ultimately progresses to renal failure [18,20]. Thus, therapies targeting the ROS would conceivably play a key role in attenuating renal injury and delaying the progression of DN.
p66Shc is a member of the adaptor protein family and it has three isoforms encoded by ShcA with relative molecular weights of 46, 52 and 66 kDa. Among them, the p66Shc protein is attracting more attention because it has an additional N-terminal region named CH2, which is responsible for its redox properties, lifespan regulation and apoptosis [11]. Previous studies from our laboratory and of others have shown that p66Shc plays a critical role in renal oxidative injury in DN [10,34,35]. Incidentally, HG and angiotensin II (Ang II) could activate PKC-β and PKC-δ, then phosphorylate p66Shc which translocates into mitochondria and increase ROS generation. So P66ShC could serve as a mediator of mitochondrial oxidative stress and apoptosis in the proximal tubule [35]. In this regard, the increased p66Shc mRNA expression has been linked to renal oxidative damage in DN patients [42]. Another molecule that can modulate the biology of p66Shc is Sirt1, an enzyme belonging to the family of sirtuins. They are highly conserved NAD +dependent deacetylases that regulate cellular energy and lifespan in mammals through histone H3 deacetylation [44], and thereby modulate the promoter activities of its target genes [33]. Sirt1 over-expression can inhibit HG-induced p66Shc expression through Ac-H3, which decreases oxidative stress and improves endothelial functions [50]. These data would indicate that Sirt1-mediated p66Shc expression regulation via Ac-H3 may be related to oxidative injury in DN. In light of these findings, we investigated the expression of p66Shc, Sirt1 and Ac-H3 in renal biopsy tissue specimens of patients with DN. We noted significantly decreased Sirt1 expression while there was an increased expression of p66Shc and Ac-H3 (Fig. 1B). Furthermore, we noted a correlation between p66Shc expression and tubulo-interstitial damage, as well as a correlation between Sirt1 and Ac-H3 expression (Fig. 1F-H), suggesting that Sirt1/Ac-H3 modulates p66Shc expression, which most likely would result in renal oxidative injury in DN. To verify whether targeting p66Shc attenuates renal oxidative damage in DN and to determine whether the Sirt1/Ac-H3 pathway is involved this process, probucol, an antioxidant, was administered to diabetic mice and its effects in in vitro cell culture studies were also assessed Among various strains of mice, C57BL/6 mice are the most widely used for hyperglycemia-induced renal injury. The glomerular injury and tubulo-interstitial fibrosis can be induced by daily intraperitoneal injection of low-dose STZ or by high-fat and high-cholesterol diet administration [3,27]. Probucol treatment may exert therapeutic effects by inhibiting oxidative injury in STZ-induced diabetic rats [24]. Likewise, we observed that after hyperglycemia induction via STZ injection, the mice exhibited high glucose levels, increased proteinuria ( Fig. 2B and C), and enhanced expression of oxidative stress markers, such as, urinary H 2 O 2 ,TBARS, serum MDA and LDL-c (Fig. 2D-G). Additionally, p66Shc mRNA expression was also increased in the kidneys of diabetic mice (Fig. 3H). We also observed a notable increase in renal fibrosis in Fig. 3. Effects of probucol on renal tubular injury, oxidative injury and apoptosis in STZ-induced diabetic mice. Panel A: Mouse kidney sections were subjected to Masson's trichrome staining (upper panels) and IHC staining with anti-fibronectin (lower panels). B-C: Bar graphs representing semi-quantified tubular injury scores and average optical densities (AODs) of FN protein expression in the kidneys of STZ-induced diabetic mice, respectively. D: Correlation between tubular interstitial damage scores and p66Shc mRNA expression (R = 0.749, p < 0.01). Panel E: Dihydroethidium (DHE) staining (upper panels) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and histochemical (TUNEL-H) staining (lower panels) in mouse kidney sections in various groups. F-G: Bar graphs representing quantification of tissues stained with DHE (F) and TUNEL-H (G). H: Relationship between p66Shc mRNA expression and relative ROS scores. I: Relationship between p66Shc mRNA expression and TUNEL positive-cells (R = 0.736, p < 0.01). Values are the mean ± SE, R: correlation coefficient, *P < 0.01 vs. control; #P < 0.01 vs STZ (magnification ×200).
STZ-induced diabetic mice (Fig. 3A). However, all of these changes were reversed by probucol treatment. Furthermore, analysis showed that p66Shc mRNA expression correlated with H 2 O 2 and LDL-c levels ( Fig. 2I and J) and is altered expression was associated with the extent of glomerular damage (Fig. 2O). We therefore surmised that probucol reduces oxidative damage by inhibiting p66Shc expression. Our results also support the findings of previous studies, which suggest that probucol is a cholesterol-lowering drug with antioxidant properties that   LG; #P < 0.01 vs. HG; $P < 0.01 vs. HG + Probucol; @ P < 0.01 vs. HG + Probucol + Ex-527. C: Cellular immunofluorescence with anti-p-AMPK, anti-Sirt1 and anti-p66Shc antibodies. High levels of p-AMPK were observed under LG conditions and were accompanied by Sirt1 expression. HG reduced this fluorescence. This effect was reversed by probucol treatment and Dorsomorphin and EX-527 treatment but was increased by AICAR treatment. Contrasting results were observed regarding p66Shc and DHE staining (lower panel). D-F: Representative Western blots (D) and densitometric analysis of Sirt1 (E) and p66Shc protein expression (F) in HK-2 cells exposed to HG conditions with or without treatment with various reagents. Quantitative analyses of the Western blotting results are included, and the data were normalized to β-actin and are presented as the mean ± SEM (n = 4). *P < 0.01 versus LG; #P < 0.01 vs. HG; $P < 0.01 vs. HG + Probucol; @ P < 0.01 vs. HG + Probucol + Ex-527. protect against free radical-induced damage in a wide variety of scenarios, including doxorubicin-induced cardiotoxicity [7], HG-induced podocyte injury [49] and tubular epithelial cell injury [6]. The mechanism by which probucol protects against oxidative injury may involve increase in antioxidant gene mRNA expression and decrease in apoptosis via improving mitochondrial oxidative phosphorylation and energy production [1]. Previously studies has showed that the podocyte injury such as cytoskeletal rearrangement and podocyte effacement plays a key role in development of proteinuria of DN [29]. Probucol can reduce ROS production and attenuated podocyte injury in db/db mice [49], which may relative to its effect on inhibiting Ang II receipt expression [23]. Here, we demonstrate that probucol is not only exerts its beneficial anti-diabetic effect by regulating lipid metabolism but also can eliminating oxygen radicals in the kidneys of STZ-induced diabetic mice by p66Shc, a novel pathway, and thus the p66Shc nay play a role in these processes as the molecular targets of probucol.
It is known that the tubulo-interstitial fibrosis can occur in DN and it may occur independent of glomerular injury [37,52]. p66Shc is mainly expressed in the proximal tubules, and its up-regulation correlates with tubular damage in biopsy specimens of DN patients, as described in our previous publications [42]. Thus, we investigated the effects of probucol on tubulo-interstitial injury in STZ-induced diabetic mice. We observed a notably reduced tubular damage and interstitial fibrosis, as well as decreased oxidative damage and apoptosis, in STZ-induced diabetic mice treated with probucol ( Fig. 3A-C), Correlation analyses showed that p66Shc mRNA expression was associated with tubulo-interstitial damage scores, oxidative stress and apoptosis (Fig. 3D). In addition, probucol treatment in STZ-induced diabetic mice also Fig. 7. The effects of Probucol on Sirt1/Ac-H3 binding to the p66Shc promoter in HK-2 cells. Panels A-B: The HK-2 cells were pretreated with probucol, AMPK siRNA and Scramble siRNA, respectively, and then exposed to HG for 24 h. Sirt1 and Ac-H3 protein expression were analyzed by Western blot. C: The bar graphs represent the immunoprecipitated genomic DNA amplified by real-time PCR using different primers corresponding to specific areas of the p66Shc promoter region. D-E: Chromatin immunoprecipitation (ChIP) was carried out in HK-2 cells using normal rabbit IgG antibodies, anti-Sirt1 antibodies or anti-Ac-H3 antibodies, as indicated. Precipitated DNA was analyzed by amplifying the PCR product spanning the regions of potential Sir1/Ac-H3 binding sites in the p66Shc promoter (−508 bp to −250 bp). IgG served as a negative control. Data were normalized by total input DNA, SIRT1/input (D, bottom graph) and AcH3/input (E, bottom graph). Values are the mean ± SEM, n = 4. *P < 0.01 vs. LG; #p < 0.01 vs. HG; $p < 0.01 vs. HG + Probucol. @ p < 0.01 vs. HG + Probucol + AMPK siRNA. attenuated renal oxidative stress and apoptosis, and these changes correlated with p66Shc mRNA expression (Fig. 3E-I). These findings indicated that p66Shc over-expression causes renal oxidative stress, which plays a key role in DN progression, and also indicated that the probucol exerts an anti-oxidative and anti-apoptotic effect on the kidneys of diabetic mice that may be facilitated by p66Shc inhibition.
We subsequently attempted to elucidate the mechanisms by which p66Shc transcription is regulated in the kidneys of diabetic mice following probucol treatment. Abnormal Sirt1 and Ac-H3 expression has been noted in patients with DN, and the expression of these proteins has been correlated with p66Shc up-regulation (Fig. 1). It has been demonstrated that HG-induced oxidative stress and apoptosis are associated with AMPK/SIRT1 signaling. AICAR, an AMPK activator, activates Sirt1 and prevents oxidative stress and apoptosis in cultured mesangial cells exposed to HG conditions [21,47]. In addition, AMPK phosphorylation is decreased in STZ-induced diabetic [13] and db/db mice [15], as well as in cultured mesangial cells exposed to HG ambience [14]. Furthermore, Wu et al. found that probucol treatment prevents nonalcoholic steatohepatitis development by up-regulating p-AMPK expression [40], which may be facilitated by probucol-enhanced AMPK-Thr172 phosphorylation though hepatic kinase B1(LKB1) phosphorylation at Ser 428 [30].
Based on these findings, we hypothesized that the probucol protects against renal oxidative damage by inhibiting p66Shc expression though the AMPK/Sirt1/Ac-H3 signaling pathway. To confirm this hypothesis, we investigated the relevance of two important metabolic sensors and stress regulators in the kidneys of STZ-induced diabetic mice. As shown in Fig. 4, probucol administration restored p-AMPK expression and activity, as well as of Sirt1 expression and activity. In addition, Ac-H3 expression, which was notably increased in STZ-induced diabetic mice, was decreased by probucol treatment (Fig. 4A-F). Further analysis showed that p66shc mRNA expression was negatively correlated with p-AMPK and Sirt1 expression and positively correlated with Ac-H3 expression ( Fig. 4G and H). Similar results were noted regarding p-AMPK and Sirt1 activity. These results were also supported by both IHC and Western blot analysis with respect to protein expression. These findings indicate that probucol-mediated p66Shc mRNA suppression most likely occurs via the AMPK/Sirt1 pathway.
To confirm these findings, we performed in vitro studies using HK-2 cells, a human proximal tubular cell line. Increased apoptosis has been observed in the proximal and distal tubular epithelial cells of patients with diabetes [25]. Also, apoptosis is seen in the proximal tubular epithelial cells of kidney tissues exposed to HG conditions [35]. In addition, probucol has been shown to attenuate methylguanidine-induced HK-cell apoptosis [39]. In this study, we observed that HG treatment significantly decreased p-AMPK expression and activity which was partially ameliorated by probucol treatment. Similar results were observed for Sirt1 expression. Furthermore, probucol treatment of HK-2 cells reversed HG-mediated Ac-H3 up-regulation (Fig. 5). Additional studies were performed using Dorsomorphin, a cell-permeable and reversible ATP-competitive AMPK inhibitor, and also Ex527, a Sirt1 inhibitor. Both the agents reversed the effects of probucol on p66Shc expression. Concomitant treatment with probucol and AICRA, an activator of AMPK, augmented the above mentioned decreases in p66SShc mRNA expression in HK-2 cells exposed to HG ambience. Sirt1 and p66Shc exhibited contrasting changes in their protein expression patterns, and also MDA and ROS revealed contrasting changes in their levels as well (Fig. 6). These findings suggest that probucol inhibits p66Shc expression and thus modulates renal tubular oxidative injury in diabetes via the AMPK-SIRT signaling pathway.
The next question that we addressed in this study was the epigenetic regulation of p66Shc. The p66Shc increase has been described to be associated with increased histone acetylation [45], which is necessary for p66Shc transcription [22]. Promoter analysis showed that the highest transcription levels in the p66Shc promoter were localized to a region between −434 and −101 bp upstream of ATG upon transfection of cells lacking endogenous p66Shc [38]. Another interesting point described in the literature is that epigenetic p66Shc gene expression regulation is modulated by histone modifications that are dependent on Sirt1 binding to the p66Shc promoter, and as a result there is an aberrant acetyl-HistoneH3 (Lys9) expression [45]. In addition, it has been shown that SIRT1 affects the p66Shc promoter in hyperglycemiainduced endothelial dysfunction through histone H3 modification [50]. Recent studies by Bock et al. demonstrated that the coagulation protease activated protein C (aPC) exerts cyto-protective effects in DN. The mechanism by which it reverses glucose-induced hyperacetylation of the p66Shc promoter in podocytes may involve the suppression of HGinduced p66Shc expression and the reduction of glucose-induced mitochondrial p66Shc translocation, as well as mitochondrial ROS production [2]. We thus explored the epigenetic factors which may be involved in the regulation of p66Shc expression in HK-2 cells treated with probucol. As shown in Fig. 7A and B, significantly decreased Sirt1 expression was observed in HK-2 cells treated with HG. Sirt1 expression was restored by pretreatment with probucol but was partially blocked by AMPK siRNA co-transfection. Contrasting results were noted regarding Ac-H3 expression, as determined by Western blot analysis. Furthermore, to delineate the Sirt1 binding region in the p66Shc gene promoter, ChIP assay was performed in HK-2 cells after nuclear chromatin immunoprecipitation with normal rabbit IgG or antibodies against Sirt1. Within the p66Shc gene promoter (−1641 to + 45 bp), only one region (−508 to −250 bp) could be amplified by Chip-PCR. These results are consistent with the observations made by Zhou et al. [50], indicating that Sirt1 affects the p66Shc promoter through Ac-H3 modification in HK-2 cells and that the effects of probucol may be modulated by inhibiting p66Shc transcription under HG conditions. It has been demonstrated that H3 acetylation within the p66Shc promoter is induced by glucose [2]. Similar results were observed in cells treated with AICAR, but not following AMPK siRNA treatment. In contrast, Ac-H3 binding was dramatically increased under HG ambience and was reduced by probucol treatment (Fig. 7), indicating that probucol regulates p66shc transcription by facilitating Sirt1 activity and modulation of Ac-H3 acetylation.
Our study showed that there was significantly positive correlation between p66Shc expression and renal tubulo-interstitial damage in DN patients. Probucol administration could effectively alleviate renal tubular oxidative injury by inhibiting p66Shc expression, accompany with enhanced the expression of p-AMPK and Sirt1, and decreased Ac-H3 expression both in vivo and in vitro. Further analysis showed that p66shc expression was negatively correlated with p-AMPK and Sirt1 expression while positively correlated with Ac-H3 expression. Furthermore, both AMPK inhibitor and Sirt1 inhibitor could reverse the anti-oxidative injury effects of probucol and the regulation of p66Shc expression, while AMPK activator could enhance the reduction of p66Shc and AcH3 expression. Moreover, ChIP analysis showed that probucol could inhibit p66Shc transcription by reducing Ac-H3 binding to p66Shc promoter (−508 to −250 bp) via facilitating Sirt1 activity and modulating of Ac-H3 acetylation. Take together, these findings indicated that probucol suppresseed HG-induced tubular oxidative injury, fibrosis and apoptosis by inhibiting p66Shc expression via activating the AMPK-SIRT1-AcH3 pathway (shown in Fig. 8).
In summary, we demonstrated that probucol ameliorates oxidative stress in kidneys of STZ-induced diabetic mice and it suppresses HGinduced tubular oxidative injury by inhibiting p66Shc expression, and it seems that these effects are mediated by activating the AMPK/Sirt1 pathway. Because probucol has other effects, such as, alleviation of renal vasoconstriction [19] and reduction of inflammatory responses [28], additional studies are needed to determine if probucol also exerts such beneficial therapeutic in the amelioration of DN.

Kidney morphological analysis
Human kidney biopsy tissues were obtained from patients with a 10-15-years history of class III DN (n = 15). These patients were confirmed to have pathological and clinical findings consistent with DN (including both type 1 and type 2 diabetic nephropathy), and an equal number of patients of non-diabetic renal disease (NDRD, n = 15) with minimal change nephropathy as control were recruited for this study. Renal biopsy tissue sections were subjected to hematoxylin-eosin (H & E), periodic acid-Schiff (PAS), periodic acid-silver methenamine (PASM) and Masson's trichrome staining. Glomerular damage and tubulo-interstitial lesion indices were assessed using a semi-quantitative scoring system, as described previously [46].

Animal experimental design
C57BL/6 mice (~20 g B.W) were purchased from Better Biotechnology Co., Ltd, Nanjing, China, and provided by the Second Xiangya Hospital Animal Center. The animals were housed at 22°C under a 12 h light-dark cycle, and they were allowed free access to food and water. A total of 60 adult C57BL/6 mice aged 8 weeks were used, and they were divided into 4 groups of 15 animals. The first group was injected with sodium citrate buffer (25 mM/L pH 4.0) only and served as a control group. The second group was injected intraperitoneally (i.p.) with 40 mg/kg body wt. streptozotocin (STZ, Sigma-AIdrich, St. Louis, MO). The solution was freshly made in sodium citrate buffer and injected for 5 consecutive days. Tail vein blood glucose levels were tested 1 week after the last STZ injection, and mice with glucose levels > 250 mg/dI were considered diabetic. The third group of STZinduced diabetic mice received DMSO (10 ml/kg). The fourth group of STZ-induced diabetic mice received probucol every other day for 20 weeks. For probucol-treated mice, a probucol dose of 10 mg/kg body wt IP was calculated based on average daily liquid ingestion, as descried previously [51]. All animals were fed a high-fat high-cholesterol Western diet (WD,TD88137) from Harlan-Teklad (Madison, WI) after the onset of diabetes, and they were sacrificed at 20 weeks following STZ administration. Their sera and kidneys were saved for various biochemical and morphological studies. All animal experiments were Fig. 8. Schematic diagram depicting the conceivable protective effects of probucol on the progression of DN. SIRT1 is downregulated in tubular cells under high glucose (HG) ambience, SIRT1 down-expression can increase HG-induced p66Shc expression through Ac-H3, which then increases oxidative stress, aggravates renal tubular cells damage and exacerbate the progression of diabetic nephropathy. Probucol administration could enhance the expression of p-AMPK and Sirt1, and then inhibit p66Shc transcription under HG conditions through affecting the Ac-H3 modification of p66Shc promoter region. In short, probucol suppresseed HG-induced tubular oxidative injury, fibrosis and apoptosis by inhibiting p66Shc expression, and these effects are mediated by activating the AMPK-SIRT1-AcH3 pathway. performed in accordance with the regulations established by the Institutional Committee for Care & Use of Laboratory Animals at Central South University, China.

Morphological studies
Kidney tissues were fixed with 4% buffered paraformaldehyde and embedded in paraffin, and 4 μm-thick sections were prepared. The sections were then subjected to hematoxylin-eosin (H & E), Periodic acid-Schiff (PAS) and Masson's trichrome staining. A semi-quantitative scoring system was used to evaluate tubulo-interstitial injury severity, as previously described [36].

Serum biochemistry
Blood glucose levels were measured using a blood glucose monitor (Boehringer Mannheim, Germany). Serum triglyceride and cholesterol levels were measured by an automated biochemical analyzer (OLYMPUS AU100, Japan).

Urinary and blood chemistry
Mice were placed in individual metabolic cages for 24 h urine collection. Urine samples were centrifuged at 2000g for 5 min, and the supernatants were saved. Urine albumin was measured by enzymelinked immunosorbent assay (ELISA; Bethyl Laboratories, USA) [36]. Urine creatinine (Cr) concentrations were determined using an ELISA kit from Exocell (Philadelphia, USA). Urinary thiobarbituric acid reactive substances (TBARS) and H 2 O 2 levels were measured using kits from Thermo Fisher Scientific (Rockford, USA) and Bioassay Systems (Hayward, USA), respectively.

Kidney AMPK activity
Activated AMPKα1 levels in renal cortical tissues were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R & D systems, USA & D systems, UR & D systems, USAA), following manufacturer's instructions [48].

SIRT1 deacetylase activity assay
Renal tissue and HK-2 cell Sirt1 deacetylase activity was determined using the Sirt1 deacetylase activity assay kit (Genmed Scientifics Inc., USA), according to the manufacturer's instructions [43]. Activity was expressed as a percentage of the control.

Renal tissue immunohistochemistry (IHC) and apoptosis assay
Human and mouse kidney tissue samples were used for IHC analysis. Tissue Section (3 µm thick) were prepared, mounted on glass slides, de-paraffinized, rehydrated and blocked with 3% H 2 O 2 solution. Following antigen retrieval in a microwave oven, the sections were then incubated with antibodies against p66Shc (1:50 dilution), SIRT1 (1:100 dilution), Ac-H3, phosphorylated AMPK (1:100 dilution) and AMPK (1:100 dilution). The sections were then subjected to DAB reaction, in accordance with the manufacturer's instructions. Apoptotic renal cells were identified via TUNEL staining and an In Situ Cell Death Detection Kit. They were cover-slipped using a drop of mounting medium and then examined with a microscope.

Cell culture and treatment
Human proximal tubular epithelial cells (HK-2) were purchased from ATCC and cultured in Dulbecco's modified Eagle medium (DMEM)-F12 medium supplemented with 10% fetal bovine serum (FBS). After the cells achieved 80% confluence, the medium was changed to FBS-free DMEM, and the culture was maintained in an atmosphere of 5% CO 2 /95% air at 37°C. Four sets of experiments involving the following cells were performed: cells maintained in 5 mM Dglucose (LG), cells maintained in 30 mM D-glucose (HG), cells treated with HG plus 20-80 µM probucol, and cells maintained in LG medium and DMSO. Dorsomorphin, AICAR and EX-527 were added individually to the culture medium at concentrations of 20 µM, 1 mM and 1 µM, respectively, before HG treatment at different time points.

Cells immunofluorescence
Cells were grown on coverslips, washed three times with PBS, fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 and then incubated in blocking buffer. They were subsequently incubated in primary antibody solution overnight. Then, the cells were incubated with FITC or Rhodamine-conjugated secondary antibodies and examined with a Zeiss fluorescence microscope equipped with a UV epi-illumination [36].

Cellular AMPK measurement
HK-2 cells were subjected to various treatments, as indicated. Cell lysates were prepared and centrifuged, and the supernatants were collected for AMPK ELISA. AMPK concentrations were measured using an ELISA kit, according to the manufacturer's instructions [48].

Intracellular ROS and malondialdehyde (MDA) measurements
Intracellular ROS generation was assessed by dihydroethidium (DHE) staining, as described previously [41]. Cellular lipid peroxidation injury was assessed by measuring malondialdehyde (MDA) content in the medium using a malondialdehyde (MDA) assay kit (Promega, USA), according to the manufacturer's instructions.

Statistical analysis
Statistical analyses were carried out using SPSS 16.0 software. The experimental results are expressed as the mean ± SD, and one-way analysis of variance (ANOVA) was carried out. Correlation analyses were carried out using Spearman's correlation coefficient. p < 0.05 was considered statistically significant.

Author disclosure statement
Authors have nothing to disclose