Kohei Omachi

Endoplasmic Reticulum (ER) Stress Induces Sirtuin 1 (SIRT1) Expression via the PI3K-Akt-GSK3β Signaling Pathway and Promotes Hepatocellular Injury

Sirtuin 1 (SIRT1), an NAD+-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage.

Mild Electrical Stimulation at 0.1-ms Pulse Width Induces p53 Protein Phosphorylation and G2 Arrest in Human Epithelial Cells

Background: A controlled approach as opposed to conventional toxic drugs to activate p53 is applicable for tumors and metabolic and inflammatory diseases. Results: A 0.1-ms pulse width mild electrical stimulation (MES) activated p53 function in epithelial cell lines. Conclusion: MES induced p53 phosphorylation via p38 MAPK signaling and G2 cell cycle arrest without cell death. Significance: MES works as a non-cytotoxic and controllable p53 activator. Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53−/− cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

Podocyte p53 Limits the Severity of Experimental Alport Syndrome

Alport syndrome (AS) is one of the most common types of inherited nephritis caused by mutation in one of the glomerular basement membrane components. AS is characterized by proteinuria at early stage of the disease and glomerular hyperplastic phenotype and renal fibrosis at late stage. Here, we show that global deficiency of tumor suppressor p53 significantly accelerated AS progression in X-linked AS mice and decreased the lifespan of these mice. p53 protein expression was detected in 21-week-old wild-type mice but not in age-matched AS mice. Expression of proinflammatory cytokines and profibrotic genes was higher in p53(+/-) AS mice than in p53(+/+) AS mice. In vitro experiments revealed that p53 modulates podocyte migration and positively regulates the expression of podocyte-specific genes. We established podocyte-specific p53 (pod-p53)-deficient AS mice, and determined that pod-p53 deficiency enhanced the AS-induced renal dysfunction, foot process effacement, and alteration of gene-expression pattern in glomeruli. These results reveal a protective role of p53 in the progression of AS and in maintaining glomerular homeostasis by modulating the hyperplastic phenotype of podocytes in AS.

Bromide supplementation exacerbated the renal dysfunction, injury and fibrosis in a mouse model of Alport syndrome

A seminal study recently demonstrated that bromide (Br-) has a critical function in the assembly of type IV collagen in basement membrane (BM), and suggested that Br- supplementation has therapeutic potential for BM diseases. Because salts of bromide (KBr and NaBr) have been used as antiepileptic drugs for several decades, repositioning of Br- for BM diseases is probable. However, the effects of Br- on glomerular basement membrane (GBM) disease such as Alport syndrome (AS) and its impact on the kidney are still unknown. In this study, we administered daily for 16 weeks 75 mg/kg or 250 mg/kg (within clinical dosage) NaBr or NaCl (control) via drinking water to 6-week-old AS mice (mouse model of X-linked AS). Treatment with 75 mg/kg NaBr had no effect on AS progression. Surprisingly, compared with 250 mg/kg NaCl, 250 mg/kg NaBr exacerbated the progressive proteinuria and increased the serum creatinine and blood urea nitrogen in AS mice. Histological analysis revealed that glomerular injury, renal inflammation and fibrosis were exacerbated in mice treated with 250 mg/kg NaBr compared with NaCl. The expressions of renal injury markers (Lcn2, Lysozyme), matrix metalloproteinase (Mmp-12), pro-inflammatory cytokines (Il-6, Il-8, Tnf-α, Il-1β) and pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) were also exacerbated by 250 mg/kg NaBr treatment. Notably, the exacerbating effects of Br- were not observed in wild-type mice. These findings suggest that Br- supplementation needs to be carefully evaluated for real positive health benefits and for the absence of adverse side effects especially in GBM diseases such as AS.

The effect of mild electrical stimulation with heat shock on diabetic KKAy mice

Diabetes mellitus (DM) is a growing health burden worldwide, and the number of patients with metabolic syndrome is increasing as well. The therapeutic strategies for DM and metabolic syndrome, and prevention of disease progression are therefore urgently needed. Insulin resistance is the main pathology in DM and metabolic syndrome. We previously showed that co-treatment of mild electrical stimulation (MES) with heat shock (HS) enhances insulin sensitivity in vitro and in diabetic mouse models. Moreover, MES+HS ameliorated the pathophysiology in human subjects with DM or metabolic syndrome in clinical trials. Despite the growing evidence that MES+HS could be a novel therapeutic approach, it is unclear whether long-term MES+HS treatment from the early stage of disease has preventive effect for DM. Thus, we assessed the effect of MES+HS on the pathophysiology of maturity-onset obesity using diabetic KKAy mice. Here, we showed that long-term treatment of MES+HS alleviated diabetic condition of KKAy mice. Hyperglycemia and insulin resistance were improved by MES+HS treatment. MES+HS also tended to suppress renal hypertrophy and increased the expression of podocyte-specific genes in the kidney. Collectively, our study suggests that long-term treatment of MES+HS is an effective and well-tolerated therapeutic strategy to decelerate the progression of DM and metabolic syndrome at least in part due to enhanced insulin sensitivity and podocyte function. Abbreviations: DM: diabetes mellitus, MES: mild electrical stimulation, HS: heat shock Introduction Type 2 diabetes mellitus (T2DM) is a serious health problem worldwide, and the number of patients with T2DM is rapidly increasing [1]. About 80-90% of T2DM patients are diagnosed as obese. Obesity, caused by the accumulation of visceral fat, is the fundamental cause of the development of metabolic syndrome [2]. Chronic metabolic syndrome exhibits high-blood pressure, hyperglycemia and insulin resistance, leading to T2DM, arteriosclerosis and cardiovascular defects [3]. Especially, progression of T2DM leads to intercurrent diabetic retinopathy and diabetic nephropathy [4,5]. Therefore, treatment that can control the progression of metabolic syndrome and T2DM at the primary stage of these diseases is needed. Previously, we optimized the conditions of mild electrical stimulation (MES) that induce several physiological response signals and molecules such as the insulin receptor (IR)-Akt pathway, MKK3b/6b-p38MAPK, JNK, p53, AMPK and HSP72 [6-9]. Co-treatment with MES and 42°C of heat shock (HS) (MES+HS) ameliorated visceral obesity and diabetic pathophysiology in high-fat diet-induced and leptin receptor mutant (db/db) mice [6]. Moreover, MES+HS treatment reduced the metabolic abnormalities and inflammation in subjects with metabolic syndrome or T2DM in clinical trials [10,11]. The accumulation of IR in the lipid rafts within cell plasma membrane, followed by enhanced sensitivity to insulin, by MES+HS was suggested to contribute to the improvement of insulin resistance in T2DM [12]. Hence, MES+HS can be a useful treatment for metabolic syndrome and T2DM. However, it is unclear whether long-term treatment with MES+HS from the early stage of disease is tolerable and effective in preventing the development of maturityonset obesity and T2DM. Here, we performed long-term treatment (28 weeks) with MES+HS on diabetic KKAy mice. KKAy is a well-established mouse model of metabolic syndrome with maturity-onset obesity, and spontaneously develops hyperglycemia, high blood pressure, and insulin resistance, which are quite similar to those observed in human T2DM [13,14]. The present study showed that long-term treatment with MES+HS decreased the blood glucose levels at the late stage of disease and also improved insulin resistance compared to sham-treated group. Additionally, long-term MES+HS treatment improved renal hypertrophy that is the hallmark of primary stage diabetic nephropathy and increased the expression of glomerular visceral epithelial cells and podocyte marker genes expression. This study revealed the beneficial Correspondence to: Hirofumi Kai, Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto City 862-0973, Japan. Tel: +81-96-371-4405; Fax: +81-96371-4405; E-mail: hirokai@gpo.kumamoto-u.ac.jp

STAT3 inhibition attenuates the progressive phenotypes of Alport syndrome mouse model

Background Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS. Method Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules. Results Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-β, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-β) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-β signaling. Conclusion STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS.