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Non-alcoholic steatohepatitis (NASH) occasionally occurs under obesity; however, factors modulating the natural history of fatty liver disease remain unknown. Since hypothalamic orexin that regulates physical activity and autonomic balance prevents obesity, we investigate its role in NASH development. Male orexin-deficient mice fed a high-fat diet (HFD) show severe obesity and progression of NASH with fibrosis in the liver. Hepatic fibrosis also develops in ovariectomized orexin-deficient females fed an HFD but not ovariectomized wild-type controls. Moreover, long-term HFD feeding causes hepatocellular carcinoma (HCC) in orexin-deficient mice. Intracerebroventricular injection of orexin A or pharmacogenetic activation of orexin neurons acutely activates hepatic mTOR-sXbp1 pathway to prevent endoplasmic reticulum (ER) stress, a NASH-causing factor. Daily supplementation of orexin A attenuates hepatic ER stress and inflammation in orexin-deficient mice fed an HFD, and autonomic ganglionic blocker suppresses the orexin actions. These results suggest that hypothalamic orexin is an essential factor for preventing NASH and associated HCC under obesity.

Muscle biology is important topic in diabetes research. We have reported that a diet with ketogenic amino acids rich replacement (KAAR) ameliorated high-fat diet (HFD)-induced hepatosteatosis via activation of the autophagy system. Here, we found that a KAAR ameliorated the mitochondrial morphological alterations and associated mitochondrial dysfunction induced by an HFD through induction of the AKT/4EBP1 and autophagy signaling pathways in both fast and slow muscles. The mice were fed with a standard HFD (30% fat in food) or an HFD with KAAR (HFDKAAR). In both the gastrocnemius and the soleus, HFDKAAR ameliorated HFD-impaired mitochondrial morphology and mitochondrial function, characterized by decreased mitofusin 2, optic atrophy 1, peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α and PPARα levels and increased dynamin-related protein 1 levels. The decreased levels of phosphorylated AKT and 4EBP1 in the gastrocnemius and soleus of HFD-fed mice were remediated by HFDKAAR. Furthermore, the HFDKAAR ameliorated the HFD-induced autophagy defects in the gastrocnemius and soleus. These findings suggest that KAAR may be a novel strategy to combat obesity-induced mitochondrial dysfunction, likely through induction of the AKT/4EBP1 and autophagy pathways in skeletal muscle.

Background: Dietary interventions, including a low-protein diet (LPD) and methionine (Met) restriction, has shown longevity and metabolic health. LPD has a reno-protective effect against diabetic kidney disease (DKD) in rats with type 2 diabetes and obesity; however, it is unclear whether the beneficial effect of LPD is mediated by a low-Met intake. Methods: Rats were divided into four groups at 24 weeks of age: 1) non-diabetic Wistar lean (fa/-) rats fed a standard diet including 0.64% Met (STD) (Control); 2) Wistar fatty (fa/fa) rats with type 2 diabetes/obesity (WFRs) fed STD (0.64% Met) (Diabetes); 3) WFRs fed LPD (0.15% Met) (LPD); and 4) WFRs fed LP+Met (0.64% Met) (LP+Met). The dietary intervention was performed for 20 weeks. Findings: Accumulation of S-adenosylmethionine (SAM) in renal tubular cells of diabetic rats, associated with reduced glycine Nmethyltransferase (Gnmt) expression, led to mechanistic target of rapamycin complex1 (mTORC1) activation and impaired autophagy, resulting in the progression of DKD through mitochondrial abnormalities, oxidative stress, inflammation, tubular cell damage and tubulointerstitial fibrosis. Additionally, cystathionine γ-lyase (CGL) expression was decreased in the diabetic renal cortex. LPD reversed all of these alterations of the diabetic kidney; however, the LP+Met diet abrogated the effects of LPD in diabetic rats. Furthermore, LPD partially improved the diabetic state, while the LP+Met diet exacerbated it and was accompanied by changes in the LPD-induced expression of plasma fibroblast growth factor 21 (FGF21). Interpretation: LPD could exert a reno-protective effect through low-Met intake-mediated suppression of mTORC1 and restored autophagy, which is associated with a decrease in the accumulation of SAM due to reduced expression of Gnmt in diabetic kidneys. Funding Statement: This work was financially supported through a Grant-in-Aid for Scientific Research KAKENHI (C) (24591218), a grant for collaborative research with Kowa Hakko Kirin to MK. Declaration of Interests: Boehringer Ingelheim, Mitsubishi Tanabe Pharma, Kyowa Hakko Kirin, Taisho Toyama Pharmaceutical Co. and Ono Pharmaceutical Co. contributed to establishing the Division of Anticipatory Molecular Food Science and Technology. The authors declare that there is no duality of interest associated with this manuscript. Ethics Approval Statement: The Research Center for Animal Life Science of Kanazawa Medical University approved all experiments, and all experiments were performed in accordance with the relevant guidelines and regulations.

Background: The sodium glucose co-transporter 2(SGLT2) inhibitors are beneficial in halting diabetic kidney disease, however complete mechanisms are not elucidated yet. Epithelial to mesenchymal transition (EMT) program has been associated with suppression of sirtuin 3 (Sirt3) and aberrant glycolysis. Here, we hypothesized that SGLT2 inhibitor empagliflozin restored normal kidney histology and function, associated with the inhibition of aberrant glycolysis in diabetic kidney. Methods: In vivo, streptozotocin-induced diabetic CD-1 mice, the model exhibiting prominent kidney fibrosis compared to other strains, were utilized and intervention with empagliflozin or insulin was performed. In vitro analysis was performed in HK-2 tubular cells. Results: CD-1 mice with streptozotocin-induced diabetes displayed kidney fibrosis associated with EMT program at 4 months after diabetes induction. Empagliflozin-intervention for 1 month restored all; blood glucose adjusted by insulin did not. Empagliflozin normalized suppressed Sirt3 levels and aberrant glycolysis characterized by hypoxia-inducible factor-1I± accumulation, hexokinase 2 induction and pyruvate kinase isozymes M2 dimer formation in diabetic kidney. Empagliflozin also suppressed accumulation of by-products of glycolysis in diabetic kidney. High-glucose media induced EMT, associated with Sirt3 suppression and aberrant glycolysis induction, in HK2 proximal tubule cell line; SGLT2-knockdown suppressed EMT with restorations of all. Conclusions: Taken together, SGLT2 inhibitors exhibit reno-protective potential that is partially dependent on the inhibition of glucose-reabsorption, which could be utilized as substrate for aberrant glycolysis in kidney tubule. Funding: Empagliflozin was provided by Boehringer Ingelheim through an MTA. This study was essentially supported by the grant from Japan Diabetes Foundation (2016). This work was partially supported by grants from the Japan Society for the Promotion of Science for KK (23790381 and 23790381) and DK (25282028 and 25670414). This work was partially supported by a Grant for Collaborative Research awarded to DK (C2011-4 and C2012-1) and a Grant for Promoted Research awarded to KK (S2015-3, S2016-3, and S2017-1) from Kanazawa Medical University. Boehringer Ingelheim, Mitsubishi-Tanabe Pharma and Ono Pharmaceutical contributed to establishing the Division of Anticipatory Molecular Food Science and Technology. Declaration of Interest: KK and DK received lecture honoraria from Boehringer Ingelheim and Eli Lilly. KK is under a consultancy agreement with Boehringer Ingelheim. Ethical Approval: Animal experiment was approved by IACUC of Kanazawa Medical University (protocol number 2015-59 & 2017-73).

Hypoxia causes proximal tubular cell damage in diabetes, even though proximal tubular cells have an adaptive system to combat hypoxia involving induction of hypoxia factor-1 (HIF-1) and inhibition of mechanistic target of rapamycin complex 1 (mTORC1). Here, we examined the interference effect of altered glucose and lipid metabolism on the hypoxia responses in proximal tubular cells. In culture, hypoxia alone induced HIF-1 and inhibited mTORC1, preventing death in proximal tubular cells. However, hypoxia with high glucose and palmitate increased mTORC1 activity and promoted apoptosis in proximal tubular cells, which was inhibited by pharmacological and genetic inactivation of mTORC1. Since inhibition of all mTORC1's physiological functions regulated by growth factors including insulin causes various adverse effects, we screened for a microRNA that can inhibit only pro-apoptotic effects of mTORC1 to discover a safe therapeutic target. This screen found microRNA-148b-3p was able to specifically inhibit mTORC1-dependent apoptosis in hypoxic proximal tubular cells exposed to high glucose and palmitate, without affecting insulin-dependent mTORC1 activation. Furthermore, tumor necrosis factor receptor (TNFR) 2 was the target of microRNA-148b-3p and its suppression inhibited apoptosis. Finally, enhanced apoptosis with TNFR2 overexpression was found in hypoxic and mTORC1-activated proximal tubular cells in diabetic rats. Thus, diabetes activated mTORC1 even in hypoxic proximal tubular cells, leading to apoptosis by reducing microRNA-148b-3p expression. Modulating this pathogenic pathway may be a novel therapy for proximal tubular cell damage in diabetes.

Free fatty acid-bound albumin (FFA-albumin)-related oxidative stress is involved in the pathogenesis of proximal tubular cell (PTC) damage and subsequent renal dysfunction in patients with refractory proteinuria. Nicotinamide adenine dinucleotide (NAD) metabolism has recently been focused on as a novel therapeutic target for several modern diseases, including diabetes. This study was designed to identify a novel molecule in NAD metabolism to protect PTCs from lipotoxicity-related oxidative stress. Among 19 candidate enzymes involved in mammalian NAD metabolism, the mRNA expression level of nicotinamide n-methyltransferase (NNMT) was significantly increased in both the kidneys of FFA-albumin-overloaded mice and cultured PTCs stimulated with palmitate-albumin. Knockdown of NNMT exacerbated palmitate-albumin-induced cell death in cultured PTCs, whereas overexpression of NNMT inhibited it. Intracellular concentration of 1-Methylnicotinamide (1-MNA), a metabolite of NNMT, increased and decreased in cultured NNMT-overexpressing and -knockdown PTCs, respectively. Treatment with 1-MNA inhibited palmitate-albumin-induced mitochondrial reactive oxygen species generation and cell death in cultured PTCs. Furthermore, oral administration of 1-MNA ameliorated oxidative stress, apoptosis, necrosis, inflammation, and fibrosis in the kidneys of FFA-albumin-overloaded mice. In conclusion, NNMT-derived 1-MNA can reduce lipotoxicity-mediated oxidative stress and cell damage in PTCs. Supplementation of 1-MNA may have potential as a new therapy in patients with refractory proteinuria.

Autophagy process is essential for maintaining intracellular homeostasis and consists of autophagosome formation and subsequent fusion with lysosome for degradation. Although the role of autophagosome formation in the pathogenesis of diabetes has been recently documented, the role of the latter process remains unclear. This study analyzed high-fat diet (HFD)-fed mice lacking lysosome-associated membrane protein-2 (lamp-2), which is essential for the fusion with lysosome and subsequent degradation of autophagosomes. Although lamp-2 deficient mice showed little alteration in glucose metabolism under normal diet feeding, they showed a resistance against high-fat diet (HFD)-induced obesity, hyperinsulinemic hyperglycemia and tissues lipid accumulation, accompanied with higher energy expenditure. The expression levels of thermogenic genes in brown adipose tissue were significantly increased in HFD-fed lamp-2-deficient mice. Of some serum factors related to energy expenditure, the serum level of fibroblast growth factor (FGF) 21 and its mRNA expression level in the liver were significantly higher in HFD-fed lamp-2-deficient mice in an ER stress-, but not PPARα-, dependent manner. In conclusion, a lamp-2-depenedent fusion and degradation process of autophagosomes is involved in the pathogenesis of obese diabetes, providing a novel insight into autophagy and diabetes.

Ketogenic amino acid (KAA) replacement diet has been shown to cure hepatic steatosis, a serious liver disease associated with diverse metabolic defects. In this study, we investigated the effects of KAA replacement diet on nutrition sensing signaling pathway and analyzed whether induction of hepatic autophagy was involved. Mice are fed with high fat diet (HFD) or KAA replacement in high-fat diet (30% fat in food; HFD)-fed (HFD KAAR ) and sacrificed at 8, 12, 16 weeks after initiation of experimental food. Hepatic autophagy was analyzed in protein expression of several autophagy-associated molecules and in light chain-3 green fluorescent protein (LC-3 GFP) transgenic mice. HFD KAAR showed increased AMP-activated protein kinase (AMPK) phosphorylation and enhanced liver kinase B1 (LKB1) expression compared to control HFD-fed mice. The KAA-HFD-induced activation of AMPK was associated with an increased protein expression of sirtuin 1 (Sirt1), decreased forkhead box protein O3a (Foxo3a) level, and suppression of mammalian target of rapamycin (mTOR) phosphorylation compared with the HFD-fed mice. The intervention study revealed that a KAA-replacement diet also ameliorated all the established metabolic and autophagy defects in the HFD-fed mice, suggesting that a KAA-replacement diet can be used therapeutically in established diseases. These results indicate that KAA replacement in food could be a novel strategy to combat hepatic steatosis and metabolic abnormalities likely involvement of an induction of autophagy.

There is little evidence regarding the target blood pressure level in patients with type 2 diabetes mellitus without overt proteinuria.We followed 608 Japanese patients with type 2 diabetes without apparent cardiovascular disease and overt proteinuria who underwent cerebral magnetic resonance imaging for a mean of 7.5 years. The patients were categorized according to their mean systolic blood pressure during the follow-up period (strict:130 mm Hg, moderate: ≥130 and140 mm Hg, poor: ≥ 140 mm Hg). The risks for the primary composite outcome of death or end-stage renal disease were not different among the three groups. The renal risk of the doubling of serum creatinine for the poor group was significantly higher than those in other groups. In addition, among the patients without silent cerebral infarction (SCI), the renal risk was significantly lower in the strict group than in the moderate group. Further, in both the SCI and non-SCI groups, strict blood pressure control slowed the progression of albuminuria.In nonproteinuric diabetic patients without SCI, strict blood pressure control was associated with improved renal outcomes. There may be different effects of intensive blood pressure control on the renoprotection of diabetic patients according to their complications.

Free fatty acid (FFA)-bound albumin, which is filtrated through the glomeruli and reabsorbed into proximal tubular cells, is one of the crucial mediators of tubular damage in proteinuric kidney disease. In this study, we examined the role of each kind of FFA on renal tubular damage in vitro and tried to identify its molecular mechanism. In cultured proximal tubular cells, a saturated fatty acid, palmiate, increased the expression of monocyte chemoattractant protein-1 (MCP-1), but this effect was abrogated by co-incubation of monounsaturated fatty acid, oleate, or ω-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA). Palmitate led to intracellular accumulation of diacylglycerol (DAG) and subsequent activation of protein kinase C protein family. Among the several PKC inhibitors, rottlerin, a PKCθ inhibitor, prevented palmitate-induced MCP-1 expression via inactivation of NFB pathway. Overexpression of dominant-negative PKCθ also inhibited palmitate-induced activation of MCP-1 promoter. Furthermore, palmitate enhanced PKCθ-dependent mitochondrial apoptosis, which was also prevented by co-incubation with oleate or EPA through restoration of pro-survival Akt pathway. Moreover, oleate and EPA inhibited palmitate-induced PKCθ activation through the conversion of intracellular DAG to triglyceride with the restoration of diacylglycerol acyltransferase 2 expression. These results suggest that oleate and EPA have protective effects against the palmitate-induced renal tubular cell damage by inhibiting PKCθ activation.

To investigate the efficacy and tolerability of vildagliptin, a potent and selective dipeptidyl peptidase-4 inhibitor, as add-on to glimepiride in Japanese patients with Type 2 diabetes mellitus (T2DM) who were inadequately controlled.This 12-week, randomized, double-blind, placebo-controlled study compared vildagliptin 50mg twice-daily (n=102) with placebo (n=100) when added to a stable dose of glimepiride (or=1mg/d).Treatment groups were balanced at baseline (glycosylated hemoglobin [HbA(1c)], 7.9%; fasting plasma glucose, 163.8 mg/dL). During treatment HbA(1c) decreased progressively with vildagliptin, but remained unchanged with placebo. The adjusted mean change (AMDelta) at endpoint was -1.0+/-0.1 and -0.1+/-0.1% in vildagliptin- and placebo-treated patients (between-group Delta=-1.0+/-0.1%, P0.001). A greater proportion of vildagliptin-treated patients had HbA(1c)or=6.5% compared to placebo-treated patients (45% vs. 3%, P0.001). The AMDelta FPG was -20.9+/-2.8 mg/dL with vildagliptin compared to 6.3+/-2.8 mg/dL with placebo (between-group Delta=-27.2+/-3.9 mg/dL, P0.001). Patients in vildagliptin and placebo groups reported similar incidences of adverse events (AEs) (59.8% vs. 57.0%), serious AEs (0% vs. 2.0%), suspected drug-related AEs (21.6% vs. 23.0%), and discontinuation due to AEs (1.0% vs. 3.0%). Hypogylcaemia was reported in two (vildagliptin) and one (placebo) patient.Vildagliptin is effective and well tolerated as an add-on to glimepiride in Japanese patients with T2DM.

The improvement of salt-sensitive hypertension is a therapeutic target for various vascular diseases. Glucagon-like peptide 1 (GLP-1), an incretin peptide, has been reported to have natriuretic effect as well as blood glucose lowering effect, although its exact mechanism and clinical usefulness remain unclear. Here, we examined anti-hypertensive effect of exendin-4, a GLP-1 analog, in salt-sensitive obese db/db mice and angiotensin II (angII)-infused C57BLK6/J mice. The treatment of exendin-4 for 12 weeks inhibited the development of hypertension in db/db mice. In db/db mice, the urinary sodium excretion was delayed and blood pressure was elevated in response to a high-salt load, whereas these were attenuated by exendin-4. In db/db mice, intra-renal angII concentration was increased. Furthermore, exendin-4 prevented angII-induced hypertension in non-diabetic mice and inhibited angII-induced phosphorylation of ERK1/2 in cultured renal cells. Considered together, our results indicate that exendin-4 has anti-hypertensive effects through the attenuation of angII-induced high-salt sensitivity.

There is little clinical evidence when AST-120 should be prescribed for subjects with early stage overt diabetic nephropathy. We therefore designed a prospective, randomized, controlled study for subjects with type 2 diabetes (serum creatinine1.5mg/dl and urinary protein0.5g/day) in November, 2001. The primary end point was defined as exceeding 2mg/dl of serum creatinine, and the secondary end point was defined as introducing a hemodialysis. Twenty-two subjects were selected, and after excluding 6 drop-out subjects, 16 subjects (10 in the control group; 6 in the KRM group) finally entered the study. Mean follow-up periods were 37 and 34 months in the control and KRM groups, respectively. There was no difference in clinical characteristics including renal dysfunction at baseline between the two groups. There was a significant reduction in urinary indoxyl sulfate at month 12 in the KRM group than in the control group. A significant difference was observed in changes in mean levels of serum creatinine versus time between the two groups. The primary end points were counted in 7 (70%) of the control subjects, while only 1 (17%) of the KRM group, and the Kaplan-Meier analysis was statistically significant. Although 4 (40%) of the control group and 1 (17%) of the KRM group were initiated hemodialysis as the secondary end point, the difference did not reach a statistical significance. Thus, we concluded that administration of AST-120 initiated in early stage overt diabetic nephropathy stunts the progression of renal dysfunction.

Oxidative stress-induced apoptosis of renal glomerular cells is an important factor for the development of various kidney diseases. Identification of molecules that modulate this process could lead to the development of new strategies for preventing kidney diseases. In this study, we evaluated whether mammalian silent information regulator 2 (SIRT1), which has been recently identified as a cell survival factor countering various stressors, is a key regulator of oxidative stress-induced mesangial cell apoptosis. Morphological features of apoptotic cell death (nuclear condensation) and the expression of biochemical proapoptotic markers [cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP)] were assessed in murine mesangial cells (MMCs) exposed to hydrogen peroxide (H(2)O(2)). H(2)O(2) increased mesangial cell apoptosis, predominantly through p53 activation by acetylation, which is a posttranscriptional modification for p53 activation. H(2)O(2)-induced apoptosis was significantly attenuated in SIRT1-overexpressing MMCs, but enhanced in SIRT1-knockdown MMCs. Although SIRT1 did not affect H(2)O(2)-mediated phosphorylation of mitogen-activated protein (MAP) kinase, it interacted with p53 and inhibited H(2)O(2)-mediated p53 acetylation but not phosphorylation in MMCs. Our results indicate that SIRT1 can prevent oxidative stress-induced apoptosis through p53 deacetylation in mesangial cells. Upregulation of SIRT1 may provide a new strategy for preventing kidney glomerular diseases.

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-β in rat mesangial cells. Background Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-β (TGF-β)-nduced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-β-induced rat PAI-1 expression in rat mesangial cells. Methods TGF-β1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay. Results Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-β1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-β1, and the disruption of this AP-1-like sequence suppressed basal and TGF-β1-induced promoter activation. TGF-β1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-β1-induced rat PAI-1 promoter activity. Moreover, interferon-γ (IFN-γ) inhibited TGF-β1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway. Conclusion These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-β1-induced PAI-1 expression is unique to rat mesangial cells.

Despite the potentially protective effects of estrogen on bone and cardiovascular tissue as well as against kidney diseases, its effects on diabetic nephropathy are unknown. Here, we examined the therapeutic effectiveness of 17beta-estradiol and raloxifene, a selective estrogen receptor modulator, for preventing functional and histological alterations in the kidneys of db/db mice, a model of type 2 diabetes. In the first experiment, ovariectomized female db/db mice were treated with 17beta-estradiol for 8 weeks. The treatment significantly ameliorated albuminuria, attenuated weight gain, and reduced hyperglycemia in diabetic ovariectomized db/db mice. Histologically, the increases in mesangial area and the accumulation of fibronectin were significantly inhibited by 17beta-estradiol. In the second experiment, mice were administered vehicle or raloxifene hydrochloride (3 mg/kg/day) for 8 weeks. Raloxifene significantly reduced mesangial expansion and fibronectin accumulation in db/db mice, but in contrast to 17beta-estradiol, it failed to affect body weight or hyperglycemia. An in vitro experiment further demonstrated that raloxifene inhibited transforming growth factor beta-1-induced fibronectin transcription and AP-1 activity. Thus, our findings suggest that raloxifene, which lacks the harmful effects of estrogen, is useful for the treatment of diabetic nephropathy.

15-Deoxy-Δ 12,14 -prostaglandin J 2 inhibits IL-1β–induced cyclooxygenase-2 expression in mesangial cells . Background Cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins, is induced in mesangial cells in response to proinflammatory cytokines. Recently, 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), one of the natural ligands of peroxisome proliferator-activated receptor γ (PPARγ), has been reported to have an anti-inflammatory effect. Therefore, we examined the effect of 15d-PGJ 2 on COX-2 expression in cultured rat mesangial cells. Methods Mesangial cells were incubated with 15d-PGJ 2 for 30 minutes and then exposed to interleukin-1β (IL-1β). The expression of COX-2 mRNA and proteins was determined by Northern blot and immunoblot analyses, respectively. Accumulation of prostaglandin E 2 (PGE 2 ) was measured by an enzyme-linked immunosorbent assay (ELISA). Activities of mitogen-activated protein kinases (MAPKs) were evaluated by an immunoblot analysis. DNA binding activities of activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) were examined by an electrophoretic mobility shift assay (EMSA). The activities of PPAR responsive elements (PPRE) and COX-2 promoter were measured by a luciferase reporter assay. Results 15D-PGJ 2 significantly suppressed IL-1β–induced COX-2 expression and PGE 2 production, but thiazolidinediones, synthetic PPARγ ligands, did not affect COX-2 expression. Moreover, the cells transfected with a PPRE luciferase reporter did not respond to 15d-PGJ 2 . IL-1β rapidly activated extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK), which were involved in the up-regulation of COX-2 induction, but 15d-PGJ 2 inhibited the activation of these kinases. 15d-PGJ 2 inhibited the IL-1β–induced increase in binding activities of nuclear proteins to consensus AP-1 site and AP-1–like site of COX-2 promoter but not of NF-κB. IL-1β was unable to activate the COX-2 promoter when the AP-1–like site was mutated. Conclusions These data suggest that 15d-PGJ 2 inhibits IL-1β–induced COX-2 expression, independent of PPARγ activation, by suppression of ERK and JNK pathways and AP-1 activation in mesangial cells. Thus, 15d-PGJ 2 may play an important role in the negative feedback mechanism of COX-2 expression in renal inflammation and may be useful as an anti-inflammatory agent.

Dinucleotide repeat polymorphism of matrix metalloproteinase-9 gene is associated with diabetic nephropathy.BackgroundAlthough genetic susceptibility has been proposed as an important factor for the development and progression of diabetic nephropathy, the definitive gene has not been identified. To identify the genetic marker for diabetic nephropathy, we examined the association between the (A-C)n dinucleotide repeat polymorphism upstream of the matrix metalloproteinase-9 (MMP-9) gene and diabetic nephropathy in a group of Japanese patients with type 2 diabetes.MethodsPatients were divided into three groups based on their urinary albumin excretion rate (AER) and the stage of diabetic retinopathy as follows: uncomplicated group (U), normal albuminuria (AER

Platelet-derived growth factor (PDGF) was found to contribute to the pathophysiological process in the development and progression of glomerulosclerosis characterized by mesangial cell proliferation and accumulation of extracellular matrix. To examine the role of PDGF in the development of diabetic nephropathy, we conducted immunohistochemical analysis for PDGF B-chain (PDGF-B) and PDGF beta-receptor (PDGFR-beta) in the glomeruli of streptozotocin-induced diabetic rats. At 2, 4, and 12 weeks after the onset of diabetes, the expression of PDGF-B in glomeruli of diabetic rats was increased significantly as compared to control or diabetic rats treated with insulin. Similar changes were observed on PDGFR-beta immunostaining. The immunostaining of mirror sections revealed the existence of PDGF-B or PDGFR-beta not only in mesangial cells but also in visceral epithelial cells. Glomerular volume was significantly increased in diabetes. This early glomerular abnormality was prevented by an inhibition of PDGF system with trapidil as well as by the treatment of insulin. Our results suggest that the activation of the PDGF system in glomerular cells might play an important role in the development of early glomerular lesion in diabetes.

We have reported that d-alpha-tocopherol can prevent hyperglycemia-induced activation of DAG and PKC levels in vascular tissues as well as normalizing retinal blood flow and renal hyperfiltration. The mechanism of this effect, however, is not clear. Aside from alpha-tocopherol's principal role as an antioxidant agent, it has also been shown to act as a membrane stabilizer. Another possibility is that the effect of alpha-tocopherol is focused on the activation of DAG kinase, which is a key enzyme in the metabolism of DAG. Therefore, in this study, we examined the effect of alpha-tocopherol on the DAG kinase activity in vascular smooth muscle cell. We have also examined the effect of alpha-tocopherol, its analogues, and probucol on DAG kinase activities and expression. The present study showed that d-alpha-tocopherol's inhibitory effect on DAG-PKC pathway is by increasing DAG kinase activity in rat and human vascular smooth muscle cell (VSMC). Total DAG level was increased by 40 +/- 10% (mean +/- S.E.) (P < 0.05) in human VSMC, after exposure to 22 vs 5 mM glucose. This increase was normalized by d-alpha-tocopherol treatment in a concentration-dependent manner. In parallel, DAG kinase activation by d-alpha-tocopherol was also induced in a time- and dose-dependent manner. DAG kinase activity was increased by 57 +/- 19% (P < 0.05) in human VSMC and 112 +/- 35% (P < 0.05) in rat VSMC after 24 h of incubation with d-alpha-tocopherol (100 microg/ml). Another lipophilic antioxidant, probucol, also increased DAG kinase activity by 124 +/- 34%, but other vitamin E analogues with much less antioxidant potencies were ineffective. Western blots of various DAG kinase isoforms were not changed by d-alpha-tocopherol treatment. These results provide strong and detailed evidence that d-alpha-tocopherol can prevent hyperglycemia induced DAG-PKC activation by enhancing DAG kinase activity, probably through an antioxidant effect.

Introduction Women with preeclampsia (PE) exhibit hypersensitivity on the pressor response against angiotensin II (AngII). Catechol-O-methyltransferase (COMT) metabolizes 2-hydroxyestradiol (2-OHE2) into 2-methoxyestradiol (2-ME) and COMT deficiency has shown to be associated with PE. Objectives We have hypothesized that COMT deficiency is reasonable to explain the molecular mechanisms of the hypersensitivity on the pressor response against AngII. Methods We utilized C57BL/6 male mice for all experiments. Mice were subjected to COMT inhibitor (COMTi: 25 mg/kg/day) or olive oil (Control) for 4 weeks, with or without low-dose AngII infusion (ANGII: 70 ng/kg/min) for last 3 weeks. The AngII-infused mice were treated with 2-ME (10 ng/day), 2-OHE2 (10 ng/day), or vehicle for last 1 week. At the end of the protocol, we obtained following 6 experimental groups; Control, ANGII, COMTi, COMTi+ANGII, COMTi+ANGII+2-ME, and COMTi+ANGII+2-OHE2. Also we performed similar experiments utilizing in vivo administration of siRNA of COMT (20 nmol/week) instead of COMTi. Results Neither ANGII nor COMTi exhibited significant alteration in blood pressure through the experimental protocol compared with Control. When compared to ANGII or COMTi, COMTi+ANGII displayed significantly higher blood pressure; COMTi+ANGII+2-ME decreased in blood pressure significantly when compared to COMTi+ANGII. 2-OHE2 treatments did not suppress the blood pressure of COMTi+ANGII. When COMT protein was analyzed in several organs of siRNA-administered mice (COMTsi); COMT protein level was significantly suppressed in liver when compared to control siRNA treated mice (Csi): either heart, kidney, or aorta was not affected. There was no difference in blood pressure between COMTsi and Csi; AngII infusion increased blood pressure in COMTsi but not in Csi: 2-ME administration significantly suppressed blood pressure of COMTsi+ANGII. The levels of soluble fms-related tyrosine kinase 1and placental growth factor were not significantly associated with blood pressure of any groups of mice; plasminogen activator inhibitor(PAI)-1 level was significantly higher in both AngII-infused COMT deficient mice (COMTi+ANGII and COMTsi+ANGII) compared to control mice of each experiment. Such PAI-1 induction was suppressed by 2-ME. Conclusion Deficiency in COMT and 2-ME are associated with the hypersensitivity on the pressor response against AngII infusion. Similar mechanisms could be relevant to pregnant status and PE.

Background: Diabetic nephropathy, a kidney disease caused by diabetes, is the most devastating and money-consuming complication in patients with diabetes throughout the world. The cardinal lesion of diabetic nephropathy resides in renal glomeruli and is called diabetic glomerulosclerosis. Hyperglycemia is responsible for the development and progression of diabetic nephropathy through metabolic derangements, including increased oxidative stress, renal polyol formation, activation of protein kinase C (PKC)-mitogen-activated protein kinases (MAPKs), and accumulation of advanced glycation end products, as well as such hemodynamic factors as systemic hypertension and increased intraglomerular pressure. Methods: We examined whether inhibition of the PKC-MAPK pathway could inhibit functional and pathological abnormalities in glomeruli from diabetic animal models and cultured mesangial cells exposed to high glucose condition and/or mechanical stretch. Results: Direct inhibition of PKC by PKC β inhibitor prevented albuminuria and mesangial expansion in db/db mice, a model of type 2 diabetes. We also found that inhibition of MAPK by PD98059, an inhibitor of MAPK, or mitogen-activated extracellular regulated protein kinase kinase prevented enhancement of activated protein-1 (AP-1) DNA binding activity and fibronectin expression in cultured mesangial cells exposed to mechanical stretch in an in vivo model of glomerular hypertension. Conclusion: These findings highlight the important role of PKC-MAPK pathway activation in mediating the development and progression of diabetic nephropathy. Am J Kidney Dis 41 (S1):S19-S21. © 2003 by the National Kidney Foundation, Inc.

Alteration of mesangial response to ANP and angiotensin II by glucose. To test the hypothesis that the function of glomerular mesangial cells is impaired in diabetes, we examined the responsiveness of mesangial cells cultured under high concentrations of glucose to atrial natriuretic peptide (ANP 1 ) and angiotensin II (Ang II). The ANP-induced accumulation of cGMP was enhanced in mesangial cells cultured under high glucose conditions, possibly due to the activation of particulate guanylate cyclase. Ang II action in mesangial cells was evaluated by measuring the ability of Ang II to inhibit ANP-induced cGMP accumulation through both activating phosphodiesterase (initial phase) and inhibiting guanylate cyclase (maintenance phase). The inhibition of both ANP-induced cellular cGMP accumulation and particulate guanylate cyclase activity by Ang II was significantly reduced in mesangial cells cultured under high concentrations of glucose. Moreover, in the cells exposed to high concentrations of glucose, both basal and Ang II-stimulated levels of inositol 1,4,5-trisphosphate (IP 3 ) were significantly reduced. These results indicate that, in high glucose conditions, the actions of ANP and Ang II are modulated differently, resulting in the impairment of contractile responsiveness of mesangial cells.

The mechanism of anti-proliferative action of atrial natriuretic peptide (ANP) was examined in cultured mesangial cells. HS-142-1, a selective antagonist for ANP biological receptors, inhibited ANP-induced cellular cGMP accumulation and prevented ANP-induced inhibition of both serum-stimulated incorporation of 3H-thymidine and cell proliferation. Des-[Gln18, Ser19, Gly20, Leu21, Gly22]-ANP4-23-NH2 (C-ANP) failed to inhibit 3H-thymidine incorporation. These results indicate that anti-proliferative action of ANP is mediated by the biological receptors in cultured mesangial cells.

Dual mechanism of angiotensin II inhibits ANP-induced mesangial cGMP accumulation. To evaluate an interaction between vasoconstrictive (Ang II) 1 and vasodilating (Ang) peptides, we examined the effect of Ang II on ANP-induced accumulation of cGMP in cultured glomerular mesangial cells. ANP rapidly increased intracellular cGMP levels, with a peak stimulation at one minute in the absence of IBMX and at ten minutes in the presence of IBMX. The ANP-induced cGMP accumulation was significantly inhibited when the cells were treated with Ang II simultaneously with ANP for one minute in the absence of IBMX. This inhibitory effect of Ang II was completely abolished by IBMX and significantly reduced in calcium-free media or by W7, but not affected by H7. Similar inhibitory effect was observed when cells were treated with A23187 but not with TPA for one minute. In the presence of IBMX, Ang II inhibited ANP-induced cGMP accumulation when cells were treated with Ang II for 15 minutes prior to the stimulation by ANP. This inhibition by Ang II was blocked by H7. ANP-induced increase in particulate guanylate cyclase activity was significantly reduced in the cells treated with Ang II or TPA. This reduction of enzyme activity was also prevented by H7. These results indicate that Ang II inhibits ANP-induced cGMP accumulation in cultured glomerular mesangial cells through at least two mechanisms; one is the activation of calcium-dependent, calmodulin-stimulated cyclic nucleotide phosphodiesterase in the initial phase, and the other is the inhibition of guanylate cyclase resulting from protein kinase C activation in the maintenance phase.

In order to clarify the mechanism of mesangial cell dysfunction in diabetes, we examined the activities of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), important kinases in various cellular functions, and also evaluated the isoenzymes of PKC in mesangial cells cultured under high glucose conditions. Exposure of cells to high concentrations (27.8 mM) of glucose for 5 days resulted in a significant elevation of PKC activities in the membrane fraction. MAPK was also activated in cells cultured under high glucose conditions. Of the PKC isoenzymes, the levels of PKC alpha and zeta were significantly increased in the membrane fraction after 5 days of exposure to high concentrations of glucose. These results indicate that the translocation of PKC alpha and zeta and the activation of MAPK under high glucose conditions might be underlying mechanisms of the functional disturbance of mesangial cells in diabetes.