Role of degradation products of chlorogenic acid in the antioxidant activity of roasted coffee.

Antioxidant activities of brewed coffees prepared from six commercial brands ranged from 63.13 ± 1.01 to 96.80 ± 1.68% at the highest levels tested. Generally, the degree of antioxidant activity of the brewed coffee was inversely proportional to the total chlorogenic acid concentration. A sample obtained from the major chlorogenic acid, 5-caffeoylquinic acid (5-CQA), heated at 250 °C exhibited potent antioxidant activity (79.12 ± 2.49%) at the level of 10 µg/mL, whereas unheated 5-CQA showed only moderate antioxidant activity (44.41 ± 0.27%) at the level of 100 µg/mL. Heat produced relatively high levels of pyrocatechol (2,809.3 µg/g) and 2-methoxy-4-vinylphenol (46.4 µg/g) from 5-CQA, and their antioxidant activity levels were 76.57 ± 3.00 and 98.63 ± 0.01%, respectively. The results of the present study suggest that roasting degrades chlorogenic acids to form potent antioxidants and thus plays an important role in the preparation of high-antioxidant low-acid coffee.

Detailed localization of augmented angiotensinogen mRNA and protein in proximal tubule segments of diabetic kidneys in rats and humans.

In the intrarenal renin-angiotensin system, angiotensinogen levels are well known to be increased in diabetes, and these enhanced intrarenal angiotensinogen levels may initiate the development and accelerate the progression of diabetic nephropathy. However, the specific localization of the augmented angiotensinogen in proximal tubule segments in diabetes is still unknown. We investigated the detailed localization of angiotensinogen in 3 proximal tubule segments in the diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. We also prepared OLETF rats treated with angiotensin II type 1 receptor blocker, olmesartan or with a combination of vasodilator agents. Moreover, biopsied samples of human kidney cortex were used to confirm the results of animal studies. We examined the co-localization of angiotensinogen with segment-specific markers by double staining using fluorescence in situ hybridization and/or immunofluorescence. Angiotensinogen mRNA expression was barely detectable in segment 1. In segment 3, the area of angiotensinogen mRNA expression was augmented in the OLETF rats compared with the LETO rats. Angiotensinogen protein expression areas in segments 1 and 3 were also increased in the OLETF rats compared with the LETO rats. Chronic treatment with olmesartan ameliorated these areas of augmented angiotensinogen expression. Biopsied human kidney samples showed similar results. These data suggest that the augmented angiotensinogen mRNA levels in segment 3 and angiotensinogen protein levels in segments 1 and 3 may contribute to the progression of diabetic nephropathy.

Oxidative stress/angiotensinogen/renin-angiotensin system axis in patients with diabetic nephropathy.

Although recent studies have proven that renin-angiotensin system (RAS) blockades retard the progression of diabetic nephropathy, the detailed mechanisms of their reno-protective effects on the development of diabetic nephropathy remain uncertain. In rodent models, it has been reported that reactive oxygen species (ROS) are important for intrarenal angiotensinogen (AGT) augmentation in the progression of diabetic nephropathy. However, no direct evidence is available to demonstrate that AGT expression is enhanced in the kidneys of patients with diabetes. To examine whether the expression levels of ROS- and RAS-related factors in kidneys are increased with the progression of diabetic nephropathy, biopsied samples from 8 controls and 27 patients with type 2 diabetes were used. After the biopsy, these patients were diagnosed with minor glomerular abnormality or diabetes mellitus by clinical and pathological findings. The intensities of AGT, angiotensin II (Ang II), 4-hydroxy-2-nonenal (4-HNE), and heme oxygenase-1 (HO-1) were examined by fluorescence in situ hybridization and/or immunohistochemistry. Expression levels were greater in patients with diabetes than in control subjects. Moreover, the augmented intrarenal AGT mRNA expression paralleled renal dysfunction in patients with diabetes. These data suggest the importance of the activated oxidative stress/AGT/RAS axis in the pathogenesis of diabetic nephropathy.

Cardinal role of the intrarenal renin-angiotensin system in the pathogenesis of diabetic nephropathy.

Diabetes mellitus is one of the most prevalent diseases and is associated with increased incidence of structural and functional derangements in the kidneys, eventually leading to end-stage renal disease in a significant fraction of afflicted individuals. The renoprotective effects of renin-angiotensin system (RAS) blockade have been established; however, the mechanistic pathways have not been fully elucidated. In this review article, the cardinal role of an activated RAS in the pathogenesis of diabetic nephropathy (DN) is discussed with a focus on 4 themes: (1) introduction to RAS cascade, (2) intrarenal RAS in diabetes, (3) clinical outcomes of RAS blockade in DN, and (4) potential of urinary angiotensinogen as an early biomarker of intrarenal RAS status in DN. This review article provides a mechanistic rational supporting the hypothesis that an activated intrarenal RAS contributes to the pathogenesis of DN and that urinary angiotensinogen levels provide an index of intrarenal RAS activity.

Divergent localization of angiotensinogen mRNA and protein in proximal tubule segments of normal rat kidney.

OBJECTIVES: Angiotensinogen in the kidneys is formed primarily in the proximal tubule cells and is secreted into the tubular fluid. Structurally, proximal tubules can be divided into three segments. The first segment, segment 1 (S1) is mainly confined to the pars convoluta, the second segment, segment 2 (S2) comprises the end of pars convoluta, and the third segment, segment 3 (S3) includes the major part of the pars recta. There are some reports describing angiotensinogen localization in kidneys; however, it remains uncertain which proximal tubule segments express angiotensinogen. To determine the detailed localization of angiotensinogen in the three proximal tubule segments, we established multistaining methods using segment-specific protein markers. METHODS: Using kidneys from Wistar-Kyoto rats, we performed immunohistochemistry and double or triple staining by fluorescence in-situ hybridization and/or immunofluorescence. RESULTS: Our results show that angiotensinogen mRNA and protein are expressed in the cortex and outer medulla of the normal rat kidney. Angiotensinogen mRNA was hardly detected in S1, detected weakly in S2 and strongly in S3 segments. In contrast, angiotensinogen protein was detected in S1 at high levels and less in S2 and S3 segments. CONCLUSION: These data indicate divergence of angiotensinogen mRNA transcription and angiotensinogen protein synthesis and metabolism in different segments of the normal rat proximal tubules.

Isolation and antioxidant activity of zeylaniin A, a new macrocyclic ellagitannin from Syzygium zeylanicum leaves.

Methanol extract obtained from Syzygium zeylanicum leaves exhibited potent antioxidant activity. The water extract obtained from this methanol extract by sequential extraction with hexane, chloroform, ethylacetate, and n-butanol also showed the strongest antioxidant activity among extracts. This water extract was further fractionated by column chromatography with various concentrations of methanol solutions. Among the 6 resultant fractions, the fraction developed with 20% methanol exhibited the most potent antioxidant activity. The one peak among the three major HPLC peaks in this fraction was isolated and purified using a preparative HPLC. The structure of a pure compound was elucidated as a novel macrocyclic ellagitannin using a (1)H/(13)C NMR and a high-resolution electrospray ionization mass spectrometer. This newly isolated compound, which was named zeylaniin A, exhibited potent antioxidant activities in the assays of DPPH, oxygen radical absorbance capacity, and malonadehyde/gas chromatography. S. zeylanicum leaves can be a possible source of natural antioxidants.

Urinary angiotensinogen as a novel early biomarker of intrarenal renin-angiotensin system activation in experimental type 1 diabetes.

Urinary excretion of albumin (UAlb) is used clinically as a marker of diabetic nephropathy (DN). Although DN was thought to be a unidirectional process, recent studies demonstrated that a large proportion of patients diagnosed with DN reverted to normoalbuminuria. Moreover, despite the normoalbuminuria, one-third of them exhibited reduced renal function even during the microalbuminuric stage. This study was performed to investigate whether urinary angiotensinogen (UAGT) level may serve as a useful marker of the early stage of experimental type 1 diabetes (T1DM). T1DM was induced by a single intraperitoneal injection of streptozotocin. Control mice were injected with citrate buffer. Two days after streptozotocin injection, half of the mice received continuous insulin treatment. Our data showed that UAlb excretion was increased 6 days after streptozotocin injection compared to controls, whereas UAGT excretion was increased at an earlier time point. These increases were reversed by insulin treatment. The UAGT to UAlb ratio was increased in diabetic mice compared to control mice. Furthermore, the increased AGT expression in the kidneys was observed in diabetic mice. These data suggest that UAGT might be useful as a novel early biomarker of activation of the renin-angiotensin system in experimental type 1 diabetes.

Flavonoids with potent antioxidant activity found in young green barley leaves.

Saponarin, a flavonoid found in young green barley leaves, possesses potent antioxidant activities, which are determined by its inhibition of malonaldehyde (MA) formation from various lipids oxidized by UV light or Fenton's reagent. Lipids used were squalene, ethyl linoleate, ethyl linolenate, ethyl arachidonate, octadecatetraenoic acid (ODTA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), cod liver oil, lecithin I, lecithin II, and blood plasma. The addition of saponarin inhibited the formation of MA from squalene upon UV irradiation at the level of 2 µmol/mL by almost 100%, whereas BHT inhibited its formation by 75% at the same level. Saponarin showed potent antioxidant activity toward fatty acid ethyl esters at levels >100 µg/mL. Saponarin inhibited MA formation in ODTA by 60%, in EPA by 50%, and in DHA by 43% at the level of 15 µmol/mL. Saponarin exhibited strong antioxidant activities with dose-response levels toward cod liver oil and lipoproteins (lecithins I and II), higher than those of α-tocopherol. A mixture of saponarin/lutonarin (4.5:1, w/w) inhibited MA formation appreciably from all lipids tested with dose response. This mixture exhibited highest effect toward cod liver oil (86%), followed by DHA, lecithin II, blood plasma, EPA, and lecithin I. Supplementation of young green barley leaves containing saponarin should be beneficial to health and may prevent diseases caused by oxidative damage such as various cancers, inflammations, and cardiovascular diseases.

The establishment of a primary culture system of proximal tubule segments using specific markers from normal mouse kidneys.

The proximal tubule contains the highest expression of angiotensinogen mRNA and protein within the kidney and plays a vital role in the renal renin-angiotensin system. To study the regulation of angiotensinogen expression in the kidney in more detail, the proximal tubule needs to be accurately isolated from the rest of the nephron and separated into its three segments. The purpose of this study was to design a novel protocol using specific markers for the separation of proximal tubule cells into the three proximal tubule segments and to determine angiotensinogen expression in each segment. Kidneys were removed from C57BL/6J mice. The proximal tubules were aspirated from region of a Percoll gradient solution of the appropriate density. The proximal tubule was then separated into its three segments using segment-specific membrane proteins, after which each segment was characterized by a different specific marker (sodium-glucose transporter 2 for Segment 1; carbonic anhydrase IV for Segment 2; ecto-adenosine triphosphatase for Segment 3). The isolation of proximal tubules into three segments was successful, and angiotensinogen mRNA in Segment 2 and 3 and angiotensinogen protein in all three segments were confirmed. This protocol will be helpful for future studies of the detailed mechanisms of the intrarenal renin-angiotensin system.

The link between the renin-angiotensin-aldosterone system and renal injury in obesity and the metabolic syndrome.

Obesity is a risk factor for type 2 diabetes mellitus (DM) and is associated with chronic kidney disease. Activation of the renin-angiotensin-aldosterone system (RAAS) is common in obesity. The RAAS is an important mediator of hypertension. Mechanisms involved in activation of the RAAS in obesity include sympathetic stimulation, synthesis of adipokines in the RAAS by visceral fat, and hemodynamic alterations. The RAAS is known for its role in regulating blood pressure and fluid and electrolyte homeostasis. The role of local/tissue RAAS in specific tissues has been a focus of research. Urinary angiotensinogen (UAGT) provides a specific index of the intrarenal RAAS. Investigators have demonstrated that sex steroids can modulate the expression and activity of the different components of the intrarenal RAAS and other tissues. Our data suggest that obese women without DM and hypertension have significantly higher levels of UAGT than their male counterparts. These differences existed without any background difference in the ratio of microalbumin to creatinine in the urine or the estimated glomerular filtration rate, raising a question about the importance of baseline gender differences in the endogenous RAAS in the clinical spectrum of cardiovascular diseases and the potential utility of UAGT as a marker of the intrarenal RAAS. Animal studies have demonstrated that modifying the amount of angiotensin, the biologically active component of the RAAS, directly influences body weight and adiposity. This article reviews the role of the RAAS in renal injury seen in obesity and the metabolic syndrome.

Effects of equol on oxidized low-density lipoprotein-induced apoptosis in endothelial cells.

AIM: Equol is the main active product of daidzein metabolism, produced via specific microflora in the gut. This study aimed to clarify the effects of equol on oxidized low-density lipoprotein (OX-LDL)-stimulated apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were cultured in the presence of OX-LDL, and cell apoptosis was monitored by evaluating of DNA fragmentation and the production of cytoplasmic histone-associated DNA fragments. We simultaneously evaluated the level of cellular superoxide and nitric oxide (NO) and the effects of the anti-oxidant activity of equol on apoptosis. RESULTS: We found that equol inhibited the induction of apoptosis in response to exposure of HUVECs to OX-LDL. Treatment of cells with equol led to a significant reduction in superoxide production by NAD(P)H oxidase and also to a significant increase in NO production. We further observed an effect of equol on the suppression of OX-LDL uptake. CONCLUSIONS: These results suggested that equol might contribute to a reduced level of OX-LDL-stimulated apoptosis linked to the reduced generation of intracellular reactive oxygen species (ROS).

Inhibition of low-density lipoprotein oxidation by Nagano purple grape (Vitis viniferaxVitis labrusca).

The Nagano Purple grape (Vitis (V.) viniferaxV. labrusca) is a hybrid created by a cross between Kyoho (V. viniferaxV. labrusca) and Rosario Bianco (V. vinifera) grapes. The grape, including its skin, can be eaten and contains no seeds because of gibberellin treatment. The skins of various fruits have been shown to contain antioxidant activity. However, it is unclear whether the Nagano Purple grape contains antioxidant activity. We prepared the skins and dried fruits (including the skins) of the Nagano Purple grape, so as to assay for the presence of an antioxidant activity. We examined the concentration of polyphenols in the grape and further assayed whether components in the grape inhibited the oxidation of low-density lipoprotein (LDL). We detected the presence of cyanidin-3-glucoside (Cy-3-glc), five anthocyanidins and resveratrol in the skins. A trace of resveratrol was detected in the pulp. LDL collected from human subjects 1 h following the consumption of the skins or dried fruits revealed significant inhibition of LDL oxidation compared to that observed in fasting venous blood samples. We further observed the antioxidant activity of Cy-3-glc. Our results suggest that the consumption of the Nagano Purple grape can give rise to resistance to LDL oxidation.

Polymorphisms in the 3' UTR in the neurocalcin delta gene affect mRNA stability, and confer susceptibility to diabetic nephropathy.

Using a large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese type 2 diabetic patients, we have identified a gene encoding neurocalcin delta (NCALD) as a candidate for a susceptibility gene to diabetic nephropathy; the landmark SNP was found in the 3' UTR of NCALD (rs1131863: exon 4 +1340 A vs. G, P = 0.00004, odds ratio = 1.59, 95% CI 1.27-1.98). We also discovered two other SNPs in exon 4 of this gene (+999 T/A, +1307 A/G) that showed absolute linkage disequilibrium to the landmark SNP. Subsequent in vitro functional analysis revealed that synthetic mRNA corresponding to the disease susceptible haplotype (exon 4 +1340 G, +1307 G, +999 A) was degraded faster than mRNA corresponding to the major haplotype (exon 4 +1340 A, +1307 A, +999 T), and allelic mRNA expression of the disease susceptibility allele was significantly lower than that of the major allele in normal kidney tissues. In an experiment using a short interfering RNA targeting NCALD, we found that silencing of the NCALD led to a considerable enhancement of cell migration, accompanied by a significant reduction in E-cadherin expression, and by an elevation of alpha smooth muscle actin expression in cultured renal proximal tubular epithelial cells. We also identified the association of the landmark SNP with the progression of diabetic nephropathy in a 8-year prospective study (A vs. G, P = 0.03, odds ratio = 1.91, 95% CI 1.07-3.42). These results suggest that the NCALD gene is a likely candidate for conferring susceptibility to diabetic nephropathy.

Wnt5b partially inhibits canonical Wnt/beta-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes.

To elucidate the functional roles of Wnt5b in adipogenesis, we characterized gene expression profiles in Wnt5b overexpressing 3T3-L1 cells using microarray analysis. Of the approximately 20,000 genes screened, we found that 85 genes were up-regulated and 211 genes were down-regulated in 3T3-L1 cells overexpressing Wnt5b. Among the genes regulated by Wnt5b, the expressions of insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-C (VEGF-C), and WNT1 inducible signaling pathway protein 1 (WISP-1), which were known to be up-regulated by Wnt1/beta-catenin signaling, were decreased in the Wnt5b overexpressing cells. This result was subsequently confirmed by real-time quantitative RT-PCR (IGF-1; 0.74+/-0.08 and 0.56+/-0.08, WISP-1; 0.71+/-0.03 and 0.56+/-0.08, and VEGF-C; 0.67+/-0.01 and 0.80+/-0.07, mean+/-SEM, compared with the control at zero and two days after induction of differentiation, respectively). We also found that Wnt5b overexpression in 3T3-L1 preadipocytes was able to partially prevent the inhibitory effect of Wnt3a on adipogenesis. Furthermore, the overexpression of Wnt5b was able to inhibit Wnt3a-induced activation of the canonical Wnt/beta-catenin pathway as evidenced by the reduced translocation of beta-catenin into the nucleus. These findings indicate that Wnt5b may promote adipogenesis in 3T3-L1 cells, at least in part, by antagonizing the canonical Wnt/beta-catenin pathway.

Contribution of Rho A and Rho kinase to platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells.

In order to identify small G protein (s) which contributes to the proliferation of vascular smooth muscle cells (VSMCs), we examined the effect of an HMG-CoA reductase inhibitor (cerivastatin), a farnesyltransferase inhibitor (FTI-277), a geranyl geranyl transferase inhibitor (GGTI-286) and a Rho kinase inhibitor (Y-27632) on the proliferation of cultured rat VSMCs stimulated with 20ng/ml platelet-derived growth factor (PDGF)-BB. Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced activation of extracellular signal related kinase (ERK1/2). The inhibitory effect of cerivastatin on the PDGF-BB-induced activation of ERK1/2 was fully recovered by the addition of geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP). Cerivastatin and GGTI-286, but not FTI-277, suppressed the PDGF-BB-induced [3H] thymidine incorporation and activation of ornitine decarboxylase (ODC), both of which were fully recovered by the addition of GGPP, but not FPP. These data indicate that the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs depend upon geranylgeranylated small G protein. Immunoblotting analysis revealed the upregulation of Rho A protein in the membrane fractions of VSMCs stimulated by PDGF-BB. Furthermore, Y-27632 suppressed the PDGF-BB-induced activation of ERK1/2 and proliferation of VSMCs. On the basis of these data, we conclude that PDGF-BB stimulates the proliferation of VSMCs via the activation of Rho A. Rho kinase plays an important role in this process as an effector of Rho A.