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ABSTRACT: We investigated the relationship between oxidative stress, hypertension, renal injury and angiotensin-converting enzyme-2 (Ace2) expression in type 1 diabetic Akita mice. Blood glucose, blood pressure and albuminuria were monitored for up to 5 months in adult male Akita and Akita catalase (Cat) transgenic (Tg) mice specifically overexpressing Cat, a key antioxidant enzyme in their renal proximal tubular cells (RPTCs). Same-age non-Akita littermates and Cat-Tg mice served as controls. In separate studies, adult male Akita mice (14 weeks) were treated with Ang 1-7 (500 μg/kg-1.day-1, subcutaneously) ± A-779, an antagonist of Mas receptor (10 mg/kg-1.day-1) and euthanized at the age of 18 weeks. The left kidneys were processed for histology and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess protein and gene expression. Urinary angiotensinogen (Agt), angiotensin II (Ang II) and Ang 1-7 were quantified by specific ELISAs. Overexpression of Cat attenuated renal oxidative stress, prevented hypertension, normalized RPTC Ace2 expression and urinary Ang 1-7 levels (both were low in Akita mice), ameliorated glomerular filtration rate, albuminuria, kidney hypertrophy, tubulointerstitial fibrosis and tubular apoptosis, and suppressed profibrotic and proapoptotic gene expression in RPTCs of Akita Cat-Tg mice compared with Akita mice. Furthermore, daily administration of Ang 1-7 normalized systemic hypertension in Akita mice, which was reversed by A-779. These data demonstrate that Cat overexpression prevents hypertension and progression of nephropathy and highlight the importance of intrarenal oxidative stress and Ace2 expression contributing to hypertension and renal injury in diabetes.
AJP Renal Physiology 04/2013; · 4.42 Impact Factor
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ABSTRACT: We investigated the impact of heterogeneous nuclear ribonucleoprotein F (hnRNP F) overexpression on angiotensinogen (Agt) gene expression, hypertension, and renal proximal tubular cell (RPTC) injury in high-glucose milieu both in vivo and in vitro. Diabetic Akita transgenic (Tg) mice specifically overexpressing hnRNP F in their RPTCs were created, and the effects on systemic hypertension, Agt gene expression, renal hypertrophy, and interstitial fibrosis were studied. We also examined immortalized rat RPTCs stably transfected with control plasmid or plasmid containing hnRNP F cDNA in vitro. The results showed that hnRNP F overexpression attenuated systemic hypertension, suppressed Agt and transforming growth factor-β1 (TGF-β1) gene expression, and reduced urinary Agt and angiotensin II levels, renal hypertrophy, and glomerulotubular fibrosis in Akita hnRNP F-Tg mice. In vitro, hnRNP F overexpression prevented the high-glucose stimulation of Agt and TGF-β1 mRNA expression and cellular hypertrophy in RPTCs. These data suggest that hnRNP F plays a modulatory role and can ameliorate hypertension, renal hypertrophy, and interstitial fibrosis in diabetes. The underlying mechanism is mediated, at least in part, via the suppression of intrarenal Agt gene expression in vivo. hnRNP F may be a potential target in the treatment of hypertension and kidney injury in diabetes.
Diabetes 06/2012; 61(10):2597-608. · 8.29 Impact Factor
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Garnet J Lau,
Nicolas Godin,
Hasna Maachi, Chao-Sheng Lo,
Shyh-Jong Wu,
Jian-Xin Zhu,
Marie-Luise Brezniceanu,
Isabelle Chénier,
Joelle Fragasso-Marquis,
Jean-Baptiste Lattouf,
Jean Ethier,
Janos G Filep,
Julie R Ingelfinger,
Viji Nair,
Matthias Kretzler,
Clemens D Cohen,
Shao-Ling Zhang,
John S D Chan
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ABSTRACT: This study investigated the mechanisms underlying tubular apoptosis in diabetes by identifying proapoptotic genes that are differentially upregulated by reactive oxygen species in renal proximal tubular cells (RPTCs) in models of diabetes. Total RNAs isolated from renal proximal tubules (RPTs) of 20-week-old heterozygous db/m+, db/db, and db/db catalase (CAT)-transgenic (Tg) mice were used for DNA chip microarray analysis. Real-time quantitative PCR assays, immunohistochemistry, and mice rendered diabetic with streptozotocin were used to validate the proapoptotic gene expression in RPTs. Cultured rat RPTCs were used to confirm the apoptotic activity and regulation of proapoptotic gene expression. Additionally, studies in kidney tissues from patients with and without diabetes were used to confirm enhanced proapoptotic gene expression in RPTs. Bcl-2-modifying factor (Bmf) was differentially upregulated (P<0.01) in RPTs of db/db mice compared with db/m+ and db/db CAT-Tg mice and in RPTs of streptozotocin-induced diabetic mice in which insulin reversed this finding. In vitro, Bmf cDNA overexpression in rat RPTCs coimmunoprecipated with Bcl-2, enhanced caspase-3 activity, and promoted apoptosis. High glucose (25 mmol/L) induced Bmf mRNA expression in RPTCs, whereas rotenone, catalase, diphenylene iodinium, and apocynin decreased it. Knockdown of Bmf with small interfering RNA reduced high glucose-induced apoptosis in RPTCs. More important, enhanced Bmf expression was detected in RPTs of kidneys from patients with diabetes. These data demonstrate differential upregulation of Bmf in diabetic RPTs and suggest a potential role for Bmf in regulating RPTC apoptosis and tubular atrophy in diabetes.
Diabetes 12/2011; 61(2):474-84. · 8.29 Impact Factor
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ABSTRACT: We investigated the effects of dual renin-angiotensin system (RAS) blockade on angiotensin-converting enzyme-2 (Ace2) expression, hypertension, and renal proximal tubular cell (RPTC) apoptosis in type 1 diabetic Akita angiotensinogen (Agt)-transgenic (Tg) mice that specifically overexpress Agt in their RPTCs. Adult (11 wk old) male Akita and Akita Agt-Tg mice were treated with two RAS blockers (ANG II receptor type 1 blocker losartan, 30 mg·kg(-1)·day(-1)) and angiotensin-converting enzyme (ACE) inhibitor perindopril (4 mg·kg(-1)·day(-1)) in drinking water. Same-age non-Akita littermates and Agt-Tg mice served as controls. Blood pressure, blood glucose, and albuminuria were monitored weekly. The animals were euthanized at age 16 wk. The left kidneys were processed for immunohistochemistry and apoptosis studies. Renal proximal tubules were isolated from the right kidneys to assess gene and protein expression. Urinary ANG II and ANG 1-7 were quantified by ELISA. RAS blockade normalized renal Ace2 expression and urinary ANG 1-7 levels (both of which were low in untreated Akita and Akita Agt-Tg), prevented hypertension, albuminuria, tubulointerstitial fibrosis and tubular apoptosis, and inhibited profibrotic and proapoptotic gene expression in RPTCs of Akita and Akita Agt-Tg mice compared with non-Akita controls. Our results demonstrate the effectiveness of RAS blockade in preventing intrarenal RAS activation, hypertension, and nephropathy progression in diabetes and support the important role of intrarenal Ace2 expression in modulating hypertension and renal injury in diabetes.
AJP Renal Physiology 12/2011; 302(7):F840-52. · 4.42 Impact Factor
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ABSTRACT: Up-regulation of atrial natriuretic peptide (ANP) mRNA in the kidneys in several disorders has been demonstrated; however, evidence that ANP synthesized by the kidney exerts a local function has never been produced. Therefore, we investigated whether endogenous ANP could modulate high glucose-stimulated TGF-beta1, collagen type I and nuclear factor-kappaB (NF-kappaB) in NRK-52E cells using transfection of ANP and ANP small interfering RNA (siANP). NRK-52E cells were grown with or without transfection with ANP plasmid; cells were also transfected with ANP siRNA or control siRNA. These cells were then stimulated with a high glucose concentration to modulate ANP, TGF-beta1, collagen type I, NF-kappaB and IkappaB-alpha, and the results showed that ANP, TGF-beta1, collagen type I and NF-kappaB significantly increased in untransfected cells, and the transfection of ANP significantly attenuated high glucose-activated TGF-beta1, collagen I and NF-kappaB expression. ANP siRNA knocked-down ANP but significantly increased TGF-beta1 and collagen I under normal glucose conditions; ANP siRNA decreased IkappaB-alpha but strongly enhanced high glucose-activated TGF-beta1, collagen type I and NF-kappaB. In contrast, medium from ANP-transfected cells attenuated high glucose-activated TGF-beta1 and collagen type I expression in NRK-52E cells transfected with siANP. In conclusion, our results demonstrated that siANP increased activation of TGF-beta1, collagen type I and NF-kappaB in NRK-52E cells under high glucose conditions, and medium from ANP-transfected cells attenuated high glucose-activated TGF-beta1 and collagen type I. This is the first study to demonstrate the auto/paracrine action of endogenous ANP in renal tubular cells on the attenuation of hyperglycemia-activated TGF-beta1 and NF-kappaB expression. J. Cell. Physiol. 219: 776-786, 2009. (c) 2009 Wiley-Liss, Inc.
Journal of Cellular Physiology 03/2009; 219(3):776-86. · 3.87 Impact Factor
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ABSTRACT: Apolipoprotein E (apo E) regulates the metabolism of lipoproteins. Three different apo E alleles (ε2, ε3 and ε4) produce the apo E isoproteins, apo E2, apo E3 and apo E4. Previous studies had revealed that lipid abnormalities might contribute to the development and progression of kidney diseases. Thus, the apo E gene polymorphism may be associated with end-stage renal disease. to investigate the distribution of apo E genotype in patients under haemodialysis in Taiwan, we examined 205 dialysis patients and 103 age-matched normal controls. the apo E gene polymorphism was analysed by the use of polymerase chain reaction. Our study revealed that the frequency of apo E2 allele was significantly higher in dialysis patients (8.0%) than in normal controls (1.9%; P<0.01). the E3 and E4 allele frequencies were not significantly different between the groups. Our findings indicate that apo E polymorphism was apparently associated with the development of end-stage renal disease in Taiwan.
Nephrology 06/2008; 7(6):277 - 280. · 1.31 Impact Factor
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ABSTRACT: Atrial natriuretic peptide, besides its role in the regulation of volume homeostasis, has been noted to exert cytoprotective effects in several cell types from hypoxia. The present study was performed to explore the effect of ANP on high glucose-activated transforming growth factor-beta1 (TGF-beta1), Smad and collagen synthesis in renal proximal epithelial cells. Cultured NRK-52E cells were divided into five groups: (1) normal glucose (5.5 mM), (2) high glucose (35 mM), (3) D-mannitol (29.5 mM), (4) high glucose plus ANP (10(-6)-10(-9) M), and (5) high glucose plus ANP (10(-6) M) and guanylate cyclase inhibitor LY83583 (10(-7) M) groups. Messenger RNA levels of TGF-beta1, Smad2, and collagens were measured by RT-PCR. ELISA, immunocytochemistry and Western blotting were used to detect protein levels of TGF-beta1, Smad2, phospho-Smad 2/3 and collagen type 1. We found high glucose to significantly increase mRNA levels of TGF-beta1, Smad 2, collagen types I and III and protein levels of TGF-beta1, phospho-Smad 2/3 and collagen type 1, but mannitol did not affect their expression. The addition of ANP significantly attenuated high glucose-enhanced mRNA and protein levels of TGF-beta1, Smad and collagens. LY83583 blocked the influence of ANP on high glucose-activated TGF-beta1, Smad and collagen synthesis. This is the first study to demonstrate that activation of TGF-beta1, Smad and collagen synthesis stimulated by high glucose can also be inhibited by exogenous ANP in renal tubular epithelial cells.
Journal of Cellular Biochemistry 05/2008; 103(6):1999-2009. · 2.87 Impact Factor
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Ming-Chia Hsieh,
Kai-Jen Tien,
Shun-Jen Chang, Chao-Sheng Lo,
Shih-Chieh Hsin,
Jeng-Yueh Hsiao,
Shih-Chieh Hsu,
Hui-Ting Liang,
Hung-Chun Chen,
Shyi-Jang Shin,
Shiu-Ru Lin
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ABSTRACT: Diabetes is known to be a high-risk factor for coronary artery disease (CAD), and lipid abnormalities have been found to possibly contribute to CAD in diabetic patients. Cholesteryl ester transfer protein (CETP) gene TaqIB polymorphism is associated with lipid profile variability, and this polymorphism may be a risk factor for CAD in diabetic patients. To clarify the relationship between CETP TaqIB gene polymorphism and CAD, we enrolled in our study 365 Taiwanese with type 2 diabetes mellitus (101 with CAD and 264 without CAD). The genotype of the subjects for TaqIB polymorphism of CETP in intron 1 was analyzed by using polymerase chain reaction-restriction fragment length polymorphism. The CETP B1B1 genotype (18.8% vs 8.5%, P = .002) and B1 allele (42.1% vs 29.7%, P = .002) were significantly more frequent in diabetic patients with CAD than those without CAD. Logistic regression analysis revealed that the CETP B1B1 genotype was associated with CAD in patients with type 2 diabetes mellitus (odds ratio, 3.18; 95% confidence interval, 1.54-6.54; P = .002). Interestingly, in diabetic patients, serum creatinine levels higher than 1.4 mg/dL were also associated with increased risk for CAD (odds ratio, 2.09; 95% confidence interval, 1.12-3.91; P = .02). Our results suggest that the CETP B1B1 genotype is a strong genetic predictor of CAD in Taiwanese with type 2 diabetes mellitus.
Metabolism 07/2007; 56(6):745-50. · 2.66 Impact Factor
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ABSTRACT: Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2. Cardiotoxin III-induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub-G1 formation) with an IC(50) of 4 mg/mL at 48 h. 3. Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase-9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl-2, protein in the cells. Moreover, the CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. 4. However, CTX III did not generate the formation of reactive oxygen species and anti-oxidants, including N-acetylcysteine, and catalase could not block CTX III-induced apoptosis in the Colo205 cells. 5. Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial- and caspase-dependent mechanism and alteration of Bax/Bcl-2 ratio in human colorectal Colo205 cancer cells.
Clinical and Experimental Pharmacology and Physiology 04/2006; 33(3):177-82. · 1.85 Impact Factor
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ABSTRACT: Increased oxidative stress has been observed to contribute the development of insulin resistance. Oxidative stress is known to increase the conversion of deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG). Human 8-oxoguanine glycosylase (hOGG1) is the key component responsible for the removal of 8-OHdG from oxidatively damaged DNA. The repair activity of the hOGG1 Ser326Cys gene variant has been demonstrated to be lower than that of the hOGG1 Ser/Ser genotype. Therefore, the possible association of the hOGG1 Ser326Cys gene variant with insulin sensitivity was investigated in 279 normal glucose-tolerant subjects without history of cancer. Allele frequency was 21.5% for the Ser/Ser genotype (n = 60), 45.9% for the Ser/Cys genotype (n = 128), and 32.6% for the Cys/Cys genotype (n = 91). Subjects carrying the Cys/Cys genotype had significantly lower insulin sensitivity levels, assessed by homeostasis model assessment-insulin resistance (HOMA-IR), compared with the Ser/Ser and Ser/Cys genotypes (P < 0.001 and P < 0.001, respectively). In a multiple linear regression analysis, the Cys/Cys genotype was a significant determinant of HOMA-IR, independent of age, sex, body mass index, fasting plasma cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, or hypertension. The present study indicates that the hOGG1 gene Cys/Cys variant is associated with a significant decrease in insulin sensitivity in subjects with normal glucose tolerance.
Journal of Human Genetics 02/2006; 51(2):124-8. · 2.57 Impact Factor
