Effects of ferulic acid on diabetic nephropathy in a rat model of type 2 diabetes

Diabetic nephropathy is the most serious complication in diabetes mellitus. It is known that oxidative stress and inflammation play a central role in the development of diabetic nephropathy. In this study, we investigated that ferulic acid (FA) known as anti-oxidative agent could effect on diabetic nephropathy by anti-oxidative and anti-inflammatory mechanism. We examined the effects of FA in obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats. We treated FA to experimental rats from 26 to 45 weeks of age. We evaluated ACR, MDA and MCP-1 in 24 h urine and examined renal histopathology and morphologic change in extracted kidneys from rats. Also, we evaluated the ROS production and MCP-1 levels in cultured podocyte after FA treatment. In the FA-treated OLETF rats, blood glucose was significantly decreased and serum adiponectin levels were increased. Urinary ACR was significantly reduced in FA-treated OLETF rats compared with diabetic OLETF rats. In renal histopathology, FA-treated OLETF rats showed decreased glomerular basement membrane thickness, glomerular volume, and mesangial matrix expansion. FA treatment decreased oxidative stress markers and MCP-1 levels in 24 h urine of rats and supernatants of cultured podocyte. In conclusion, it was suggested that FA have protective and therapeutic effects on diabetic nephropathy by reducing oxidative stress and inflammation.     

Interference with TGFβ1-Mediated Inflammation and Fibrosis Underlies Reno-Protective Effects of the CB1 Receptor Neutral Antagonists AM6545 and AM4113 in a Rat Model of Metabolic Syndrome

The role of cannabinoid receptors in nephropathy is gaining much attention. This study investigated the effects of two neutral CB1 receptor antagonists, AM6545 and AM4113, on nephropathy associated with metabolic syndrome (MetS). MetS was induced in rats by high-fructose high-salt feeding for 12 weeks. AM6545, the peripheral silent antagonist and AM4113, the central neutral antagonist were administered in the last 4 weeks. At the end of study, blood and urine samples were collected for biochemical analyses while the kidneys were excised for histopathological investigation and transforming growth factor beta 1 (TGFβ1) measurement. MetS was associated with deteriorated kidney function as indicated by the elevated proteinuria and albumin excretion rate. Both compounds equally inhibited the elevated proteinuria and albumin excretion rate while having no effect on creatinine clearance and blood pressure. In addition, AM6545 and AM4113 alleviated the observed swelling and inflammatory cells infiltration in different kidney structures. Moreover, AM6545 and AM4113 alleviated the observed histopathological alterations in kidney structure of MetS rats. MetS was associated with a ten-fold increase in urine uric acid while both compounds blocked this increase. Furthermore, AM6545 and AM4113 completely prevented the collagen deposition and the elevated expression of the TGFβ1 seen in MetS animals. In conclusion, AM6545 and AM4113, possess reno-protective effects by interfering with TGFβ1-mediated renal inflammation and fibrosis, via peripheral action.     

Angiotensin-I converting enzyme gene polymorphism in Turkish type 2 diabetic patients

Non-insulin dependent diabetes mellitus is often associated with some complications such as nephropathy, retinopathy and neuropathy. Genes of the renin angiotensin system are potential candidate genes for diabetic complications. We investigated the relationship between angiotensin converting enzyme (ACE) gene polymorphism in type 2 diabetic patients with and without diabetic nephropathy. Seventy five patients (25 type 2 diabetic patients with nephropathy, 50 type 2 diabetic patients without nephropathy) and 37 healthy controls were studied. Gene polymorphism of ACE was determined by PCR (polymerase chain reaction) amplification using allele-spesific primers. The frequencies of ACE DD, ID and II genoypes among the patients with type 2 diabetic patients were found 48%, 42%, 10% whereas in control subjects, 27%, 60%, 13% respectively. Type 2 diabetic patients carrying DD genotype without nephropathy increased 1.77 fold than control subjects (P < 0.05). There is no significant correlation between diabetic nephropathy and ACE gene polymorphism. But we found that ACE DD genotype increased significantly in type 2 diabetic patients compared to control subjects (P <.05).     

Altered expression of insulins I and II and their mRNAs in the islets of Langerhans in dexamethasone-induced diabetic rats

Rats have two isomeric insulins (insulins I and II). There have been no reports on the expression of the isomeric insulins in glucocorticoid-induced diabetic rats. To clarify the relation of the expression of each insulin and its mRNAs in dexamethasone-induced diabetic rats, the amounts of the isomeric insulins and mRNAs in the islets of Langerhans were determined in vivo and in vitro. A sensitive and selective HPLC-fluorescence determination method for the isomeric insulins and a newly developed real-time quantitative RT-PCR method for their mRNAs were used. There was a greater reduction of insulin II than insulin I in the islets of Langerhans in dexamethasone-induced diabetic rats. This alteration may be caused by a disproportionate expression of the respective mRNA for the isomeric insulins that resulted from the direct effect of dexamethasone. In addition, continuous hyperglycemia may also suppress the expression of the insulin II mRNA. The overall effects of dexamethasone and hyperglycemia may cause a greater reduction of insulin II than insulin I in the dexamethasone-induced diabetic rat. Conversely, an elevated ratio of insulin I to II in the islets could suggest a diabetic condition.     

Myricitrin,a Glycosyloxyflavone in Myrica esculenta Bark Ameliorates Diabetic Nephropathy via Improving Glycemic Status,Reducing Oxidative Stress,and Suppressing Inflammation

The present study evaluated the therapeutic potential of myricitrin (Myr), a glycosyloxyflavone extracted from Myrica esculenta bark, against diabetic nephropathy. Myr exhibited a significant hypoglycemic effect in high fat-fed and a single low-dose streptozotocin-induced type 2 diabetic (T2D) rats. Myr was found to improve glucose uptake by the skeletal muscle via activating IRS-1/PI3K/Akt/GLUT4 signaling in vitro and in vivo. Myr significantly attenuated high glucose (HG)-induced toxicity in NRK cells and in the kidneys of T2D rats. In this study, hyperglycemia caused nephrotoxicity via endorsing oxidative stress and inflammation resulting in the induction of apoptosis, fibrosis, and inflammatory damages. Myr was found to attenuate oxidative stress via scavenging/neutralizing oxidative radicals and improving endogenous redox defense through Nrf-2 activation in both in vitro and in vivo systems. Myr was also found to attenuate diabetes-triggered renal inflammation via suppressing NF-κB activation. Myr inhibited hyperglycemia-induced apoptosis and fibrosis in renal cells evidenced by the changes in the expressions of the apoptotic and fibrotic factors. The molecular docking predicted the interactions between Myr and different signal proteins. An in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) study predicted the drug-likeness character of Myr. Results suggested the possibility of Myr to be a potential therapeutic agent for diabetic nephropathy in the future.     

Thymoquinone Lowers Blood Glucose and Reduces Oxidative Stress in a Rat Model of Diabetes

The aim of the present study was to assess the short-term effects of Thymoquinone (TQ) on oxidative stress, glycaemic control, and renal functions in diabetic rats. DM was induced in groups II and III with a single dose of streptozotocin (STZ), while group I received no medication (control). The rats in groups I and II were then given distilled water, while the rats in group III were given TQ at a dose of 50 mg/kg body weight/day for 4 weeks. Lipid peroxidase, nitric oxide (NO), total antioxidant capacity (TAC), glycated haemoglobin (HbA1c), lipid profiles, and renal function were assessed. Moreover, the renal tissues were used for histopathological examination. STZ increased the levels of HbA1c, lipid peroxidase, NO, and creatinine in STZ-induced diabetic rats in comparison to control rats. TAC was lower in STZ-induced diabetic rats than in the control group. Furthermore, rats treated with TQ exhibited significantly lower levels of HbA1c, lipid peroxidase, and NO than did untreated diabetic rats. TAC was higher in diabetic rats treated with TQ than in untreated diabetic rats. The histopathological results showed that treatment with TQ greatly attenuated the effect of STZ-induced diabetic nephropathy. TQ effectively adjusts glycaemic control and reduces oxidative stress in STZ-induced diabetic rats without significant damaging effects on the renal function.     

An antioxidant modulates expression of receptor activator of NF-kappaB in asthma

Oxidative stress plays critical roles in airway inflammation that is usually accompanied by increased vascular permeability and plasma exudation. VEGF increases vascular permeability and leads to airway inflammation. In addition, VEGF has been shown to enhance receptor activator of NF-kappaB (RANK) expression in endothelial cells. An aim of the study was to determine the potential role of antioxidant in the regulation of RANK expression in murine model of asthma. We have used a C57BL/6 mouse model of allergic asthma to evaluate the effect of L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, which acts as an antioxidant, and VEGF receptor inhibitor on RANK mRNA expression. The mice develop the following pathophysiological features of asthma in the lungs: increased expression of RANK mRNA, increased number of inflammatory cells of the airways, increased vascular permeability, and increased levels of VEGF. Administration of OTC and VEGF receptor inhibitor markedly reduced plasma extravasation and VEGF levels in allergen-induced asthmatic lungs. We also showed that the increased RANK mRNA expression at 72 h after ovalbumin inhalation were reduced by the administration of OTC or VEGF receptor inhibitor. The results indicate that OTC and VEGF receptor inhibitor which inhibit up-regulation of VEGF expression modulate RANK expression that may be in association with the regulation of vascular permeability, and suggest that VEGF may regulate the RANK expression. These findings provide a crucial molecular mechanism for the potential use of antioxidants to prevent and/or treat asthma and other airway inflammatory disorders.     

Orphan nuclear receptor small heterodimer partner inhibits angiotensin II-stimulated PAI-1 expression in vascular smooth muscle cells

Angiotensin II is a major effector molecule in the development of cardiovascular disease. In vascular smooth muscle cells (VSMCs), angiotensin II promotes cellular proliferation and extracellular matrix accumulation through the upregulation of plasminogen activator inhibitor-1 (PAI-1) expression. Previously, we demonstrated that small heterodimer partner (SHP) represses PAI-1 expression in the liver through the inhibition of TGF-β signaling pathways. Here, we investigated whether SHP inhibited angiotensin II-stimulated PAI-1 expression in VSMCs. Adenovirus-mediated overexpression of SHP (Ad-SHP) in VSMCs inhibited angiotensin II- and TGF-β-stimulated PAI-1 expression. Ad-SHP also inhibited angiotensin II-, TGF-β- and Smad3-stimulated PAI-1 promoter activity, and angiotensin II-stimulated AP-1 activity. The level of PAI-1 expression was significantly higher in VSMCs of SHP^-/- mice than wild type mice. Moreover, loss of SHP increased PAI-1 mRNA expression after angiotensin II treatment. These results suggest that SHP inhibits PAI-1 expression in VSMCs through the suppression of TGF-β/Smad3 and AP-1 activity. Thus, agents that target the induction of SHP expression in VSMCs might help prevent the development and progression of atherosclerosis.     

The reno-protective effect of a phosphoinositide 3-kinase inhibitor wortmannin on streptozotocin-induced proteinuric renal disease rats

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 ± 0.56 mg/day, whereas wortmannin group was 1.77 ± 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 ± 10.82 mg/g, whereas wortmannin group was 20.27 ± 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 ± 0.02), podocin (87.81 ± 0.03) and Rac1/Cdc42 (86.12 ± 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 ± 0.03), podocin (53.40 ± 0.06) and Rac1/Cdc42 (54.05 ± 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.     

Proteome analysis of altered proteins in streptozotocin‐induced diabetic rat kidney using the fluorogenic derivatization–liquid chromatography–tandem mass spectrometry method

To find new molecular markers for early diagnosis of diabetic nephropathy, we applied fluorogenic derivatization–liquid chromatography–tandem mass spectrometry to identify the differentially expressed proteins in the kidney of control and streptozotocin‐induced diabetic rats. The Sprague–Dawley rats were injected with the sodium citrate buffer or streptozotocin and then killed after 1, 4, 12 and 24 weeks. The results showed that seven proteins were significantly changed after 1 week of injection. Only one protein had significantly changed after 4 weeks of injection. However, after 12 weeks of injection, the number of altered proteins rose to 10. After 24 weeks of injection, 18 proteins had altered significantly. Five common proteins were significantly altered at week 12 and 24 after injection, respectively. Importantly, these proteins appeared prior to microalbuminuria and may serve as new biomarkers that are able to improve early detection of and new drug development for diabetic‐related nephropathy. Copyright © 2012 John Wiley & Sons, Ltd.     

High resolution UPLC-MS/MS method for simultaneous separation and determination of six flavonoids from semen cuscutae extract in rat plasma: application to comparative pharmacokinetic studies in normal and kidney-deficient rats

Abstract

Semen Cuscutae, which mainly consisted of flavonoids, is a traditional Chinese herbal medicine and used for nourishing the liver and kidneys. The aim of this study was to develop a sensitive and selective UPLC-MS/MS method for simultaneous separation and determination of six main active renoprotective components of Hyperoside, Astragalin, Isoquercitrin, Quercitrin, Quercetin, and Kaempferol from Semen Cuscutae in rat plasma, and to reveal the pharmacokinetic differences between normal and kidney deficient rats. The validated method has been successfully applied to comparing pharmacokinetic profiles of the six analytes in rat plasma. The results indicated that there was significant difference in pharmacokinetic parameters of the six analytes between two groups, while absorptions in kidney deficient group were significantly lower than those in normal group. This study would be helpful for evaluating the Semen Cuscutae as renoprotective drug candidates for pre-clinical and clinical research.     

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS).     

糖肾方对自发性Ⅱ型糖尿病鼠磷脂代谢的影响

利用正相液相色谱飞行时间质谱建立了大鼠血浆磷脂的轮廓谱。通过对磷脂轮廓谱的PLS-DA判别分析及14种化合物的定量,考察了中药糖肾方对自发性II型糖尿病鼠磷脂代谢的影响,并对比了糖肾方与西药蒙诺在糖尿病发展过程中的不同作用。结果表明：与正常对照组相比,糖尿病鼠的血浆磷脂代谢发生异常。糖肾方能够调节糖尿病鼠的磷脂代谢紊乱,蒙诺则加剧了病鼠的磷脂代谢异常。此外,8种磷脂化合物可能成为糖尿病发展过程中的潜在生物标志物。     

Potential Effect of Polyphenolic-Rich Fractions of Corn Silk on Protecting Endothelial Cells against High Glucose Damage Using In Vitro and In Vivo Approaches

Endothelial cell dysfunction is considered to be one of the major causes of vascular complications in diabetes. Polyphenols are known as potent antioxidants that can contribute to the prevention of diabetes. Corn silk has been reported to contain polyphenols and has been used in folk medicine in China for the treatment of diabetes. The present study aims to investigate the potential protective role of the phenolic-rich fraction of corn silk (PRF) against injuries to vascular endothelial cells under high glucose conditions in vitro and in vivo. The protective effect of PRF from high glucose toxicity was investigated using human umbilical vein endothelial cells (HUVECs). The protective effect of PRF was subsequently evaluated by using in vivo methods in streptozotocin (STZ)-induced diabetic rats. Results showed that the PRF significantly reduced the cytotoxicity of glucose by restoring cell viability in a dose-dependent manner. PRF was also able to prevent the histological changes in the aorta of STZ-induced diabetic rats. Results suggested that PRF might have a beneficial effect on diabetic patients and may help to prevent the development and progression of diabetic complications such as diabetic nephropathy and atherosclerosis.     

微量元素锌、血清胱抑素 C在糖尿病肾病早期诊断的意义

目的：探讨早期糖尿病肾病患者（ DN）的微量元素锌、血清胱抑素C（ CysC）、血清肌酐（ SCr）和血清尿素（ BUN）水平的变化程度及意义。方法将糖尿病肾病患者分为3组：正常蛋白尿组,微量蛋白尿组,临床蛋白尿组。所有患者和对照组均空腹抽取静脉血检测微量元素锌、血清胱抑素C、血清尿素、血清肌酐。结果正常蛋白尿组、微量蛋白尿组、临床蛋白尿组微量元素锌均明显低于正常对照组,差异有统计学意义（ P＜0.05）。正常蛋白尿组、微量蛋白尿组、临床蛋白尿组血清胱抑素C水平均明显高于正常对照组,差异有统计学意义（ P＜0.05）。微量蛋白尿组、临床蛋白尿组血清肌酐检测值和正常对照组有明显差异,差异有统计学意义（ P＜0.05）。结论检测微量元素锌、血清胱抑素C对糖尿病肾病的早期肾损害有较高的临床价值,可作为早期糖尿病肾病诊断的参考指标。     

Emodin ameliorates high-glucose induced mesangial p38 over-activation and hypocontractility via activation of PPAR��

Early stage diabetic nephropathy is characterized by elevated glomerular filtration. Recent studies have identified high-glucose induced p38 MAPK (p38) over-activation in mesangial cells. Mesangial hypocontractility is the major underlying mechanism, however, no ameliorating agents are currently available. We investigated the protective effects of emodin on high-glucose induced mesangial cell hypocontractility. Mesangial cells were cultured under normal (5.6 mM) and high glucose (30 mM) conditions. Emodin was administrated at doses of 50 mg/l and 100 mg/l. Angiotension II stimulated cell surface reductions were measured to evaluate cell contractility. p38 activity was detected using Western blotting. To further explore the possible mechanism of emodin, expression of the peroxisome proliferator-activated receptor γ (PPARγ) was measured and its specific inhibitor, gw9662, was administrated. Our results showed: (1) high-glucose resulted in a 280% increase in p38 activity associated with significant impairment of mesangial contractility; (2) emodin treatment dose-dependently inhibited high-glucose induced p38 over-activation (a 40% decrease for 50 mg/l emodin and a 73% decrease for 100 mg/l emodin), and mesangial hypocontractility was ameriolated by emodin; (3) both the PPARγ mRNA and protein levels were elevated after emodin treatment; (4) inhibition of PPARγ using gw9662 effectively blocked the ameliorating effects of emodin on high-glucose induced p38 over-activation and mesangial hypocontractility. Emodin effectively ameliorated p38 over-activation and hypocontractility in high-glucose induced mesangial cells, possibly via activation of PPARγ.     

Momordica charantia Extract Protects against Diabetes-Related Spermatogenic Dysfunction in Male Rats: Molecular and Biochemical Study

More than 90% of diabetic patients suffer from sexual dysfunction, including diminished sperm count, sperm motility, and sperm viability, and low testosterone levels. The effects of Momordica charantia (MC) were studied by estimating the blood levels of insulin, glucose, glycosylated hemoglobin (HbA1c), testosterone (TST), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in diabetic rats treated with 250 and 500 mg/kg b.w. of the total extract. Testicular antioxidants, epididymal sperm characteristics, testicular histopathology, and lesion scoring were also investigated. Testicular mRNA expression of apoptosis-related markers such as antiapoptotic B-cell lymphoma-2 (Bcl-2) and proapoptotic Bcl-2-associated X protein (Bax) were evaluated by real-time PCR. Furthermore, caspase-3 protein expression was evaluated by immunohistochemistry. MC administration resulted in a significant reduction in blood glucose and HbA1c and marked elevation of serum levels of insulin, TST, and gonadotropins in diabetic rats. It induced a significant recovery of testicular antioxidant enzymes, improved histopathological changes of the testes, and decreased spermatogenic and Sertoli cell apoptosis. MC effectively inhibited testicular apoptosis, as evidenced by upregulation of Bcl-2 and downregulation of Bax and caspase-3. Moreover, reduction in apoptotic potential in MC-treated groups was confirmed by reduction in the Bax/Bcl-2 mRNA expression ratio.     

An integrated proteomic approach to studying glomerular nephrotoxicity

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.     

Heat shock protein 90 regulates IκB kinase complex and NF-κB activation in angiotensin II-induced cardiac cell hypertrophy

Heat shock protein 90 (HSP90), one of the most abundant proteins in the cardiac cells is essential for cell survival. Previous studies have shown that angiotensin II induces cardiac cell hypertrophy. However, the role of HSP90 in the angiotensin II-induced cardiac hypertrophy is unclear. In this study, we showed that HSP90 regulated angiotensin II-induced hypertrophy via maintenance of the IκB kinase (IKK) complex stability in cardiac cells. An HSP90 inhibitor, geldanamycin (GA), significantly suppressed angiotensin II-induced [3H]leucine incorporation and atrial natriuretic factor expression in cardiac cells. GA also inhibited the NF-κB activation induced by angiotensin II. Importantly, treatment with GA caused a degradation of IKKα/β; on the other hand, a proteasome-specific inhibitor restored the level of IKKα/β. We also found that GA prevented HSP90-IKKs complex induced by angiotensin II in cardiac cells. The small interfering RNA (siRNA)-mediated knockdown of HSP90 expression significantly inhibited angiotensin II-induced cell hypertrophy and NF-κB activation. These results suggest that angiotensin II-induced cardiac hypertrophy requires HSP90 that regulates the stability and complex of IKK.