Application of GC-MS coupled with chemometrics for scanning serum metabolic biomarkers from renal fibrosis rat.

Renal interstitial fibrosis closely relates to chronic kidney disease and is regarded as the final common pathway in most cases of end-stage renal disease. Metabolomic biomarkers can facilitate early diagnosis and allow better understanding of the pathogenesis underlying renal fibrosis. Gas chromatography-mass spectrometry (GC/MS) is one of the most promising techniques for identification of metabolites. However, the existence of the background, baseline offset, and overlapping peaks makes accurate identification of the metabolites unachievable. In this study, GC/MS coupled with chemometric methods was successfully developed to accurately identify and seek metabolic biomarkers for rats with renal fibrosis. By using these methods, seventy-six metabolites from rat serum were accurately identified and five metabolites (i.e., urea, ornithine, citric acid, galactose, and cholesterol) may be useful as potential biomarkers for renal fibrosis.

Picrorhiza scrophulariiflora improves accelerated atherosclerosis through inhibition of redox-sensitive inflammation.

BACKGROUND: Accumulation of advanced glycation end products (AGEs) or advanced oxidation protein products (AOPPs) has been identified as a risk factor for accelerated atherosclerosis seen in diabetes and chronic kidney disease. However, little is known about the intervention for atherogenesis associated with these oxidized proteins. The rhizome of Picrorhiza scrophulariiflora (PS) has long been used to treat inflammatory diseases as a traditional medication. The study was performed to test the hypothesis that ethanol extraction of PS (EPS) may improve AGEs- or AOPPs-induced accelerated atherosclerosis in vivo. METHODS AND RESULTS: Hypercholesterolemic or normal rabbits were randomly assigned to 8 groups treated with intravenous injection of AGEs- or AOPPs-modified rabbit serum albumin (AGEs-RSA or AOPPs-RSA), unmodified RSA or vehicle in the presence or absence of EPS (10 mg/kg/2 days) gavage for 10 weeks. Compared with hypercholesterolemic rabbits without EPS treatment, EPS administration significantly decreased the aortic plaque volume and oxidized low density lipoprotein (Ox-LDL) deposition in hypercholesterolemic animals. This was accompanied by significant histological improvement including decrease of intimal and smooth muscle cell proliferation and macrophage influx in affected areas. EPS administration almost completely abolished the accelerated atherosclerosis induced by chronic treatment of AGEs- or AOPPs-RSA in both hypercholesterolemic and normal rabbits. EPS administration significantly restored the AGEs- or AOPPs-induced redox imbalance and inflammation, evidenced by decrease of plasma Ox-LDL, thiobarbituric acid reactive substances and TNF-alpha, and increase of glutathione peroxidase activity. CONCLUSION: These data suggested that EPS may improve atherosclerosis, particularly that induced by AGEs or AOPPs, through inhibition of redox-sensitive inflammation.

Renoprotection of Optimal Antiproteinuric Doses (ROAD) Study: a randomized controlled study of benazepril and losartan in chronic renal insufficiency.

The Renoprotection of Optimal Antiproteinuric Doses (ROAD) study was performed to determine whether titration of benazepril or losartan to optimal antiproteinuric doses would safely improve the renal outcome in chronic renal insufficiency. A total of 360 patients who did not have diabetes and had proteinuria and chronic renal insufficiency were randomly assigned to four groups. Patients received open-label treatment with a conventional dosage of benazepril (10 mg/d), individual uptitration of benazepril (median 20 mg/d; range 10 to 40), a conventional dosage of losartan (50 mg/d), or individual uptitration of losartan (median 100 mg/d; range 50 to 200). Uptitration was performed to optimal antiproteinuric and tolerated dosages, and then these dosages were maintained. Median follow-up was 3.7 yr. The primary end point was time to the composite of a doubling of the serum creatinine, ESRD, or death. Secondary end points included changes in the level of proteinuria and the rate of progression of renal disease. Compared with the conventional dosages, optimal antiproteinuric dosages of benazepril and losartan that were achieved through uptitration were associated with a 51 and 53% reduction in the risk for the primary end point (P = 0.028 and 0.022, respectively). Optimal antiproteinuric dosages of benazepril and losartan, at comparable BP control, achieved a greater reduction in both proteinuria and the rate of decline in renal function compared with their conventional dosages. There was no significant difference for the overall incidence of major adverse events between groups that were given conventional and optimal dosages in both arms. It is concluded that uptitration of benazepril or losartan against proteinuria conferred further benefit on renal outcome in patients who did not have diabetes and had proteinuria and renal insufficiency.

Level of asymmetric dimethylarginine and carotid atherosclerosis in patients with chronic kidney disease.

OBJECTIVE: To determine the association between asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, with atherosclerosis in patients with chronic kidney disease (CKD). METHODS: One hundred thirty-eight CKD patients were enrolled in this study. Serum levels of L-arginine, ADMA, and SDMA were measured by high-performance liquid chromatography (HPLC). Common carotid arteries intimae-medial thickness (CCA-IMT), cross-sectional calculated intimae-medial thickness (cIM area) and atherosclerotic plaque were detected by noninvasive high-resolution B-mode ultrasonography. RESULTS: Serum levels of ADMA and SDMA were significantly increased in CKD patients (n=138) compared with age matched healthy subjects (n=42, P<0.01). ADMA and SDMA levels increased with the progression of renal dysfunction and were negatively related to creatinine clearance (Ccr) in pre-dialysis patients (r=-0.315, P<0.05; r=-0.426, P<0.01). Serum levels of ADMA and SDMA in dialysis patients (n=74) were significantly higher than those in pre-dialysis patients (P<0.05). Patients with carotid artery plaques showed significantly higher levels of ADMA compared with those without plaques. Serum levels of ADMA closely correlated with the mean IMT (r=0.471, P<0.01) and cIM area value (r=0.430, P<0.01). These correlations remained significant even after adjusting GFR, age, gender ,and other risk factors for atherosclerosis in the multiple regression analysis. CONCLUSION: Serum levels of ADMA increased with the progression of CKD and may play a role in the pathogenesis of accelerated atherosclerosis in CKD patients.

Advanced oxidation protein products accelerate atherosclerosis through promoting oxidative stress and inflammation.

OBJECTIVE: Increased level of plasma advanced oxidation protein products (AOPPs) has been found in patients with uremia and nonuremic subjects with coronary artery disease. This study was conducted to test the hypothesis that AOPPs play a causal role in atherosclerosis. METHODS AND RESULTS: Hypercholesterolemic (0.5% wt/wt diet) or normal rabbits received either repeated intravenous injections of AOPPs modified rabbit serum albumin (AOPPs-RSA) or unmodified RSA for 8 weeks. Compared with RSA- or vehicle-treated hypercholesterolemic rabbits, AOPPs-RSA-treated animals displayed increased atherosclerotic plaque area oxidized low-density lipoprotein (oxLDL) deposition, macrophage infiltration, and smooth muscle cell proliferation. Aortic sections from AOPPs-RSA-treated normal rabbits showed significant focal intima proliferation and mild Oil-Red-O staining lipid deposition in the affected areas, a phenomenon not observed in the RSA- or vehicle-treated controls. Plasma AOPPs levels in AOPPs-treated groups significantly increased in both hypercholesterolemic and normal rabbits compared with their relevant controls. Close correlations were found between plasma levels of AOPPs and the parameters of oxidative stress, eg, oxLDL and thiobarbituric acid reactive substances levels, or glutathione peroxidase activity. A highly significant correlation was also observed between plasma AOPPs and tumor necrosis factor (TNF)-alpha levels. CONCLUSIONS: This study provides in vivo evidence for a causal relationship between chronic AOPPs accumulation and atherosclerosis.

Efficacy and safety of benazepril for advanced chronic renal insufficiency.

BACKGROUND: Angiotensin-converting-enzyme inhibitors provide renal protection in patients with mild-to-moderate renal insufficiency (serum creatinine level, 3.0 mg per deciliter or less). We assessed the efficacy and safety of benazepril in patients without diabetes who had advanced renal insufficiency. METHODS: We enrolled 422 patients in a randomized, double-blind study. After an eight-week run-in period, 104 patients with serum creatinine levels of 1.5 to 3.0 mg per deciliter (group 1) received 20 mg of benazepril per day, whereas 224 patients with serum creatinine levels of 3.1 to 5.0 mg per deciliter (group 2) were randomly assigned to receive 20 mg of benazepril per day (112 patients) or placebo (112 patients) and then followed for a mean of 3.4 years. All patients received conventional antihypertensive therapy. The primary outcome was the composite of a doubling of the serum creatinine level, end-stage renal disease, or death. Secondary end points included changes in the level of proteinuria and the rate of progression of renal disease. RESULTS: Of 102 patients in group 1, 22 (22 percent) reached the primary end point, as compared with 44 of 108 patients given benazepril in group 2 (41 percent) and 65 of 107 patients given placebo in group 2 (60 percent). As compared with placebo, benazepril was associated with a 43 percent reduction in the risk of the primary end point in group 2 (P=0.005). This benefit did not appear to be attributable to blood-pressure control. Benazepril therapy was associated with a 52 percent reduction in the level of proteinuria and a reduction of 23 percent in the rate of decline in renal function. The overall incidence of major adverse events in the benazepril and placebo subgroups of group 2 was similar. CONCLUSIONS: Benazepril conferred substantial renal benefits in patients without diabetes who had advanced renal insufficiency. (ClinicalTrials.gov number, NCT00270426.)

[Changes of serum asymmetric dimethylarginine in essential hypertension before and after the treatment].

OBJECTIVE: To investigate the relationship between serum asymmetric dimethylarginine (ADMA) and blood pressure as well as target organ damage in essential hypertension, and to evaluate the effects of enalapril and losartan on them. METHODS: Forty-two newly diagnoszed patients with essential hypertension were randomly divided into enalapril-treated group and losartan-treated group. Serum ADMA, L-arginine, and nitric oxide( NO) were measured before and after the treatment for 8 weeks. Twenty-three healthy volunteers were included as control subjects. RESULTS: The concentrations of ADMA and L-arginine in serum were significantly higher but the level of nitric oxide was relatively lower ( P < 0.01 ) in hypertensive patients than those in control subjects. Serum ADMA was higher in different levels of blood pressure and target organ damage. Treatment with enalapril or losartan for 8 weeks not only reduced blood pressure but also decreased serum ADMA (P <0.01 ). Furthermore, treatment with these drugs also increased the level of serum nitric oxide but didn't change the level of L-arginine. CONCLUSION: The concentrations of serum ADMA and L-arginine were increased, but the level of nitric oxide was decreased in the early stage of essential hypertension. Both enalapril and losartan could ameliorate the endothelial function by reducing the concentration of ADMA.

[Advanced glycation end products accelerate atherosclerosis via enhancement of oxidative stress].

OBJECTIVE: To investigate the effect of advanced glycation end products (AGE) on atheromatous plaque formation and its possible mechanisms. METHODS: Fifty rabbits were randomly divided into five groups of 10 rabbits: group A, fed with hypercholesterolemic diet and injected intravenously with AGE modified rabbit serum albumin (AGE-RSA); group B, fed with hypercholesterolemic diet and injected with unmodified RSA; group C, fed with hypercholesterolemic diet; group D, fed with normal diet alone: and group E, fed with normal diet and injected with AGE-RSA. Ten weeks after the rabbits were killed. Their aortas were taken out and stained with Sudan red IV. The extent of atheromatous plaques in the aortas en face was evaluated by computer-assisted morphometry and by histologic examination. Photoshop system was used to measure the percentage of atheromatous plaques in the area of tunica intima. The depositions of AGE, malondialdehyde modified low-density lipoprotein (MDA-LDL), oxidized low-density lipoprotein (ox-LDL) and expression of receptor of AGE (RAGE) in aortic tissue were detected by using immunohistological staining. The circulating AGE, blood lipids, serum selenium glutathione peroxydase (SeGSHPx) activity, malonyldialdehyde (MDA), and oxidized LDL (ox-LDL) were detected before the experiment and after the rabbits were killed. RESULTS: (1) The relative plaque area was significantly increased in group A (50% +/- 8%) compared with in group B (21% +/- 7%) and group C (29% +/- 6%). No plaque could be found in animals fed with normal diet (group D) even in those receiving repeated injections of AGE-RSA (group E). Depositions of ox-LDL, MDA-LDL and AGE in atherosclerotic lesions increased and RAGE expression were upregulated in the rabbits fed with hypercholesterolemic diet and injected with AGE-RSA (group A) compared with the other four groups. (2) All hypercholesterolemic rabbits showed comparable serum levels of triglyceride and cholesterol. However, the serum levels of AGE, ox-LDL and MDA were significantly higher and the serum level of SeGSHPx was relatively lower in group A compared with those in the other four groups. (3) The serum level of AGE was directly correlated with the serum ox-LDL (r = 0.459, P < 0.01) or serum MDA concentration (r = 0.423, P < 0.05), and inversely correlated with the serum level of SeGSHPx (r = - 0.448, P < 0.01). A close correlation was found between the serum level of AGE and endothelium RAGE expression (r = 0.384, P < 0.05) and deposition area of AGE (r = 0.468, P < 0.05) in aorta. CONCLUSION: AGE accelerates the atheromatous plaque formation through induction of oxidative stress and upregulation of RAGE.

[Proteins modified with glycation or oxidation products accelerate atherosclerosis in experimental hypercholesterolemic rabbits].

OBJECTIVE: To investigate whether advanced glycation end products (AGE) or advanced oxidative protein products (AOPP) contributes to atherogenesis in experimental hypercholesterolemic rabbits. METHODS: Hypercholesterolemic (0.5% wt/wt diet) rabbits received repeated intravenous injections of either AGE modified rabbit serum albumin (AGE-RSA) or AOPP modified RSA (AOPP-RSA) for 10 weeks. Three control groups were set as follows: a group fed with high cholesterol diet alone, a group fed with high cholesterol diet and injected with normal RSA, and a group fed with normal diet. The animals were sacrificed and aortas were dissected and stained with Sudan red IV. Atheromatous plaques in the aortas en face were evaluated by computer-assisted morphometry and by histologic examination. Hematological parameters (triglyceride, cholesterol, AGE and AOPP) were also tested. RESULTS: (1) The relative plaque area in rabbits receiving repeated injections of AGE or AOPP was 50.1%+/-7.4% and 62.4%+/-8.8% respectively, both were significantly larger than either that of hypercholesterolemic rabbits (29.8%+/-6.3%, P<0.05) or that of hypercholesterolemic rabbits injected with unmodified RSA (20.9%+/-6.4%, P<0.05). In aortic arch, thoracic aorta and abdominal aorta, atherosclerotic lesions in AGE group and AOPP group were more severe than in any of the control groups. (2)The average thickness of the plaques on thoracic aortas in AGE group [(138.1+/-13.0) microm] and in AOPP group [(147.7+/-13.1) microm ] were significantly thicker than either of that in the group treated with hypercholesterolemic diet alone [(85.7+/-15.0) microm] or that in the group treated with non modified RSA [(95.5+/-15.7) microm]. (3) Glyceride and cholesterol levels in all animals on hypercholesterolemic diet were similar. AGE and AOPP serum levels in groups injected with AGE or AOPP were significantly higher than that in any of the control groups. AGE serum concentrations were positively correlated with the atheromatous plaque area(r=0.408, P=0.005), so were the AOPP serum concentrations (r=0.595, P=0.000). CONCLUSION: AGE or AOPP modified proteins accelerate the formation of atherosclerosis and may contribute to the accelerated atherosclerosis in end stage renal diseases.

[Serum nitric oxide and D-dimer before and after administering antihypertensive drugs in essential hypertension].

OBJECTIVE: To investigate the relationship among serum nitric oxide, D-dimer, blood pressure, dangerous states and target organ damage in essential hypertension and to evaluate the effects of enalapril and terazosin. METHODS: Fifty-five patients were randomized into the enalapril group and terazosin group, each group receiving enalapril and terazosin respectively for 8 weeks. Serum nitric oxide and D-dimer were measured before and after the treatment. RESULTS: Serum nitric oxide was lower (P < 0.001) but D-dimer was higher (P < 0.01) in the hypertensions than those in the controls. Serum nitric oxide and D-dimer were related to different levels of blood pressure, dangerous states and target organ damage; Both enalapril and terazosin could reduce the level of blood pressure; Both could increase the serum nitric oxide (P < 0.001) and decrease the D-dimer(P < 0.01). CONCLUSION: Endothelial dysfunction and microthrombin can be found in the early stage of essential hypertension. Both enalapril and terazosin could improve the endothelial function and prothrombotic state by promoting the synthesis of nitric oxide.

[Relationship between serum asymmetric dimethylarginine and blood pressure in patients with chronic renal failure].

OBJECTIVE: To investigate the relationship between serum assymmetric dimethylarginine and blood pressure in patients with chronic renal failure. METHODS: We examined the levels of asymmetric dimethylarginine (ADMA) in the serum and urine of 29 hemodialysis patients using the HPLC method, and assessed the relationship between serum ADMA and blood pressure. RESULTS: The level of serum ADMA was higher, but the level of urinary ADMA was lower in the patients with chronic renal failure than those in the controls. The serum ADMA was positively related to blood pressure. CONCLUSION: The accumulation of ADMA may be related to hypertension in patients with chronic renal failure.