Relationship between a Urine Protein-to-creatinine Ratio of 150 mg/gram Creatinine and Dipstick Grade in the Health Checkup: Substantial Number of False-negative Results for Chronic Kidney Disease.

OBJECTIVE: Proteinuria is a marker for cardiovascular diseases and all-cause mortality. In the Specific Health Checkups in Japan, when subjects show trace proteinuria (grade±) on dipstick assay, further examination is recommended to them. Although 150 mg/gCr is a threshold for diagnosing chronic kidney disease (CKD), little data on the relationship between dipstick grade± and the protein-creatinine ratio have been reported. METHODS: A cross-sectional study using urine specimens obtained in a single institute, JCHO Saitama Northern Medical Center, was performed from October 2014 to March 2016. The level of proteinuria was measured in fresh morning urine samples from 819 volunteer participants of the Specific Health Checkups by two methods: Eiken Uropaper III to detect and qualitatively grade proteinuria, and total protein concentration by the pyrogallol red method. RESULTS: Sensitivity, specificity, and the positive likelihood ratio to detect proteinuria of 30 mg/dL by 1+ were 90.3%, 97.8%, and 41.9, whereas 150 mg/gCr by ± were 45.3%, 81.4%, and 2.4, respectively. Therefore, screening for 150 mg/gCr by dipstick grade± had a false-negative rate of 54.7% and false-negative rate was significantly higher in women (8.0%) than in men (1.7%) (p < 0.0001). CONCLUSIONS: Although the dipstick assay is useful to detect clinically significant proteinuria, substantial numbers of false-negative results occur in checkups for identifying subjects with a risk of CKD.

Angiotensin II Type 1 Receptor Blockers Reduce Urinary Angiotensinogen Excretion and the Levels of Urinary Markers of Oxidative Stress and Inflammation in Patients with Type 2 Diabetic Nephropathy.

OBJECTIVE: To demonstrate that the administration of an angiotensin (Ang) II type 1 receptor (AT1R) blocker (ARB) inhibits the vicious cycle of high glucose (HG)-reactive oxygen species (ROS)-angiotensinogen (AGT)-Ang II-AT1R-ROS by suppressing ROSs and inflammation, thus ameliorating diabetic nephropathy (DN). RESEARCH DESIGN AND METHODS: Thirteen hypertensive DN patients were administered ARBs, and the following parameters were evaluated before and 16 weeks after the treatment: urinary AGT (UAGT), albumin (albumin-creatinine ratio: ACR), 8-hydroxyde-oxyguanosine (8-OHdG), 8-epi-prostaglandin F2alpha(8-epi-PGF2alpha), monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-10. RESULTS: ARB treatment reduced the blood pressure and urinary levels of AGT, ACR, 8-OHdG, 8-epi-PGF2alpha, MCP-1, and IL-6 but increased the urinary levels of IL-10. The reduction rate of UAGT correlated with the reduction rate of blood pressure; the reduction rates of the urinary ACR, 8-OHdG, 8-epi-PGF2alpha, MCP-1, and IL-6 levels; and the increase rate of the urinary IL-10 levels. Moreover, subjects who had high UAGT values at baseline exhibited higher reduction rates of urinary albumin excretion. CONCLUSIONS: ARB-induced blockade of the abovementioned vicious cycle contributes to the renoprotective effects of ARBs in DN. The urinary levels of AGT could represent a predictive factor for reduced ACR in patients receiving ARB treatment.

Characteristics of the antibodies of two patients who developed daytime hyperglycemia and morning hypoglycemia because of insulin antibodies.

We encountered two patients who developed daytime hyperglycemia and early morning hypoglycemia because of insulin antibody (IA) that the affinity was extremely lower and the capacity extremely higher than those of IA in the insulin autoimmune syndrome, after their insulin treatment were changed from human insulin to analog insulin.

Reduced albuminuria with sarpogrelate is accompanied by a decrease in monocyte chemoattractant protein-1 levels in type 2 diabetes.

BACKGROUND AND OBJECTIVES: Sarpogrelate has been shown to reduce albuminuria in diabetic nephropathy. For examination of whether this is based on the same mechanisms as angiotensin II receptor blockers or thiazolidinedione, effects of sarpogrelate on atherosclerotic inflammatory molecules and their relations to albuminuria in patients who had diabetes and had already been treated with angiotensin II receptor blockers and with or without thiazolidinedione were examined. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Forty patients who had diabetes with nephropathy and arteriosclerosis obliterans and had already been treated with angiotensin II receptor blocker (n = 40) were randomly assigned to sarpogrelate (300 mg/d; n = 20) or aspirin group (100 mg/d; n = 20). Plasma monocyte chemoattractant protein-1 and urinary albumin-to-creatinine ratio and monocyte chemoattractant protein-1 were measured at baseline and 16 wk after administration. RESULTS: Only the sarpogrelate group showed increases in plasma adiponectin and decreases in both plasma and urinary monocyte chemoattractant protein-1 and albumin-to-creatinine ratio levels. Moreover, percentage change of monocyte chemoattractant protein-1 level correlated positively to that of albumin-to-creatinine ratio. Even when the sarpogrelate group was further divided into two groups with (n = 9) or without thiazolidinedione (n = 11), changes in monocyte chemoattractant protein-1 or albumin-to-creatinine ratio did not differ. CONCLUSIONS: Sarpogrelate can reduce albuminuria and plasma and urinary monocyte chemoattractant protein-1 levels while increasing plasma adiponectin in diabetic nephropathy. These effects seem to be mediated via mechanisms that are different from those of angiotensin II receptor blocker or thiazolidinedione.

20-Hydroxyeicosatetraenoic acid stimulates nuclear factor-kappaB activation and the production of inflammatory cytokines in human endothelial cells.

Endothelial dysfunction is associated with endothelial cell activation, i.e., up-regulation of surface cell adhesion molecules and the release of proinflammatory cytokines. 20-Hydroxyeicosatetraenoic acid (HETE), a major vasoactive eicosanoid in the microcirculation, has been implicated in the regulation of endothelial cell function through its angiogenic and pro-oxidative properties. We examined the effects of 20-HETE on endothelial cell activation in vitro. Cells transduced with adenovirus containing either CYP4A1 or CYP4A2 produced higher levels of 20-HETE, and they demonstrated increased expression levels of the adhesion molecule intercellular adhesion molecule (ICAM) (4-7-fold) and the oxidative stress marker 3-nitrotyrosine (2-3-fold) compared with cells transduced with control adenovirus. Treatment of cells with 20-HETE markedly increased levels of prostaglandin (PG) E(2) and 8-epi-isoprostane PGF(2alpha), commonly used markers of activation and oxidative stress, and most prominently, interleukin-8, a potent neutrophil chemotactic factor whose overproduction by the endothelium is a key feature of vascular injury. 20-HETE at nanomolar concentrations increased inhibitor of nuclear factor-kappaB phosphorylation by 2 to 5-fold within 5 min, which was followed with increased nuclear translocation of nuclear factor-kappaB (NF-kappaB). Likewise, 20-HETE activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway by stimulating phosphorylation of ERK1/2. Inhibition of NF-kappaB activation and inhibition of ERK1/2 phosphorylation inhibited 20-HETE-induced ICAM expression. It seems that 20-HETE triggers NF-kappaB and MAPK/ERK activation and that both signaling pathways participate in the cellular mechanisms by which 20-HETE activates vascular endothelial cells.

Vascular cytochrome P450 4A expression and 20-hydroxyeicosatetraenoic acid synthesis contribute to endothelial dysfunction in androgen-induced hypertension.

Epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. It has been shown that 5alpha-dihydrotestosterone (DHT) administration (56 mg/kg of body weight per day IP for 14 days) increases blood pressure, cytochrome P450 4A expression, and 20-hydroxyeicosatetraenoic acid synthesis in rats. We examined whether increased vascular 20-hydroxyeicosatetraenoic acid synthesis underlies endothelial dysfunction and hypertension in DHT-treated male Sprague-Dawley rats by using HET0016, a selective cytochrome P450 4A inhibitor. Coadministration of HET0016 (10 mg/kg per day IP for 14 days) to DHT-treated rats markedly reduced DHT-induced interlobar arterial production of 20-hydroxyeicosatetraenoic acid (14.3+/-1.5 versus 1.5+/-0.5 ng/mg of protein per hour; P<0.05), superoxide anion (246+/-47 versus 31+/-8 cpm/microg of protein), and the levels of gp91-phox, p47-phox, and 3-nitrosylated proteins. Moreover, the maximal relaxing response to acetylcholine in phenylephrine-preconstricted renal interlobar arteries from DHT-treated rats (42.8+/-4.8%) significantly (P<0.05) increased in the presence of HET0016 (81.5+/-10.8%). Importantly, the administration of HET0016 negated DHT-induced hypertension; systolic blood pressure was reduced from 146+/-2 mm Hg in DHT-treated rats to 130+/-1 mm Hg (P<0.05). The results strongly implicate vascular cytochrome P450 4A-derived 20-hydroxyeicosatetraenoic acid in the development of androgen-induced endothelial dysfunction and hypertension.

Expression of cytochrome P-450 4 enzymes in the kidney and liver: regulation by PPAR and species-difference between rat and human.

Members of the cytochrome P-450 4 (CYP4) family catalyze the omega-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARalpha was expressed in the liver, proximal tubule and TAL. PPARgamma was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARalpha agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARgamma agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.

Endothelial dysfunction and hypertension in rats transduced with CYP4A2 adenovirus.

Vascular cytochrome P450 (CYP) 4A enzymes catalyze the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid which participates in the regulation of vascular tone by sensitizing the smooth muscle cells to constrictor and myogenic stimuli. This study was undertaken to investigate the consequences of CYP4A overexpression on blood pressure and endothelial function in rats treated with adenoviral vectors carrying the CYP4A2 construct. Intravenous injection of Adv-CYP4A2 increased blood pressure (from 114+/-1 to 133+/-1 mm Hg, P<0.001), and interlobar renal arteries from these rats displayed decreased relaxing responsiveness to acetylcholine, which was offset by treatment with an inhibitor of CYP4A. Relative to data in control rats, arteries from Adv-CYP4A2-transduced rats produced more 20-HETE (129+/-10 versus 97+/-7 pmol/mg protein, P<0.01) and less nitric oxide (NO; 4.2+/-1.6 versus 8.4+/-1 nmol nitrite+nitrate/mg; P<0.05). They also displayed higher levels of oxidative stress as measured by increased generation of superoxide anion and increased expression of nitrotyrosine and gp91phox. Collectively, these findings demonstrate that augmentation in vascular 20-HETE promotes the development of hypertension and causes endothelial dysfunction, a condition characterized by decreased NO synthesis and/or bioavailability, imbalance in the relative contribution of endothelium-derived relaxing and contracting factors, and enhanced endothelial activation.

[Role of androgens in the renal production of 20-hydroxyeicosatetraenoic acid in spontaneously hypertensive rats].

The role of androgens in the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was determined in the kidney of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The renal production of 20-HETE and blood pressure were higher in males than in females at 9 weeks of age. The renal production of 20-HETE was significantly greater in male SHR than in male WKY, whereas it was significantly lower in female SHR than in female WKY. The differences in the renal production of 20-HETE were consistent with the cytochrome P-450 4A protein levels. Plasma free-testosterone levels in male SHR were twice as high as those in male WKY. Castration and treatment with the androgen receptor antagonist, flutamide, reduced blood pressure, the renal production of 20-HETE, and P-450 4A protein levels in both strains. The renal production of 20-HETE was significantly lower in castrated SHR than in castrated WKY. These results indicate that the renal production of 20-HETE and the expression of P-450 4A have gender and strain-differences, and high levels of plasma androgens induce the expression of P-450 4A and the production of 20-HETE in the kidney of male SHR. The androgen-induced production of 20-HETE may be associated with hypertension in male SHR.

Precision and detection limit of quality test for amorphous drug in powder X-ray diffractometry.

This report puts forward a method of powder X-ray diffractometry to estimate the precision and detection limit of the crystalline component in an amorphous drug. Cefditoren pivoxil (CP) was employed as a model drug. The major error source of the measurement at low crystal contents is shown to be the random noise in a diffraction pattern (halo pattern) of the amorphous material. For the analysis of the noise, the obstructive halo pattern should be eliminated from the observed pattern. The subtraction of the observed halo pattern from another one derived from the same material, extracts the random noise alone, although the noise is amplified by square root 2 times. The noise in the powder X-ray diffractometry was identified as the white noise. On the basis of the stochastic properties of the extracted noise and signal parameters (peak area) of CP, the relative standard deviations (R.S.D.) of the area measurements of the crystalline diffraction peaks were estimated over a wide range of crystal contents without repeated experiments. The detection limit was determined such that the crystal content at detection limit produced 30% R.S.D. of the measurements. The R.S.D. and detection limit obtained from FUMI theory were in good agreement with the results from the repeated measurements.

Regulation of cytochrome P-450 4A activity by peroxisome proliferator-activated receptors in the rat kidney.

The localization of cytochrome P-450 4A, peroxisome proliferator-activated receptor (PPAR) alpha, and PPARgamma proteins, and the inducibility of P-450 4A expression and activity by PPAR agonists were determined in the rat kidney. The expressions of these proteins in isolated nephron segments were evaluated by immunoblot analysis, and the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was measured as P-450 4A activity. P-450 4A proteins were expressed predominantly in the proximal tubule (PT), with lower expression in the preglomerular arteriole (Art), glomerulus (Glm), and medullary thick ascending limb (mTAL), but their expression was not detected in the inner medullary collecting duct (IMCD). PPARalpha protein was expressed in the PT and mTAL, and PPARgamma protein was expressed in the IMCD and mTAL. Treatment with clofibrate, the PPARalpha agonist, increased P-450 4A protein levels and the production of 20-HETE in microsomes prepared from the renal cortex, whereas treatment with pioglitazone, the PPARgamma agonist, affected neither of them. These results indicate that PPARalpha and PPARgamma proteins are localized in different nephron segments and the inducibility of P-450 4A expression and activity by the PPAR agonists correlates with the nephron-specific localization of the respective PPAR isoforms.