Recombinant human erythropoietin ameliorated endothelial dysfunction and macrophage infiltration by increasing nitric oxide in hypertensive 5/6 nephrectomized rat aorta.

Recombinant human erythropoietin (rHuEPO), used clinically for renal anemia, reportedly exhibits pleiotropic properties in various tissues. To test whether it ameliorates vascular injury, rHuEPO (75U/kg) was administered subcutaneously every 3days for 10days to 5/6 nephrectomized hypertensive rats (5/6Nx) treated with 1% NaCl. rHuEPO had no effect on increased systolic blood pressure or decreased hematocrit values, but normalized levels of proteinuria and creatinine clearance. Vasodilation in response to acetylcholine in the aortic ring was impaired in the 5/6Nx, and improved by treatment with rHuEPO. Immunohistochemical analysis revealed that the infiltration of adventitial areas by macrophages and expression of osteopontin were enhanced in the 5/6Nx aorta and the overexpression was suppressed by rHuEPO. rHuEPO also attenuated medial hyperplasia. Akt signaling was activated by the increased expression of phosphorylated Akt and GSK-3ß in aorta from rHuEPO-treated 5/6Nx. rHuEPO restored plasma NOx (NO(2)(-)+NO(3)(-)) levels and endothelial nitric oxide synthase (eNOS) content in the 5/6Nx aorta. Treatment with an eNOS substrate, l-arginine, which caused a similar increase in plasma NOx levels as the rHuEPO treatment, resulted in a normalization of endothelial dysfunction and vascular inflammation. These results suggest that a low dose of rHuEPO exerted vasoprotective effects in rats with hypertensive renal failure.

L/N-type calcium channel blocker cilnidipine ameliorates proteinuria and inhibits the renal renin-angiotensin-aldosterone system in deoxycorticosterone acetate-salt hypertensive rats.

Cilnidipine, an N/L-type calcium channel blocker, has been reported to inhibit sympathetic nerve activity and has a greater renoprotective effect than L-type calcium channel blockers. To investigate the hypothesis that cilnidipine might ameliorate advanced hypertensive nephropathy and inhibit the renal renin-angiotensin-aldosterone system, cilnidipine (1 mg per kg per day) or amlodipine (1 mg per kg per day) was administered to uninephrectomized deoxycorticosterone (DOCA)-salt hypertensive rats (DOCA-salt) for 4 weeks by gavage. Although the blood pressure in the DOCA-salt group was higher than that of control, neither cilnidipine nor amlodipine had any effect on the increase in blood pressure in the DOCA-salt group. The DOCA (40 mg per kg per week, subcutaneously (s.c.)) and salt (1% NaCl in drinking water) treatment significantly aggravated the levels of urinary protein excretion and creatinine clearance and increased glomerulosclerosis and collagen deposition in the tubulointerstitial area of the kidney. These effects were attenuated by cilnidipine treatment. Reverse transcription-polymerase chain reaction analysis revealed that the renal expression of mRNA for collagen I/IV and transforming growth factor-ß was enhanced in the DOCA-salt group and that the overexpression of these molecules was suppressed by cilnidipine. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide production in the kidney and urinary norepinephrine excretion, which were enhanced in the DOCA-salt group, were suppressed by cilnidipine. Cilnidipine also decreased the activity and expression of angiotensin-converting enzyme (ACE) and the aldosterone concentration in the renal homogenate. Although neither cilnidipine nor amlodipine had any effect on the increased blood pressure in the DOCA-salt group, these renal changes were not induced by treatment with amlodipine. In conclusion, cilnidipine inhibited renal dysfunction, sympathetic nerve activity and renal renin-angiotensin-aldosterone system in the DOCA-salt group.

Automated single nucleotide polymorphism typing using bead array in capillary tube.

A low-cost and simple on-site technique for genotyping single nucleotide polymorphisms (SNPs) was developed. The technique is based on allele-specific primer PCR and the recently developed bead arrays in a single tip technique. The performance of the method was verified by genotyping four SNPs that correlate with cardiovascular diseases.

Chronic treatment with recombinant human erythropoietin exerts renoprotective effects beyond hematopoiesis in streptozotocin-induced diabetic rat.

Recombinant human erythropoietin (rHuEPO), which has been used clinically for the management of renal anemia, is reported to exert pleiotropic beneficial properties against acute ischemic/reperfusion injury in various tissues. To investigate the hypothesis that chronic treatment with rHuEPO might ameliorate diabetic nephropathy beyond hematopoiesis, rHuEPO (150 U/kg, subcutaneously) was administered three times per week to the streptozotocin-induced diabetic rats for 4 weeks. Streptozotocin (65 mg/kg, intravenously) significantly increased urinary protein excretion and collagen deposition in glomerular and tubulointerstitial areas in the kidney, which were attenuated by rHuEPO. rHuEPO normalized the levels of creatinine clearance, serum creatinine, and blood urea nitrogen of diabetic rats. RT-PCR analysis revealed that the expressions of mRNA for transforming growth factor-beta, osteopontin and adhesion molecules were enhanced in the diabetic rat kidney and that the overexpression of these molecules was suppressed by rHuEPO. rHuEPO exerted antioxidant properties by inhibiting renal activation and overexpression of NADPH oxidase. We found the activation of the Akt signaling pathway by the increased expression of phosphorylated Akt and GSK-3beta and a reduction of TUNEL-positive apoptotic cell death in renal tissue from rHuEPO-treated diabetic group. We also demonstrated that rHuEPO restored the endothelial nitric oxide synthase (eNOS) content in the diabetic rat kidney. On the other hand, treatment with rHuEPO did not affect blood glucose level, blood pressure, or hematocrit in diabetic rats. These results suggest that chronic treatment with rHuEPO attenuated renal injury beyond hematopoiesis and regulated apoptosis and eNOS expression, which might be due to the activation of Akt pathway.

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G).

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.

Development of an automated SNP analysis method using a paramagnetic beads handling robot.

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.

Pretreatment of polyamide monofilament with hydrochloric acid improves sensitivity of three-dimensional microarray, Bio-Strand.

A single-nucleotide-polymorphism-typing method using a novel three-dimensional DNA microarray, Bio-Strand, is promising because it is rapid, inexpensive and easily automated. It has been developed with the intent to overcome the drawbacks of conventional DNA microarrays, which use flat surfaces and impermeable materials such as glass slides; Bio-Strand as a novel DNA microarray, with its permeability, has a significantly improved stability compared with conventional DNA microarrays that use impermeable materials. In this study, we have developed a simple method of pretreating a polyamide monofilament to increase its surface area and to make it permeable, which makes Bio-Strand more sensitive and stable, allowing it to be adapted for clinical diagnostic applications. The fluorescence signal obtained with a nylon 6 monofilament pretreated under optimal conditions (hydrolysis by 5 M HCl/ethanol followed by washing with 50% ethanol and 100% ethanol) was significantly stronger than that obtained with an untreated monofilament.

Biomimetic Oxidation of Aldehyde with NAD+ Models: Glycolysis-Type Hydrogen Transfer in an NAD+ /NADH Model System.

Just like in biological systems, the GAPDH-catalyzed oxidation of aldehyde to carboxylate proceeds in conjunction with 1,4-selective reduction of NAD+ to NADH model compounds [Eq. (1)]. The combination of GAPDH- and LDH-type transfer reactions is also described here as a system mimic for the NAD+ /NADH redox cycle in anaerobic glycolysis. GAPDH=D-glyceraldehyde-3-phosphate dehydrogenase, LDH=L-lactate dehydrogenase.