-
[show abstract]
[hide abstract]
ABSTRACT: Genome-wide screening using a small interfering RNA (siRNA) library has revealed novel molecules that are involved in a wide range of physiological responses. The expression of vascular endothelial growth factor (VEGF) is increased under hypoxic conditions, and plays a crucial role in tumor angiogenesis and tissue responses to ischemia. Here, we used a siRNA expression vector library to elucidate molecules that modify VEGF expression. Screening using an siRNA library revealed that MAPKKK6 (MEKK6/MAP3K6) regulates VEGF expression under both normoxic and hypoxic conditions in vitro, although the biological function of MAP3K6 remains unknown. Attenuation of VEGF expression by MAP3K6 inhibition was demonstrated by transient transfection of double-stranded RNA as well as by stable transfection of short hairpin RNA-expressing vectors against MAP3K6. Conditioned medium of MAP3K6-knocked down cells attenuated both endothelial proliferation and capillary network formation in a VEGF-dependent manner in vitro. In addition, tumor cells with down-regulation of MAP3K6 expression showed significant suppression of tumor growth in vivo, which was accompanied by significant repression of vessel formation and VEGF expression in these tumors. The results of this study suggest that MAP3K6 regulates VEGF expression in both normoxia and hypoxia, and that regulation of VEGF by MAP3K6 may play a crucial role in both angiogenesis and tumorigenesis.
American Journal Of Pathology 03/2009; 174(4):1553-63. · 4.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent studies have established that erythropoietin (EPO) is a pleiotropic cytokine. In this study we investigated whether pleiotropic effects of EPO may involve regulation of heme oxygenase (HO)-1, an anti-oxidative stress protein. A stimulatory effect of EPO on HO-1 expression was demonstrated in cultured renal endothelial cells, in which EPO decreased intracellular oxidative stress and provided cytoprotection against H(2)O(2). These beneficial effects were partially reversed by a HO-1 inhibitor. We then evaluated whether EPO induces HO-1 and ameliorates renal injury in vivo. Administration of EPO to Dahl salt-sensitive (DS) rats with low salt diet, a model of chronic tubulointerstitial injury, reduced proteinuria, and renal injury including peritubular capillaries rarefaction as compared to vehicle-treated DS rats. This renoprotection was associated with up-regulation of HO-1 in the kidney. In conclusion, EPO-induced HO-1 expression is likely to provide cytoprotection against oxidative stress.
Biochemical and Biophysical Research Communications 09/2007; 359(4):928-34. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Inorganic phosphate in food is absorbed two ways, the transcellular route via the brush border membrane and the paracellular route via tight junctions. NaPi, a sodium-dependent inorganic phosphate transporter, is expressed in rat and human intestine. However, the relative contribution of NaPi to total carrier-mediated transport of physiological concentrations of inorganic phosphate in rat intestine is not clear. Here, we characterized inorganic phosphate transport across the rat small intestine using a voltage-clamp analysis which allowed the diffrentiation of inorganic phosphate permeation through these two (transcellular and paracellular) routes. Results showed that, under a physiologically normal transmucosal electrical potential difference (about 2 mV), permeation of inorganic phosphate by the transcellular route was greater than that by the paracellular route. Further, transport was significantly decreased by the addition to the incubation medium of phosphonoformic acid, a sodium-dependent phosphate transporter inhibitor, and severely inhibited under sodium-free conditions. Similar results were obtained without the voltage-clamp. Together, these results suggest that NaPi-mediated transcellular permeation is the dominant route in the absorption of inorganic phosphate across the small intestine.
Drug Metabolism and Pharmacokinetics 07/2006; 21(3):217-21. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.
Journal of Biochemistry 06/2006; 139(5):821-9. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The characteristic features of thrombotic microangiopathy (TMA) include glomerular and peritubular capillary endothelial cell injury in association with loss of heparan sulfate proteoglycans on the cell surface and thrombus formation, followed by subsequent ischemic tubulointerstitial damage. It therefore was hypothesized that dextran sulfate (DXS) may protect the kidney against endothelial damage in a model of TMA. TMA was induced in rats by renal artery perfusion of an antiglomerular endothelial antibody, followed by the administration of DXS or vehicle. Renal damage was assessed by histologic analysis and measurements of blood urea nitrogen and creatinine. Whereas control rats developed severe renal failure with extensive glomerular and tubular injury, administration of DXS significantly protected renal function and preserved the glomerular endothelium and peritubular capillaries. The beneficial effect of DXS could be attributed to the ability of DXS to protect endothelial cells from coagulation and complement activation, as demonstrated by the histologic analysis. In addition, binding of the administered DXS to the surface of the glomerular endothelium was confirmed in TMA rats, suggesting that DXS acts as a "repair coat" of injured glomerular endothelium. In conclusion, DXS protects the kidney from experimental TMA. This protection may be mediated by DXS's binding directly to the surface of glomerular endothelium and amelioration of coagulation, complement activation, and cellular matrix loss.
Journal of the American Society of Nephrology 11/2005; 16(10):2997-3005. · 9.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nicotinamide has been shown to inhibit intestinal sodium-dependent phosphate transport activity in normal rats. It was reported recently that type IIb sodium-dependent phosphate co-transporter (NaPi-2b) is a carrier of intestinal phosphate absorption, and that its expression level is regulated by serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and Pi levels in normal rats. However, in chronic renal failure (CRF), serum 1,25(OH)(2)D and Pi levels are often abnormal. In a rat model of CRF, we investigated whether short-term nicotinamide administration was effective in reducing intestinal phosphate absorption and, if so, whether the effect was mediated by intestinal NaPi-2b.
Adenine-induced CRF rats were given a single daily intrapenitoneal administration of nicotinamide or vehicle solution for 6 days, and time course changes in serum Pi, Ca, blood urea nitrogen (BUN) and creatinine levels were monitored. Intestinal phosphate absorption was examined by oral administration of radiolabelled phosphate on the final day. In addition, NaPi-2b protein content in jejunum brush border membranes was determined.
Nicotinamide prevented the progressive increase in serum Pi associated with renal failure and significantly inhibited intestinal Pi absorption as assessed by the influx of orally administered radiolabelled phosphate into the circulation. This effect was accompanied by a decrease in NaPi-2b expression in jejunum brush border membranes. In addition, nicotinamide treatment was also associated with less marked elevations in BUN and serum creatinine and a higher creatinine clearance.
Nicotinamide inhibited intestinal Pi absorption in a rat model of CRF, at least in part by inhibiting the expression of NaPi-2b, and appeared to protect against the deterioration of renal function.
Nephrology Dialysis Transplantation 08/2005; 20(7):1378-84. · 3.40 Impact Factor
-
Tsuyoshi Mikkaichi,
Takehiro Suzuki,
Tohru Onogawa,
Masayuki Tanemoto,
Hiroya Mizutamari,
Masahiro Okada,
Tatsuji Chaki,
Satohiro Masuda,
Taro Tokui, Nobuaki Eto,
Michiaki Abe,
Fumitoshi Satoh,
Michiaki Unno,
Takanori Hishinuma,
Ken-Ichi Inui,
Sadayoshi Ito,
Junichi Goto,
Takaaki Abe
[show abstract]
[hide abstract]
ABSTRACT: Digoxin, which is one of the most commonly prescribed drugs for the treatment of heart failure, is mainly eliminated from the circulation by the kidney. P-glycoprotein is well characterized as a digoxin pump at the apical membrane of the nephron. However, little is known about the transport mechanism at the basolateral membrane. We have isolated an organic anion transporter (OATP4C1) from human kidney. Human OATP4C1 is the first member of the organic anion transporting polypeptide (OATP) family expressed in human kidney. The isolated cDNA encodes a polypeptide of 724 aa with 12 transmembrane domains. The genomic organization consists of 13 exons located on chromosome 5q21. Its rat counterpart, Oatp4c1, is also isolated from rat kidney. Human OATP4C1 transports cardiac glycosides (digoxin, K(m) = 7.8 microM and ouabain, K(m) = 0.38 microM), thyroid hormone (triiodothyronine, K(m) = 5.9 microM and thyroxine), cAMP, and methotrexate in a sodium-independent manner. Rat Oatp4c1 also transports digoxin (K(m) = 8.0 microM) and triiodothyronine (K(m) = 1.9 microM). Immunohistochemical analysis reveals that rat Oatp4c1 protein is localized at the basolateral membrane of the proximal tubule cell in the kidney. These data suggest that human OATP4C1/rat Oatp4c1 might be a first step of the transport pathway of digoxin and various compounds into urine in the kidney.
Proceedings of the National Academy of Sciences 04/2004; 101(10):3569-74. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We demonstrated that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for advanced glycation endproducts (AGE)-modified bovine serum albumin (AGE-BSA) (Kd=27.8 nM) and EN-RAGE (extracellular newly identified RAGE binding protein) (Kd=118 nM). Western blot and RT-PCR analysis showed that receptor for AGE (RAGE) was highly expressed on A549 cells, while the expression of other known AGE-receptors, e.g., galectin-3 and class A scavenger receptor (SR-A), were below the detection level. The binding of 125I-AGE-BSA to these cells was inhibited by unlabeled AGE-BSA but not by EN-RAGE. In contrast, the binding of 125I-EN-RAGE was significantly inhibited by unlabeled EN-RAGE and soluble RAGE but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one is specific for EN-RAGE and the other is specific for AGE-BSA. The latter receptor on A549 cells is novel as it is distinct from other known AGE-receptors.
International Congress Series 1245:119-124.
