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Recently, sphingosine 1-phosphate (S1P) has been highlighted as an endothelial barrier-stabilizing mediator. FTY720 is a S1P analog originally developed as a novel immunosuppressant. The phosphorylated form of FTY720 binds to S1P receptors to exert S1P-like biological effects, suggesting endothelial barrier promotion by FTY720. To elucidate whether FTY720 induces signaling events related to endothelial barrier enhancement under hyperglycemic conditions, human microvascular endothelial cells (HMVECs) preincubated with hyperglycemic (30 mM) medium were treated with 100 nM FTY720 for 3 h. Immunofluorescent microscopy and coprecipiration study revealed FTY720-induced focal adhesion kinase (FAK)-associated adherens junction (AJ) assembly at cell-cell contacts coincident with formation of a prominent cortical actin ring. FTY720 also induced transmonolayer electrical resistance (TER) augmentation in HMVEC monolayers in both normoglycemic and hyperglycemic conditions, implying endothelial barrier enhancement. Similar to S1P, site-specific FAK tyrosine phosphorylation analysis revealed FTY720-induced FAK [[Y.sup.576]] phosphorylation without phosphorylation of FAK [[Y.sup.397]/[Y.sup.925]]. Furthermore, FTY720 conditioned the phosphorylation profile of FAK [[Y.sup.397]/[Y.sup.576]][Y.sup.925]] in hyperglycemic medium to the same pattern observed in normoglycemic medium. FTY720 challenge resulted in small GTPase Rac activation under hyperglycemic conditions, whereas increased Rho activity in hyperglycemic medium was restored to the basal level. Rac protein depletion by small interfering RNA (siRNA) technique completely abolished FTY720-induced FAK [[Y.sup.576]] phosphorylation. These findings strongly suggest the barrier protective effect of FTY720 on HMVEC monolayers in hyperglycemic medium via S1P signaling, further implying the possibility of FTY720 as a therapeutic agent of diabetic vascular disorder. endothelial barrier function; human microvascular endothelial cell; diabetes mellitus; transmonolayer electrical resistance; cytoskeleton doi: 10.1152/ajpcell.00606.2008

Here we investigated the effects of mineralocorticoid in the regulation of catecholamine biosynthesis using rat pheochromocytoma PC12 cells. Expression of mineralocorticoid receptor (MR) was confirmed in undifferentiated PC12 cells. Aldosterone stimulated dopamine production by PC12 cells without any increase in cAMP activity. Aldosterone-induced dopamine accumulation was enhanced in accordance with the increase in the rate-limiting enzyme tyrosine hydroxylase (TH). Blocking MR with eplerenone suppressed aldosterone-induced increases of TH mRNA and dopamine production. A glucocorticoid receptor (GR) antagonist, RU-486, attenuated dexamethasone- but not aldosterone-induced TH expression. Cycloheximide reduced both aldosterone- and dexamethasone-induced TH mRNA. A SAPK/JNK inhibitor, SP600125, suppressed aldosterone-induced TH mRNA expression; however, the aldosterone-induced TH expression was not affected by inhibition of ERK1/2, p38-MAPK, Rho-kinase, PI 3-kinase, and PKC. It was of note that cotreatment with eplerenone and SP600125 restored aldosterone-induced TH mRNA expression to basal levels. To investigate the involvement of bone morphogenetic protein (BMP) actions in aldosterone-induced catecholamine production, we examined the effects of BMP-4 and BMP-7, which are expressed in the adrenal medulla, on catecholamine biosynthesis. BMP-4 preferentially enhanced aldosterone-induced TH mRNA and dopamine production, although BMP-4 alone did not affect TH expression. The BMP-4 enhancement of aldosterone-induced TH expression was not observed in cells treated with eplerenone. BMP-4 did not affect MR expression of PC12 cells; however, it did enhance aldosterone-induced SAPK/JNK phosphorylation. Inhibition of SAPK/JNK or Rho suppressed BMP-4 enhancement of aldosterone-induced TH expression. Collectively, our findings demonstrate that aldosterone stimulates catecholamine biosynthesis in adrenomedullar cells via MR through genomic action and partly through nongenomic action by Rho-SAPK/JNK signaling, the latter of which is facilitated by BMP-4. A functional link between MR actions and endogenous BMP may be involved in the catecholamine production. stress-activated protein kinase; c-Jun N[H.sub.2]-terminal kinase; aldosterone; eplerenone; mineralocorticoid receptor; pheochromocytoma

Edaravone is a potent scavenger of hydroxyl radicals and is quite successful in patients with acute cerebral ischemia, and several organ-protective effects have been reported. Treatment of human microvascular endothelial cells with edaravone (1.5 [micro]M) resulted in the enhancement of transmonolayer electrical resistance coincident with cortical actin enhancement and redistribution of focal adhesion proteins and adherens junction proteins to the cell periphery. Edaravone also induced small GTPase Rac activation and focal adhesion kinase (FAK; [Tyr.sup.576]) phosphorylation associated with sphingosine-1-phosphate receptor type 1 ([S1P.sub.1]) transactivation. [S1P.sub.1] protein depletion by the short interfering RNA technique completely abolished edaravoneinduced FAK ([Tyr.sub.576]) phosphorylation and Rac activation. This is the first report of edaravone-induced endothelial barrier enhancement coincident with focal adhesion remodeling and cytoskeletal rearrangement associated with Rac activation via S1[P.sub.1] transactivation. Considering the well-established endothelial barrier-protective effect of S1P, endothelial barrier enhancement as a consequence of [S1P.sub.1] transactivation may at least partly be the potent mechanisms for the organ-protective effect of edaravone and is suggestive of edaravone as a therapeutic agent against systemic vascular barrier disorder. focal adhesion; Rac; sphingosine-l-phosphate receptor type 1; transmonolayer electrical resistance

Aldosterone and angiotensin II (ANG II) contribute to the development and progression of renal damage. Here we investigated the effects of bone morphogenetic proteins (BMPs) on renal cell proliferation evoked by aldosterone and ANG II with mouse mesangial cells, which express mineralocorticoid receptors (MR), ANG II type 1 receptors, and BMP signaling molecules. Aldosterone and ANG II stimulated mesangial cell mitosis and activated ERK1/2 and SAPK/JNK signaling. These aldosterone effects were neutralized by the MR antagonist eplerenone and inhibition of transcription or translation, suggesting the involvement of genomic activation via MR. BMP-4 and BMP-7 stimulated Smad1, -5, -8 signaling more potently than BMP-2 and BMP-6, leading to suppression of mesangial cell mitosis and MR expression. MAPK inhibitors including U-0126 and SP-600125, but not SB-203580, suppressed aldosterone-induced cellular DNA synthesis, implying that ERK1/2 and SAPK/JNK pathways play crucial roles in mesangial cell proliferation. BMP-4 and BMP-7 inhibited phosphorylation of ERK1/2 and SAPK/JNK induced by aldosterone while activating p38 pathway, resulting in inhibition of aldosterone-induced cell mitosis. In contrast, aldosterone modulated the mesangial BMP system by decreasing expression of ALK-3, BMP-4, and BMP-7 while increasing inhibitory Smad6 expression. Thus novel functional cross talk between the mesangial BMP system and aldosterone signaling was uncovered, in which inhibition of MAPK signaling and MR expression by BMP-4 and BMP-7 may be involved in ameliorating renal damage due to mesangial proliferation caused by aldosterone. angiotensin II; kidney; mineralocorticoid doi:10.1152/ajprenal.00402.2006

Thiazolidinedione (TZD), a ligand for peroxisome proliferator-activated receptor-[gamma] (PPAR-[gamma]), exerts anti-inflammatory effects independently of the insulin-sensitizing effect. In the present study, we tested the hypothesis that TZD prevents the progression of diabetic nephropathy by modulating the inflammatory process. Five-week-old Sprague-Dawley rats were divided into three groups: 1) nondiabetic control rats (non-DM), 2) diabetic rats (DM), and 3) diabetic rats treated with pioglitazone (DM+pio). Diabetes was induced by injection with streptozotocin (STZ). The DM+pio group received 0.0002% pioglitazone mixed in chow for 8 wk after induction of diabetes. Blood glucose and HbA1c were elevated in diabetic rats but did not change by treatment with pioglitazone. Pioglitazone reduced urinary albumin excretion and glomerular hypertrophy, suppressed the expression of transforming growth factor (TGF)-[beta], type IV collagen, and ICAM-1, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, renal NF-[kappa]B activity was increased in diabetic rats and reduced by pioglitazone. PPAR-[gamma] was expressed in glomerular endothelial cells in the diabetic kidney and in cultured glomerular endothelial cells. High-glucose conditions increased the expression of ICAM-1 and the activation of NF-[kappa]B in cultured glomerular endothelial cells. These changes were reduced by pioglitazone, ciglitazone, and pyrrolidine dithiocarbamate, an inhibitor of NF-[kappa]B. However, pioglitazone did not show the changes in the presence of PPAR-[gamma] antagonist GW9662. Our results suggest that the preventive effects of pioglitazone may be mediated by its anti-inflammatory actions, including inhibition of NF-[kappa]B activation, ICAM-1 expression, and macrophage infiltration in the diabetic kidney. macrophages; ICAM-1

Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or [N.sup.G]-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker/ with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (B[H.sub.4]), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of B[H.sub.4] availability. These mechanisms are potential key targets for therapeutic interventions. in situ detection of nitric oxide and reactive oxygen species; tetrahydrobiopterin; low-temperature SDS-PAGE

Acatalasemia ensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis after unilateral ureteral obstruction. Am J Physiol Renal Physiol 286: F1030--F1038, 2004. First published January 13, 2004; 10.1152/ajprenal.00266.2003.--Tissue homeostasis is determined by the balance between oxidants and antioxidants. Catalase is an important antioxidant enzyme regulating the level of intracellular hydrogen peroxide and hydroxyl radicals. The effect of catalase deficiency on renal tubulointerstitial injury induced by unilateral ureteral obstruction (UUO) has been studied in homozygous acatalasemic mutant mice (C3H/AnL[Cs.sup.b][Cs.sup.b]) compared with wild-type mice (C3H/AnL[Cs.sup.a][Cs.sup.a]). Complete UUO caused interstitial cell infiltration, tubular dilation and atrophy, and interstitial fibrosis with accumulation of type IV collagen in obstructed kidneys (OBK) of both mouse groups. However, the degree of injury showed a significant increase in OBK of acatalasemic mice compared with that of wild-type mice until day 7. The deposition of lipid peroxidation products including 4-bydroxy-2-hexenal, malondialdehyde, and 4-hydroxy-2-nonenal was severer in dilated tubules of acatalasemic OBK. Apoptosis in tubular epithelial cells significantly increased in acatalasemic OBK at day 4. Expression of caspase-9, a marker of mitochondrial pathway-derived apoptosis, increased in dilated tubules of acatalasemic mice. The level of catalase activity remained low in acatalasemic OBK until day 7 without compensatory upregulation of glutathione peroxidase activity. The data indicate that acatalasemia exacerbated oxidation of renal tissue and sensitized tubular epithelial cells to apoptosis in OBK of UUO. This study demonstrates that catalase deficiency enhanced tubulointerstitial injury and fibrosis in a murine model of UUO and thus supports the protective role of catalase in this model. catalase; acatalasemic mice; free radical; cell death; caspase

Angiotensin II mediates the progression of renal disease through the type 1 receptor (A[T.sub.1]R). Recent studies have suggested that type 2 receptor (A[T.sub.2]R)-mediated signaling inhibits cell proliferation by counteracting the actions of A[T.sub.1]R. The aim of the present study was to determine the effect of A[T.sub.2]R overexpression on glomerular injury induced by 5/6 nephrectomy (5/6 Nx). A[T.sub.2]R transgenic mice (A[T.sub.2]R-Tg), overexpressing A[T.sub.2]R under the control of [alpha]-smooth muscle actin ([alpha]-SMA) promoter, and control wild-type mice (Wild) were subjected to 5/6Nx. In A[T.sub.2]-Tg mice, the glomerular expression of A[T.sub.2]R was upregulated after 5/6Nx. Urinary albumin excretion at 12 wk after 5/6Nx was decreased by 33.7% in A[T.sub.2]R-Tg compared with Wild mice. Glomerular size in A[T.sub.2]-Tg mice was significantly smaller than in Wild mice after 5/6Nx (93.1 [+ or -] 3.0 vs. 103.3 [+ or -] 1.8 [micro]m; P < 0.05). Immunohistochemistry revealed significant decreases in glomerular expression of platelet-derived growth factor-BB chain (PDGF-BB) and transforming growth factor-[[beta].sub.1] (TGF-[[beta].sub.1]) in A[T.sub.2]-Tg with 5/6Nx compared with Wild mice. Urinary excretion of nitric oxide metabolites was increased 2.5-fold in A[T.sub.2]-Tg compared with Wild mice. EMSA showed that activation of early growth response gene-1 which induces the transcription of PDGF-BB and TGF-[[beta].sub.1], was decreased in A[T.sub.2]-Tg mice. These changes in A[T.sub.2]-Tg mice at 12 wk after 5/6Nx were blocked by the A[T.sub.2]R antagonist PD-123319. Taken together, our findings suggest that A[T.sub.2]R-mediated signaling may protect from glomerular injuries induced by 5/6Nx and that overexpression of A[T.sub.2]R may serve as a potential therapeutic strategy for glomerular disorders. angiotensin II receptor; angiotensin II; transgenic mouse; nitric oxide

The chronic effects of cilazapril, an angiotensin converting enzyme inhibitor, and TCV-116, an angiotensin receptor blocker, on rats were investigated to determine the contribution of the renin-angiotensin system on glomerular pathology in hypertensive rats. Results reveal that both drugs significantly reduce urinary protein excretion although they failed to lower the blood pressure. TCV-116 had a greater reno-protective effect than did cilazapril.

The involvement of VEGF-A as well as the therapeutic efficacy of angiogenesis inhibitors in diabetic nephropathy have been reported. We recently reported the therapeutic effects of vasohibin-1 (VASH-1), an endogenous angiogenesis inhibitor, in a type 1 diabetic nephropathy model (Nasu T, Maeshima Y, Kinomura M, Hirokoshi-Kawahara K, Tanabe K, Sugiyama H, Sonoda H, Sato Y, Makino H. Diabetes 58: 2365-2375, 2009). In this study, we investigated the therapeutic efficacy of VASH-1 on renal alterations in obese mice with type 2 diabetes. Diabetic db/db mice received intravenous injections of adenoviral vectors encoding human VASH-1 (AdhVASH-1) and were euthanized 8 wk later. AdhVASH-1 treatment resulted in significant suppression of glomerular hypertrophy, glomerular hyperfiltration, albuminuria, increase in the CD31+ glomerular endothelial area, F4/80+ monocyte/macrophage infiltration, the accumulation of type IV collagen, and mesangial matrix. An increase in the renal levels of VEGF-A, VEGFR-2, transforming growth factor (TGF)-β1, and monocyte chemoattractant protein-1 in diabetic animals was significantly suppressed by AdhVASH-1 (immunoblotting). AdhVASH-1 treatment significantly recovered the loss and altered the distribution patterns of nephrin and zonula occludens (ZO)-1 and suppressed the increase in the number of fibroblast-specific protein-1 (FSP-1+) and desmin+ podocytes in diabetic mice. In vitro, recombinant human VASH-1 (rhVASH-1) dose dependently suppressed the upregulation of VEGF induced by high ambient glucose (25 mM) in cultured mouse podocytes. In addition, rhVASH-1 significantly recovered the mRNA levels of nephrin and the protein levels of ZO-1 and P-cadherin and suppressed the increase in protein levels of desmin, FSP-1, Snail, and Slug in podocytes under high-glucose condition. Taken together, these results suggest the potential use of VASH-1 as a novel therapeutic agent in type 2 diabetic nephropathy mediated via antiangiogenic effects and maintenance of podocyte phenotype in association with antiproteinuric effects. [ABSTRACT FROM AUTHOR]

Thiazolidinedione (TZD), a Iigand for peroxisome proliferator-activated receptor-γ (PPAR-γ), exerts anti-inflammatory effects independently of the insulin-sensitizing effect. In the present study, we tested the hypothesis that TZD prevents the progression of diabetic nephropathy by modulating the inflammatory process. Five-week-old Sprague-Dawley rats were divided into three groups: 1) nondiabetic control rats (non-DM), 2) diabetic rats (DM), and 3) diabetic rats treated with pioglitazone (DM+pio). Diabetes was induced by injection with streptozotocin (STZ). The DM+pio group received 0.0002% pioglitazone mixed in chow for 8 wk after induction of diabetes. Blood glucose and HbA1c were elevated in diabetic rats but did not change by treatment with pioglitazone. Pioglitazone reduced urinary albumin excretion and glomerular hypertrophy, suppressed the expression of transforming growth factor (TGF)-β, type IV collagen, and ICAM-1, and infiltration of macrophages in the kidneys of diabetic rats. Furthermore, renal NF-κB activity was increased in diabetic rats and reduced by pioglitazone. PPAR-γ was expressed in glomerular endothelial cells in the diabetic kidney and in cultured glomerular endothelial cells. High-glucose conditions increased the expression of ICAM-1 and the activation of NF-κB in cultured glomerular endothelial cells. These changes were reduced by pioglitazone, ciglitazone, and pyrrolidine dithiocarbamate, an inhibitor of NF-κB. However, pioglitazone did not show the changes in the presence of PPAR-γ antagonist GW9662. Our results suggest that the preventive effects of pioglitazone may be mediated by its anti-inflammatory actions, including inhibition of NF-κB activation, ICAM-1 expression, and macrophage infiltration in the diabetic kidney. [ABSTRACT FROM AUTHOR]