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We developed a novel technology using the photoacoustic effect that improve needle tip visibility. We evaluated whether this technology improves needle tip visibility when performing a deep peripheral nerve block in a cadaver model. A photoacoustic needle was developed using a conventional echogenic needle with an intraluminal optical fiber. A pulsed laser sends light from a source through the fiber, which is converted to ultrasound at the needle tip using the photoacoustic effect. A nerve block expert performed deep nerve blocks using the photoacoustic needle and the ultrasound views recorded, with or without photoacoustic ultrasound at the needle tip. Needle tip visibility was evaluated by questionnaire (Likert scale 1: very poor, 5: very good) completed by anesthesiologists evaluating recorded images. The score was presented as median [first quartile, third quartile]. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test. The scores of needle tip visibility with photoacoustic ultrasound from the needle tip (4.3 [4.0, 4.5]) was significantly higher than that without photoacoustic ultrasound (3.5 [3.2, 3.8]) (p Clinical trial number University Hospital Medical Information Network Center Clinical Trials Registration System (UMIN000036974).

Formalin or formaldehyde is commonly used for cadaver fixation, which is, however, not suitable for endoscopic transnasal skull base approach because of consequent hardening of the soft tissue. Several alternatives have been reported, but each of them also has some limitations. We applied a novel fixation method using N-vinyl-2-pyrrolidone (NVP), a precursor of the water-soluble macromolecular polymer, for endoscopic transnasal skull base approach in six donated cadaver specimens. In four cadavers, elasticity of the soft tissue in the nasal cavity was almost similar to that of living tissue, and a surgical approach similar to the real surgical field was possible. However, the soft tissue was moderately stiffer than living tissue in two specimens so that surgical manipulation was hindered to some extent while NVP concentration was 10% in all the cadavers. Since the brain tissue was too soft and pliable for surgical manipulation in NVP, more careful surgical manipulation than real surgical field was mandatory in order to prevent damage in the brain tissue. Therefore, this concentration of NVP was considered to be appropriate. In conclusion, NVP embalming was effective for endoscopic transnasal skull base approach in cadaver dissection, providing environment similar to the real surgical field.

Background: We developed a novel technology using the photoacoustic effect that improve needle tip visibility. We evaluated whether this technology improves needle tip visibility when performing a deep peripheral nerve block in a cadaver model.Methods: A photoacoustic needle was developed using a conventional echogenic needle with an intraluminal optical fiber. A pulsed laser sends light from a source through the fiber, which is converted to ultrasound at the needle tip using the photoacoustic effect. A nerve block expert performed deep nerve blocks using the photoacoustic needle and the ultrasound views recorded, with or without photoacoustic ultrasound at the needle tip. Needle tip visibility was evaluated by questionnaire (Likert scale 1 : very poor, 5 : very good) completed by anesthesiologists evaluating recorded images. The score was presented as median [first quartile, third quartile]. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test. Results: The scores of needle tip visibility with photoacoustic ultrasound from the needle tip (4.3 [ 4.0, 4.5 ]) was significantly higher than that without photoacoustic ultrasound (3.5 [ 3.2, 3.8 ]) (pConclusions: Ultrasound emitted at the needle tip using the photoacoustic effect improves needle tip visibility during deep peripheral nerve blocks.

Background and objective Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT). Methods We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10−7 M aldosterone for 1 h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10−7 M aldosterone for 3 h by microarray. Results 3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10−9 M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated. Conclusions We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.

Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing macrophage-derived cytokines, IL-6 and TNFα, without blocking recruitment. Our data suggest that Rac1 in macrophage is a novel target for the treatment of kidney disease through inhibition of cytokine production.

Aging is accelerated, at least in part, by pathological condition such as metabolic syndrome (MetS), and various molecular pathways such as oxidative stress are common mediators of aging and MetS. We previously developed the aging-like skin model by single ultraviolet (UV) irradiation on the MetS model mice. Recent studies revealed that mineralocorticoid receptor (MR) signaling plays a pivotal role for various tissue inflammation and damages in MetS. Although previous studies reported that MR is expressed in the skin and that overexpression of MR in the skin resulted in the skin atrophy, the physiological or pathological functions of MR in the skin are not fully elucidated. Here, we show the involvement of MR signaling in the aging-like skin changes in our own model. Elevations of oxidative stress and inflammation markers were observed in the MetS mice, and the UV-evoked aging-like skin damages were attenuated by topical antioxidant. MR expression was higher in the MetS mouse skin, and notably, expression of its effecter gene Sgk1 was significantly upregulated in the aging-like skin in the UV-irradiated MetS mice. Furthermore, topical application of MR antagonist spironolactone suppressed Sgk1 expression, oxidative stress, inflammation, and the aging-like changes in the skin. The 2-week UV onto the non-MetS mice, the more usual photoaging model, resulted in the skin damages mostly equivalent to the MetS mice with single UV, but they were not associated with upregulation of MR signaling. Our studies suggested an unexpected role of MR signaling in the skin aging in MetS status.

Overactivation of the mineralocorticoid receptor signaling is implicated in cardiovascular disease, including hypertensive heart disease. Oxidative stress is suggested to augment mineralocorticoid receptor signal transduction, but the precise mechanisms remain unclear. Mineralocorticoid receptor activity is regulated by multiple factors, in addition to plasma ligand levels. We previously identified Rac1 GTPase as a modulator of mineralocorticoid receptor activity. Here we show that oxidative stress induces mineralocorticoid receptor activation in a ligand-independent, Rac1-depenent manner in cardiomyocytes. Oxidant stress was induced in rat cultured cardiomyocytes (H9c2) by l -buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO depleted intracellular glutathione and concomitantly increased reactive oxygen species (199%; P P N -acetylcysteine. The ligand independency of BSO action was indicated using a mutant mineralocorticoid receptor that does not bind ligands. With this mutant mineralocorticoid receptor, BSO-evoked mineralocorticoid receptor activation remained intact, whereas ligand-induced mineralocorticoid receptor activation was abolished. We next examined the involvement of Rac1. BSO increased active Rac1 in a redox-dependent fashion, and Rac inhibition suppressed the enhancing effect of BSO. Constitutively active Rac1, indeed, potentiated mineralocorticoid receptor transactivation. Furthermore, mineralocorticoid receptor transactivation by BSO was accompanied by enhanced nuclear accumulation of mineralocorticoid receptor. We conclude that alteration of redox state modulates mineralocorticoid receptor–dependent transcriptional activity via Rac1 in the heart. This redox-sensitive, ligand-independent mineralocorticoid receptor activation may contribute to the processes by which oxidant stress promotes cardiac injury.

Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP–dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.

Although inhibitors of the renin-angiotensin system are effective as first-line antihypertensive drugs in hypertensive patients with chronic kidney disease, they cannot completely prevent the progression of renal injury. Many animal studies, including our own, and a few human studies suggest that mineralocorticoid receptor blockade could inhibit the ongoing renal damage in chronic kidney disease. Thus, we designed this double-blinded, randomized, placebo-controlled trial to evaluate the antialbuminuric effect of a low dose (50 mg day(-1)) of the mineralocorticoid receptor antagonist eplerenone. The study subjects will include 340 hypertensive patients (blood pressure: 130-180/80-100 mm Hg) with albuminuria (urinary albumin/creatinine ratio: 30-600 mg g(-1) in the first morning void urine), who are treated with an inhibitor of the renin-angiotensin system. Other classes of antihypertensive drugs may be added as needed to achieve the target blood pressure (130/80 mm Hg). The primary study end point is the change in the urinary albumin/creatinine ratio after a 1-year study period. This trial is expected to show whether a low dose of mineralocorticoid receptor antagonists can exert an antialbuminuric effect in patients with chronic kidney disease.

Blockade of mineralocorticoid receptor has been shown to improve the clinical outcomes of proteinuric kidney diseases. However, little is known about the regulation of mineralocorticoid receptor-dependent transcriptional activity in renal disease. Here we identify a new role for Rac1, a member of the Rho family GTPases, as a potent activator of mineralocorticoid receptor signal transduction both in vitro and in vivo. Transient transfection assays in HEK 293 cells revealed that constitutively active Rac1 (CA-Rac1) enhanced mineralocorticoid receptor-dependent reporter activity, which was accompanied by increased nuclear translocation of mineralocorticoid receptor. CA-Rac1 facilitated mineralocorticoid receptor nuclear accumulation also in podocytes via p21-activated kinase phosphorylation. In mice lacking Rho GDP-dissociation inhibitor-alpha (Arhgdia(-/-) mice), renal abnormalities, including heavy albuminuria and podocyte damage, were associated with increased Rac1 (but not RhoA) and mineralocorticoid receptor signaling in the kidney, without alteration in systemic aldosterone status. Pharmacological intervention with a Rac-specific small-molecule inhibitor diminished mineralocorticoid receptor overactivity and renal damage in this model. Furthermore, albuminuria and histological changes in Arhgdia(-/-) mice were suppressed by mineralocorticoid receptor blockade, confirming the pathological role of Rac1-mineralocorticoid receptor interaction. Our results provide evidence that signaling cross-talk between Rac1 and mineralocorticoid receptor modulates mineralocorticoid receptor activity and identify Rac1 as a therapeutic target for chronic kidney disease.

Sonic hedgehog (Shh) is a well-known morphogen indispensable in facial and nervous development, and recently it has also garnered much attention as a potent angiogenic factor. We previously created an animal model of holoprosencephaly by administration of cyclopamine, a specific inhibitor of hedgehog signaling, to the mouse embryos cultured in vitro, and found several types of angiogenic defects. In this study, we focused on other angiogenic phenotypes in the same model. When cyclopamine was added for embryonic day (E) 8.0-9.5, a pair of immature dorsal aortae, which normally fuse to form the single aorta by E9.5, remained to be separated. Expressions of vascular endothelial growth factor and bone morphogenetic protein 4, putative mediators of aortic fusion, were also reduced around the aorta by blockade of Shh signaling. When cyclopamine was added for E8.5-10.5, vessels on the surface of craniofacial region (possibly external cardinal veins) were extended and malformed. These results suggest that Shh signaling is essential for some aspects of embryonic angiogenesis, and that pathophysiology of holoprosencephaly may involve, at least in part, the Shh-dependent angiogenesis.

Recent clinical studies implicate proteinuria as a key prognostic factor for renal and cardiovascular complications in hypertensives. The pathogenesis of proteinuria in hypertension is, however, poorly elucidated. Podocytes constitute the final filtration barrier in the glomerulus, and their dysfunction may play a pivotal role in proteinuria. In the present study, we examined the involvement of podocyte injury in Dahl salt-hypertensive rats, an animal model prone to hypertensive glomerulosclerosis, and explored the effects of inhibition of aldosterone. Four-week–old Dahl salt-resistant and salt-sensitive rats were fed a 0.3% or 8.0% NaCl diet. Some salt-loaded Dahl salt-sensitive rats were treated with a selective aldosterone blocker eplerenone (1.25 mg/g diet) or hydralazine (0.5 mmol/L). After 6 weeks, salt-loaded Dahl salt-sensitive rats developed severe hypertension, proteinuria, and glomerulosclerosis. Immunostaining for nephrin, a constituent of slit diaphragm, was attenuated, whereas expressions of damaged podocyte markers desmin and B7-1 were upregulated in the glomeruli of salt-loaded Dahl salt-sensitive rats. Electron microscopic analysis revealed podocyte foot process effacement. Podocytes were already impaired at as early as 2 weeks of salt loading in Dahl salt-sensitive rats, when proteinuria was modestly increased. Both eplerenone and hydralazine partially reduced systemic blood pressure as measured by indirect and direct methods in salt-loaded Dahl salt-sensitive rats, but only eplerenone dramatically improved podocyte damage and retarded the progression of proteinuria and glomerulosclerosis. Our findings suggest that podocyte injury underlies the glomerulopathy of Dahl salt-hypertensive rats and that inhibition of aldosterone by eplerenone is protective against podocyte damage, proteinuria, and glomerulosclerosis in this hypertensive model.

Statins have been reported to confer renoprotection in several experimental models of renal disease through pleiotropic actions. The roles of statins in glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of fluvastatin on podocyte and tubulointerstitial injury in puromycin aminonucleoside (PAN)-induced nephrosis. PAN induced massive proteinuria and serum creatinine elevation on day 7, which were significantly suppressed by fluvastatin. Immunofluorescence studies of podocyte-associated proteins nephrin and podocin revealed diminished and discontinuous staining patterns in rats with PAN nephrosis, indicating severe podocyte injury. Fluvastatin treatment dramatically mitigated the abnormal staining profiles. Reduction of nephrin expression by PAN and its reversal by fluvastatin were confirmed by quantitative analyses. By electron microscopy, effacement of foot processes was ameliorated in fluvastatin-treated rats. Fluvastatin also mitigated tubulointerstitial damage in PAN nephrosis, with the repression of PAN-induced NF-kappaB and activator protein-1 activation in the kidneys. In addition, expression of activated membrane-bound small GTPase RhoA was markedly increased in the glomeruli of PAN nephrosis, which was inhibited by fluvastatin treatment. In cultured podocytes, fluvastatin suppressed PAN-evoked activation of RhoA and actin cytoskeletal reorganization. Furthermore, fasudil, a specific Rho-kinase inhibitor, successfully ameliorated PAN-induced podocyte damage and proteinuria. In summary, fluvastatin alleviated podocyte and tubulointerstitial injury in PAN nephrosis. The beneficial effects of fluvastatin on podocytes can be attributable to direct modulation of excessive RhoA activity. Our data suggest a therapeutic role for statins in clinical conditions that are relevant to podocyte injury.

LOX-1 is a novel receptor for oxidized low-density lipoprotein (LDL) isolated from vascular endothelial cells and has been suggested to be involved in the formation of atherosclerotic and hypertensive vascular lesions. We previously reported that salt loading caused glomerulosclerosis and upregulation of LOX-1 in the kidney of Dahl salt-sensitive hypertensive rats. In the present study, we investigated LOX-1 expression in the remnant kidney, an established rat model for chronic renal failure. Six weeks after 5/6 nephrectomy, the rats showed elevated blood pressure, impaired renal function and increased renal expression of type I collagen. The LOX-1 gene expression in the remnant kidney was markedly increased compared with that in control rats, and immunohistochemical analysis showed that LOX-1 was widely expressed in the interstitial cells, whereas there was almost no staining in the glomeruli or tubules. Moreover, reduction of blood pressure by the angiotensin II type 1 (AT1) receptor antagonist candesartan significantly suppressed the renal LOX-1 expression, and this suppression was accompanied by amelioration of renal injury. These results suggest that enhanced renal expression of LOX-1 might play some roles in the progression of chronic renal failure in rats.

Adrenomedullin (AM) is a potent vasodilating peptide secreted from the vasculature of various organs. It is biologically active when its C-terminus is amidated. Recently, an RIA method was developed for measurement of the active form of AM, or mature AM. We here employed this method to investigate the significance of amidation of AM in controlling cardiovascular function. Thirty-six patients under hemodialysis were recruited and divided into hypertensive (n = 25; 157/86 mmHg) and normotensive (n= 11; 116/66 mmHg) groups. Mature AM, immature AM and blood pressure were monitored during hemodialysis in all patients. There was a significant reduction in blood pressure during hemodialysis in both groups, although after hemodialysis blood pressure was still higher in hypertensives than in normotensives (139 +/-14.8/76 +/- 2.5 mmHg vs. 110 +/- 5.1/66.7 +/- 3.1 mmHg). Mature AM before hemodialysis were lower in hypertensives than normotensives and it decreased in both groups. Although mature AM decreased more in normotensives than in hypertensives (-27 +/- 8% vs. -17 +/- 5%), at the end point, its level was still higher in normotensives. The ratio of mature AM/immature AM decreased only in normotensives (-11.4 8.7%), whereas it remained stable in hypertensives (0.2 +/- 5.6%). Both groups showed similar changes in ANP, endothelin, catecholamines, cGMP, and NOx. The low level in mature AM level in hypertensives may have contributed to the higher blood pressure in this group. The attenuation of AM amidation in normotensives indicates that an unspecified amidative enzyme of AM was regulated in order to normalize blood pressure.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a recently identified receptor for oxidized low-density lipoprotein, one of the major atherogenic substances. Although LOX-1 was reported to be expressed abundantly in endothelial cells, including atheromatous lesions, the regulation of LOX-1 gene has not yet been clarified. In the present study, we isolated the rat LOX-1 gene and investigated the regulation of gene expression. The rat LOX-1 gene was encoded by a single copy gene spanning over 19 kilobases and consisted of eight exons. Exon boundaries correlated well with the functional domain boundaries of the receptor protein. The promoter region contained putative TATA and CAAT boxes and multiple cis-elements such as NF-kappaB, AP-1 and AP-2 sites, and a shear stress response element. Northern blot analysis revealed that LOX-1 gene expression was up-regulated 9-fold by shear stress, 21-fold by lipopolysaccharide, and 4-fold by tumor necrosis factor-alpha, in cultured vascular endothelial cells. LOX-1 was also expressed in macrophages but not in vascular smooth muscle cells. These data provide important information for elucidating the molecular mechanisms of LOX-1 gene regulation and suggest a role for LOX-1 in the pathophysiology of atherosclerotic cardiovascular disease.

We report the identification of a unique repetitive sequence in the rat endothelial receptor for oxidized low-density lipoprotein (LOX-1) and unexpected blood-pressure-associated regulation of its expression, a new link between lipid metabolism and blood-pressure control. A rat aorta cDNA library was constructed and screened with a probe synthesized by degenerate PCR. Rat LOX-1 cDNA encoded a protein of 364 amino acids that showed ~ 60% similarity to its bovine and human counterparts. The protein consisted of intracellular N-terminal, transmembrane and extracellular lectin-like domains. Rat LOX-1 was unique in having three repeats of a 46-amino-acid motif between the transmembrane and lectin-like regions. Two isoforms of mRNA were found to be generated by alternative use of two polyadenylation signals in a tissue-specific manner. The 3ʹ-untranslated region contained multiple A+U-rich elements for rapid degradation of mRNA. Northern-blot analysis revealed that LOX-1 mRNA was expressed predominantly in the lung. Quite unexpectedly, the expression was dramatically up-regulated in the aorta in hypertensive SHR-SP/Izm rats compared with very low levels in control WKY/Izm rats, suggesting a potential role for LOX-1 in the pathogenesis of hypertension as well as atherosclerosis.

Abstract The natriuretic peptide system is suggested to be involved in the pathogenesis of salt-sensitive hypertension; a recent report indicated that disruption of the atrial natriuretic peptide precursor gene caused salt-sensitive hypertension. However, natriuretic peptide receptor (NPR)-A knockout mice did not show enhanced salt sensitivity of blood pressure. The aim of the present study was to investigate the role of NPR-C, the other receptor for atrial natriuretic peptide, in increased salt sensitivity of blood pressure. Dahl salt-sensitive (DS) and salt-resistant (DR) rats were placed on a 0.3% or 8% NaCl diet for 4 weeks. Blood pressure was elevated by salt loading only in DS rats. RNase protection assay demonstrated that NPR-C transcript level in the kidney was reduced by chronic salt loading in both DR and DS rats, whereas expression of NPR-A and NPR-B was not altered. The reduction of NPR-C mRNA in response to salt loading was enhanced in DS compared with DR rats. In situ hybridization indicated that the salt-induced NPR-C change was attributed mainly to suppressed expression of NPR-C in the podocytes. NPR-C gene expression was regulated by salt loading in a tissue-specific manner; the marked decrease in NPR-C mRNA by salt loading was seen only in the kidney. These data suggest that the exaggerated salt-induced reduction of NPR-C in the kidney of DS rats may play an important role in the pathogenesis of salt hypertension in this animal, possibly related to impaired renal sodium excretion.

The present study was designed to clarify whether modulation of norepinephrine (NE) release by vascular angiotensin (Ang) II is involved in the increased peripheral sympathetic activity of spontaneously hypertensive rats (SHR). In the perfusion system of isolated mesenteric vascular beds, periarterial nerve stimulation (PNS)-evoked NE overflow was significantly greater in SHR than Wistar-Kyoto rats (WKY). Administration of Ang II increased PNS-induced NE overflow, which could be reversed by pretreatment with the AT1 receptor antagonist CV-11974 in both types of rats; the facilitation by Ang II was more potent in SHR. Moreover, CV-11974 by itself could attenuate PNS-evoked NE overflow, the extent of which was also significantly greater in SHR, suggesting an augmented sympatho-facilitatory effect of enodogenous Ang II in SHR. Consistently, sympatho-facilitation by Ang I, which could be abolished by the angiotensin converting enzyme (ACE) inhibitor imidaprilat, was apparently greater than that of Ang II in SHR, despite no difference in WKY. These findings suggest that the increased peripheral sympathetic activity in SHR is attributed not only to the elevated sensitivity of nerve endings to Ang II but also to the increased local generation of Ang II, an effect possibly mediated by augmented vascular ACE activity. (Hypertens Res 1996; 19: 171-177)

降圧療法の最終目標は高血圧による心血管系合併症(脳血管障害,虚血性心疾患,心不全,腎不全など)を阻止することである.現在利尿薬, β遮断薬, Ca拮抗薬, ACE阻害薬, α1遮断薬, αβ遮断薬が第一次薬に定められているが,患者の病態(年齢,性,高血圧の重症度,合併症,偶発症,他の心血管系危険因子など)に基づき,良好な長期予後をもたらすような,しかも服薬コンプライアンスやQOLをも考慮に入れた降圧薬の選択が望ましい.

A Rho family GTPase, Rac1, has emerged as an important molecule involved in cardiac remodeling. Some studies demonstrated the requirement of Rac1 in angiotensin II-induced cardiac hypertrophy and diabetic cardiomyopathy in association with generation of reactive oxygen species (ROS). However its role in pressure overload-induced cardiac remodeling is still unclear. On the other hand, we previously reported that Rac1 can activate mineralocorticoid receptor (MR) in cultured cardiomyocytes. Here we demonstrate the requirement of Rac1 in pressure-overload cardiac remodeling, and putative role of MR as a downstream pathway of Rac1. First, we performed sham or transverse aortic constriction (TAC) surgery in C57BL/6 mice, and examined the effect of Rac1 inhibitor ( NSC23766 ) and MR blocker (eplerenone). After 7 weeks, TAC caused severe hypertrophy and dysfunction of left ventricle with significant increase in active form of Rac1. In addition, the amount of MR protein in nuclear fraction, and the expression of some target genes of MR (including serpina3n and serpine-1) in left ventricle were also increased by TAC. NSC23766 significantly reduced the TAC-induced activation of Rac1, and both NSC23766 and eplerenone attenuated cardiac hypertrophy and dysfunction, along with inhibition of MR signaling. Furthermore, TAC significantly increased ROS production in left ventricle, which was also attenuated by both of the pharmacological interventions. We next generated cardiomyocyte-specific heterozygous Rac1-deficient mice (Rac1 CM +/− ) and littermate wild-type mice (WT), and performed Sham or TAC surgery. The TAC-induced hypertrophy and dysfunction of left ventricle were significantly suppressed in Rac1 CM +/− compared with WT (heart/body weight ratio: 6.4 ± 0.86 vs 10.8 ± 0.81 mg/g, ejection fraction 54.1 ± 6.5 vs 33.3 ± 7.3 %, p < 0.05), with inhibition of Rac1 activation. Nuclear translocation of MR protein and increases in expression of the target genes of MR were also significantly attenuated in Rac1 CM +/− compared with WT. These results indicate that Rac1 plays an essential role in the maladaptive cardiac hypertrophy induced by pressure overload, and that MR is an important downstream pathway of Rac1 in heart.

Experiments with hyperaldosteronemic animals suggest that, despite lowering plasma aldosterone, salt worsens renal injury by paradoxical activation of the mineralocorticoid receptor (MR). Salt and aldosterone synergistically contribute to renal impairment through Rac1-mediated activation of the MR, but whether angiotensin II also promotes renal injury through this mechanism is unknown. Here, we placed angiotensin II-overproducing double transgenic Tsukuba hypertensive mice on a low- or high-salt intake for 6 weeks and treated some animals with adrenalectomy, the MR antagonist eplerenone, the Rac inhibitor EHT1864, or hydralazine. High-salt intake, but not low-salt intake, led to hypertension and prominent kidney injury. Adrenalectomy prevented angiotensin II/salt-induced nephropathy in mice receiving high-salt intake, which was recapitulated by aldosterone supplementation, suggesting the involvement of aldosterone/MR signaling. Plasma aldosterone levels, however, were lower in high- than low-salt conditions. Instead, angiotensin II/salt-evoked MR activation associated with Rac1 activation and was not dependent on plasma aldosterone level. Both EHT1864 and eplerenone repressed the augmented MR signaling and mitigated kidney injury with partial but significant reduction in BP with high-salt intake. Hydralazine similarly reduced BP, but it neither suppressed the Rac1-MR pathway nor ameliorated the nephropathy. Taken together, these results show that angiotensin II and salt accelerate kidney injury through Rac1-mediated MR activation. Rac inhibition may be a promising strategy for the treatment of CKD.

Obesity hypertension and metabolic syndrome have become major public health concerns. Nowadays, aldosterone is recognized as an important mediator of cardiovascular and renal damage. In the kidney, aldosterone injures glomerular visceral epithelial cells (podocytes), the final filtration barrier to plasma macromolecules, leading to proteinuria and glomerulosclerosis. Mineralocorticoid receptor (MR) antagonists effectively ameliorate proteinuria in patients or in animal models of hypertension, diabetes mellitus and chronic kidney disease (CKD), as well as in patients who experience ‘aldosterone breakthrough.’ Recently, clinical and experimental studies have shown that plasma aldosterone concentration is associated with obesity hypertension and metabolic syndrome. We showed that spontaneously hypertensive rats (SHR)/cp, an experimental model of obesity hypertension and metabolic syndrome, are prone to glomerular podocyte injury, proteinuria and left ventricular diastolic dysfunction, especially when the animals are fed a high-salt diet. Inappropriate activation of the aldosterone/MR system underlies the renal and cardiac injuries. Adipocyte-derived aldosterone-releasing factors (ARFs), although still unidentified, may account for aldosterone excess and the resultant target organ complication in SHR/cp. On the other hand, recent studies have shown that MR activation triggers target organ disease even in normal or low aldosterone states. We identified a small GTP (guanosine triphosphate)-binding protein, Rac1, as a novel activator of MR, and showed that this ligand-independent MR activation by Rac1 contributes to the nephropathy of several CKD models. We expect that ARFs and Rac1 can be novel therapeutic targets for metabolic syndrome and CKD. Future large-scale clinical trials are awaited to prove the efficacy of MR blockade in patients with obesity hypertension and metabolic syndrome.

Metabolic syndrome is a highly predisposing condition for cardiovascular disease and could be a cause of excess salt–induced organ damage. Recently, several investigators have demonstrated that salt loading causes left ventricular diastolic dysfunction associated with increased oxidative stress and mineralocorticoid receptor activation. We, therefore, investigated whether excess salt induces cardiac diastolic dysfunction in metabolic syndrome via increased oxidative stress and upregulation of mineralocorticoid receptor signals. Thirteen-week-old spontaneously hypertensive rats and SHR/NDmcr-cps, the genetic model of metabolic syndrome, were fed a normal salt (0.5% NaCl) or high-salt (8% NaCl) diet for 4 weeks. In SHR/NDmcr-cps, salt loading induced severe hypertension, abnormal left ventricular relaxation, and perivascular fibrosis. Salt-loaded SHR/NDmcr-cps also exhibited overproduction of reactive oxygen species and upregulation of mineralocorticoid receptor–dependent gene expression, such as Na + /H + exchanger-1 and serum- and glucocorticoid-inducible kinase-1 in the cardiac tissue. However, in spontaneously hypertensive rats, salt loading did not cause these cardiac abnormalities despite a similar increase in blood pressure. An antioxidant, tempol, prevented salt-induced diastolic dysfunction, perivascular fibrosis, and upregulation of mineralocorticoid receptor signals in SHR/NDmcr-cps. Moreover, a selective mineralocorticoid receptor antagonist, eplerenone, prevented not only diastolic dysfunction but also overproduction of reactive oxygen species in salt-loaded SHR/NDmcr-cps. These results suggest that metabolic syndrome is a predisposed condition for salt-induced left ventricular diastolic dysfunction, possibly via increased oxidative stress and enhanced mineralocorticoid receptor signals.

Aldosterone is implicated in the pathogenesis of proteinuria and chronic kidney disease. We previously demonstrated the contribution of elevated serum aldosterone in the early nephropathy of SHR/NDmcr-cp (SHR/cp), a rat model of metabolic syndrome. In the present study, we investigated the effect of salt loading on renal damage in SHR/cps and explored the underlying mechanisms. SHR/cps fed a high-sodium diet for 4 weeks developed severe hypertension, massive proteinuria, and advanced renal lesions. High salt also worsened glomerular podocyte impairment. Surprisingly, selective mineralocorticoid receptor (MR) antagonist eplerenone dramatically ameliorated the salt-induced proteinuria and renal injury in SHR/cps. Although salt loading reduced circulating aldosterone, it increased nuclear MR and expression of aldosterone effector kinase Sgk1 in the kidney. Gene expressions of transforming growth factor-β1 and plasminogen activator inhibitor-1 were also enhanced in the kidneys of salt-loaded SHR/cps, and eplerenone completely inhibited these injury markers. To clarify the discrepancy between decreased aldosterone and enhanced MR signaling by salt, we further investigated the role of oxidative stress, a putative key factor mediating salt-induced tissue damage. Interestingly, antioxidant Tempol attenuated the salt-evoked MR upregulation and Sgk1 induction and alleviated proteinuria and renal histological abnormalities, suggesting the involvement of oxidative stress in salt-induced MR activation. MR activation by salt was not attributed to increased serum corticosterone or reduced 11β-hydroxysteroid dehydrogenase type 2 activity. In conclusion, sodium loading exacerbated proteinuria and renal injury in metabolic syndrome rats. Salt reduced circulating aldosterone but caused renal MR activation at least partially via induction of oxidative stress, and eplerenone effectively improved the nephropathy.

Accumulating evidence suggests that mineralocorticoid receptor blockade effectively reduces proteinuria in hypertensive patients. However, the mechanism of the antiproteinuric effect remains elusive. In this study, we investigated the effects of aldosterone on podocyte, a key player of the glomerular filtration barrier. Uninephrectomized rats were continuously infused with aldosterone and fed a high-salt diet. Aldosterone induced proteinuria progressively, associated with blood pressure elevation. Notably, gene expressions of podocyte-associated molecules nephrin and podocin were markedly decreased in aldosterone-infused rats at 2 weeks, with a gradual decrease thereafter. Immunohistochemical studies and electron microscopy confirmed the podocyte damage. Podocyte injury was accompanied by renal reduced nicotinamide-adenine dinucleotide phosphate oxidase activation, increased oxidative stress, and enhanced expression of aldosterone effector kinase Sgk1. Treatment with eplerenone, a selective aldosterone receptor blocker, almost completely prevented podocyte damage and proteinuria, with normalization of elevated reduced nicotinamide-adenine dinucleotide phosphate oxidase activity. In addition, proteinuria, podocyte damage, and Sgk1 upregulation were significantly alleviated by tempol, a membrane-permeable superoxide dismutase, suggesting the pathogenic role of oxidative stress. Although hydralazine treatment almost normalized blood pressure, it failed to improve proteinuria and podocyte damage. In cultured podocytes with consistent expression of mineralocorticoid receptor, aldosterone stimulated membrane translocation of reduced nicotinamide-adenine dinucleotide phosphate oxidase cytosolic components and oxidative stress generation in podocytes. Furthermore, aldosterone enhanced the expression of Sgk1, which was inhibited by mineralocorticoid receptor antagonist and tempol. In conclusion, podocytes are injured at the early stage in aldosterone-infused rats, resulting in the occurrence of proteinuria. Aldosterone can directly modulate podocyte function, possibly through the induction of oxidative stress and Sgk1.

Embryonic morphogenesis of vascular and nervous systems is tightly coordinated, and recent studies revealed that some neurogenetic factors such as Sonic hedgehog (Shh) also exhibit angiogenetic potential. Vascularization within the developing mouse neural tube depends on vessel sprouting from the surrounding vascular plexus. Previous studies implicated possible roles of VEGF/Flk-1 and Angiopoietin-1(Ang-1)/Tie-2 signaling as candidate molecules functioning in this process. Examining gene expressions of these factors at embryonic day (E) 9.5 and 10.5, we unexpectedly found that both VEGF and Ang-1 were expressed in the motor neurons in the ventral neural tube. The motor neurons were indeed located in the close vicinity of the infiltrating vessels, suggesting involvement of motor neurons in the sprouting. To substantiate this possibility, we inhibited induction of the motor neurons in the cultured mouse embryos by cyclopamine, a Shh signaling blocker. The vessel sprouting was dramatically impaired by inhibition of Shh signaling, together with nearly complete loss of the motor neurons. Expression of Ang-1, but not VEGF, within the neural tube was remarkably reduced in the cyclopamine treated embryos. These results suggest that the neural tube angiogenesis is dependent on Shh signaling, and mediated, at least in part, by the Ang-1 positive motor neurons.

Oxidized low-density lipoprotein (OxLDL) has been implicated in atherosclerosis and glomerulosclerosis. LOX-1 is a recently identified OxLDL receptor that is abundantly expressed in vascular endothelial cells. The aim of the present study was to investigate LOX-1 expression in the kidneys of hypertensive rats. Dahl salt-sensitive (DS) and salt-resistant (DR) rats were fed a 0.3% or 8% NaCl diet. Some DS 8% rats were treated with manidipine or hydralazine. LOX-1 gene expression was markedly elevated in the kidneys and glomeruli of hypertensive DS 8% rats compared with those of normotensive DR and DS 0.3% rats. Prolonged salt loading further increased the renal LOX-1 expression in DS rats. The LOX-1 upregulation in DS 8% rats was accompanied by renal overexpression of transforming growth factor-beta 1 and type I collagen, impaired renal function, and histologic glomerulosclerotic changes, all of which were ameliorated by antihypertensive treatment. LOX-1 was indeed expressed in the glomeruli in vivo and in cultured glomerular cells in vitro. However, LOX-1 expression was elevated in the aorta but not the kidneys of spontaneously hypertensive rats, which exhibited hypertension but minor glomerulosclerotic changes. In conclusion, the LOX-1 upregulation in the kidney of DS 8% rats was parallel to glomerulosclerotic changes and renal dysfunction, suggesting a possible pathogenetic role for renal LOX-1 in the progression to hypertensive glomerulosclerosis.

Retrotransposons transcribed preferentially in proximal tubules of salt-hypertensive rats. Background The kidney is considered to play an important etiologic role in salt-sensitive hypertension. The aim of the present study was to isolate genes whose expression differs between the kidneys of salt-hypertensive and control rats using an mRNA differential display method. Methods Dahl salt-sensitive (DS) and control salt-resistant rats (DR) were fed a 0.3% or 8% NaCl diet. Renal RNA was amplified by RNA arbitrarily primed polymerase chain reaction (RAP-PCR) and compared among DR 0.3%, DR 8%, DS 0.3%, and DS 8%. Gene expression and localization were examined by Northern blotting, RNase protection assay, and in situ hybridization. Full-length nucleotide sequence was determined by screening a DS rat kidney cDNA library. Results We identified one differentially displayed clone, and its expression was greater in DS than DR, which was not affected by salt loading. The sequence was 90% homologous to the 3′-noncoding region of the nicotinic acetylcholine receptor α7 subunit gene. Its expression was kidney-specific, and was localized in the proximal tubules. The transcript level was markedly increased precedent to the development of hypertension. Its expression was also high in other salt-sensitive rats, and low in normotensive Sprague-Dawley and Wistar rats. The full-length cDNA contained elements homologous to the retroviral pol gene, a primer binding site sequence for reverse transcriptase, and long-terminal repeats. Conclusion These results demonstrated that the newly identified transcripts (REPT1) belong to a novel retrotransposon family, which showed unique strain-, age-, tissue-, and cell type-specific expression pattern.

The metabolic syndrome is complicated by nephropathy in humans and rats, and males are more affected than females. We hypothesized that female rats had reduced expression of glomerular oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1), attendant glomerular oxidant injury, and renal inflammation. Three groups, obese males (OM), obese females (OF), and lean males (LM) of first-generation (F(1)) hybrid rats derived from the Zucker fatty diabetic (ZDF) strain and the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) were studied from 6 to 41 weeks of age. OM had severe renal oxidant injury and renal failure. Their glomeruli expressed the LOX-1, and exhibited heavier accumulation of the lipid peroxide 4-hydroxynonenal (4-HNE). OM had compromised mitochondrial enzyme function, more renal fibrosis, and vascular leakage. Younger LM, OM, and OF ZS (ZDF/SHHF F(1) hybrid rat) rats, studied from 6 to 16 weeks of age, showed that unutilized renal lipids were comparable in OM and OF, although young OM had worse nephropathy and inflammation. In conclusion, glomerular LOX-1 expression is coupled to deposits of 4-HNE and glomerulosclerosis in OM. We presume that LOX-1 enhances glomerular uptake of oxidized lipids and renal inflammation, causing greater oxidant stress and severe glomerulosclerosis. In OF, renal protection from lipid oxidants appears to be conferred by blunted glomerular LOX-1 expression and renal inflammation.