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Tissue-hardening effect and health-hazard issue of formaldehyde (FA) have long been a great disadvantage of this conventional fixative in anatomical research. We recently developed a FA-free embalming method for cadavers which utilizes N-vinyl-2-pyrrolidone (NVP) and enables assessment of motion kinetics by maintaining the softness of embalmed tissue. By assessing the feasibility of NVP-embalmed tissue to mimic vocalization, this study aimed to prove the potential of embalmed cadavers, which have previously been used only for the understanding of anatomical morphology, for the assessment of precise motion physiology in the human body. Ten cadavers embalmed in NVP (n = 6) and FA (n = 4) were incorporated in this study. Excised larynges underwent experimental phonation to mimic vocalization with fast and pliable vibration of vocal folds. High-speed digital imaging was utilized for the assessment of vocal fold vibration. Furthermore, acoustic analysis of the voiced sound, and reproducibility examination were also performed. Regular vocal fold vibrations successfully produced voiced sounds during experimental phonation using NVP-embalmed larynges. The vibratory frequency, vibration amplitude, and stretch rate of the vocal folds were comparable to those of living humans. Six months after the first experiment, the vocal parameters were reproduced, to suggest the long-term preservation potential of our NVP-embalming technique. On the other hand, neither voiced sound nor vocal fold vibration were observed in FA-embalmed larynges. This novel embalming technique could pioneer the next era to utilize embalmed cadavers for the examination of motion physiology in the human body.

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).

The traditional apprenticeship approach to surgical skill education for young surgeons has drastically changed to more systematic surgical training using cadavers. Cadavers fixed with formalin are not suitable for surgical training because of their associated health hazards and overhardening. Recently, we established a formalin-free soft preservation method for human cadavers using N-vinyl-2-pyrrolidone. Since 2012, 61 cadavers have been embalmed with pyrrolidone in our institution. Tissues of pyrrolidone-embalmed cadavers are soft and pliable, and their bodies can be preserved for as long as 37 months without any signs of corruption. In this review, we introduce our recent attempts to apply pyrrolidone-embalmed cadavers in surgical and medical procedure training, including endotracheal intubation, motion physiology of the vocal folds, laparoscopic surgery, endoscopic skull base surgery, and development of novel medical devices. Future research perspectives on pyrrolidone embalming are discussed.

The demand for cadavers for clinical skills training is increasing. However, conventional formalin-fixed bodies are often unsuitable for surgical training because the tissues become too hard. We recently developed a new formalin-free embalming method with N-vinyl-2-pyrrolidone (pyrrolidone) that has excellent fixative, disinfectant, and preservative properties, while still keeping tissues soft and pliable. In the study reported here we investigated the feasibility of laparoscopic manipulation using pyrrolidone-fixed cadavers. Donated cadavers were embalmed either with pyrrolidone (n = 7) or with formalin-containing fixative (n = 3). A laparoscopic 12-mm trocar was inserted into the umbilical region, and CO2 gas was insufflated. Intra-abdominal structures were observed with an endoscopic camera. In the pyrrolidone-embalmed cadavers, the abdomen remained soft and depressed. In addition, CO2 injection resulted in a marked expansion of the abdominal cavity, and it was possible to move the laparoscope freely in all directions. Clear endoscopic images of the abdominal viscera were obtained. The gallbladder and rectum were identified by grasping the surrounding organs with forceps. By contrast, in the formalin-fixed bodies, the abdominal wall was rigid, and it was difficult to move the laparoscope in the peritoneal space and observe structures in detail. The amount of CO2 and changes in abdominal diameter and circumference in response to CO2 injection were significantly larger in the pyrrolidone group. In conclusion, we successfully created a sufficient pneumoperitoneum state and obtained clear endoscopic images in the pyrrolidone-embalmed cadavers. Handling and dissection of the intra-abdominal structures with forceps closely replicated real-life surgery. These findings suggest the feasibility of laparoscopic training on cadavers embalmed with this pyrrolidone fixative.

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.

The development of formalin-free fixatives is an urgent issue in gross anatomy because of the health hazard and the tissue-hardening actions of formalin. We recently identified the fixative, antimicrobial, and preservative effects of N-vinyl-2-pyrrolidone (NVP), a precursor of the water-soluble macromolecular polymer polyvinylpyrrolidone, in animal experiments. The aim of the present study is to investigate whether NVP solution can be used as an alternative to formalin in human cadaveric dissection. Twelve donated cadavers were infused with NVP via the femoral and common carotid arteries using a peristaltic pump. Experienced teaching staff members in our department dissected the cadavers and examined their macroanatomical properties. The NVP-embalmed corpses showed no sign of decomposition or fungal growth. The bodies remained soft and flexible. Notably, the shoulder, elbow, wrist, phalangeal, hip, knee, cervical spine, and temporomandibular joints were highly mobile, almost equivalent to those of living individuals. The range of motion of most joints was greater in the NVP-fixed than formalin-fixed cadavers. Under the dermis, the subcutaneous fat was markedly reduced and the connective tissues were transparent, so the ligaments, cutaneous nerves, and veins were easily discernible. The abdominal wall and the visceral organs remained pliable and elastic, resembling those of fresh cadavers. The lungs, liver, and gastrointestinal tract were moveable in the thoracic and abdominal cavities and were readily isolated. NVP can be used successfully as a fixative and preservative solution for human cadavers; furthermore, NVP-embalmed bodies could be valuable for learning clinical skills and for training, and for developing innovative medical devices. Clin. Anat. 31:90-98, 2018. © 2017 Wiley Periodicals, Inc.

The development of formalin-free fixatives is an urgent issue in gross anatomy because of the health hazard and the tissue-hardening actions of formalin. We recently identified the fixative, antimicrobial, and preservative effects of N-vinyl-2-pyrrolidone (NVP), a precursor of the water-soluble macromolecular polymer polyvinylpyrrolidone, in animal experiments. The aim of the present study is to investigate whether NVP solution can be used as an alternative to formalin in human cadaveric dissection. Twelve donated cadavers were infused with NVP via the femoral and common carotid arteries using a peristaltic pump. Experienced teaching staff members in our department dissected the cadavers and examined their macroanatomical properties. The NVP-embalmed corpses showed no sign of decomposition or fungal growth. The bodies remained soft and flexible. Notably, the shoulder, elbow, wrist, phalangeal, hip, knee, cervical spine, and temporomandibular joints were highly mobile, almost equivalent to those of living individuals. The range of motion of most joints was greater in the NVP-fixed than formalin-fixed cadavers. Under the dermis, the subcutaneous fat was markedly reduced and the connective tissues were transparent, so the ligaments, cutaneous nerves, and veins were easily discernible. The abdominal wall and the visceral organs remained pliable and elastic, resembling those of fresh cadavers. The lungs, liver, and gastrointestinal tract were moveable in the thoracic and abdominal cavities and were readily isolated. NVP can be used successfully as a fixative and preservative solution for human cadavers; furthermore, NVP-embalmed bodies could be valuable for learning clinical skills and for training, and for developing innovative medical devices. Clin. Anat. 31:90-98, 2018. © 2017 Wiley Periodicals, Inc.

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.

The aldosterone/mineralocorticoid receptor system plays an important role in the long-term blood pressure control through Na homeostasis. Its overactivation has been implicated in salt-sensitive hypertension. Excessive salt intake augments the function of mineralocorticoid receptor, despite lowering circulating aldosterone levels, but the mechanism had long been elusive. Recently, Rac1, a member of Rho family small GTP-binding proteins, has emerged as a novel ligand-independent modulator of mineralocorticoid receptor activity. In this review, the roles of Rac1 in the pathogenesis of salt-sensitive hypertension and kidney injury have been summarized.Genetic engineering studies have highlighted the new aspects of Rac1 and its regulators in salt-sensitive hypertension and cardiac and renal disease. New evidence shows the essential roles of Rac1 in salt-evoked paradoxical mineralocorticoid receptor activation observed in salt-sensitive models and in renal tubular Na reabsorption through reduced nicotinamide-adenine dinucleotide phosphate oxidase-mediated oxidative stress or direct regulation of Na transporters.The emerging concept of 'ligand-independent aberrant mineralocorticoid receptor activation by Rac1' in the pathogenesis of salt-sensitive hypertension and kidney injury has been reviewed. Rac inhibition, in addition to mineralocorticoid receptor blockade and salt restriction, would be a new promising strategy for the treatment of salt-sensitive hypertension.

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.

Aldosterone has emerged as a deleterious hormone in the kidney, for example as a potent inducer of proteinuria. We identified the podocyte, the final filtration barrier in the glomerulus, as a novel target of aldosterone. Activation of the mineralocorticoid receptor (MR) in the podocyte disrupts the filtration barrier and induces proteinuria. Recent clinical and experimental studies have shown the efficacy of MR antagonism in reducing albuminuria in patients or rodent models of type 1 and type 2 diabetes. We assessed the pathogenic role of aldosterone in SHR/NDmcr-cp, a rat model of type 2 diabetes/metabolic syndrome. Podocyte injury and proteinuria were early manifestations of nephropathy in this model, and were exacerbated by high-salt feeding. Inappropriate activation of the aldosterone/MR system, possibly via adipocyte-derived aldosterone releasing factors, underlay the renal damage. Furthermore, we identified Rac1, a Rho family small GTPase, as a novel ligand-independent activator of MR. This alternative pathway of MR activation, indeed, contributed to podocyte injury in proteinuric kidney disease. In conclusion, MR can be activated by several different pathways, both aldosterone-dependently and -independently, leading to podocyte impairment and progression of proteinuric kidney disease. MR antagonists are promising anti-proteinuric drugs in diabetes, although hyperkalemia is a concern.

Recent clinical and experimental studies have shown that aldosterone is a potent inducer of proteinuria and that mineralocorticoid receptor (MR) antagonists confer efficient antiproteinuric effects. We identified glomerular epithelial cells (podocytes) as novel targets of aldosterone; activation of MR injures podocytes possibly via oxidative stress, resulting in disruption of glomerular filtration barrier, proteinuria, and progression of chronic kidney disease. We also demonstrated that SHR/cp, a rat model of metabolic syndrome, was susceptible to podocyte injury and proteinuria. Aldosterone excess caused by adipocyte-derived aldosterone-releasing factors was suggested to underlie the nephropathy. High salt intake augmented MR activation in the kidney and exacerbated the nephropathy. Furthermore, we identified an alternative pathway of MR activation by small GTPase Rac1. RhoGDIalpha knockout mice, a model with Rac1 activation in the kidney, showed albuminuria, podocyte injury, and glomerulosclerosis. Renal injury in the knockout mice was accompanied by enhanced MR signaling in the kidney despite normoaldosteronemia, and was ameliorated by an MR antagonist, eplerenone. Moreover, Rac-specific inhibitor significantly reduced the nephropathy, concomitantly with repression of MR activation. In vitro transfection studies provided direct evidence of Rac1-mediated MR activation. In conclusion, our findings suggest that MR activation plays a pivotal role in the pathogenesis of chronic kidney disease in metabolic syndrome, and that MR may be activated both aldosterone dependently (via aldosterone-releasing factors) and independently (via Rac1). MR antagonists are promising antiproteinuric drugs in metabolic syndrome, although long-term effects on renal outcomes, mortality, and safety need to be established.

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.

Aldosterone is traditionally viewed as a hormone regulating electrolyte and blood pressure homeostasis by acting on the distal nephron. Accumulating evidence suggests that aldosterone also plays pathogenetic roles in cardiovascular and renal injury. For example, aldosterone is a potent inducer of proteinuria. We demonstrated that podocyte injury underlies the pathogenesis of proteinuria in aldosterone-infused rats on a high salt diet. Mineralocorticoid receptor was detected in the podocytes in vivo and in vitro, and aldosterone caused induction of its effector kinase Sgk1, activation of NADPH oxidase and generation of reactive oxygen species. Selective aldosterone blocker eplerenone, as well as antioxidant tempol, ameliorated aldosterone-induced podocyte injury and proteinuria. Aldosterone was also involved in the podocyte damage and proteinuria of metabolic syndrome model SHR/NDmcr-cp. Adipocyte-derived aldosterone releasing factors were suggested to contribute to the aldosterone excess of this model. Furthermore, high salt diet markedly worsened the renal injury of SHR/NDmcr-cp. Although salt lowered serum aldosterone levels, it caused MR activation in the kidney. Accordingly, eplerenone dramatically improved the salt-evoked nephropathy. Taken together, aldosterone blockers can be an excellent therapeutic strategy for the treatment of podocyte injury, proteinuria, and cardiovascular and renal complications, not only in high aldosterone states but also in patients with activated MR signaling in the target tissue, whose circulating aldosterone level is not necessarily high. Addition of aldosterone blockers in patients treated with ACEIs or ARBs are also promising, because of "aldosterone breakthrough" phenomenon. Careful monitoring of hyperkalemia is necessary, especially in patients with impaired renal function.

The Hedgehog family of proteins are powerful morphogens mediating embryonic development as well as adult morphogenesis and carcinogenesis. For example, excess hedgehog activity has been implicated in basal cell carcinoma, medulloblastoma and rhabdomyosarcoma. More recently, hedgehog signalling has been implicated in angiogenesis. While hedgehog signalling in adult angiogenesis may constitute a simple recapitulation of that in embryonic development, it should be appreciated that Hedgehog signalling occurs in embryonic angiogenesis in different developmental contexts. This article reviews the role of Hedgehog signalling in both embryonic and postnatal vascular development. The temporal importance of a window of hedgehog dependent angiogenesis during development is emphasised and illustrated using a whole mouse embryo culture system.

Although embryonic development is inevitably affected by biophysical or biomechanical processes, it has yet to be elucidated to what extent molecular mechanisms of development are modulated by such physical factors. The hedgehog family, including Sonic hedgehog (Shh), is the most well-known morphogens involved in the developmental pattern formation of various organs, such as the nervous system, face, limbs, and skin appendages. There are several unique features in hedgehog signaling including long-range diffusion or positive and negative feedback loops, suggesting the possible modification of hedgehog signaling by biophysical or biomechanical factors. Especially, the period of embryonic day 8-10 is characterized by various biomechanically regulated processes in mouse development, such as axial rotation and vasculoangiogenesis. We executed a series of experiments using a mouse whole embryo culture system to investigate the biomechanical roles of hedgehog signaling during this period. In this review, we examine various examples in which biophysical and biomechanical aspects of hedgehog signaling in development are revealed, including our own data using the mouse whole embryo culture system.

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.

Hedgehog family was reported to be involved in murine yolk sac angiogenesis. However, it has not been clarified whether impaired angiogenesis under hedgehog signaling blockade is attributable to true defect in angiogenic process or just a sequel of earlier vasculogenic abnormalities. In the present study, we examined the effects of stage-specific inhibition of hedgehog cascade on vascular morphogenesis by applying cyclopamine or jervine to culture media of whole embryo culture system from embryonic days 8.0 until 9.5. Whole-mount immunostaining revealed that cyclopamine or jervine treatment in a narrow time window impaired angiogenic remodeling such as ramification into large and small branches and pericyte recruitment, although vasculogenesis was grossly normal. Molecular analyses suggest that indian hedgehog in the yolk sac endoderm regulates the induction of VEGF, Flk-1 and Notch-1. Our results indicate that hedgehog signaling is indispensable for mouse yolk sac angiogenesis, even when vasculogenesis is not perturbed.

Fetal alcohol syndrome (FAS) is a congenital anomaly attributable to prenatal maternal excessive intake of ethanol. The authors made a mammalian model of FAS by culturing mouse embryos with high ethanol for embryonic day 7.8 to 9.5 in the whole embryo culture system. The embryos exposed to high ethanol were smaller and less advanced in development than were the embryos in the control group and showed craniofacial abnormalities, such as a fusion defect of the neural tube. The expression patterns of CRABP-I and AP-2 as markers of the neural crest cells were mostly unchanged in the in situ hybridization. However, the density and area of the expression were decreased, possibly because of the death of the neural crest cells. The expression patterns of the Sonic hedgehog signaling cascade genes (Shh, Ptc-1 and Gli-1) were mostly unchanged in the in situ hybridization, but the quantitative expressions of Ptc-1 and Gli-1 were increased in real-time reverse transcriptase-polymerase chain reaction analyses, quite contrary to the findings of a previous study using chick embryos. These findings suggest Shh signaling also is involved in the pathogenesis of FAS in mammalian embryo, but in a mode different from that in the chick embryo.

We examined a 59-year-old woman with a capillary malformation (CM) in her thigh, which was serially excised. Interestingly, the remnant CM after the first excision was enlarged at the time of the second excision. To investigate whether this phenomenon was caused by mere passive expansion or regrowth, the CM specimens of the first operation (nonexpanded) and the second operation (expanded) were examined. Expressions of angiogenic genes Tie2 and Angiopoietin-1 were up-regulated within the expanded CM compared with the nonexpanded lesion, suggesting angiogenesis in the expanded CM. Expression pattern of the endothelial marker von Willebrand factor and the capillary densities were unchanged after the excision, suggesting that angiogenesis seen in the expanded CM resulted in reorganization of vascular networks. We consider that our data support a hypothesis that the expanded lesion in this case was caused by regrowth, not a passive expansion, of the CM.

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.

Podocyte injury is a major cause of proteinuria, a core component of chronic kidney disease. We reported that podocyte impairment underlied the early glomerulopathy in animal models of lifestyle-related diseases, such as hypertension and metabolic syndrome. Accumulating evidence suggests that overactivation of the aldosterone–mineralocorticoid receptor (MR) system has harmful effects on podocytes. We found that MR signaling was enhanced in such lifestyle-related diseases with podocyte injury and proteinuria, which were ameliorated by MR antagonist. Subsequent studies revealed that plasma aldosterone concentrations are not always increased in proteinuric conditions with renal MR activation, and the mechanisms of MR overactivation remained elusive. We recently identified a novel mechanism of Rac1-mediated podocyte impairment using RhoGDIα knockout mice; Rac1 potentiates the activity of MR in a ligand-independent manner, thereby accelerating podocyte injury. We demonstrated that the Rac1–MR pathway contributes to the ligand-independent aberrant MR activation in salt-sensitive hypertension and renal injury models. The importance of the RhoGDIα-Rac1-MR pathway in human glomerular disease is underscored by the findings that mutations in RhoGDIαgene cause nephrotic syndrome. Our results provide evidence that the Rac1-MR signal cascade as a novel therapeutic target for chronic kidney disease.

Diabetes mellitus accelerating atherosclerosis was associated with the enhanced glycoxidative modification of lipoproteins. LOX-1, the endothelial oxidized LDL receptor might be involved in the pathogenesis of diabetic atherosclerosis. In this study, we examined the vascular expression of LOX-1 in streptozotocin-induced diabetic rats. We found that LOX-1 was significantly increased in diabetic rat aorta compared with nondiabetic control. Immunohistochemistry revealed that the most distinctive staining of LOX-1 was in the endothelial cells, especially in the bifurcations of artery branches from aorta. In cultured aortic endothelial cells, diabetic rat serum and advanced glycation endproducts–BSA induced LOX-1 expression, while control rat serum along with high glucose did not. Applying a competitive inhibition assay, we found that LOX-1 ligand activity was accumulated in the diabetic rat serum, mainly in VLDL/LDL fractions. In addition, VLDL/LDL prominently increased LOX-1 among all the lipoprotein fractions of diabetic rat serum. In conclusion, diabetes markedly upregulated LOX-1 expression in the aortic endothelial cells. The enhanced glycoxidative modification of lipoproteins may contribute to the underlying mechanisms.

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.

Background. Aging is considered to modulate the salt sensitivity of blood pressure (BP), but its mechanisms have not been precisely elucidated. We investigated aging-related changes in salt sensitivity and their mechanisms in patients with essential hypertension.

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.

The authors report the rare existence of a family that includes an older sister with Pfeiffer syndrome and a younger sister with achondroplasia. Gene analysis of these patients showed a T341P mutation in the FGFR2 gene in the patient with Pfeiffer syndrome, and a G380R mutation in the FGFR3 gene in the patient with achondroplasia. Both mutations have been reported previously. Their parents had no mutation in either locus. This result suggests the possibility that there may be predisposing factors for different FGFR mutations.

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.

Objective To investigate tissue distribution and localization of the natriuretic peptide receptor (NPR) subtypes' messenger RNA (mRNA) and to compare their expression between stroke-prone spontaneously hypertensive rats (SHR-SP) and Wistar-Kyoto (WKY) rats. Methods Total RNA was extracted from organs of SHR-SP and WKY rats aged 13 weeks. The mRNA level was examined by RNase protection assay. The localization of the transcripts was determined by in-situ hybridization. Results In SHR-SP aged 13 weeks, NPR-A was expressed most abundantly in the adrenal gland, lung and aorta, in that order. NPR-B was expressed highly in the uterus and ovary, and also in the lung, adrenal, and brain. NPR-C was expressed predominantly in the atrium and mesentery, less so in the lung, vein, and kidney. In the adrenal gland, NPR-A was expressed mainly in zona glomerulosa cells. In the atrium, NPR-C was expressed throughout the wall. In the mesentery, NPR-C mRNA was detected mainly in adipocytes. In the kidney, NPR-C was found predominantly in podocytes. Whereas the levels of expression of NPR subtypes in most tissues examined did not differ between SHR-SP and WKY rats, the NPR-C mRNA level was significantly greater in the kidneys of SHR-SP than it was in those of WKY rats. Conclusions These results indicated that each NPR subtype had a distinct tissue distribution pattern and that the expression of NPR-C in the kidneys of SHR-SP was greater than that in the kidneys of WKY rats.

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.

Two different strains of the spontaneously hypertensive rat (SHR) exist, either with or without a Cd36 mutation. In the F2 population derived from a cross between these two SHR strains, the mutant Cd36 allele was tightly linked to differences in metabolic phenotypes but not to those in fat pad weight. This suggested the existence of another crucial mutation related to adiposity. Linkage analysis of this F2 population showed a significant linkage between the rat chromosome 1 region (D1Rat240-D1Wox28) and fat pad weight. By integrating both positional and expression information, we identified a donor splice site mutation in the gene for solute carrier family 22 member 18 (Slc22a18) in SHR with reduced fat pad weight. This mutation was located at the linkage peak with a maximum logarithm of odds score of 7.7 and caused skipping of the whole exon 9 that results in a complete loss of a whole membrane-spanning region of the rat Slc22a18 protein. Slc22a18 mRNA was abundantly expressed in isolated adipocytes and in a differentiation-dependent manner in 3T3-L1 cells. Knockdown of the Slc22a18 mRNA via infection of adenoviral vectors markedly inhibited both triglyceride accumulation and adipocyte differentiation in 3T3-L1 cells. By contrast, overexpression of the Slc22a18 mRNA had the opposite effects. These results reveal a novel link between Slc22a18 and fat accumulation and suggest that this gene could be a new therapeutic target in obesity.