Yoshitaka Okuhara

Impact of dietary iron restriction on the development of monocrotaline-induced pulmonary vascular remodeling and right ventricular failure in rats.

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling leading to right ventricular (RV) failure. Recently, iron deficiency is reported to be prevalent in patients with PH. However, the mechanism by which iron deficiency occurs in patients with PH remains unknown. Here, we investigated the effects of dietary iron restriction on the development of monocrotaline-induced pulmonary vascular remodeling and the involved mechanisms. Male Sprague-Dawley rats were subcutaneously injected with monocrotaline (60mg/kg). Afterwards, monocrotaline-injected rats were randomly divided into two groups and were given a normal diet (n=6) or an iron-restricted diet (n=6) for 4weeks. Saline-injected rats given a normal diet were served as controls (n=6). Monocrotaline-injected rats showed pulmonary vascular remodeling, increased RV pressure, RV hypertrophy, and decreased RV ejection fraction, followed by RV failure after 4weeks. In contrast, iron restriction attenuated the development of pulmonary vascular remodeling and RV failure. Of interest, expression of cellular iron transport protein, transferrin receptor 1 was increased in the pulmonary remodeled artery and the failing right ventricle of monocrotaline-injected rats, as compared with the controls. Moreover, a key regulator of iron homeostasis, hepcidin gene expression was increased in the failing right ventricle of monocrotaline-injected rats. Iron restriction attenuated the development of monocrotaline-induced pulmonary vascular remodeling and RV failure. Cellular iron transport might be involved in the pathophysiology of PH and PH induced RV failure.

Increased renal iron accumulation in hypertensive nephropathy of salt-loaded hypertensive rats.

Although iron is reported to be associated with the pathogenesis of chronic kidney disease, it is unknown whether iron participates in the pathophysiology of nephrosclerosis. Here, we investigate whether iron is involved in the development of hypertensive nephropathy and the effects of iron restriction on nephrosclerosis in salt- loaded stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were given either a normal or high-salt diet for 8 weeks. Another subset of SHRSP were fed a high-salt with iron-restricted diet. SHRSP given a high-salt diet developed severe hypertension and nephrosclerosis. As a result, survival rate was decreased after 8 weeks diet. Importantly, massive iron accumulation and increased iron content were observed in the kidneys of salt-loaded SHRSP, along with increased superoxide production, urinary 8-Hydroxy-2′-deoxyguanosine excretion, and urinary iron excretion; however, these changes were markedly attenuated by iron restriction. Of interest, expression of cellular iron transport proteins, transferrin receptor 1 and divalent metal transporter 1, was increased in the tubules of salt-loaded SHRSP. Notably, iron restriction attenuated the development of severe hypertension and nephrosclerosis, thereby improving survival rate in salt-loaded SHRSP. Taken together, these results suggest a novel mechanism by which iron plays a role in the development of hypertensive nephropathy and establish the effects of iron restriction on salt-induced nephrosclerosis.

Dietary iron restriction prevents further deterioration of renal damage in a chronic kidney disease rat model.

Objective: Iron accumulation is associated with the pathogenesis of chronic kidney disease (CKD). However, little is known about the effects of isolated iron restriction against CKD. We have recently reported that iron restriction prevents the development of renal damage in the well established 5/6 nephrectomy rat model of CKD. Herein, we investigated the therapeutic effects of iron restriction on preexisting hypertension and renal damage in a rat model of CKD. Methods: CKD was induced by 5/6 nephrectomy in Sprague–Dawley rats. After surgery, 5/6 nephrectomized rats were given an iron-restricted diet from 1 day to 16 weeks for prevention protocol or from 8 to 16 weeks for rescue protocol. Other CKD rats were given a normal diet. Results: At 16 weeks after surgery, CKD rats developed hypertension and renal damage. Early intervention with iron restriction prevented the development of hypertension and vascular remodeling. By contrast, late intervention with iron restriction did not remarkably ameliorate preexisting hypertension and vascular remodeling in CKD rats. On the contrary, late intervention with iron restriction prevented further progression of preexisting renal damage in CKD rats. Interestingly, iron restriction led to increased urinary sodium and decreased urinary potassium excretions in CKD rats. Moreover, iron restriction markedly attenuated renal expression of nuclear mineralocorticoid receptor and Rac1 activity in CKD rats. Conclusion: Iron restriction prevented further deterioration of preexisting renal damage. The beneficial effects of iron restriction on renal damage seem to be associated with inhibition of renal mineralocorticoid receptor signaling.

Intravenous Salt Supplementation With Low-Dose Furosemide for Treatment of Acute Decompensated Heart Failure

BACKGROUND Theoretically, salt supplementation should promote diuresis through increasing the glomerular filtration rate (GFR) during treatment of acute decompensated heart failure (ADHF) even with low-dose furosemide; however, there is little evidence to support this idea. METHODS AND RESULTS This was a prospective, randomized, open-label, controlled trial that compared the diuretic effectiveness of salt infusion with that of glucose infusion supplemented with low-dose furosemide in 44 consecutive patients with ADHF. Patients were randomly administered 1.7% hypertonic saline solution supplemented with 40 mg furosemide (salt infusion group) or glucose supplemented with 40 mg furosemide (glucose infusion group). Our major end points were 24-hour urinary volume and GFR. Urinary volume was greater in the salt infusion group than in the glucose infusion group (2,701 ± 920 vs 1,777 ± 797 mL; P < .001). There was no significant difference in the estimated GFR at baseline. Creatinine clearance for 24 h was greater in the salt infusion group than in the glucose infusion group (63.5 ± 52.6 vs 39.0 ± 26.3 mL min(-1) 1.73 m(-2); P = .048). CONCLUSIONS Salt supplementation rather than salt restriction evoked favorable diuresis through increasing GFR. The findings support an efficacious novel approach of the treatment of ADHF.

Association between renal iron accumulation and renal interstitial fibrosis in a rat model of chronic kidney disease

Iron accumulation is associated with the pathophysiology of chronic kidney disease (CKD). Renal fibrosis is a final common feature that contributes to the progression of CKD; however, little is known about the association between renal iron accumulation and renal interstitial fibrosis in CKD. Here we investigate the effects of iron chelation on renal interstitial fibrosis in a rat model of CKD. CKD was induced by 5/6 nephrectomy in Sprague–Dawley rats. At 8 weeks after operation, 5/6 nephrectomized rats were administered an oral iron chelator, deferasirox (DFX), in chow for 8 weeks. Other CKD rats were given a normal diet. Sham-operative rats given a normal diet served as a control. CKD rats exhibited hypertension, glomerulosclerosis and renal interstitial fibrosis. Iron chelation with DFX did not change hypertension and glomerulosclerosis; however, renal interstitial fibrosis was attenuated in CKD rats. Consistent with these findings, renal gene expression of collagen type III and transforming growth factor-β was increased in CKD rats compared with the controls, while iron chelation suppressed these increments. In addition, a decrease in vimentin along an increase in E-cadherin in renal gene expression was observed in CKD rats with iron chelation. CKD rats also showed increased CD68-positive cells in the kidney, whereas its increase was attenuated by iron deprivation. Similarly, increased renal gene expression of CD68, tumor necrosis factor-α and monocyte chemoattractant protein-1 was suppressed in CKD rats with iron chelation. Renal iron accumulation seems to be associated with renal interstitial fibrosis in a rat model of CKD.

Iron restriction inhibits renal injury in aldosterone/salt-induced hypertensive mice

Excess iron is associated with the pathogenesis of several renal diseases. Aldosterone is reported to have deleterious effects on the kidney, but there have been no reports of the role of iron in aldosterone/salt-induced renal injury. Therefore, we investigated the effects of dietary iron restriction on the development of hypertension and renal injury in aldosterone/salt-induced hypertensive mice. Ten-week-old male C57BL/6J mice were uninephrectomized and infused with aldosterone for four weeks. These were divided into two groups: one fed a high-salt diet (Aldo) and the other fed a high-salt with iron-restricted diet (Aldo-IR). Vehicle-infused mice without a uninephrectomy were also divided into two groups: one fed a normal diet (control) and the other fed an iron-restricted diet (IR) for 4 weeks. As compared with control and IR mice, Aldo mice showed an increase in both systolic blood pressure and urinary albumin/creatinine ratio, but these increases were reduced in the Aldo-IR group. In addition, renal histology revealed that Aldo mice exhibited glomerulosclerosis and tubulointerstitial fibrosis, whereas these changes were attenuated in Aldo-IR mice. Expression of intracellular iron transport protein transferrin receptor 1 was increased in the renal tubules of Aldo mice compared with control mice. Dietary iron restriction attenuated the development of hypertension and renal injury in aldosterone/salt-induced hypertensive mice.

Expression of interleukin-33 and ST2 in nonrheumatic aortic valve stenosis

Thenumberof patientswithnonrheumatic aortic valve stenosis (NRAS) is increasing in recent years. NR-AS generally progresses without symptoms, and severe NR-AS is related to increased morbidity and mortality. The effective treatment for severe NR-AS is aortic valve replacement [1]. Many clinical trials of medical therapy have been carried out to prevent the progression of NR-AS; however, there is still a controversy in terms of an effective medical strategy to retard the progression of NR-AS. Thus, it is extremely important to elucidate the molecular pathophysiology of NR-AS to find the prevention strategies of NR-AS. Interleukin (IL)-33 is a recently identified cytokine of the IL-1 family and a ligand for ST2 [2]. IL-33 has immunomodulatory functions. A recent study has shown that IL-33 and ST2 are crucial systems that control atherosclerosis through the inhibition of inflammatory response [3]. Although inflammationplays a pivotal role in thepathophysiologyof NR-AS, the role of IL-33 and ST2 inNR-AS has not been investigated.We, therefore, hypothesized that IL-33 and ST2 systemsmight be associated with the pathophysiology of NR-AS. In the present study, we investigated the expression and cellular localization of IL-33 and ST2 in human NR-AS and aortic regurgitation (AR) valves. Aortic valve samples were collected from 67 patients undergoing valve replacement surgery (40 NR-AS and 27 AR). Patients with significant diseases such as autoimmune diseases, infections, cancer, or renal failure were excluded. All of our protocols were approved by the ethics committee of Hyogo College of Medicine. Aortic valve tissues were collected at the time of surgical valve replacement. They were immediately fixed with 4% paraformaldehyde for paraffin embedding. In addition, aortic valve tissues were frozen in liquid nitrogen and stored at −80 °C for Western blot analysis. Sections measuring 4 μm from paraffin-embedded samples were stained with hematoxylin–eosin and were incubated with either a primary antihuman IL-33 antibody (R&D, dilution 1:1000), a primary anti-human ST2 antibody (R&D, dilution 1:1000), a primary anti-human CD68 antibody (Dako, dilution1:200) formacrophages, a primaryanti-human α-smooth muscle actin (SMA) antibody (Dako, dilution 1:200) for smooth muscle cells or myofibroblasts, and a primary anti-human von Willebrand factor (vWF) antibody (Dako, dilution 1:1000) for endothelial cells. Immunostainswere visualizedwith the use of an avidin–biotin peroxidase conjugate and 3,3′-diaminobenzidine substrate. Every section was counterstained with hematoxylin. Aortic valve tissues were homogenizedwith ice-cold lysis buffer as previously described [4]. Protein extracts from aortic valves were separated by SDS-PAGE and transferred onto PVDF membranes. The expression levels of molecules were detected by an enhanced chemiluminescence kit (Thermo Scientific). The antibodies used were against anti-human IL-33 (R&D, dilution 1:1000), anti-human ST2 (R&D, dilution 1:1000), and antihuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, dilution 1:1000). Values are reported as means±SD. Statistical analysis was performed using Mann–Whitney U test. Categorical variables were compared using chi-square statistics. Differences were considered significant at pb0.05. The clinical characteristics of patientswith NR-AS and ARwere shown in Table 1. There was no significant difference in any parameters between NR-AS andAR valves. Initially,we investigated IL-33 and ST2 expression in aortic valves byWestern blot analysis. IL-33 was expressed in both NR-AS and AR valves, and the expression levels were not significantly different betweenNR-AS andARvalves (Fig.1A). On theotherhand, ST2 expression was markedly up-regulated in NR-AS compared with AR valves. The molecular weight of ST2 was approximately 50 kDa, but not 100 kDa (Fig. 1B). Next, we assessed IL-33 and ST2 expression by histological analyses. Hematoxylin–eosin staining showed many inflammatory cells closed to calcified and neovascular lesions in NR-AS valves (Fig. 2A). In contrast, neither inflammatory cells, nor calcified lesions, nor neovascular International Journal of Cardiology 168 (2013) 529–630

Interleukin-18 gene deletion protects against sepsis-induced cardiac dysfunction by inhibiting PP2A activity

BACKGROUND Interleukin-18 (IL-18) neutralization protects against lipopolysaccharide (LPS)-induced injuries, including myocardial dysfunction. However, the mechanism is yet to be fully elucidated. The aim of the present study was to determine whether IL-18 gene deletion prevents sepsis-induced cardiac dysfunction and to elucidate the potential mechanisms underlying IL-18-mediated cardiotoxicity by LPS. METHODS AND RESULTS Ten-week-old male wild-type (WT) and IL-18 knockout (IL-18 KO) mice were intraperitoneally administered LPS. Serial echocardiography showed better systolic pump function and less left ventricular (LV) dilatation in LPS-treated IL-18 KO mice compared with those in LPS-treated WT mice. LPS treatment significantly decreased the levels of phospholamban (PLN) and Akt phosphorylation in WT mice compared with those in saline-treated WT mice, while the LPS-induced decrease in the phosphorylation levels was attenuated in IL-18 KO mice compared with that in WT mice. IL-18 gene deletion also attenuated an LPS-induced increase of type 2 protein phosphatase 2A (PP2A) activity, a molecule that dephosphorylates PLN and Akt. There was no difference in type 1 protein phosphatase (PP1) activity. To address whether IL-18 affects PLN and Akt phosphorylation via PP2A activation in cardiomyocytes, rat neonatal cardiac myocytes were cultured and stimulated using 100ng/ml of recombinant rat IL-18. Exogenous IL-18 decreased the level of PLN and Akt phosphorylation in cardiomyocytes. PP2A activity but not PP1 activity was increased by IL-18 stimulation in cardiomyocytes. CONCLUSIONS IL-18 plays a pivotal role in advancing sepsis-induced cardiac dysfunction, and the mechanisms underlying IL-18-mediated cardiotoxicity potentially involve the regulation of PLN and Akt phosphorylation through PP2A activity.

Transferrin Receptor 1 in Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

BACKGROUND Iron is associated with the pathophysiology of several cardiovascular diseases, including pulmonary hypertension (PH). In addition, disrupted pulmonary iron homeostasis has been reported in several chronic lung diseases. Transferrin receptor 1 (TfR1) plays a key role in cellular iron transport. However, the role of TfR1 in the pathophysiology of PH has not been well characterized. In this study, we investigate the role of TfR1 in the development of hypoxia-induced pulmonary vascular remodeling. METHODS PH was induced by exposing wild-type (WT) mice and TfR1 hetero knockout mice to hypoxia for 4 weeks and evaluated via assessment of pulmonary vascular remodeling, right ventricular (RV) systolic pressure, and RV hypertrophy. In addition, we assessed the functional role of TfR1 in pulmonary artery smooth muscle cells in vitro. RESULTS The morphology of pulmonary arteries did not differ between WT mice and TfR1 hetero knockout mice under normoxic conditions. In contrast, TfR1 hetero knockout mice exposed to 4 weeks hypoxia showed attenuated pulmonary vascular remodeling, RV systolic pressure, and RV hypertrophy compared with WT mice. In addition, the depletion of TfR1 by RNA interference attenuated human pulmonary artery smooth muscle cells proliferation induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. CONCLUSIONS These results suggest that TfR1 plays an important role in the development of hypoxia-induced pulmonary vascular remodeling.

Interleukin-18 disruption suppresses hypoxia-induced pulmonary artery hypertension in mice

Article history: Received 31 July 2015 Received in revised form 24 September 2015 Accepted 28 September 2015 Available online 03 October 2015 atria, RV free wall was removed from left ventricle (LV). Each ventricle was weighed and the ratio of RV weight to LV plus septum weight (RV/LV + Sp) was calculated. Lungs were fixed with 4% buffered paraformaldehyde for 24 h, embedded in paraffin, and cut into 4-μm-thick sections. Hematoxylineosin (HE) and Elastica van Gieson (EVG) stainings were performed using standard protocols. Immunofluorescence staining was performed using a primary anti-α-smooth muscle actin (α-SMA) mouse antibody