Bilateral renal subcapsular hematoma caused by polyarteritis nodosa: a case report.

Polyarteritis nodosa, which is a systemic vasculitis of small- and medium-sized arteries, can cause arterial aneurysms in various organs, sometimes resulting in aneurysm rupture and hemorrhage. A kidney is one of the major targets of polyarteritis nodosa. Here, we report a 73-year-old woman who presented with sudden-onset high fever, diarrhea, and renal injury with bilateral renal subcapsular hematoma shown on contrast-enhanced computed tomography scan. She did not have trauma and significant medical history other than breast cancer in remission. Serological and immunological tests except for anti-Sjögren's syndrome-A and anti-Sjögren's syndrome-B were all negative. Digital subtraction angiography revealed bilateral intrarenal micro aneurysms, which allowed us to diagnose the patient with polyarteritis nodosa. As continuous monitoring of bilateral intrarenal hematoma by ultrasonography and computed tomography scan did not detect progression of intrarenal hemorrhage and extra renal hematoma, transcatheter arterial embolization and nephrectomy were not performed. Although hemodialysis therapy was required temporarily for acute kidney injury with anuria, her general condition and kidney function remarkably improved after receiving systemic immunosuppressive therapy with corticosteroids and cyclophosphamide. In conclusion, this is a rare case of polyarteritis nodosa manifesting as spontaneous bilateral subcapsular renal hemorrhage with deteriorated renal function, which was successfully treated with immunosuppressive therapy.

α1-Adrenergic receptor mediates adipose-derived stem cell sheet-induced protection against chronic heart failure after myocardial infarction in rats.

Cell-based therapy using adipose-derived stem cells (ADSCs) has emerged as a novel therapeutic approach to treat heart failure after myocardial infarction (MI). The purpose of this study was to determine whether inhibition of α1-adrenergic receptors (α1-ARs) in ADSCs attenuates ADSC sheet-induced improvements in cardiac functions and inhibition of remodeling after MI. ADSCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ADSCs, we determined the mRNA levels of vascular endothelial growth factor (VEGF)-A and α1-AR under normoxia or hypoxia and the effects of norepinephrine and an α1-blocker, doxazosin, on the mRNA levels of angiogenic factors. Hypoxia increased α1-AR and VEGF mRNA levels in ADSCs. Norepinephrine further increased VEGF mRNA expression under hypoxia; this effect was abolished by doxazosin. Tube formation of human umbilical vein endothelial cells was promoted by conditioned media of ADSCs treated with the α1 stimulant phenylephrine under hypoxia but not by those of ADSCs pretreated with phenylephrine plus doxazosin. In in vivo studies using rats with MI, transplanted ADSC sheets improved cardiac functions, facilitated neovascularization, and suppressed fibrosis after MI. These effects were abolished by doxazosin treatment. Pathway analysis from RNA sequencing data predicted significant upregulation of α1-AR mRNA expression in transplanted ADSC sheets and the involvement of α1-ARs in angiogenesis through VEGF. In conclusion, doxazosin abolished the beneficial effects of ADSC sheets on rat MI hearts as well as the enhancing effect of norepinephrine on VEGF expression in ADSCs, indicating that ADSC sheets promote angiogenesis and prevent cardiac dysfunction and remodeling after MI via their α1-ARs.

Protective Effects of Topiroxostat on an Ischemia-Reperfusion Model of Rat Hearts.

BACKGROUND: Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion. CONCLUSIONS: Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.

Erlotinib attenuates the progression of chronic kidney disease in rats with remnant kidney.

Background: Increasing evidence indicates that epidermal growth factor receptor (EGFR) has a pathogenic role in renal fibrosis. Currently no effective treatment can completely halt the progression of chronic kidney disease (CKD). This study was undertaken to investigate the renoprotective effects of erlotinib, a tyrosine kinase inhibitor that can block EGFR activity in the progression of CKD and the mechanisms involved. Methods: Sprague Dawley rats with 5/6 nephrectomy were administered either erlotinib or vehicle from 2 weeks after surgery and for a period of 8 weeks. Blood pressure, proteinuria and serum creatinine were measured periodically. Renal morphological investigations were performed at sacrifice. In vitro, we used normal human mesangial cells (NHMCs) and human proximal tubular cells to investigate the inhibitory effects of erlotinib on renal fibrosis-associated signaling pathways by western blotting. Results: Erlotinib treatment significantly blunted the progression of CKD as evidenced by reduced levels of serum creatinine, proteinuria and renal cortical profibrogenic genes and scores of glomerulosclerosis and tubulointerstitial damage. Tubulointerstitial macrophage infiltration and multiple pro-inflammatory cytokine gene expression levels were also attenuated by erlotinib treatment. In vitro, heparin-binding epidermal growth factor-like growth factor-induced Akt and extracellular-regulated kinase (ERK) 1/2 activation in normal human mesangial cells and human proximal tubular cells was inhibited by pretreatment with erlotinib. Conclusions: EGFR blocking by erlotinib protected against renal fibrosis in 5/6 nephrectomized rats via inhibition of Akt and ERK 1/2 signaling pathways, which are associated with renal fibrosis. Erlotinib also has anti-inflammatory properties, which may contribute to its renoprotective effects. Erlotinib represents a potential novel therapeutic strategy for the treatment of CKD.

Smooth muscle cell sheet transplantation preserve cardiac function and minimize cardiac remodeling in a rat myocardial infarction model.

BACKGROUND: We examined whether a vascular smooth muscle cell (SMC) sheet is effective in the treatment of a rat myocardial infarction (MI) model. METHODS: We examined the effect of SMC sheet on the cardiac function and cardiac remodeling in a rat MI model in comparison with their effect of dermal fibroblast (DFB) sheet in vivo. Furthermore, we estimated the apoptosis and secretion of angiogenic factor of SMC under hypoxic condition in comparison with DFB. Seven days after MI, monolayer cell sheets were transplanted on the infarcted area (SMC transplantation group, SMC-Tx; DFB transplantation group, DFB-Tx; no cell sheet transplantation group, Untreated; neither MI nor cell sheet transplantation group, Sham). We evaluated cardiac function by echocardiogram, degree of cardiac remodeling by histological examination, and secretion of angiogenic growth factor by enzyme immunoassay. RESULTS: Twenty-eight days after transplantation, SMC-Tx showed the following characteristics compared with the other groups: 1) significantly greater fractional area shortening (SMC-Tx, 32.3 ± 2.1 %; DFB-Tx, 23.3 ± 2.1 %; untreated, 25.1 ± 2.6 %), 2) suppressed left ventricular dilation, smaller scar expansion, and preserved wall thickness of the area at risk and the posterior wall, 3) decreased fibrosis, preserved myocardium in the scar area, and greater number of arterioles in border-zone, 4) tight attachment of SMC sheets on the scarred myocardium, and less apoptotic cell death. In in vitro experiments, SMCs secreted higher amounts of basic fibroblast growth factor (SMC, 157.7 ± 6.4 pg/ml; DFB, 3.1 ± 1.0 pg/ml), and showed less apoptotic cell death under hypoxia. CONCLUSIONS: Our results illustrate that transplantation of SMC sheets inhibited the progression of cardiac remodeling and improve cardiac function. These beneficial effects may be due to superior SMC survival.

Nimesulide, a cyclooxygenase-2 selective inhibitor, suppresses obesity-related non-alcoholic fatty liver disease and hepatic insulin resistance through the regulation of peroxisome proliferator-activated receptor γ.

Cyclooxygenase (COX)-2 selective inhibitors suppress non-alcoholic fatty liver disease (NAFLD); however, the precise mechanism of action remains unknown. The aim of this study was to examine how the COX-2 selective inhibitor nimesulide suppresses NAFLD in a murine model of high-fat diet (HFD)­induced obesity. Mice were fed either a normal chow diet (NC), an HFD, or HFD plus nimesulide (HFD-nime) for 12 weeks. Body weight, hepatic COX-2 mRNA expression and triglyceride accumulation were significantly increased in the HFD group. Triglyceride accumulation was suppressed in the HFD-nime group. The mRNA expression of hepatic peroxisome proliferator-activated receptor Î³ (PPARγ) and the natural PPARγ agonist 15-deoxy-Δ12,14-prostaglandin J2 (15d­PGJ2) were significantly increased in the HFD group and significantly suppressed in the HFD-nime group. Glucose metabolism was impaired in the HFD group compared with the NC group, and it was significantly improved in the HFD-nime group. In addition, the plasma insulin levels in the HFD group were increased compared with those in the NC group, and were decreased in the HFD-nime group. These results indicate that HFD-induced NAFLD is mediated by the increased hepatic expression of COX-2. We suggest that the production of 15d-PGJ2, which is mediated by COX-2, induces NAFLD and hepatic insulin resistance by activating PPARγ. Furthermore, the mRNA expression of tissue inhibitor of metalloproteinases-1 (TIMP­1), procollagen-1 and monocyte chemoattractant protein-1 (MCP-1), as well as the number of F4/80-positive hepatic (Kupffer) cells, were significantly increased in the HFD group compared with the NC group, and they were reduced by nimesulide. In conclusion, COX-2 may emerge as a molecular target for preventing the development of NAFLD and insulin resistance in diet-related obesity.

Anti-Phospholipase A2 Receptor (PLA2R) Antibody and Glomerular PLA2R Expression in Japanese Patients with Membranous Nephropathy.

The phospholipase A2 receptor (PLA2R) is the major target antigen (Ag) in idiopathic membranous nephropathy (IMN). Recently, several types of immunoassay systems for anti-PLA2R antibody (Ab) have been developed. However, the correlation of serum anti-PLA2R Abs and glomerular expression of PLA2R Ag, and their association with clinicopathological characteristics have yet to be proven in Japanese patients. We examined serum anti-PLA2R Abs by both ELISA and cell-based indirect immunofluorescence assay (CIIFA), and glomerular PLA2R expression by immunofluorescence (IF) in 59 biopsy-proven MN patients including IMN (n = 38) and secondary MN (SMN) (n = 21). In this study, anti-PLA2R Abs were present in 50% of IMN patients, but was absent in SMN patients. The concordance rate between ELISA and CIIFA was 100%. Serum IgG levels were significantly lower in anti-PLA2R Ab-positive patients. Serum albumin levels correlated inversely with serum anti-PLA2R Ab titers. The prevalence and intensity of glomerular staining for IgG4 by IF were significantly higher in anti-PLA2R Ab-positive patients than in -negative patients. Glomerular PLA2 Ag expression evaluated by IF was positive in 52.6% of IMN patients, but was absent in SMN patients. The concordance rate between the prevalence of glomerular PLA2R Ag expression and anti-PLA2R Ab was 84.2%. The prevalence of anti-PLA2R Abs measured by ELISA/CIIFA was equivalent to previous Japanese studies evaluated using Western blotting. These analyses showed an excellent specificity for the diagnosis of IMN, and anti-PLA2R positivity was associated with some clinicopathological features, especially glomerular IgG4-dominant deposition.

Therapeutic effects and mechanism of conditioned media from human mesenchymal stem cells on anti-GBM glomerulonephritis in WKY rats.

Recent studies have demonstrated that conditioned media derived from mesenchymal stem cells (MSC-CM) have therapeutic effects in various experimental diseases. However, the therapeutic mechanism is not fully understood. In the present study, we investigated the therapeutic effects and mechanism of MSC-CM in experimental antiglomerular basement membrane glomerulonephritis. We administered either MSC-CM or vehicle from day 0 to day 10 after the induction of nephrotoxic serum nephritis in Wistar-Kyoto rats. In vitro, we analyzed the effects of MSC-CM on TNF-α-mediated cytokine production in cultured normal human mesangial cells, proximal tubular (HK-2) cells, human umbilical vein endothelial cells, and monocytes (THP-1 and peripheral blood mononuclear cells). Compared with vehicle treatment, MSC-CM treatment improved proteinuria and renal dysfunction. Histologically, MSC-CM-treated rats had reduced crescent formation and glomerular ED1(+) macrophage infiltration and increased glomerular ED2(+) macrophage infiltration. Increased serum monocyte chemoattractant protein (MCP)-1 levels were observed in MSC-CM-treated rats. Renal cortical mRNA expression levels of proinflammatory cytokines, such as TNF-α and IL-6, and of the T helper cell 1 cytokine interferon-γ were greatly decreased by MSC-CM treatment. In vitro, pretreatment with MSC-CM blocked TNF-α-mediated IL-8 release in normal human mesangial cells and HK-2 cells. TNF-α-mediated MCP-1 release was enhanced by pretreatment with MSC-CM in human umbilical vein endothelial cells and HK-2 cells and was strikingly enhanced in THP-1 cells. Stimulation of peripheral blood mononuclear cells with a combination of MCP-1 and IL-4 enhanced the expression of M2-associated genes compared with IL-4 alone. We demonstrated that MSC-CM had therapeutic effects in experimental antiglomerular basement membrane glomerulonephritis that were mediated through anti-inflammatory effects that were partly due to acceleration of M2 macrophage polarization, which might be mediated by MCP-1 enhancement.

Streptococcal Infection-related Nephritis (SIRN) Manifesting Membranoproliferative Glomerulonephritis Type I.

We herein report the case of an 18-year-old boy who developed nephrotic syndrome and hypertension after upper airway inflammation. Post-streptococcal acute glomerulonephritis was diagnosed on the basis of a high antistreptolysin O titer, hypocomplementemia, proteinuria, and microscopic hematuria. A renal biopsy was performed due to persistent proteinuria, and the pathological diagnosis was membranoproliferative glomerulonephritis (MPGN) type I. Glomeruli showed positive staining for nephritis-associated plasmin receptor (NAPlr), a nephritogenic group A streptococcal antigen, and plasmin activity was found in a similar distribution as NAPlr deposition. This rare case of streptococcal infection-related nephritis (SIRN) manifesting MPGN type I supports the histological diversity of SIRN.

Adipose stem cell sheets improved cardiac function in the rat myocardial infarction, but did not alter cardiac contractile responses to ß-adrenergic stimulation.

Adipose stem cells (ASCs) are a source of regenerative cells available for autologous transplantation to hearts. We compared protective actions of ASC sheets on rat myocardial infarction (MI) in comparison with those of skeletal myoblast cell sheets. Their effects on infarcted hearts were evaluated by biological, histochemical as well as physiological analyses. ASC sheets secreted higher concentrations of angiogenic factors (HGF, VEGF, and bFGF; P < 0.05) under normoxic and hypoxic conditions than those of myoblast cell sheets, associated with reduction of cell apoptosis (P < 0.05). Like myoblast cell sheets, ASC sheets improved cardiac function (P < 0.05) and decreased the plasma level of ANP (P < 0.05) in MI hearts. ASC sheets restored cardiac remodeling characterized by fibrosis, cardiac hypertrophy and impaired angiogenesis (P < 0.05), which was associated with increases in angiogenic factors (P < 0.05). In isolated perfused rat hearts, ASC sheets improved both systolic and diastolic functions, which was comparable to cardiac functions of myoblast cell sheets, while both cell sheets failed to restore cardiac contractile response to either isoproterenol, pimobendan or dibutyryl cAMP. These results indicated that ASC sheets improved cardiac function and remodeling of MI hearts mediated by their paracrine action and this improvement was comparable to those by myoblast cell sheets.

Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD that encodes uromodulin. Topiroxostat, a novel non-purine analog, selectively inhibits xanthine oxidase and reduces the serum uric acid levels and the urinary albuminuria. METHODS: Genomic DNA of a patient was extracted from peripheral white blood. Exons and flanking sequences of UMOD were amplified by PCR with primers. Mutation analysis was performed by direct sequencing of the PCR products. The wild-type and mutant uromodulin were expressed in HEK293 cells and analyzed by western blotting, immunoprecipitation, immunofluorescence, and flow cytometry. RESULTS: We identified an FJHN patient who carried a novel UMOD mutation G335A (C112Y). The levels of both cytosolic and secreted C112Y protein were significantly decreased compared with the wild-type, whereas the level of ubiquitination was higher in C112Y than that in the wild type. The half-life of C112Y was shortened and it was restored by a proteasome inhibitor MG132. Immunofluorescence revealed decreased levels of C112Y in the Golgi apparatus and on the plasma membrane. Expression of C112Y induced cellular apoptosis as revealed by flow cytometry. Apoptosis induced by C112Y was suppressed by topiroxostat. CONCLUSION: C112Y causes its protein instability resulting cellular apoptosis which could be suppressed with topiroxostat.

Epidermal growth factor receptor inhibition with erlotinib partially prevents cisplatin-induced nephrotoxicity in rats.

The effects of blocking the epidermal growth factor receptor (EGFR) in acute kidney injury (AKI) are controversial. Here we investigated the renoprotective effect of erlotinib, a selective tyrosine kinase inhibitor that can block EGFR activity, on cisplatin (CP)-induced AKI. Groups of animals were given either erlotinib or vehicle from one day before up to Day 3 following induction of CP-nephrotoxicity (CP-N). In addition, we analyzed the effects of erlotinib on signaling pathways involved in CP-N by using human renal proximal tubular cells (HK-2). Compared to controls, rats treated with erlotinib exhibited significant improvement of renal function and attenuation of tubulointerstitial injury, and reduced the number of apoptotic and proliferating cells. Erlotinib-treated rats had a significant reduction of renal cortical mRNA for profibrogenic genes. The Bax/Bcl-2 mRNA and protein ratios were significantly reduced by erlotinib treatment. In vitro, we observed that erlotinib significantly reduced the phosphorylation of MEK1 and Akt, processes that were induced by CP in HK-2. Taken together, these data indicate that erlotinib has renoprotective properties that are likely mediated through decreases in the apoptosis and proliferation of tubular cells, effects that reflect inhibition of downstream signaling pathways of EGFR. These results suggest that erlotinib may be useful for preventing AKI in patients receiving CP chemotherapy.

Instability of KCNE1-D85N that causes long QT syndrome: stabilization by verapamil.

BACKGROUND: A KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed-rectifier K(+) channel current (IKs ). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them. METHODS: KCNE1-D85N or the wild-type protein was coexpressed in COS7 cells with KCNQ1 to form K(+) channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch-clamp techniques. RESULTS: The protein level of KCNE1-D85N was lower than that of the wild-type, in spite of the comparable levels of their mRNA. KCNE1-D85N was highly ubiquitinated and rapidly degraded as compared to the wild-type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady-state level. Both KCNE1-D85N and the wild-type proteins were co-immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1-D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch-clamp experiments demonstrated that the membrane current corresponding to IKs was much smaller in cells expressing KCNE1-D85N than in those expressing the wild-type. Verapamil (0.5-10 µM) increased the protein level of KCNE1-D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1-D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil. CONCLUSION: KCNE1-D85N is less stable than the wild-type protein, and is rapidly degraded through the ubiquitin-proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1-D85N.

Effects of azelnidipine on uric acid metabolism in patients with essential hypertension.

PURPOSE: To examine effects of a long-acting calcium channel blocker (CCB) azelnidipine on uric acid metabolism in hypertensive patients. METHODS: Azelnidipine was administered to 72 patients at a daily dose of 8 mg or 16 mg. In 22 cases out of the 72 patients, a different CCB was switched to azelnidipine. Blood pressure was measured and biochemical parameters of blood and urine were evaluated before and 2-3 months after the administration. RESULTS: Azelnidipine significantly decreased both systolic and diastolic blood pressure and the heart rate. It decreased both serum urate levels and the urinary uric acid to creatinine ratio (Uur/Ucr), but did not affect the uric acid clearance to creatinine clearance ratio (Cur/Ccr). Azelnidipine decreased both Uur/Ucr and Cur/Ccr in patients with Uur/Ucr ≥ 0.5 or ≥ 0.34, although it did not change these clearance parameters in patients with Uur/Ucr <0.5 or <0.34. Azelnidipine decreased the serum urate levels and Uur/Ucr in hyperuricemic patients with uric acid levels ≥ 7.0 mg/dL in males and ≥ 6.0 mg/dL in females. It did not change these parameters in normouricemic patients with serum urate levels <7.0 mg/dL in males and <6.0 mg/dL in females. Azelnidipine decreased Uur/Ucr and Cur/Ccr in hyperuricemic patients with normal or over excretion of uric acid, although it did not change these clearance parameters in hyperuricemic patients with uric acid hypoexcretion. CONCLUSIONS: Azelnidipine decreased the serum urate acid levels and Uur/Ucr, and this response was most prominent in hyperuricemic patients or patients with normal and over excretion of uric acid.

VEGF secretion by adipose tissue-derived regenerative cells is impaired under hyperglycemic conditions via glucose transporter activation and ROS increase.

Transplantation of cultured adipose-derived regenerative cells (ADRCs) into ischemic tissues promotes neovascularization and blood perfusion recovery. These effects are attenuated in diabetes patients. We examined the effects of hyperglycemia on the angiogenic capacity of ADRCs derived from Wistar rats both in vivo and in vitro. Cultured ADRCs were predominantly composed of CD90 positive cells; prevalence of CD90 positive cells was not affected by hyperglycemia. mRNA and protein levels of vascular endothelial growth factor (VEGF) were significantly decreased in ADRCs under hyperglycemic conditions independent of osmolarity, whereas mRNA levels of hepatocyte growth factor and fibroblast growth factor were unaffected. Since ADRCs express glucose transporter proteins GLUT1, 3 and 4, we examined the effects of the glucose transporter inhibitor phloretin on reactive oxygen species (ROS) and angiogenic factors. Phloretin decreased the glucose uptake rate, reduced ROS, and increased VEGF mRNA in ADRCs exposed to a hyperglycemic condition. In vivo transplantation of ADRCs cultured under hyperglycemic conditions into mouse ischemic limbs resulted in significantly decreased blood perfusion and capillary density in ischemic regions compared with transplantation of ADRCs cultured under normoglycemic conditions. These results suggest that hyperglycemia impaired VEGF production in ADRCs via an increase of ROS, impairing the angiogenic capacity of ADRCs transplanted into ischemic limbs.

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

AIMS: We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. METHODS AND RESULTS: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. CONCLUSION: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

Therapeutic effects of human mesenchymal stem cells in Wistar-Kyoto rats with anti-glomerular basement membrane glomerulonephritis.

INTRODUCTION: Multipotent mesenchymal stem cells (MSCs) have become a promising therapeutic approach in many clinical conditions. The hypothesis that MSCs can provide a potential therapy for human anti-glomerular basement membrane (GBM) glomerulonephritis (GN) was tested. METHODS: Nephrotoxic serum nephritis was induced in Wistar-Kyoto rats on day 0. Groups of animals were given either human MSCs (hMSCs, 3×10(6)) or vehicle by intravenous injection on day 4; all rats were sacrificed at either day 7 or day 13. RESULTS: Fluorescently labeled hMSCs were localized in glomeruli and tubulointerstitium 5 h after hMSC administration and persisted until 48 h, but hMSCs were barely detectable after 7 days. hMSC-treated rats had decreased kidney weight, proteinuria, and glomerular tuft area at each time point. The serum creatinine level and degree of glomerular crescent formation were decreased by hMSC treatment on day 13. ED1-positive macrophages, CD8-positive cells, and TUNEL-positive apoptotic cells in glomeruli were reduced by hMSC treatment on day 7, and this trend in apoptotic cells persisted to day 13. Renal cortical mRNA for TNF-α, IL-1ß, and IL-17, and the serum IL-17A level were decreased, whereas renal cortical mRNA for IL-4 and Foxp3 and the serum IL-10 level were increased in the MSC-treated group on day 7. Collagen types I and III and TGF-ß mRNA were decreased by hMSC treatment on day 13. CONCLUSION: The present results demonstrated that anti-inflammatory and immunomodulatory effects were involved in the mechanism of attenuating established experimental anti-GBM GN by hMSCs. These results suggest that hMSCs are a promising therapeutic candidate for the treatment of anti-GBM GN.

Transplantation of freshly isolated adipose tissue-derived regenerative cells enhances angiogenesis in a murine model of hind limb ischemia.

Therapeutic angiogenesis has emerged as one of the most promising therapies for severe ischemic cardiovascular diseases with no optional therapy. Several investigators have reported that transplantation of cultured adipose-derived regenerative cells (cADRCs) to ischemic tissues promotes neovascularization and blood perfusion recovery; however, cell therapy using cultured cells has several restrictions. To resolve this problem, the angiogenic capacity of freshly isolated ADRCs (fADRCs) obtained from Lewis rats was compared with cADRCs, both in vivo and in vitro. Flow cytometric analysis showed that fADRCs contained several cell types such as endothelial progenitor cells and endothelial cells; however, these cells were present in a very small proportion in cADRCs. Transplantation of fADRCs in mice significantly improved blood perfusion, capillary density, and production of several angiogenic factors in transplanted ischemic limbs compared with a saline-injected group, whereas these effects were not observed in the cADRCs-injected group. fADRCs also showed significantly higher expression levels of angiogenic factors than cADRCs in the in vitro study. Furthermore, fADRC stimulated tube formation more remarkably than cADRC in an in vitro tube formation assay. These results suggested that fADRCs have an effective angiogenic capacity, and they would be more valuable as a source for cell-based therapeutic angiogenesis than cADRCs or other stem/progenitor cells.

Enhancing effects of salicylate on quinidine-induced block of human wild type and LQT3 related mutant cardiac Na+ channels.

It is unknown whether salicylate enhances the action of antiarrhythmic agents on human Na+ channels with state dependency and tissue specificity. We therefore investigated effects of salicylate on quinidine-induced block of human cardiac and skeletal muscle Na+ channels. Human cardiac wild-type (hH1), LQT3-related mutant (ΔKPQ), and skeletal muscle (hSkM1) Na+ channel α subunits were expressed in COS7 cells. Effects of salicylate on quinidine-induced tonic and use-dependent block of Na+ channel currents were examined by the whole-cell patch-clamp technique. Salicylate enhanced the quinidine-induced tonic and use-dependent block of both hH1 and hSkM1 currents at a holding potential (HP) of -100 mV but not at -140 mV. Salicylate decreased the IC50 value for the quinidine-induced tonic block of hH1 at an HP of -100 mV, and produced a negative shift in the steady-state inactivation curve of hH1 in the presence of quinidine. According to the modulated receptor theory, it is probable that salicylate decreases the dissociation constant for quinidine binding to inactivated-state channels. Furthermore, salicylate significantly enhanced the quinidine-induced tonic and use-dependent block of the peak and steady-state ΔKPQ channel currents. The results suggest that salicylate enhances quinidine-induced block of Na+ channels via increasing the affinity of quinidine to inactivated state channels.

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction.

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K(v)1.5, Na(v)1.5, Hcn4, Ca(v)3.2, Ca(v)1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca(2+) transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca(2+) transients were recapitulated by a simulation model. Although a Ca(2+) antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca(2+) transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.