Reiichi Murakami

Intracellular Signaling for Vasoconstrictor Coupling Factor 6. Novel Function of β-Subunit of ATP Synthase as Receptor

Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to &bgr;-subunit of ATP synthase at the cell surface, but unaffected that for the &agr;-subunit antibody. A significant displacement of radioligand was observed at 3×10−9 through 10−7 M unlabeled CF6, and the Kd was 7.6 nM. Adenosine diphosphate (ADP) at 10−7 M and &bgr;-subunit antibody suppressed the binding of 125I-CF6 by 81.3±9.7% and 32.0±2.0%, respectively, whereas the &agr;-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10−7 M, and efrapeptin at 10−5 M, an inhibitor of ATP synthase, blocked it. CF6 at 10−7 M decreased intracellular pH in 2′,7′-bis(carboxyethyl-5 (6))-carboxyfluorescein-loaded HUVEC. Amyloride at 10−4 M augmented the pH decrease in response to CF6, whereas efrapeptin at 10−5 M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10−5 M or &bgr;-subunit antibody or ADP at 10−7 M. The &bgr;-subunit antibody suppressed coupling factor 6–induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.

Effect of vasoconstrictor coupling factor 6 on gene expression profile in human vascular endothelial cells: enhanced release of asymmetric dimethylarginine.

Background Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. Methods and results The increased gene expression after 24-h exposure to CF6 at 10−7 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 ± 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 ± 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 ± 0.24, P = 0.06) and estrogen receptor β (1.74 ± 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 ± 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 ± 2% (n = 10, P < 0.01) at 48 h after CF6 at 10−7 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 ± 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 ± 5% (n = 5, P < 0.01) and 19 ± 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 ± 8% and NOS activity was decreased by 13 ± 1% (both n = 8, P < 0.05) by CF6. Conclusions CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.

Coupling factor 6 downregulates platelet endothelial cell adhesion molecule-1 via c-Src activation and acts as a proatherogenic molecule.

Coupling factor 6 (CF6), a component of ATP synthase, suppresses the generation of prostacyclin and nitric oxide (NO). Platelet endothelial cell adhesion molecule-1 (PECAM-1) is involved in shear-induced NO production. To investigate the linkage between the actions of CF6 and PECAM-1, we examined the effects of CF6 on PECAM-1 expression and shear-mediated NO release, comparatively with those of angiotensin II (AngII). Treatment of human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells (HAEC) with CF6 at 10(-7)M or AngII at 10(-7)M for 24h suppressed PECAM-1 gene and protein expression. CF6 or AngII activated c-Src at 15 min in HUVEC, and blockade of c-Src with PP1, its specific inhibitor, restored them. Efrapeptin, an inhibitor of ATPase, attenuated CF6-induced suppression of PECAM-1 gene expression by blockade of acidification, whereas superoxide dismutase or apocinin, an inhibitor of NADPH oxidase, blocked AngII-induced suppression of PECAM-1. Exposure of the cells to shear stress at 25 dynes/cm(2) for 30 min enhanced phosphorylation of eNOS at Ser(1177) and NO release. Pretreatment with CF6 or AngII for 24h attenuated them in HUVEC and HAEC. These suggest that CF6 downregulates PECAM-1 expression via c-Src activation and attenuates shear-induced NO release presumably by suppressing eNOS phosphorylation.

Nephrotic syndrome associated with interferon-β-1b therapy for multiple sclerosis

A 43-year-old woman with multiple sclerosis (MS) had nephrotic syndrome 21 months after starting treatment with interferon (IFN)-β-1b (subcutaneous administration). She had taken no drug except for the IFN-β-1b. Because nephrotic syndrome may be induced by IFN therapy, the IFN was stopped. Percutaneous renal biopsy revealed that she had minimal change nephrotic syndrome. As nephrotic-range proteinuria, hypoalbuminemia, and general edema were worsening even 2 weeks after cessation of the drug, oral corticosteroid therapy (prednisolone 40 mg/day) was started. The nephrotic syndrome was treated successfully with prednisolone. The dosage of prednisolone was tapered, without a relapse, and then the corticosteroid therapy was stopped. IFN-β-1b therapy was then resumed, and the patient is in remission for both nephrotic syndrome and MS. Though proteinuria and nephrotic syndrome is a rare adverse effect of IFN-β-1b therapy, physicians treating MS patients with this agent should pay careful attention to new clinical symptoms and laboratory findings.

BK Virus–Associated Urothelial Carcinoma of a Ureter Graft in a Renal Transplant Recipient: A Case Report

BACKGROUND Urothelial carcinomas of ureter grafts in renal transplant patients are rare. Here we report our experience with a case of BK virus-associated urothelial carcinoma in a ureter graft. CASE REPORT A 47-year-old man developed chronic renal failure secondary to diabetes mellitus and started maintenance hemodialysis in September 2007. Two months later, the patient received a renal transplant from his 70-year-old mother. The patient developed BK virus-associated nephropathy 1 year after transplantation and presented with a decline in renal function and hydronephrosis in the transplanted kidney 4 years 6 months after transplantation. Cystoscopy and retrograde pyelography revealed an irregular filling defect in the ureter graft. Cytologic diagnosis of his urine revealed a high-grade urothelial carcinoma. Computerized tomography showed a cT2 ureteral tumor and no involvement of other organs. The patient subsequently underwent a transplant nephroureterectomy with bladder cuff resection. Histopathologic findings revealed a high-grade urothelial carcinoma, pT2, in the ureter graft with SV40-positive staining. The patient was closely observed without adjuvant chemotherapy therapy and remained disease free 1 year after surgery. Renal transplant recipients with BK virus infection are at high risk of developing urologic malignancies. Close attention is necessary to diagnose post-transplantation urologica malignancies as early as possible.

A case of myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA)-associated glomerulonephritis and concurrent membranous nephropathy

BackgroundMyeloperoxidase anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (MPO-ANCA-GN) and concurrent membranous nephropathy (MN) are very rare combination. Their causal relationship has been suggested, but not determined.Case presentationA 73-years-old male with 5-year history of proteinuria underwent an operation for his sigmoid colon cancer. Seven months later, he was referred to a nephrology division due to an exacerbating renal function and hypoalbuminemia. Laboratory examination revealed positive MPO-ANCA in the serum. A renal biopsy revealed a necrotizing extracapillary proliferative glomerulonephritis with crescents, demonstrating MPO-ANCA-GN. Whereas, immunofluorescent staining documented granular deposition of immumoglobulin (Ig) G and C3 along the capillary wall and electron microscopy showed subepithelial deposits in the glomerular basement membrane demonstrating MN. Immunofluorescent staining of IgG subclass showed positive IgG1, IgG2, negative IgG3 and weak positive IgG4 suggested the possibility of malignancy-associated MN.ConclusionCombination of MPO-ANCA-GN and MN are rare. Although the causal relationship has been suggested in some cases, we should consider all the possibilities including idiopathic MN and secondary MN associated with malignancy, drug use or infection.

Extracellular matrix metalloproteinase inducer is expressed in the proximal tubular epithelial cells of the human kidney

Aim:  Matrix metalloproteinases (MMP) affect matrix remodelling, and extracellular matrix metalloproteinase inducer (EMMPRIN) has been reported to increase the levels of several MMP. However, the expression of EMMPRIN in the human kidney and its regulatory mechanisms are not well known. In this study, we examined EMMPRIN expression in the human kidney with the biopsied specimens, cultured proximal tubular epithelial cells (PTEC) and human mesangial cells (HMC).

Thrombin enhances the production of monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 in cultured rat glomerular epithelial cells

BACKGROUND Glomerular crescents play an important role in progressive glomerular injury. The lesions consist of epithelial cells, macrophages and deposits of fibrin and extracellular matrix. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) are members of chemokine subfamilies. MCP-1 and MIP-2 are potent chemoattractant leukocyte cytokines, and they may be involved in crescent formation. Thrombin participates in fibrin formation. We hypothesized that thrombin stimulates the production of MCP-1 and MIP-2 by glomerular epithelial cells (GECs). METHODS Cultured rat GECs from the 19th to the 24th passage were used. We incubated GECs with or without thrombin to examine the effect of thrombin on the production of MCP-1 and MIP-2. The levels of MCP-1 and MIP-2 were measured in the cell supernatants by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of MCP-1 and MIP-2 were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). We also examined the inhibitory effect of argatroban, a synthetic thrombin inhibitor, and prednisolone in the production of MCP-1 and MIP-2 stimulated by thrombin. RESULTS Thrombin stimulated the production of MCP-1 and MIP-2 in a dose- and time-dependent manner. Thrombin also enhanced the mRNA expressions of MCP-1 and MIP-2 in the GECs. The stimulating effect of thrombin on the production of MCP-1 and MIP-2 was inhibited by the addition of argatroban or prednisolone. CONCLUSIONS We demonstrated a novel role of thrombin: it stimulates the production of MCP-1 and MIP-2 by GECs. It is clinically important that the inhibition of these chemokines leads to the improvement of crescentic glomerulonephritis. Anti-thrombin drugs and prednisolone may be useful in treating crescentic glomerulonephritis.

Post-transplant Renal Function and Cardiovascular Events Are Closely Associated With the Aortic Calcification Index in Renal Transplant Recipients

INTRODUCTION The aortic calcification index (ACI) is reported to be closely associated with renal dysfunction and cardiovascular events; however, its implication in renal transplant recipients has not been well examined. In this study, we investigated the relationship between pretransplant ACI, ACI progression, post-transplant renal function, and post-transplant cardiovascular events in renal transplant recipients. PATIENTS AND METHODS The study from June 1996 to Jan 2012 included 61 renal transplant recipients (living donors, 47; cadaveric donors, 14). The median follow-up period was 60 months. ACI was quantitatively measured on abdominal computed tomography. The relationship between age, dialysis period, estimated glomerular filtration rate (eGFR), and pre- and post-transplant ACI was longitudinally evaluated. Risk factors for post-transplant ACI progression were determined by logistic regression analysis. Patient background and the incidence of post-transplant cardiovascular events were also assessed. RESULTS The pretransplant ACI (median 4.2%) significantly correlated with age at transplant, dialysis period, and diabetes mellitus. ACI gradually increased up to 2.8 times at 10 years after transplantation. Post-transplant eGFR significantly correlated with ACI progression in patients with chronic kidney disease of stage ≥ 3. Logistic regression analyses showed that age at transplantation, post-transplant period, cadaveric donors, and post-transplant chronic kidney disease stage 3 were risk factors for post-transplant ACI progression. The pretransplant ACI was higher (median 66%) in 3 patients who experienced post-transplant cardiovascular events. CONCLUSIONS ACI progression closely correlates with age and post-transplant renal function. A high pretransplant ACI is a risk factor for post-transplant cardiovascular events in renal transplant recipients.

Roxithromycin inhibits transforming growth factor-β production by cultured human mesangial cells

Background:  Transforming growth factor‐β (TGF‐β) plays an important role in progression of renal injury. However, few materials which inhibit TGF‐β have been known. Roxithromycin (ROX), macrolide antibiotics, is known to have anti‐inflammatory, immunomodulatory and tissue reparative effects besides its bacteriostatic activity, although the exact mechanism of its anti‐inflammatory and immunomodulatory effects was not defined. We examined the effect of ROX on production of TGF‐β and type IV collagen by cultured human mesangial cells (HMC).