Homare Shimohata

MafA Is a Key Regulator of Glucose-Stimulated Insulin Secretion

ABSTRACT MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic β cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.

Hyperglycemia induces oxidative and nitrosative stress and increases renal functional impairment in Nrf2-deficient mice

The transcription factor Nrf2 regulates the expression of antioxidant genes. Hyperglycemia‐induced oxidative stress is involved in the pathogenesis of diabetes and its complications. However, little is known about the protective role of Nrf2 in diabetes. To gain insight into the protective role of Nrf2 in diabetes we treated Nrf2 knockout (Nrf2 KO) mice with streptozotocin (STZ). The STZ Nrf2 KO mice did not develop renal hyperfiltration, which was observed in the STZ‐treated wild‐type (STZ WT) mice, but renal function gradually deteriorated over the 10‐week observation period. Urinary excretion of nitric oxide metabolites and the occurrence of 8‐nitroguanosine, which was detected in glomerular lesions, were increased in STZ Nrf2 KO mice during the early stages after treatment. In vivo electron paramagnetic resonance analysis revealed an accelerated rate of decay of the 3‐carbamoyl‐2,2,5,5‐tetramethylpyrrolidine‐1‐oxyl spin probe signal in STZ Nrf2 KO mice. The addition of superoxide dismutase prolonged the half‐life of the signal, which suggested that increased oxygen radical formation occurred in the STZ Nrf2 KO mice. These results suggested that hyperglycemia increased oxidative and nitrosative stress and accelerated renal injury in the Nrf2 KO mice and that Nrf2 serves as a defense factor against some diabetic complications.

Overexpression of c-Maf Contributes to T-Cell Lymphoma in Both Mice and Human

c-Maf translocation or overexpression has been observed in human multiple myeloma. Although c-maf might function as an oncogene in multiple myeloma, a role for this gene in other cancers has not been shown. In this study, we have found that mice transgenic for c-Maf whose expression was direct to the T-cell compartment developed T-cell lymphoma. Moreover, we showed that cyclin D2, integrin beta(7), and ARK5 were up-regulated in c-Maf transgenic lymphoma cells. Furthermore, 60% of human T-cell lymphomas (11 of 18 cases), classified as angioimmunoblastic T-cell lymphoma, were found to express c-Maf. These results suggest that c-Maf might cause a type of T-cell lymphoma in both mice and humans and that ARK5, in addition to cyclin D2 and integrin beta(7), might be downstream target genes of c-Maf leading to malignant transformation.

Th1 and Type 1 Cytotoxic T Cells Dominate Responses in T-bet Overexpression Transgenic Mice That Develop Contact Dermatitis

Contact dermatitis in humans and contact hypersensitivity (CHS) in animal models are delayed-type hypersensitivity reactions mediated by hapten-specific T cells. Recently, it has become clear that both CD4+ Th1 and CD8+ type 1 cytotoxic T (Tc1) cells can act as effectors in CHS reactions. T-bet has been demonstrated to play an important role in Th1 and Tc1 cell differentiation, but little is known about its contribution to CHS. In the present study, we used C57BL/6 mice transgenic (Tg) for T-bet to address this issue. These Tg mice, which overexpressed T-bet in their T lymphocytes, developed dermatitis characterized by swollen, flaky, and scaly skin in regions without body hair. Skin histology showed epidermal hyperkeratosis, neutrophil, and lymphocyte infiltration similar to that seen in contact dermatitis. T-bet overexpression in Tg mice led to elevated Th1 Ig (IgG2a) and decreased Th2 Ig (IgG1) production. Intracellular cytokine analyses demonstrated that IFN-γ was increased in both Th1 and Tc1 cells. Furthermore, Tg mice had hypersensitive responses to 2,4-dinitrofluorobenzene, which is used for CHS induction. These results suggest that the level of expression of T-bet might play an important role in the development of contact dermatitis and that these Tg mice should be a useful model for contact dermatitis.

Serum Levels of BAFF and APRIL in Myeloperoxidase Anti-Neutrophil Cytoplasmic Autoantibody-Associated Renal Vasculitis: Association with Disease Activity

Background: A proliferation-inducing ligand (APRIL) and the B cell activation factor belonging to the tumor necrosis factor family (BAFF) have proven to be key factors in the selection and survival of B cells, and a higher concentration of BAFF has been shown to contribute to autoreactive B cell survival and elevated autoantibody production. Here, serum BAFF and APRIL levels were investigated to analyze their association with disease activity in myeloperoxidase anti-neutrophil cytoplasmic autoantibody (MPO-ANCA)-associated renal vasculitis. Methods: APRIL and BAFF levels in serum obtained from 37 patients with MPO-ANCA-associated vasculitis were measured by ELISA. Samples were taken from active vasculitis patients, inactive vasculitis patients and inactive vasculitis patients with infectious complications. Results: Although there was no difference in serum APRIL among the active vasculitis, inactive vasculitis and infectious complication patients, serum BAFF was higher in active vasculitis patients than in inactive vasculitis, infectious complication and control patients (for all, p < 0.001). There was no significant correlation between serum APRIL and ANCA titers, but there was a significant correlation between serum BAFF and ANCA titers (r = 0.465, p < 0.001). Conclusion: Excessive BAFF production in MPO-ANCA-associated vasculitis may be one of the factors for autoimmune B cell tolerance, resulting in MPO-ANCA production.

Transgenic Overexpression of GATA-3 in T Lymphocytes Improves Autoimmune Glomerulonephritis in Mice with a BXSB/MpJ-Yaa Genetic Background

A T helper 1 (Th1)/Th2 imbalance is thought to contribute to the pathogenesis of autoimmune diseases. The differentiation of T cells into Th1 or Th2 subtypes is under the regulation of several transcription factors. Among these, transcription factor GATA-3 is thought to play an indispensable role in the development of T cells and the differentiation of Th2 cells. To examine how a Th1/Th2 imbalance affects the development of autoimmune disease, GATA-3 was overexpressed in the T lymphocytes of C57BL/6 x BXSB/MpJ-Yaa F(1) (Yaa) mice. Yaa mice developed autoimmune nephritis similarly to BXSB/MpJ-Yaa mice, which are commonly used as a model for Th1-dominant murine lupus. GATA-3 overexpression in T cells improved the 50% mortality incidence time for GATA-3-transgenic Yaa mice (41.6 wk), compared with Yaa mice (30.9 wk), and reduced proteinuria, serum creatinine levels, and the severity of glomerulonephritis in GATA-3-transgenic Yaa mice. GATA-3 overexpression in Yaa mice led to simultaneously elevated Th2 Ig (IgG1) and decreased Th1 Ig (IgG2a and IgG3) production and serum IFN-gamma levels. Although IL-4 production remained unchanged, intracellular cytokine analyses demonstrated that IL-5 was induced and IFN-gamma was suppressed in stimulated T cells from the GATA-3-transgenic Yaa mice. These results indicated that abundant GATA-3 was unable to stimulate complete differentiation of Th2 cells but did counteract the dominance of Th1 cells and alleviated the disease severity in Yaa mice. These data suggest that transcriptional regulation therapy may have potential as an effective strategy for treating autoimmune glomerulonephritis.

Overexpression of Mafb in Podocytes Protects against Diabetic Nephropathy

We previously showed that the transcription factor Mafb is essential for podocyte differentiation and foot process formation. Podocytes are susceptible to injury in diabetes, and this injury leads to progression of diabetic nephropathy. In this study, we generated transgenic mice that overexpress Mafb in podocytes using the nephrin promoter/enhancer. To examine a potential pathogenetic role for Mafb in diabetic nephropathy, Mafb transgenic mice were treated with either streptozotocin or saline solution. Diabetic nephropathy was assessed by renal histology and biochemical analyses of urine and serum. Podocyte-specific overexpression of Mafb had no effect on body weight or blood glucose levels in either diabetic or control mice. Notably, albuminuria and changes in BUN levels and renal histology observed in diabetic wild-type animals were ameliorated in diabetic Mafb transgenic mice. Moreover, hyperglycemia-induced downregulation of Nephrin was mitigated in diabetic Mafb transgenic mice, and reporter assay results suggested that Mafb regulates Nephrin directly. Mafb transgenic glomeruli also overexpressed glutathione peroxidase, an antioxidative stress enzyme, and levels of the oxidative stress marker 8-hydroxydeoxyguanosine decreased in the urine of diabetic Mafb transgenic mice. Finally, Notch2 expression increased in diabetic glomeruli, and this effect was enhanced in diabetic Mafb transgenic glomeruli. These data indicate Mafb has a protective role in diabetic nephropathy through regulation of slit diaphragm proteins, antioxidative enzymes, and Notch pathways in podocytes and suggest that Mafb could be a therapeutic target.

Osteomalacia due to Fanconi’s syndrome and renal failure caused by long-term low-dose adefovir dipivoxil

To the Editor Acquired Fanconi’s syndrome is a disorder of the proximal tubules and causes hypophosphatemic osteomalacia, glucosuria, aminoaciduria and metabolic acidosis. The several causes of adult Fanconi’s syndrome include dysproteinemias, glomerular disease, after acute tubular necrosis, tetracycline, cancer chemotherapy agents and toxins [1]. The acyclic nucleoside phosphonates also cause Fanconi’s syndrome as well as renal damage. Among them, adefovir dipivoxil is often used to treat lamivudine-resistant hepatitis B and its nephrotoxicity is dose dependent. Therefore, low-dose use of this drug is recommended to avoid renal tubular damage. Here, we report a case of osteomalacia due to Fanconi’s syndrome caused by low-dose adefovir. A 56-year-old male with chronic hepatitis B infection was referred to our hospital in July 2011 to investigate the cause of renal impairment. He also had a history of cirrhosis and hepatocellular carcinoma. Adefovir at 10 mg a day had been added to lamivudine 100 mg a day in 2007 because of lamivudine resistance. Laboratory data on his first visit to our hospital showed elevated serum creatinine (2.23 mg/dL; 0.6–1.1 mg/dL) and low serum uric acid level (2.7 mg/dL; 3.0–7.5 mg/dL). Hemoglobin, serum albumin and platelet count were 12.6, 4.6 g/dL and 10.9 9 10/lL, respectively. C-reactive protein, alanine, aspartate aminotransferase, blood glucose, and serum levels of immunoglobin G, A, M were all normal. Urinalysis showed proteinuria (726 mg/gCre) and glucosuria with phosphaturia and general aminoaciduria. Urinary N-acetylglucosaminidase and b-2 microglobulin were 19.2 U/L (\11.5 U/L), and 45942 lg/L (\200 lg/L), respectively. Blood gas analysis showed metabolic acidosis. Computed tomography scan of the abdomen showed normal kidney size with some small calcification. From these results, we diagnosed acquired Fanconi’s syndrome in this patient. The patient also had a history of bone pain and waddling gait due to osteomalacia and had been treated by administration of alfacalcidol 3 lg and dibasic calcium phosphate dehydrate. His serum alkaline phosphatase level was elevated to 674 U/L (104–338 U/L), but serum creatine phosphokinase level was normal. At the first visit to our hospital, the reason for his osteomalacia was not clear, but he was diagnosed with osteomalacia associated with Fanconi’s syndrome after renal examination. Concomitant with this diagnosis, we suspected that adefovir was the cause of the Fanconi’s syndrome, and this drug was reduced to three times a week. After reduction of adefovir, his bone pain and waddling gait was alleviated gradually during ambulatory care. Seven months after the reduction, his proteinuria, glucosuria, phosphaturia and general aminoaciduria had disappeared and renal function was improved (serum creatinine was1.5 mg/dL). From these results, we considered that adefovir might be a cause of renal impairment. Several hypophosphatemic osteomalacia cases induced by low-dose adefovir therapy (10 mg daily) have been reported [2, 3]. In previously reported cases, the symptoms H. Shimohata Y. Ogawa K. Hirayama M. Kobayashi (&) Department of Nephrology, Tokyo Medical University Ibaraki Medical Center, 3-20-1 Chuo, Ami, Inashiki, Ibaraki 300-0395, Japan e-mail: masaki-k@tokyo-med.ac.jp

Serum ratio of soluble triggering receptor expressed on myeloid cells-1 to creatinine is a useful marker of infectious complications in myeloperoxidase-antineutrophil cytoplasmic antibody-associated renal vasculitis

BACKGROUND The contribution of infections to the mortality of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis patients is important and should induce early and careful control of these events. However, the differentiation of infection from active vasculitis is often difficult. The usefulness of serum-soluble triggering receptor expressed on myeloid cells-1 (TREM-1) for detecting the presence of infectious complications regardless of disease activity was investigated. METHODS Soluble TREM-1 in serum obtained from 41 patients with myeloperoxidase (MPO)-ANCA-associated vasculitis was measured by an enzyme-linked immunosorbent assay. Twenty-nine samples were from active vasculitis patients, 27 samples from inactive vasculitis patients without infection and 17 samples from inactive vasculitis patients with infectious complications. Serum-soluble TREM-1 was also measured in 10 patients with acute pyelonephritis and 30 patients with chronic kidney disease (CKD). RESULTS There was a significant correlation between serum levels of soluble TREM-1 and serum creatinine levels among all patients (r = 0.554, P < 0.0001). The serum-soluble TREM-1/creatinine ratio was higher in inactive vasculitis patients with infectious complications than in active vasculitis, inactive vasculitis without infection and CKD patients (P = 0.0005, P < 0.0001 and P < 0.0001, respectively), but not significantly different to that in acute pyelonephritis patients. On receiver-operating-characteristic curve analysis, a lower-limit value of 9.40 ng/mg for this ratio had a sensitivity of 84.6% and a specificity of 90.8% in differentiating patients with infection from those without infection. CONCLUSIONS The serum ratio of soluble TREM-1 to creatinine may be a useful marker for detection of infectious complications in MPO-ANCA-associated vasculitis.

Angiopoietin balance in septic shock patients treated by direct hemoperfusion with polymyxin b-immobilized fiber.

Capillary permeability is a tightly regulated feature of microcirculation in all organ beds; however, in sepsis this feature is fundamentally altered. We previously reported elevated levels of vascular endothelial growth factor and its receptor (fms‐like tyrosine kinase‐1) in patients with septic shock, then investigated two kinds of angiopoietins in those patients. An enzyme‐linked immunoassay was used to measure serum angiopoietin‐1 and ‐2 levels in 12 patients with septic shock who were treated by direct hemoperfusion with a polymyxin B‐immobilized fiber column (DHP‐PMX). The angiopoietin‐1 level was lower in patients with septic shock (7.01 ± 10.08 ng/mL) than in controls (28.24 ± 11.61 ng/mL, P < 0.001), but the angiopoietin‐2 level was higher in septic shock patients (40.83 ± 30.13 ng/mL vs. 2.47 ± 1.78 ng/mL, P < 0.001). Between seven survivors and five non‐survivors there was no significant difference in angiopoietin‐1 levels before DHP‐PMX therapy. During DHP‐PMX therapy, however, the angiopoietin‐2 level was significantly decreased in survivors (31.52 ± 26.15 ng/mL vs. 17.32 ± 22.46 ng/mL, P = 0.035). Moreover, at the end of the therapy, the angiopoietin‐1 level was significantly lower in non‐survivors (1.14 ± 1.30 ng/mL vs. 10.43 ± 13.56 ng/mL, P = 0.042), but the angiopoietin‐2 level in non‐survivors was significantly higher (70.79 ± 40.47 ng/mL vs. 17.32 ± 22.46 ng/mL, P = 0.019). The angiopoietin‐2 level may be associated with vascular permeability in septic patients, and angiopoietins may be suitable markers of disease severity and mortality.