Joichi Usui

Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors

Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

Podocytes, parietal cells, and glomerular pathology: the role of cell cycle proteins

Abstract. Podocytes are highly differentiated cells that play a central role in glomerular function. Cell cycle quiescence sustained by the tight regulation of cell cycle molecules seems to be the basis of podocyte differentiation, as reflected in reciprocal expression of cyclins and cyclin kinase inhibitors (CKIs) during glomerulogenesis, concurrent with the expression of podocyte-specific markers. In addition, cell cycle re-entry by podocytes, accompanied by down-regulation of CKIs, leads to either nuclear division without cytokinesis or to marked cell proliferation, both of which result in heavy proteinuria and progressive glomerulosclerosis. These observations suggest that proper cell cycle regulation in podocytes is pivotal for their role in glomerular function and that dysregulation of this system plays a role in glomerular pathology. This review discusses the role of cell cycle molecules in the differentiation, function, and pathology of podocytes.

Tubulointerstitial nephritis and uveitis syndrome associated with hyperthyroidism

We report a 17-year-old male patient with tubulointerstitial nephritis and uveitis (TINU) associated with hyperthyroidism. He presented with a 2-month history of fatigue, loss of appetite, low-grade fever, and a 12-kg weight loss when he was admitted to our hospital. He had iritis, which was complicated by fibrin in the anterior chamber, diagnosed by slit-lamp examination. On laboratory examinations, deteriorated renal function (blood urea nitrogen level was 25.9 mg/dl and creatinine level was 2.82 mg/dl) and elevated urinary levels of N-acetyl-β-D-glucosaminidase (33.1 U/l) and β2-microglobulin (78 600 µg/l) were observed. Serum thyroid-stimulating hormone (TSH) was undetectable, at less than 0.01 µIU/ml, and free triiodothyronine and free thyroxine were elevated, up to 5.23 pg/ml and 2.85 ng/dl, respectively. The titers of antithyroglobulin and antithyroid microsomal and TSH-receptor antibodies were not elevated. Abdominal and thyroidal ultrasonography showed evident bilateral enlargement of the kidneys and diffuse enlargement of the thyroid gland. Iodine-123 scintigraphy showed low uptake in the thyroid gland. The biopsied renal specimen showed mild edema and severe diffuse infiltration of mononuclear cells and few eosinophils in the interstitium, without any glomerular or vascular abnormalities. Based on the clinical features and pathological findings, a diagnosis of TINU syndrome with associated hyperthyroidism was made. Treatment was started with 30 mg/day of prednisolone. The iritis disappeared, and the patients clinical status improved remarkably. This case suggests the possibility of thyroid dysfunction in some patients with TINU syndrome, and we believe thyroid function should be measured in all TINU patients. Moreover, histopathological diagnosis of the thyroid glands before treatment is necessary for TINU patients with thyroid dysfunction.

In situ expression of connective tissue growth factor in human crescentic glomerulonephritis

Connective tissue growth factor (CTGF) has recently been recognized as an important profibrotic factor and is up-regulated in various renal diseases with fibrosis. The present study describes the sequential localization of CTGF mRNA and its association with transforming growth factor (TGF)-β1 in human crescentic glomerulonephritis (CRGN). Furthermore, we examined the phenotype of CTGF-expressing cells using serial section analysis. Kidney biopsy specimens from 18 CRGN patients were examined using in situ hybridization and immunohistochemistry. CTGF mRNA was expressed in the podocytes and parietal epithelial cells (PECs) in unaffected glomeruli. In addition, it was strongly expressed in the cellular and fibrocellular crescents, particularly in pseudotubule structures. Serial sections revealed that the majority of CTGF mRNA-positive cells in the crescents co-expressed the epithelial marker cytokeratin, but not a marker for macrophages. Moreover, TGF-β1, its receptor TGF-β receptor-I, and extracellular matrix molecules (collagen type I and fibronectin) were co-localized with CTGF mRNA-positive crescents. Our results suggest that CTGF is involved in extracellular matrix production in PECs and that it is one of the mediators promoting the scarring process in glomerular crescents.

Localization of intercellular adherens junction protein p120 catenin during podocyte differentiation

To reveal the role of cadherin complex in podocyte differentiation, the present study describes the localization of the cadherin complex, including p120 catenin (p120ctn), in the developing and in the aminonucleoside nephrosis (PAN nephrosis) rat kidney, by immunofluorescence microscopy and immunogold electron microscopy. p120ctn and β-catenin were co-localized at the apical part of lateral cell membranes in presumptive podocytes of the S-shaped body, and their localization shifted to the basal margin of lateral cell membranes in the capillary loop stage. There was no expression of the cadherin complex at the slit diaphragm, the intercellular junction of mature podocytes. After the regression of the podocyte junctional structure in PAN nephrosis, the cadherin complex was not re-expressed. The dynamic changes in the localization of the cadherin complex suggest that the it plays an important role during podocyte differentiation, including the rearrangement of the intercellular junction and the formation of the slit diaphragm.

Renal pathology of ANCA-related vasculitis: proposal for standardization of pathological diagnosis in Japan

BackgroundIn Japan, systematic evaluation of the histologic parameters of anti-neutrophil cytoplasmic autoantibodies (ANCA)-related vasculitis has been performed according to the Japanese classification by Shigematsu et al. However, this classification is quite different from that of the European Vasculitis Study Group (EUVAS) classification. Therefore, a histological common basis is needed to compare Japanese histological data with the international database.MethodHistological parameters concerning glomerular, tubulointerstitial, and vascular lesions of ANCA-related vasculitis, which are indispensable for clinical management, were elucidated and defined by reviewing, utilizing the merits of, and amending the two scoring systems.Results and conclusionA new comprehensive and standardized scoring system, by which histological quantitative assessment can provide evidence for therapy planning, has been developed for renal biopsy of Japanese ANCA-related vasculitis.

Glomerular crescents predominantly express cadherin–catenin complex in pauci-immune-type crescentic glomerulonephritis

Aims:  To investigate the expression of the cadherin complex in human crescentic glomerulonephritis to elucidate the role of intercellular adherens junction molecules in crescent formation.

Vascular endothelial growth factor and soluble fms-like tyrosine kinase-1 in septic shock patients treated with direct hemoperfusion with a polymyxin B-immobilized fiber column.

Abstract:  Sepsis is characterized by a systemic inflammatory response to a microbial pathogen. In sepsis, capillary permeability is a tightly regulated feature of microcirculation in all organ beds and is fundamentally altered. We investigated the vascular endothelial growth factor (VEGF) level as a vascular permeability factor and the soluble fms‐like tyrosine kinase‐1 (Flt‐1) level as an antagonist of the VEGF receptors. Serum VEGF and soluble Flt‐1 levels in 21 patients with septic shock, who were treated with direct hemoperfusion with a polymyxin B‐immobilized fiber column (DHP‐PMX), were measured by enzyme‐linked immunoassay. The VEGF and the soluble Flt‐1 levels were more elevated in patients with septic shock than in controls. Between 14 survivors and 7 non‐survivors, there was no significant difference in VEGF level before the DHP‐PMX therapy, but the soluble Flt‐1 level of survivors was significantly lower than that of non‐survivors. Although there was no significant difference between starting and ending VEGF levels in survivors, in non‐survivors the VEGF level at the end of DHP‐PMX therapy was significantly lower than that at the start. In survivors, the soluble Flt‐1 level at the end of DHP‐PMX therapy was significantly lower than that at the start. On the other hand, in non‐survivors, there was no significant difference between the ending and starting soluble Flt‐1 levels. The soluble Flt‐1 level may be a suitable marker of disease severity and mortality.

Angiopoietin Balance in Septic Shock Patients With Acute Kidney Injury: Effects of Direct Hemoperfusion With Polymyxin B-Immobilized Fiber.

Acute lung injury (ALI) in sepsis is characterized by an increase in microvascular permeability, resulting in pulmonary edema. Several studies have suggested that angiopoietin‐1 and ‐2 play a contributory role in the pathogenesis of ALI. Polymyxin B‐immobilized fiber column hemoperfusion is effective for sepsis‐induced ALI. We investigated the angiopoietin levels before and after direct hemoperfusion with polymyxin B‐immobilized fiber column (PMX) therapy. Enzyme‐linked immunoassay was used to measure the serum angiopoietin‐1 and ‐2 levels in 25 patients with septic shock treated with PMX. Eleven of the 25 patients were diagnosed with ALI. There was a significant positive correlation between the angiopoietin‐1 level and the PaO2/FiO2 ratio, but there was a significant inverse correlation between the angiopoietin‐2 level and the PaO2/FiO2 ratio. The mean angiopoietin‐1 level before PMX therapy in the ALI group was significantly lower and the mean angiopoietin‐2 level was significantly higher than in the non‐ALI group. The mean angiopoietin‐1 level of the ALI patients in response to PMX therapy was increased during PMX therapy, but that of the non‐ALI patients with newly occurring ALI showed a decreased angiopoietin‐1 level. On the other hand, the mean angiopoietin‐2 level of the responders was decreased during PMX therapy, but that of patients with newly occurring ALI showed an increased angiopoietin‐2 level. This result suggested that each angiopoietin‐1 and ‐2 level may play a role in the pathogenesis of ALI and that PMX therapy ameliorates the angiopoietin balance in patients with ALI in sepsis.

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.