Katsutoshi Kojima

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.

Adrenal reserve function after unilateral adrenalectomy in patients with primary aldosteronism.

Objective: After unilateral adrenalectomy (uADX) in patients with a unilateral aldosterone-producing adenoma (APA), the remaining contralateral adrenal gland is generally considered sufficient to support life. However, few studies have compared adrenal reserve function before and after uADX. Therefore, we closely evaluated adrenal cortisol secretory function before and after uADX in patients with unilateral APA. Methods: Patients who were diagnosed with APA and underwent uADX for unilateral APA were initially included in this study. Patients with subclinical Cushings syndrome (SCS) or Cushings syndrome were excluded on suspicion of autonomous cortisol secretion. Fourteen patients were finally evaluated. Morning basal serum cortisol and plasma adrenocorticotropin hormone (ACTH) levels were measured, and ACTH stimulation tests under 1-mg dexamethasone suppression (dex-ACTH test) were performed before and after uADX. Results: No patient developed clinical adrenal insufficiency. Basal cortisol levels were not significantly different before and after uADX. However, basal ACTH levels were significantly elevated after uADX. In addition, peak cortisol levels on the dex-ACTH test decreased in all patients after uADX. The peak cortisol level after uADX was 86.6 (81.4–92.4)% of the level before uADX. Conclusion: The adrenal cortisol secretory response to ACTH stimulation is mildly reduced after uADX in patients with unilateral APA without SCS or Cushings syndrome, although their basal cortisol level is sustained by elevated ACTH. These data will be important as a point of discussion when patients with unilateral APA consider either uADX or specific pharmacotherapy as treatment options.

Evaluation of quantitative parameters for distinguishing pheochromocytoma from other adrenal tumors

Adrenal tumors are increasingly found incidentally during imaging examinations. It is important to distinguish pheochromocytomas from other adrenal tumors because of the risk of hypertensive crisis. Although catecholamines and their metabolites are generally used to diagnose pheochromocytoma, false-positive test results are common. An effective screening method to distinguish pheochromocytoma from adrenal incidentalomas is needed. We analyzed 297 consecutive patients with adrenal incidentalomas. Our findings included 162 non-functioning tumors, 47 aldosterone-producing adenomas, 26 metastases, 22 cases of subclinical Cushing’s syndrome, 21 pheochromocytomas, 12 cases of Cushing’s syndrome, and 7 adrenocortical cancers. We checked quantitative parameters such as age, blood, and urine catecholamines and their metabolites, neuron-specific enolase, size and computed tomography (CT) attenuation values. Among catecholamine-related parameters, the sum of urine metanephrine and normetanephrine (urineMNM) levels produced the highest area under the receiver operating characteristic curve regarding discrimination of pheochromocytoma from other lesions. Size and CT attenuation values also differed significantly. However, size was correlated with catecholamine levels. CT attenuation was not correlated with other factors. The optimal thresholds were 19 Hounsfield units (HU) for CT attenuation (sensitivity, 100%; specificity, 60%) and 0.43 mg/24 h for urineMNM (sensitivity, 89%; specificity, 96%). No pheochromocytomas were evident when CT attenuation values were under 19 HU. Even in adrenal tumors with CT attenuation values ≥ 19 HU, when urineMNM was < 0.43 mg/24 h, the frequency of pheochromocytoma was only 4.3%, when urineMNM was ≥ 0.43 mg/24 h, the frequency of pheochromocytoma was 93% and when urineMNM was > 0.77 mg/24 h the frequency of pheochromocytoma was 100%. CT attenuation value and urineMNM represented the most useful combination for diagnosis of pheochromocytoma.

Significance of dopamine D 1 receptor signalling for steroidogenic differentiation of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) are expected to be both a revolutionary cell source for regenerative medicine and a powerful tool to investigate the molecular mechanisms underlying human cell development in vitro. In the present study, we tried to elucidate the steroidogenic differentiation processes using hiPSC-derived intermediate mesoderm (IM) that is known to be the origin of the human adrenal cortex and gonads. We first performed chemical screening to identify small molecules that induce steroidogenic differentiation of IM cells expressing Odd-skipped related 1 (OSR1), an early IM marker. We identified cabergoline as an inducer of 3β-hydroxysteroid dehydrogenase, an essential enzyme for adrenogonadal steroidogenesis. Although cabergoline is a potent dopamine D2 receptor agonist, additional experiments showed that cabergoline exerted effects as a low-affinity agonist of D1 receptors by increasing intracellular cyclic AMP. Further analysis of OSR1+ cells transfected with steroidogenic factor-1/adrenal 4 binding protein revealed that D1 receptor agonist upregulated expression of various steroidogenic enzymes and increased secretion of steroid hormones synergistically with adrenocorticotropic hormone. These results suggest the importance of dopamine D1 receptor signalling in steroidogenic differentiation, which contributes to effective induction of steroidogenic cells from hiPSCs.

MPS 01-09 THE APPLICATION OF VASCULAR CELL DIFFERENTIATION SYSTEM FROM HUMAN ES/iPS CELLS FOR VASCULAR BIOLOGY

Objective: Previously, we have succeeded in inducing and isolating vascular endothelial cells (ECs) and mural cells (MCs) from human iPS cells. Recently, we modified our vascular differentiation system and we have induced vascular cells more effectively and stably from various ES/iPS cell clones. Our vascular differentiation system could be applied for the clarification of vascular biology or pathology, because we can observe the change of gene expressions in the process of human vascular cell differentiation, not mouse or other species. Design and Method: We have researched about the expressions of cardiovascular hormones and their receptors through our vascular differentiation kinetics, aiming to research the meanings of these hormones into vascular cells. At first, we examined mRNA expressions in undifferentiated ES/iPS cells, ES/iPS-derived immature vascular cells, ES/iPS-derived ECs, ES/iPS-derived MCs. Also we used cell line aortic ECs and smooth muscle cells(SMCs) as control. Results: hES/iPS derived ECs expressed adrenomedullin and endothelin, which mean that we succeed to induce mature ECs endocrinologically. Also I examined the expressions of natriuretic peptides family. Interestingly, the mRNA expression of brain natriuretic peptide (BNP) was highly detected only in sorted Flk1-positive cells, which are considered as immature vascular cells. After differentiation, these BNP expressions were decreased. Also we could measure BNP at high concentration in the culture medium of Flk1 positive cells. By doing fraction assay, the majority of measured BNP turned out to be not BNP-32 but pro-BNP. As for NP receptors, GC-A were expressed only in ES or iPS – derived EC. Conclusions: These results suggest the possibility that BNP (which would be secreted from immature vascular cells) has some paracrine functions to vascular endothelial cells and now we examine the function of BNP for vascular differentiation/development. Also I would introduce other application of our vascular differentiation system, using patient specific-iPS cells.