Shintaro Yamaguchi

The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells

BACKGROUND Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs. METHODS AND RESULTS Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase. CONCLUSION We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.

Kidney Specific Protein-Positive Cells Derived from Embryonic Stem Cells Reproduce Tubular Structures In Vitro and Differentiate into Renal Tubular Cells

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

miR-34c attenuates epithelial-mesenchymal transition and kidney fibrosis with ureteral obstruction

micro RNAs (miRNAs) are small non-coding RNAs that act as posttranscriptional repressors by binding to the 3′-UTR of target mRNAs. On the other hand, mesenchymal-epithelial transition (EMT) and kidney fibrosis is a pathological process of chronic kidney disease (CKD), and its relationship to miRNAs is becoming recognized as a potential target for CKD therapies. To find new miRNAs involved in EMT, we examined miRNA expression in experimental models of EMT and renal epithelialization using microarray, and found that miR-34c attenuates EMT induced by TGF-β in a mouse tubular cell line. To confirm the effects of miR-34c in vivo, we administered the precursor of miR-34c to mice with unilateral ureteral obstruction, and miR-34c decreased kidney fibrosis area and the expression of connective tissue growth factor, α-SMA, collagen type 1, collagen type 3 and fibronectin. In conclusion, our study showed miR-34c attenuates EMT and kidney fibrosis of mice with ureteral obstruction.

Renal Microcirculation and Calcium Channel Subtypes

It has recently been reported that voltage-dependent Ca channel subtypes, e.g., L-, T-, N-, and P/Q-type, are expressed in renal arterioles and renal tubules, and the inhibition of these channels exerts various effects on renal microcirculation. For example, selective blockade of L-type Ca channels with nifedipine preferentially dilates the afferent arteriole and potentially induces glomerular hypertension. On the other hand, recently developed Ca channel blockers (CCBs) such as mibefradil and efonidipine block both T-type and L-type Ca channels and consequently dilate both afferent and efferent arterioles, leading to lowering of intraglomerular pressure. Interestingly, aldosterone has recently been recognized as a factor exacerbating renal diseases, and its secretion from adrenal gland is mediated by T-type Ca channels. Furthermore, T-type CCBs were shown to ameliorate renal dysfunction by suppressing inflammatory processes and renin secretion. On the basis of histological evaluations, N-type Ca channels are present in peripheral nerve terminals innervating both afferent and efferent arterioles. Further, it was suggested that N-type CCBs such as cilnidipine suppress renal arteriolar constriction induced by enhanced sympathetic nerve activity, thereby lowering intraglomerular pressure. Taken together, various Ca channel subtypes are present in the kidney and blockade of selective channels with distinct CCBs exerts diverse effects on renal microcirculation. Inhibition of T-type and N-type Ca channels with CCBs is anticipated to exert pleiotropic effects that would retard the progression of chronic kidney disease through modulation of renal hemodynamic and non-hemodynamic processes.

Generation of kidney tubular organoids from human pluripotent stem cells

Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3β inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.

miR-363 induces transdifferentiation of human kidney tubular cells to mesenchymal phenotype

BackgroundmicroRNAs (miRNAs) are non-coding small RNAs that regulate embryonic development, cell differentiation and pathological processes via interaction with mRNA. Epithelial–mesenchymal transition (EMT) is pathological process that involves in a variety of diseases such as cancer or fibrosis.MethodsIn this study, we identified miR-363 as a potent inducer of EMT by microarray analysis in human kidney tubular cells, and analyzed the function and mechanisms of miR-363.ResultsOverexpression of miR-363 induced mesenchymal phenotypes with loss of epithelial phenotypes in human kidney tubular cells. In addition, in vitro scratch assay demonstrated that miR-363 promotes cell migration of primary culture of human kidney tubular cells. We identified TWIST/canonical WNT pathway as the downstream effecter of miR-363, and inhibition of canonical WNT by small molecule, IWR-1, attenuated EMT induced by miR-363.ConclusionmiR-363 induces transdifferentiation of human kidney tubular cells via upregulation of TWIST/canonical WNT pathway.

A case of severe osteomalacia caused by Tubulointerstitial nephritis with Fanconi syndrome in asymptomotic primary biliary cirrhosis

BackgroundPrimary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease, characterized by increased concentrations of serum IgM and the presence of circulating anti-mitochondrial antibodies. Although bone diseases such as osteoporosis or osteodystrophy are commonly associated with PBC, osteomalacia which is caused by abnormal vitamin D metabolism, mineralization defects, and phosphate deficiency has not been recognized as a complication of PBC.Case presentationWe report the case of a 49-year-old Japanese woman who complained of multiple fractures. Hypophosphatemic osteomalacia was diagnosed from a low serum phosphorus level, 1,25-dihydroxyvitamin D3 level, high levels of bone specific alkaline phosphatase and the findings of bone scintigraphy, although a bone biopsy was not performed. Twenty four hour urine demonstrated a low renal fractional tubular reabsorption of phosphate, increased fractional excretion of uric acid and generalized aminoaciduria. An intravenous bicarbonate loading test suggested the presence of proximal renal tubular acidosis (RTA). These biochemical data indicated Fanconi syndrome with proximal RTA. A kidney biopsy demonstrated the features of tubulointerstitial nephritis (TIN).The patient was also suspected as having primary biliary cirrhosis (PBC) because of high levels of alkaline phosphatase, IgM and the presence of anti-mitochondrial M2 antibody, though biochemical liver function was normal. Sequential liver biopsy was compatible with PBC and the diagnosis of PBC was definite. After administration of 1,25 dihydroxyvitamin D3, neutral potassium phosphate, sodium bicarbonate for osteomalacia and subsequent predonizolone for TIN, symptoms of fractures were relieved and renal function including Fanconi syndrome was ameliorated.ConclusionIn this case, asymptomatic PBC was shown to induce TIN with Fanconi syndrome with dysregulation of electrolytes and vitamin D metabolism, which in turn led to osteomalacia with multiple fractures. Osteomalacia has not been recognized as a result of the renal involvement of PBC. PBC and its rare complication of TIN with Fanconi syndrome should be considered in adult patients with unexplained osteomalacia even in the absence of liver dysfunction.

Triglyceride‐rich lipoproteins in chronic kidney disease patients undergoing maintenance haemodialysis treatment

Objective:  Plasma triglyceride (TG) levels were reported to be high in chronic kidney disease (CKD) patients undergoing haemodialysis (HD) treatment. One of the atherogenic causes of hypertriglyceridemia is the increase in TG‐rich lipoprotein remnants, which are equivalent to remnant‐like particle cholesterol (RLP‐C). Here, we compared the plasma levels of TG, a representative indicator of TG‐rich lipoproteins and RLP‐C, as well as the TG/RLP‐C ratio between CKD patients undergoing HD and controls, in an effort to elucidate the atherogenicity of TG‐rich lipoproteins in CKD patients on HD.

Gastrointestinal pseudo-obstruction after debulking surgery of malignant pheochromocytoma, improved by intravenous administration of α-adrenergic receptor blocker, phentolamine

Gastrointestinal pseudo-obstruction after debulking surgery of malignant pheochromocytoma, improved by intravenous administration of α-adrenergic receptor blocker, phentolamine

Obesity-induced kidney injury is attenuated by amelioration of aberrant PHD2 activation in proximal tubules

The involvement of tissue ischemia in obesity-induced kidney injury remains to be elucidated. Compared with low fat diet (LFD)-mice, high fat diet (HFD)-fed mice became obese with tubular enlargement, glomerulomegaly and peritubular capillary rarefaction, and exhibited both tubular and glomerular damages. In HFD-fed mice, despite the increase in renal pimonidazole-positive areas, the expressions of the hypoxia-responsive genes such as Prolyl-hydroxylase PHD2, a dominant oxygen sensor, and VEGFA were unchanged indicating impaired hypoxic response. Tamoxifen inducible proximal tubules (PT)-specific Phd2 knockout (Phd2-cKO) mice and their littermate control mice (Control) were created and fed HFD or LFD. Control mice on HFD (Control HFD) exhibited renal damages and renal ischemia with impaired hypoxic response compared with those on LFD. After tamoxifen treatment, HFD-fed knockout mice (Phd2-cKO HFD) had increased peritubular capillaries and the increased expressions of hypoxia responsive genes compared to Control HFD mice. Phd2-cKO HFD also exhibited the mitigation of tubular damages, albuminuria and glomerulomegaly. In human PT cells, the increased expressions of hypoxia-inducible genes in hypoxic condition were attenuated by free fatty acids. Thus, aberrant hypoxic responses due to dysfunction of PHD2 caused both glomerular and tubular damages in HFD-induced obese mice. Phd2-inactivation provides a novel strategy against obesity-induced kidney injury.