Tetsuhiko Yasuno

Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells

A method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm. Here we show the establishment of an efficient system of homologous recombination in human pluripotent stem cells by means of bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection. This system allowed us to generate human-induced pluripotent stem cell lines containing green fluorescence protein knocked into OSR1, a specific intermediate mesoderm marker. We have also established a robust induction protocol for intermediate mesoderm, which produces up to 90% OSR1(+) cells. These human intermediate mesoderm cells can differentiate into multiple cell types of intermediate mesoderm-derived organs in vitro and in vivo, thereby supplying a useful system to elucidate the mechanisms of intermediate mesoderm development and potentially providing a cell source for regenerative therapies of the kidney.

Functional analysis of iPSC-derived myocytes from a patient with carnitine palmitoyltransferase II deficiency

INTRODUCTION Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving β-oxidation of long-chain fatty acids (FAO), which leads to rhabdomyolysis and subsequent acute renal failure. The detailed mechanisms of disease pathogenesis remain unknown; however, the availability of relevant human cell types for investigation, such as skeletal muscle cells, is limited, and the development of novel disease models is required. METHODS We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a Japanese patient with CPT II deficiency. Mature myocytes were differentiated from the patient-derived hiPSCs by introducing myogenic differentiation 1 (MYOD1), the master transcriptional regulator of myocyte differentiation. Using an in vitro acylcarnitine profiling assay, we investigated the effects of a hypolipidemic drug, bezafibrate, and heat stress on mitochondrial FAO in CPT II-deficient myocytes and controls. RESULTS CPT II-deficient myocytes accumulated more palmitoylcarnitine (C16) than did control myocytes. Heat stress, induced by incubation at 38°C, leads to a robust increase of C16 in CPT II-deficient myocytes, but not in controls. Bezafibrate reduced the amount of C16 in control and CPT II-deficient myocytes. DISCUSSION In this study, we induced differentiation of CPT II-deficient hiPSCs into mature myocytes in a highly efficient and reproducible manner and recapitulated some aspects of the disease phenotypes of CPT II deficiency in the myocyte disease models. This approach addresses the challenges of modeling the abnormality of FAO in CPT II deficiency using iPSC technology and has the potential to revolutionize translational research in this field.

Mutations of carnitine palmitoyltransferase II (CPT II) in Japanese patients with CPT II deficiency

Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving β‐oxidation of long‐chain fatty acids. CPT II deficiency is a wide‐spectrum disorder that includes a lethal neonatal form, an infantile form, and an adult‐onset form. However, the ethnic characteristics and the relationship between genotype and clinical manifestation are not well understood. We investigated three non‐consanguineous Japanese patients with CPT II deficiency and examined cell lines from 4 unrelated patients and 50 healthy donors. The CPT 2 gene was typed by direct DNA sequencing of polymerase chain reaction‐amplified gene products. Case 1 (infantile form) was heterozygous for a phenylalanine to tyrosine substitution at position 383 (p.F383Y) and a novel valine to leucine substitution at 605 (p.V605L). Cases 2, 4, and 5 (infantile form) and case 3 (adult‐onset form) were heterozygous for a single mutation at F383Y. Case 6 (adult‐onset form) was compound heterozygous at the CPT 2 locus, with deletion of cytosine and thymine at residue 408, resulting in a stop signal at 420 (p.Y408fsX420), and an arginine to cysteine substitution at position 631 (p.R631C). Case 7 (adult‐onset form) was homozygous for the p.F383Y mutation. In conclusion, we identified p.F383Y mutations in six of seven patients with CPT II deficiency and two novel variants of the coding gene: p.Y408fsX420 and p.V605L. These mutations differ from those in Caucasian patients, who commonly harbor p.S113L, p.P50H, and p.Q413fsX449 mutations; therefore, our data and those of other Japanese groups suggest that the p.F383Y mutation is significant in Japanese patients with CPT II deficiency.

A JAPANESE ADULT FORM OF CPT II DEFICIENCY ASSOCIATED WITH A HOMOZYGOUS F383Y MUTATION

Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited autosomal-recessive disorder of the fatty acid metabolism.1 CPT II deficiency has three phenotypes: a lethal neonatal form, an infantile form, and an adult form. The adult form of CPT II deficiency, which is characterized by recurrent episodes of muscle pain and rhabdomyolysis, is the most common disorder of the fatty acid metabolism affecting skeletal muscles. We describe a Japanese adult form of CPT II deficiency with a homozygous F383Y mutation. The patient was a 21-year-old woman born of nonconsanguineous parents. Her brother had died as an infant, and Reye syndrome was suspected based on his liver biopsy. Her sister also died as an infant. At age 2 years, she had a hypoglycemic episode (blood sugar 9 mg/dL). When she caught a cold at age 19 years, myalgia of the right upper limb appeared and progressed rapidly to other muscles. Laboratory data showed a massively increased creatine kinase (CK) level (101,790 IU/L). Her blood urea nitrogen (BUN) was normal (19.7 mg/dL), whereas the creatinine (CREA) had increased (3.84 mg/dL). …

Macrophage impairment produced by Fc receptor gamma deficiency plays a principal role in the development of lipoprotein glomerulopathy in concert with apoE abnormalities

BACKGROUND To obtain a clear understanding of the pathogenesis of lipoprotein glomerulopathy (LPG), we studied the role of the deficiency of Fc receptor gamma chain (FcRγ) for the development of LPG in concert with apolipoprotein E (apoE) abnormalities. METHODS We generated apoE and FcRγ double-knockout (FcRγ/apoE-KO) mice, and subsequently introduced several kinds of human recombinant apoE genes. At 21 days after infection, the mice were sacrificed and histologically examined. Peritoneal macrophages were evaluated for their response to modified lipids. RESULTS In the FcRγ/apoE-KO mice, the human apoE3-injected mice showed the most drastic LPG-like changes, as well as prominent hypertriglyceridemia. Meanwhile, relative to the human apoE3-injected mice, the FcRγ/apoE-KO mice showed greater lipoprotein deposition and less macrophage infiltration into the mesangial area. Moreover, the peritoneal macrophages in the apoE/FcRγ-KO mice were impaired in lipid uptake and secretion of the cytokines monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted, after the uptake of oxidized low-density lipoprotein. CONCLUSIONS These results suggest that the impairment of macrophage function resulting from FcRγ deficiency plays a principal role in the development of LPG in the presence of apoE abnormalities.

Identification of MMP1 as a novel risk factor for intracranial aneurysms in ADPKD using iPSC models

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca2+ entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.

Spondylodiscitis Complicated by Chronic, Treatment-Dependent NephroticSyndrome

Spondylodiscitis develops in people with attenuated resistance to infection, such as patients undergoing steroid treatment and those with diabetes mellitus. Most patients with nephrotic syndrome are administered long-term steroids and immunosuppressants use, thus exposing patients to high risk of spondylodiscitis. However, no report has been published thus far on this issue. We here report a case of spondylodiscitis complicated by chronic, treatment-dependent nephrotic syndrome due to focal glomerular sclerosis.

Distinct cytokine mRNA expression pattern in immunoglobulin G4-related kidney disease associated with renal cell carcinoma

We treated a 61-year-old man with immunoglobulin (Ig)G4-related kidney disease (IgG4-RKD). He had a history of allergic diseases and an allergic reaction and had received a diagnosis of autoimmune pancreatitis (AIP). He had also received a diagnosis of renal cell carcinoma (RCC) and had undergone segmental resection of the left kidney at 59 years of age. His serum amylase level and number of peripheral eosinophils increased after RCC development. We hypothesized that the RCC may have induced AIP and IgG4-RKD and we therefore examined the excised RCC tissue; typical findings of IgG4-RKD associated with RCC were recognized. We next evaluated the mRNA expression of cytokines in the excised tissues of this case and ten other ordinary RCC cases. In all cases, notable levels of IL-10 mRNA and high levels of TGF-β mRNA were seen. Although prominent differences were not observed in the mRNA expression of Th1, Th17 and Treg cytokines in all cases, the present case alone showed increased production of the Th2 cytokines IL-4 and IL-5, which were not detected in ordinary RCC cases. Although the mechanism underlying IgG4-RKD development has not yet been determined, Th2 and Treg cells are thought to play a prominent role in the pathogenesis. It is therefore likely that in this case, the association of these two diseases was not coincidental, and a distinct immune response against RCC may trigger IgG4-RKD development.

Three novel mutations in the carnitine–acylcarnitine translocase ( CACT ) gene in patients with CACT deficiency and in healthy individuals

Carnitine–acylcarnitine translocase (CACT) and carnitine palmitoyltransferase II (CPT2) are key enzymes for transporting long-chain fatty acids into mitochondria. Deficiencies of these enzymes, which are clinically characterized by life-threatening non-ketotic hypoglycemia and rhabdomyolysis, cannot be distinguished by acylcarnitine analysis performed using tandem mass spectrometry. We had previously reported the CPT2 genetic structure and its role in CPT2 deficiency. Here, we analyzed the CACT gene in 2 patients diagnosed clinically with CACT deficiency, 18 patients with non-traumatic rhabdomyolysis and 58 healthy individuals, all of whom were confirmed to have normal CPT2 genotypes. To facilitate CACT genotyping, we used heat-denaturing high-performance liquid chromatography (DHPLC), which helped identify five distinct patterns. The abnormal heteroduplex fragments were subjected to CACT-specific DNA sequencing. We found that one patient with CACT deficiency, Case 1, carried c.576G>A and c.199-10t>g mutations, whereas Case 2 was heterozygous for c.106-2a>t and c.576G>A. We also found that one patient with non-traumatic rhabdomyolysis and one healthy individual were heterozygous for c.804delG and the synonymous mutation c.516T>C, respectively. In summary, c.576G>A, c.106-2a>t and c.516T>C are novel CACT gene mutations. Among the five mutations identified, three were responsible for CACT deficiency. We have also demonstrated the successful screening of CACT mutations by DHPLC.

Clopidogrel-induced Thrombotic Microangiopathy in a Patient with Hypocomplementemia

Clopidogrel was administered to a 67-year-old Japanese man to prevent the recurrence of cerebral infarction. Twelve weeks later, he was admitted to our hospital with acute renal failure, hemolytic anemia and thrombocytopenia, and was diagnosed with clopidogrel-induced thrombotic microangiopathy. Clopidogrel was immediately discontinued and corticosteroid and plasma exchange therapy were administered simultaneously. Thereafter, the patients condition gradually improved. The patient had a decreased serum complement C3 level. This suggests that the activated alternative pathway is related to thrombotic microangiopathy (TMA). TMA is a critical drug-associated adverse reaction that clinicians should always be vigilant about, because clopidogrel is widely prescribed.