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 ß-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.

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.

Anemia and hypertension are risk factors for both renal prognosis and survival in patients with diabetes mellitus.

BACKGROUND: Diabetic nephrosclerosis is the most common cause of renal failure in the industrialized countries. At the same time, the mortality rate of patients with diabetes mellitus is high. METHODS: To clarify the factors influencing the prognosis and survival of patients with diabetic nephrosclerosis, we carried out a retrospective follow-up study of 166 cases (age, 55.6 +/- 1.0 years; male/female, 110/56) by simple and multifactorial analyses of clinical data recorded at time of renal biopsy, including survival after diagnosis of diabetic mellitus (months), body mass index (BMI) (kg/m(2)) [body weight/(body height)(2)], age (years), mean blood pressure (mBP) (mmHg) [diastolic BP + (systolic BP - diastolic BP)/3], serum levels of albumin (mg/dl), urea nitrogen (BUN) (mg/dl), serum creatinine (s-Cr) (mg/dl), total cholesterol (mg/dl), triglyceride (mg/dl), and fasting blood sugar (FBS) (mg/dl), hematocrit (%), HbA1c (%), urinary protein secretion (g/day), insulin resistance, BP control (good, <140/90 mmHg or poor, > or =140/90 mmHg) after biopsies, and pathomorphological parameters at the biopsy. RESULTS: We found a significant association between renal prognosis and several factors, e.g., hypoalbuminemia, anemia, high levels of BUN and s-Cr, hypercholesteremia, hypertriglyceridemia at biopsy, poor control of BP after biopsies, Kimmelstiel-Wilson nodule, and severe glomerular and tubulointerstitial damages at the biopsy. In addition, associations between survival and factors such as low value of BMI, elderly age at the biopsy, and poor control of BP after biopsies were significant. By multivariate analysis we also found a significant association of renal prognosis with anemia, BUN, severe glomerular damage at the biopsy, and poor control of BP after biopsies. At the same time, poor control of BP after biopsies had a significant association with survival. On Kaplan-Meier analysis, anemia at biopsy and hypertension after biopsies are risk factors for both renal prognosis and survival in diabetes mellitus patients. CONCLUSIONS: Our data strongly suggest that good control of BP after biopsies and anemia at the biopsy play pivotal roles in the prognosis and survival of patients with diabetic glomerulosclerosis.

Membranoproliferative glomerulonephritis-like glomerular disease and concurrent tubulointerstitial nephritis complicating IgG4-related autoimmune pancreatitis.

Autoimmune pancreatitis is characterized by diffuse enlargement of the pancreas, irregular narrowing of the pancreatic duct, high serum levels of IgG4, and lymphoplasmacytic infiltration in the pancreatic parenchyma. Accumulating evidence suggests that this autoimmune disease could present with diffuse infiltration of IgG4-positive plasmacytes in multiple organs. Recently, a new concept of IgG4-related systemic disease including autoimmune pancreatitis, characterized by high serum IgG4 level and tissue infiltration by IgG4-positive plasma cells, has been proposed. Renal lesions in IgG4-related diseases have been reported recently. Most of them are tubulointerstitial nephritis; however, glomerulonephritis associated with IgG4-related diseases is very rare. We describe here a patient with membranoproliferative glomerulonephritis-like glomerular disease, together with tubulointerstitial nephritis, idiopathic thrombocytopenic purpura, and autoimmune pancreatitis. An 80-year-old Japanese man was referred to our hospital with a 14-month history of proteinuria, and a progressively rising serum creatinine level. Renal biopsy revealed membranoproliferative glomerulonephritis-like glomerular disease and concurrent tubulointerstitial nephritis. Immunolabeling of renal tissue showed numerous IgG4-positive plasma cells in the interstitium. The rare association between glomerulonephritis and IgG4-related systemic disease is discussed.

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

BACKGROUND: In 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. METHOD: Histological 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 CONCLUSION: A 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.

Effect of alendronate on glucocorticoid-induced osteoporosis in Japanese women with systemic autoimmune diseases: versus alfacalcidol.

Glucocorticoids-induced osteoporosis is a serious problem for patients with systemic autoimmune disease requiring relatively long-term glucocorticoid treatment. Effectiveness of alendronate for the prevention of glucocorticoids-induced osteoporosis was evaluated in comparison with that of alfacalcidol in Japanese women with autoimmune disease excluding rheumatoid arthritis. Loss of bone mass was evaluated with bone mineral density (BMD) of lumber vertebrae, bone resorption was with urinary N-telopeptide for type I collagen (NTX), and bone formation was with serum bone-specific alkaline phosphatase (B-ALP). A total of 33 patients who were treated with oral glucocorticoids (>or=5 mg/day of prednisolone equivalence) for more than 6 months were randomized into two groups; alendronate group (n = 17) received 5 mg/day of alendronate, and alfacalcidol group (n = 16) received 1.0 mug/day of alfacalcidol for 24 months with glucocorticoids. The dose of alendronate was the maximal dose approved in Japan. BMD had tendency to decrease with alfacalcidol, while increase with alendronate. The difference in BMD change between the two groups was significant by 4.3% at 18 months and by 4.2% at 24 months (both P < 0.05). Bone resorption was significantly reduced only with alendronate; NTX was decreased by 28 to 35% at 6 to 24 months (P < 0.05), but not changed with alfacalcidol at 24 months. The bone formation was found to be unchanged according to the B-ALP measured between the two groups. In conclusion, the treatment of 5 mg alendronate daily is more effective than alfacalcidol for preventing the glucocorticoid-induced osteoporosis by the mechanism of reducing bone resorption in Japanese women with systemic autoimmune disease.

Prognostic factors for renal amyloidosis: a clinicopathological study using cluster analysis.

OBJECTIVE: There is no standardized therapy for renal amyloidosis, which shows rapid progression and poor prognosis. Here, we used cluster analysis to examine the correlation between amyloid-related renal damage and prognosis, and determined the clinicopathological prognostic factors for renal amyloidosis. METHODS AND PATIENTS: We analyzed 125 patients with renal amyloidosis (men/women: 43/82; mean age at renal biopsy: 58.8+/-11.1 years, +/-SD; range: 21-78 years). Cluster analysis was performed using clinical parameters, renal histological findings, type of renal amyloidosis, and follow-up data. We also analyzed survival data. RESULTS: We divided 125 cases (prognosis was checked in 97 [77.6%] cases) into three groups by cluster analysis. In the cluster groups, accelerated progression correlated with serum creatinine (s-Cr) levels at renal biopsy and histological grade of renal damage by amyloid deposition (p<0.0001). The most important prognostic factors were glomerular, tubulointerstitial, and vascular lesions induced by amyloid deposition at biopsy (p<0.0001). We also found that amyloid-A (AA) type amyloidosis correlated is more significantly with amyloid-mediated vascular (P=0.0010) and tubulointerstitial lesions (p=0.0705) than with amyloid-L (AL) type amyloidosis. Proteinuria and nephrotic syndrome were more severe in AL than AA amyloidosis (p=0.0836). The 10-year individual survival rate was about 20%, and most deaths were due to cardiovascular disease and infection. CONCLUSION: Our results indicate that the quantity of amyloid deposition in the kidney, and the extent of glomerular, tubulointerstitial, and vascular damage are significant renal prognostic factors in amyloidosis.

Carnitine palmitoyltransferase II deficiency due to a novel gene variant in a patient with rhabdomyolysis and ARF.

Adult patients deficient in carnitine palmitoyltransferase II (CPT II) cannot generate sufficient amounts of energy, which results in rhabdomyolysis and acute renal failure (ARF). Its genetic basis has been recognized; but histopathologic changes, especially electron microscopic changes, have scarcely been described. The study subject is a patient with ARF caused by repetitive nontraumatic rhabdomyolysis. The acylcarnitine profile of serum and enzyme assay on skin fibroblasts confirmed the diagnosis of CPT II deficiency. Renal biopsy specimens were examined microscopically and immunohistochemically. The histological diagnosis was interstitial nephritis with acute tubular necrosis caused by rhabdomyolysis. Myoglobin in tubules was detected by means of immunohistochemistry and electron microscopy. The genetic structure of CPT II was analyzed in the patient and his family. Eight pairs of polymerase chain reaction (PCR) primers were designed to cover the coding region. Each PCR-amplified gene product was subjected to DNA sequencing, which unveiled heterozygosity at the CPT II locus consisting of a deletion of cytosine and thymine at codon 408, resulting in a stop signal at 420, as well as a mutation of arginine to cysteine at codon 631. The frame shift at 408 has never been described before. DNA sequencing of the family showed the deletion mutation from the mother and the point mutation from the father. We describe renopathological findings in a patient with CPT II deficiency associated with rhabdomyolysis, which suggested the pathological role of myoglobin casts in the development of tubular necrosis. Genetic analysis of the patient identified a novel variant of the CPT II gene.

Pure red-cell aplasia caused by the antibody to recombinant erythropoietin, epoetin-beta, in a Japanese patient with chronic renal failure.

A 68-year-old male with chronic renal failure and anemia received recombinant human erythropoietin (rHuEPO), epoetin beta, for approximately 1 year. Although the agent was initially effective for improving anemia, anemia refractory to EPO administration appeared and then worsened later, and pure red-cell aplasia (PRCA) was diagnosed. Anti-EPO antibody was detected by radioimmunoprecipitation (RIP) assay in the patient's serum. The antibody inhibited the proliferation of EPO-dependent cell line in a dose-dependent manner neutralizing EPO activity. The antibody also reacted with the other epoetin alfa products. The antibody did not recognize the carbohydrate moieties or denatured epoetin beta. The result suggested that the antibody recognized the conformational epitope of epoetin beta peptide molecule. Withdrawal of EPO and administration of cyclosporine decreased the titers of antibody; however, erythroid progenitor has not yet regenerated although the requirement for red blood cell transfusion is decreasing.

Relationship of predialytic intact parathyroid hormone on secondary hyperparathyroidism in chronic maintenance haemodialysis patients.

METHODS AND RESULTS: In order to clarify the predialytic factors influencing the onset of secondary hyperparathyroidism (SHPT) in patients on chronic maintenance haemodialysis, the time-course changes of serum levels of intact-PTH (i-PTH) during haemodialysis for 5 years were investigated. The subjects were 69 non-diabetic patients who had a serum aluminium level of less than 1.85 nmol/L at the end of observation. Patients were divided into two groups based on i-PTH levels obtained at the start of dialysis; the high group (H group) consisted of patients whose i-PTH levels were more than 22.00 pmol/L, the low group (L group) had levels less than 22.00 pmol/L. In the H group, i-PTH was 41.46 +/- 2.87 pmol/L at the start of dialysis (vs L group, P < 0.0001) and 15.82 +/- 2.85 pmol/L after haemodialysis initiation. In the L group, i-PTH levels did not significantly change and was 11.69 +/- 2.50 pmol/L 12 months after the start of dialysis (at the 12th month). However, at the 60th month, the i-PTH level was 33.24 +/- 5.30 pmol/L in the H group, and 9.85 +/- 2.13 pmol/L in the L group (P < 0.005). CONCLUSION: It is suggested that control of i-PTH levels in the predialytic period may be important to suppress SHPT throughout haemodialysis.