Noriyoshi Hashimoto

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis (jvs; ref. 6). Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 (ref. 9). Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

RAGE Control of Diabetic Nephropathy in a Mouse Model Effects of RAGE Gene Disruption and Administration of Low-Molecular Weight Heparin

Diabetic nephropathy is a major microvascular complication in long-standing diabetic patients who eventually undergo renal dialysis or transplantation. To prevent development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. In this study, we examined whether inhibition of the receptor for advanced glycation end products (RAGE) could attenuate changes in the diabetic kidney. Here, we show that inactivation of the RAGE gene in a mouse model of diabetic nephropathy results in significant suppression of kidney changes, including kidney enlargement, increased glomerular cell number, mesangial expansion, advanced glomerulosclerosis, increased albuminuria, and increased serum creatinine compared with wild-type diabetic mice. The degree of kidney injury was proportional to RAGE gene dosage. Furthermore, we show that low–molecular weight heparin (LMWH) can bind RAGE at a mean equilibrium dissociation constant (Kd) value of ∼17 nmol/l and act as an antagonist to RAGE. LMWH treatment of mice significantly prevented albuminuria and increased glomerular cell number, mesangial expansion, and glomerulosclerosis in a dose-dependent manner; it also significantly improved the indexes of advanced-stage diabetic nephropathy. This study provides insight into the pathological role of RAGE in both early- and advanced-phase diabetic nephropathy and suggests that RAGE antagonists will be a useful remedy in the treatment of diabetic nephropathy.

Functional relevance of carnitine transporter OCTN2 to brain distribution of l‐carnitine and acetyl‐l‐carnitine across the blood–brain barrier

Transport of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine at the blood–brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl‐l‐[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl‐l‐carnitine and in the absence of sodium ions. Similar transport properties for l‐[3H]carnitine and/or acetyl‐l‐[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine by RBEC1 were sodium ion‐dependent, saturable with Km values of 33.1 ± 11.4 µm and 31.3 ± 11.6 µm, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT‐PCR method. Furthermore, the uptake of acetyl‐l‐[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of l‐[3H]carnitine and acetyl‐l‐[3H]carnitine in jvs mice were slightly lower than those of wild‐type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of l‐carnitine and acetyl‐l‐carnitine from the circulating blood to the brain across the BBB.

Effect of γ-butyrobetaine on fatty liver in juvenile visceral steatosis mice

We pharmacokinetically examined the effect of γ‐butyrobetaine, a precursor of l‐carnitine, on the change of fatty acid metabolism in juvenile visceral steatosis (JVS) mice, which have systemic l‐carnitine deficiency due to lack of l‐carnitine transporter activity. The concentrations of total free fatty acid (FFA), palmitic acid and stearic acid in the liver of JVS mice were significantly higher than those in wild‐type mice. After intravenous administration of γ‐butyrobetaine (50 mg kg−1), the concentration of l‐carnitine in the plasma of JVS mice reached about twice that of the control level and levels in the brain, liver and kidney were also significantly increased, whereas those in wild‐type mice hardly changed. Although the plasma concentrations of FFA in both types of mice were unchanged after administration of γ‐butyrobetaine, the concentrations of palmitic acid and stearic acid were significantly decreased. In particular, the liver concentration of FFA in JVS mice was decreased to the wild‐type control level, accompanied by significant decreases in long‐chain fatty acids, palmitic acid and stearic acid, whereas those in wild‐type mice were not changed. These results suggest that γ‐butyrobetaine can be taken up into organs, including the liver, of JVS mice, and transformed to l‐carnitine. Consequently, administration of γ‐butyrobetaine may be more useful than that of l‐carnitine itself for treatment of primary deficiency of carnitine due to a functional defect of the carnitine transporter.

Mice lacking asparaginyl endopeptidase develop disorders resembling hemophagocytic syndrome

Asparaginyl endopeptidase (AEP or legumain) is a lysosomal cysteine protease that cleaves protein substrates on the C-terminal side of asparagine. AEP plays a pivotal role in the endosome/lysosomal degradation system and is implicated in antigen processing. The processing of the lysosomal proteases cathepsins in kidney is completely defective in AEP-deficient mice with accumulation of macromolecules in the lysosomes, which is typically seen in lysosomal disorders. Here we show that mutant mice lacking AEP develop fever, cytopenia, hepatosplenomegaly, and hemophagocytosis, which are primary pathological manifestations of hemophagocytic syndrome/hemophagocytic lymphohistiocytosis (HLH). Moreover, AEP deficiency provokes extramedullary hematopoiesis in the spleen and abnormally enlarged histiocytes with ingested red blood cells (RBCs) in bone marrow. Interestingly, RBCs from AEP-null mice are defective in plasma membrane components. Further, AEP-null mice display lower natural killer cell activity, but none of the major cytokines is substantially abnormal. These results indicate that AEP might be a previously unrecognized component in HLH pathophysiology.

Gene-Dose Effect on Carnitine Transport Activity in Embryonic Fibroblasts of JVS Mice as a Model of Human Carnitine Transporter Deficiency

Recently, the marked decline in renal carnitine reabsorption has been thought to account fotr the systemic carnitine deficiency in juvenile visceral steatosis (JVS) mice. We have conducted a kinetic analysis using embryonic fibroblasts derived from normal, heterozygous, and homozygous jvs mice and found that the high-affinity carnitine transporter (Km = 5.5 microM), which shows Na+ and temperature dependency and stereospecificity, is defective in homozygous jvs mice. Moreover, a gene dose-dependent decrease of carnitine transport activity, which was due to a decrease in the number of the transporter molecules, was found in heterozygous jvs mice. Similar phenomena have been observed in human primary carnitine deficiency. Therefore, JVS mice may be useful for understanding this extremely rare human hereditary disorder.

Receptor for Advanced Glycation End Products Is a Promising Target of Diabetic Nephropathy

Abstract: Advanced glycation end products (AGEs) and the receptor for AGE (RAGE) interactions have been implicated in the development of diabetic vascular complications, which cause various disabilities and shortened life expectancy, and reduced quality of life in patients with diabetes. Diabetes‐induced RAGE‐overexpressing transgenic mice exhibited the exacerbation of the indices of nephropathy, and this was prevented by the inhibition of AGE formation. We also created RAGE‐deficient mice by homologous recombination. They showed marked amelioration of diabetic nephropathy as compared with wild‐type mice. Through an analysis of vascular polysomal poly(A)+ RNA, we identified a novel splice variant coding for a soluble RAGE protein and named it endogenous secretory RAGE (esRAGE). esRAGE was able to protect AGE‐induced vascular cell injuries as a decoy receptor and was actually detected in human circulation. We conclude that RAGE plays an active role in the development of diabetic vascular complications, especially nephropathy, and is a promising target for overcoming this disease. The esRAGE, an endogenous decoy receptor, may be related to individual variations in resistance to the development of diabetic vascular complications.

SCID-bg mice as xenograft recipients

SCID-bg (scid/scid, beige/beige) is a strain of double-mutant mice with impaired lymphoid development and reduced natural killer (NK) cell activity. The present study was undertaken to evaluate the usefulness of SCID-bg mice as xenograft recipients. Fetal guineapig tissues (liver, thymus, spleen) were transplanted under the kidney capsule of the mice and their serum guineapig IgG levels were measured weekly thereafter. C.B.-17-scid and anti-asialo GM1 antiserum-treated (NK-depleted) C.B.-17-scid (C.B.-17-scid-AGM1) mice that received the identical transplants were used as controls. Throughout the experimental period (1, 2, and 3 weeks after transplantation), the average serum guineapig IgG concentrations was highest in C.B.-17-scid-AGM1 mice followed by SCID-bg mice and lowest in C.B.-17-scid mice without antiserum treatment, though we could not find any statistical significance among these groups. However, SCID-bg mice always showed the smallest within-group variance (individual differencel in the serum guineapig IgG concentrations (P <0.05, versus C.B.-17-scid-AGM1 mice at 1,2, and 3 weeks and versus C.B.-17-scid mice at 2 weeks). The graft size was not significantly different among these three groups, but the spleen grafts in C.B.-17-scid mice contained fewer nucleate cells than the other two groups. These results indicate that the reduced NK cell activity by beige mutation is not crucial for the success of xenogenic transplantation, though SCID-bg mice may be useful as xenograft recipients with a consistent potential to retain the viability and function of engrafted tissues.

Development of IgA Nephropathy-Like Disease with High Serum IgA Levels and Increased Proportion of Polymeric IgA in Β-1,4-Galactosyltransferase-Deficient Mice

The glycosylation of glycoproteins is important for their biological activity, conformation and stability. Recent studies indicate that aberrant glycosylation causes various human disorders. Here we report that mice lacking beta-1,4-galactosyltransferase-I (beta4GalT-I), which transfers galactose from UDP-Gal to terminal GlcNAc of N- and O-glycans in a beta-1,4- linkage, developed IgA nephropathy (IgAN)-like disease. Urinary albumin levels were significantly increased in the beta4GalT-I-deficient mice. Hematuria was detected in some of the beta4GalT-I-deficient mice, suggesting impaired renal function. Furthermore, histological and immunohistochemical examination showed expanded mesangial matrix, IgA deposition with mesangial pattern and electron-dense deposits in the paramesangial regions in the beta4GalT-Ideficient mice. These results demonstrate that the beta4GalT-I-deficient mice developed IgANlike disease. Furthermore, high serum IgA levels with increased polymeric forms were detected. In humans, serum IgA derived from patients with IgAN has aberrant beta3-galactosylation and sialylation on its O-linked glycans of the hinge region. Mouse IgA does not have O-glycans of the hinge region and has several N-glycans. As expected, beta4-galactosylation on the N-glycans of the serum IgA of the beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We suggest that aberrant beta4-galactosylation of serum IgA participates in the Nishie/Miyaishi/Azuma/Kameyama/Naruse/Hashimoto/Yokoyama/Narimatsu/Wada/Asano 126 development of IgAN, including deposition of IgA, polymerization of IgA, and glomerular injury after IgA deposition.

TCRβ-independent development of CD4 + CD8 + thymocytes observed in a strain of scid mice

Mice homozygous for severe combined immunodeficiency (scid) mutation usually halt their thymocyte development at the CD4−CD8− double negative (DN) stage due to their inability of TCR gene rearrangement. In this study, we report that SCID‐bg mice, which were originally generated by mating CB‐17‐scid mice with KSN‐bg mice. spontaneously develop dominant CD4+CD8+ double positive (DP) thymocytes. Their thymi were mainly composed of DN, CD4−CD8+ and DP cells, and the majority of them did not present CD3. Similarly, they lacked TCRβ expression both on cell surface and in cytoplasm, which suggests that the thymocyte development to the DP stage observed m SCID‐bg mice was independent of CD3 and TCRβ expression. In spite of significant DP thymocytes in SCID‐bg mice, the histology of their thymi was not so different from those of CB‐17‐scid mice. Analysis of bone marrow cells in SCID‐bg mice showed that the development of B lineage cells was not altered when compared with CB‐17‐scid mice. These findings point out the fact that thymocytes in SCID‐bg mice have a peculiar characteristic compared to CB‐17‐scid mice, and provide evidence of TCRβ‐independent development of thymocytes to the DP stage.