Yoshihiro Motomiya

Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22phox and gp91phox, membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22PHOX and gp91PHOX. This led to blockade of the AGE-elicited Ras activation and NF-κB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.

A Circulating β2-Microglobulin Intermediate in Hemodialysis Patients

Background/Aims: A misfolded β₂-microglobulin (β₂m) is a principle component in dialysis-related amyloidosis. However, no such conformational variant of β₂m has yet been reported in a clinical setting. Capillary electrophoresis is a tool that can identify the conformational variant of β₂m. Methods: Capillary electrophoresis was used to measure a transitional intermediate from native β₂m (N-β₂m) to the amyloid β₂m. This technique was utilized to assay for intermediate β₂m (I-β₂m) in serum from 31 hemodialysis (HD) patients before and after HD, 5 patients with non-dialysis chronic renal failure (CRF), and 5 healthy persons. Results:The predialysis values of serum I-β₂m and N-β₂m were 2.7 ± 1.4 and 29.4 ± 6.8 mg/l, respectively, in the HD patients. The presence of serum I-β₂m correlated weakly with the total serum β₂m concentration in all HD patients. The serum N-β₂m concentration decreased significantly during two types of dialysis treatment: by 32.8% on HD using a polymethylmethacrylate (PMMA) membrane and by 71.2% on online hemodiafiltration (HDF) with a polysulfone (PS) membrane. On the other hand, a dialysis-associated change in serum I-β₂m varied from –36.4 to +203.5% in HD patients using PMMA and from –70.8 to +62.5% in online HDF patients using PS. Moreover, a rebound β₂m profile suggested that I-β₂m might be immobilized in the extracellular space. Conclusion: This study demonstrated that two or three conformational isomers of β₂m were probably ubiquitously recognized in human serum. Though no progressive increase in serum I-β₂m concentration could be found along with HD, this study shows a significantly poor removal of I-β₂m in comparison to N-β₂m in patients receiving ongoing dialysis treatment, even with online HDF.

Marked Increases in Macrophage Colony-Stimulating Factor and Interleukin-18 in Maintenance Hemodialysis Patients: Comparative Study of Advanced Glycation End Products, Carboxymethyllysine and Pentosidine

Background: Recent studies have demonstrated the crucial involvement of advanced glycation end products (AGEs) in major complications of long-term hemodialysis (HD) patients. HD, in a clinical setting, is characterized by increased production of proinflammatory cytokines. AGEs and cytokines are presumed to be responsible for the development of major complications in long-term HD. We therefore investigate here the relationship between a newly identified cytokine, interleukin-18 (IL-18), and two AGEs, carboxymethyllysine-hemoglobin (CML-Hb) and pentosidine. Methods: CML-Hb, pentosidine macrophage colony-stimulating factor (M-CSF), and IL-18 were evaluated in 35 patients undergoing stable maintenance HD. CML-Hb and pentosidine were measured by a dot blot and competitive ELISA. Cytokines were measured with a cytokine-specific ELISA. Results: Circulating levels of CML-Hb and pentosidine were elevated in HD patients as compared to controls. The serum values of M-CSF and IL-18 were significantly increased in the HD patients in comparison to controls. Moreover, these two AGEs and serum values of M-CSF, M-CSF and IL-18 showed significant correlation by simple and multiple regression analysis. Conclusion: Elevation of circulating IL-18 levels was demonstrated in maintenance HD patients relative to controls. A correlative increase in M-CSF and IL-18 suggests the presence of a primed state of monocytes/macrophages in HD patients.

A possible role for leptin in normo- or hypoparathyroid uremic bone in postmenopausal dialysis women.

Abstract Leptin has been proposed to be a key molecule involved in energy regulation. Based on the generally acknowledged concept that a heavier person has a higher bone density, leptin is thought to be potentially involved in bone metabolism. Serum leptin, various bone markers, and bone mineral density (BMD) were studied in 51 dialysis patients (26 men and 25 women). The serum concentrations of leptin in dialysis patients ranged from 0.7 to 10.4 ng/ml (mean 3.2 ± 2.1 ng/ml) for males and from 1.4 to 44.6 ng/ml (mean 11.8 ± 10.4 ng/ml) for females. There was a good correlation between the body mass index (BMI) and leptin concentration in both male and female patients (P < 0.05). Serum leptin levels also correlated well with the age-adjusted z-score for BMD (P < 0.02) and were inversely correlated with levels of the carboxy-terminal propeptide of type I procollagen (P < 0.05) in females patients, but not in male patients. In conclusion, these results suggest an actual contribution of serum leptin in maintaining bone density in postmenopausal female dialysis patients.

Role of Extracellular Superoxide Dismutase in Patients under Maintenance Hemodialysis

Background: It has been well documented that free radical injury is involved in the progression of chronic renal failure. Extracellular superoxide dismutase (EC-SOD), localized on the endothelial cell surface, plays an important role in reducing oxidative stress especially in the vessels by binding to the endothelial cell surface via the heparin-binding domain. Although EC-SOD Arg213Gly, which cannot bind on endothelial cells, has been considered a polymorphism, the effect of EC-SOD on hemodialysis patients has not been well examined. Methods: In 178 hemodialysis patients, the following examinations were performed. EC-SOD Arg213Gly was examined by polymerase chain reaction (PCR)-induced mutation restriction analysis (PCR-IMRA). As indexes of atherosclerosis, the annual progression in intima-media thickness (ΔIMT), plaque score, pulse wave velocity (PWV) and plasma-oxidized low-density lipoprotein (OxLDL) values were examined. Results: PCR-IMRA revealed that 20 of 178 patients possessed the mutation (11.2%), and the incidence was about twice as high as that in a previously reported Japanese population. Although there were no statistical differences in plaque score and PWV with and without EC-SOD Arg213Gly, ΔIMT and plasma OxLDL values in patients with EC-SOD Arg213Gly were significantly higher than those in patients without the mutation. Conclusion: EC-SOD Arg213Gly is an accelerating factor for the progression of renal failure and atherosclerosis.

Lipid droplets are present in amyloid deposits in familial amyloidotic polyneuropathy and dialysis related amyloidosis

It has been well documented that transthyretin (TTR) shows an affinity for lipoproteins and amyloid is deposited around adipocytes in patients with familial amyloidotic polyneuropathy (FAP). We examined the involvement of lipids in amyloid fibrils in the tissues by histopathologic methods. Sudan black B staining for frozen tissues of autopsied FAP patients and biospied dialysis related amyloidosis (DRA) patients revealed colocalization of lipids in the tissue sections where Congo red staining was positive, while no such positive staining was observed in paraffin embedded tissues. Immunohistochemical study using lipoprotein antibodies revealed that only anti-high density lipoprotein (HDL) antibody showed immunoreactivity in both types of amyloid specimens where Congo red was positively stained. Measurement of the components of lipids in the frozen cardiac samples from an FAP patient and an amyotrophic lateral sclerosis (ALS) patient revealed that concentrations of triglyceride and cholesterol in each lipoprotein, except for HDL-triglyceride, were markedly elevated in the FAP patients material, compared to that of the ALS patient. These results suggest that interaction of amyloid fibrils with HDL may play an important role in amyloid formation in FAP.

Role of conformational change in the C-terminus of β2-microglobulin in dialysis-related amyloidosis

Background β2-Microglobulin (β2m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying β2m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of β2m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded β2m in the C-terminus may be prone to form amyloid fibrils. Methods To evaluate roles of C-terminal amino acids in β2m-induced amyloid formation, we generated six types of recombinant β2m with amino acid substitutions in the C-terminal region. To investigate their conformational change and amyloidogenicity, we measured circular dichroism spectra, the fluorescence intensity of tryptophan and thioflavin-T (ThT) of the recombinant β2m. To analyse morphological change of β2m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance β2m destabilization of the protein. Results β2M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with β2m wild-type (WT). With ultrasonication, β2m Trp95Leu and Arg97Ala generated more amyloid fibrils than did β2m WT even in physiological solution. EM showed that β2m formed amorphous debris containing typical amyloid fibrils at 24 hours, when ThT fluorescence intensity was three-fold lower than that at six hours. Conclusions Conformational changes in the C-terminus of β2m may play an important role in DRA and that ultrasonication is useful for analysis of β2m amyloidogenesis.

Transplantation of nonvascularized kidney tissue fragments into the rat liver with the aim of preserving renal function.

For research in regenerative medicine, not only the study of cellular pluripotency but also knowledge of the reorganization of tissue structure is crucial. However, the latter will probably be more difficult to acquire. When small fragments of kidney (approx. 1 × 1 mm) were implanted in the liver of syngeneic LEW rats, the tissue survived at least 2 weeks with retention of normal structure including glomeruli and tubules. In contrast, no kidney structure survived when transplanted to subcutaneous sites, omentum, or spleen. Molecules involved in renal tubular function, such as megalin and glut2 transporter protein, were detectable in the implanted tissue by immunohistochemistry, suggesting that the cells were biologically active. Survival of cortex, medulla, and calyx tissues was then compared. All three components were still detectable 8 weeks after transplantation but cortex and medulla were replaced by granuloma at 6 months. Only calyx tissue survived for up to 12 months after transplantation. There was no marked difference in tissue survival, either when the recipient liver was partially resected or when infantile donor kidney was implanted instead of adult kidney. The present method opens new avenues in the development of regenerative medicine (i.e., tissue transplantation) as an intermediate modus between organ transplantation and cell transplantation.

Potential value of CML-Hb in predicting the progression of bone cysts in dialysis-related amyloidosis.

Background/Aim: Carboxymethyllysine is one of the common advanced glycation end products in vivo. In a previous report, we were the first to describe the increase of circulating carboxymethyllysine-hemoglobin (CML-Hb) levels in hemodialysis patients, particularly in patients with dialysis-related amyloidosis (DRA). The aim of this study was to investigate the predictive value of CML-Hb in the progression of DRA using computed tomography images of 2-year or 3-year follow-up periods of patients with amyloid bone cysts at the hip joint. Methods: Circulating CML-Hb levels were measured as previously reported, and computed tomography scanning was conducted from 1996 to 1998 or 1999 in 57 hemodialysis patients whose original renal disease had been confirmed to be nondiabetic. Patients who showed a new growth of cysts or a growth rate of 30% or more were classified as progressive cases, while the other patients were classified as nonprogressive cases. Results: (1) The circulating CML-Hb levels showed a strong correlation with the DRA score by multiple regression analysis, and (2) patients with progression of amyloid cysts showed a significantly higher circulating CML-Hb level than patients without progression. Conclusion: Based upon these results, we conclude that measurement of circulating CML-Hb levels has a potential value in both judgment of the clinical state and prediction of progression of DRA in hemodialysis patients.

C-terminal unfolding of an amyloidogenic β2-microglobulin fragment: Δn6β2-microglobulin

Abstract Objectives: A β2-microglobulin (β2m) fragment that lacks the first six amino acids, i.e., ΔN6β2-microglobulin (ΔN6β2m), is an endogenous, proteolytically derived, amyloidogenic fragment of β2m, the precursor protein in Aβ2M amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of β2m, in this study we aimed to investigate conformational characteristics of ΔN6β2m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of β2m with C-terminal unfolding (β2m92-99) in serum samples from 10 patients undergoing hemodialysis (HD). Methods: We utilized capillary electrophoretic analysis, surface plasmon resonance and enzyme-linked immunosorbent assay. Results and conclusions: We confirmed the normal core structure of ΔN6β2m with a commercial monoclonal anti-β2m antibody. In addition, using the specific monoclonal antibody for the C-terminal peptide, i.e. mAb 92-99, we confirmed unfolding in the C-terminal region of ΔN6β2m. On the basis of these findings, we established an ELISA to measure β2m92-99 using ΔN6β2m as a standard molecule in circulation. However, we did not detect β2m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum.