Effects of N-acetyl-L-tryptophan on desorption of the protein-bound uremic toxin indoxyl sulfate and effects on uremic sarcopenia.

INTRODUCTION: Indoxyl sulfate (IS) is a protein-bound uremic toxin that causes uremic sarcopenia. IS has poor dialysis clearance; however, the addition of a binding competitor improves its removal efficiency. METHODS: Dialysis experiments were performed using N-acetyl-l-tryptophan (L-NAT) instead of l-tryptophan (Trp) using pooled sera obtained from dialysis patients. The molecular structures of L-NAT and Trp were similar to that of IS. Therefore, we examined whether Trp and L-NAT were involved in muscle atrophy in the same manner as IS by performing culture experiments using a human myotube cell line. RESULTS: The removal efficiency of L-NAT was the same as that of Trp. However, L-NAT concentrations in the pooled sera increased at the end of the experiment. Trp (1 mM) decreased the area of human myocytes, similar to IS, whereas L-NAT did not. CONCLUSION: L-NAT is a binding competitor with the ability to remove protein-bound IS while preventing sarcopenia.

Chitin powder enhances growth factor production and therapeutic effects of mesenchymal stem cells in a chronic kidney disease rat model.

Previously, we fabricated a device with polylactic acid nonwoven filters and mesenchymal stem cells (MSCs), which effectively reduced urinary protein levels in a rat model of chronic kidney disease (CKD) but could not suppress CKD progression. Therefore, to improve the therapeutic effects of MSCs, in this study, we analyzed the ability of rat adipose tissue-derived MSCs (ADSCs) in contact with chitin nonwoven filters or chitin powder to produce growth factors and examined their therapeutic effect in an adriamycin (ADR)-induced CKD rat model. Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) production was significantly enhanced by ADSCs cultured in a medium containing chitin powder (C-ADSCs) compared with that by ADSCs cultured in a standard medium without chitin (N-ADSCs). However, the production of HGF and VEGF by ADSCs on chitin nonwoven filters was not significantly enhanced compared with that by the control. Intravenous C-ADSC injection significantly increased podocin expression and improved proteinuria compared with those in saline-treated CKD rats; however, no such improvements were observed in the N-ADSC-treated group. These results showed that ADSCs cultured in a medium supplemented with chitin powder suppressed proteinuria via enhanced HGF and VEGF production in ADR-induced CKD rats to mitigate podocyte damage, offering a new strategy to reduce the dose of MSC therapy for safe and effective treatment of kidney disease.

Suppression of inflammation during cell-free concentrated ascites reinfusion therapy using a blood purification device.

In recent years, cell-free concentrated ascites reinfusion therapy has been used to treat patients with malignant ascites. However, concentrated ascites reinfusion therapy involves enrichment and reinfusion of useful proteins and inflammatory cytokines. Therefore, fever is a primary side effect and significant problem for patients with ascites. We removed IL-6, an inflammatory cytokine, by mixing malignant ascites and the hexadecyl group adsorbent from a ß2 -microglobulin-adsorbing column (Lixelle S-15). As a result, the hexadecyl group adsorbent did not adsorb the albumin of malignant ascites but adsorbed 43% of IL-6. To investigate the effect of the hexadecyl group adsorbent on hepatocytes, the adsorbed ascites was added to a human hepatoma cell line (HepG2), and the gene expression levels of albumin and serum amyloid A protein were examined. After absorption, ascites showed significantly suppressed serum amyloid A protein expression and significantly increased albumin gene expression compared to before adsorption. Our results suggest that incorporation of Lixelle to filter and concentrate malignant ascites can suppress inflammatory responses and reduce the inhibition of albumin synthesis in the liver after reinfusion.

A novel cell-containing device for regenerative medicine: biodegradable nonwoven filters with peripheral blood cells promote wound healing.

The efficacy of skin regeneration devices consisting of nonwoven filters and peripheral blood cells was investigated for wound healing. We previously found that human peripheral blood cells enhanced their production of growth factors, such as transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor, when they were captured on nonwoven filters. Cells on biodegradable filters were expected to serve as a local supply of growth factors and cell sources when they were placed in wounded skin. Nonwoven filters made of biodegradable polylactic acid (PLA) were cut out as 13-mm disks and placed into cell-capturing devices. Mouse peripheral blood was filtered, resulting in PLA filters with mouse peripheral blood cells (m-PBCs) at capture rates of 65.8 ± 5.2%. Then, the filters were attached to full-thickness surgical wounds in a diabetic db/db mouse skin for 14 days as a model of severe chronic wounds. The wound area treated with PLA nonwoven filters with m-PBCs (PLA/B+) was reduced to 8.5 ± 12.2% when compared with day 0, although the non-treated control wounds showed reduction only to 60.6 ± 27.8%. However, the PLA filters without m-PBCs increased the wound area to 162.9 ± 118.7%. By histopathological study, the PLA/B+ groups more effectively accelerated formation of epithelium. The m-PBCs captured on the PLA filters enhanced keratinocyte growth factor (FGF-7) and TGF-ß1 productions in vitro, which may be related to wound healing. This device is useful for regeneration of wounded skin and may be adaptable for another application.

Appropriate nonwoven filters effectively capture human peripheral blood cells and mesenchymal stem cells, which show enhanced production of growth factors.

Scaffolds, growth factors, and cells are three essential components in regenerative medicine. Nonwoven filters, which capture cells, provide a scaffold that localizes and concentrates cells near injured tissues. Further, the cells captured on the filters are expected to serve as a local supply of growth factors. In this study, we investigated the growth factors produced by cells captured on nonwoven filters. Nonwoven filters made of polyethylene terephthalate (PET), biodegradable polylactic acid (PLA), or chitin (1.2-22 µm fiber diameter) were cut out as 13 mm disks and placed into cell-capturing devices. Human mesenchymal stem cells derived from adipose tissues (h-ASCs) and peripheral blood cells (h-PBCs) were captured on the filter and cultured to evaluate growth factor production. The cell-capture rates strongly depended on the fiber diameter and the number of filter disks. Nonwoven filter disks were composed of PET or PLA fibers with fiber diameters of 1.2-1.8 µm captured over 70% of leukocytes or 90% of h-ASCs added. The production of vascular endothelial growth factor (VEGF), transforming growth factor ß1, and platelet-derived growth factor AB were significantly enhanced by the h-PBCs captured on PET or PLA filters. h-ASCs on PLA filters showed significantly enhanced production of VEGF. These enhancements varied with the combination of the nonwoven filter and cells. Because of the enhanced growth factor production, the proliferation of human fibroblasts increased in conditioned medium from h-PBCs on PET filters. This device consisting of nonwoven filters and cells should be investigated further for possible use in the regeneration of impaired tissues.

Specificity of two monoclonal antibodies against a synthetic glycopeptide, an analogue to the hypo-galactosylated IgA1 hinge region.

Increased levels of hypo-galactosylated immunoglobulin (Ig)A1 (HG-IgA1) in IgA nephropathy (IgAN) have been detected using a Helix aspersa agglutinin lectin enzyme-linked immunosorbent assay (ELISA). In this study, we developed monoclonal antibodies to evaluate the HG-IgA1 in IgA nephropathy, aiming to gain a more consistent and reproducible assay. As an analogue to the HG-IgA1 hinge region, a 19 mer synthetic peptide with five GalNAc (sHGP) residues at positions 4, 7, 9, 11 and 15 [VPST(GalNAc)PPT(GalNAc)PS(GalNAc)PS(GalNAc)TPPT (GalNAc)PSPS-NH2] was synthesized. Two monoclonal antibodies against sHGP (35A12 and 44H8) that reacted with human IgA were developed. Also, their reactivities to serum IgA from IgAN patients (n = 49), patients with other forms of kidney diseases (OKD, n = 48), and healthy controls (HC, n = 41) were evaluated using ELISA assays. The binding levels of the two monoclonal antibodies against serum IgA were significantly higher (all comparisons, p < 0.0001, Steel-Dwass non-parametric test) in IgAN patients compared to HC and OKD patients. In each individual, there was a close correlation of IgA binding levels between 35A12 and 44H8 (R(2) = 0.737). These results indicate that the monoclonal antibodies recognize similar epitopes in HG IgA1, which is found predominantly in IgAN patients. The developed antibodies are proposed as a clinically useful tool for IgAN screening.

A potential therapeutic system for Alzheimer's disease using adsorbents with alkyl ligands for removal of blood amyloid ß.

Amyloid beta proteins (Aß) in the brain are the main cause of Alzheimer's disease. Peripheral administration of Aß-binding substances, which may act as a sink for Aß from the brain, has been reported to reduce brain Aß. We previously found C16-cellulose beads had high Aß-removal activity in vitro. In this study, we investigated the optimum surface properties of adsorbents for removal of Aß in vitro and in humans. Batch analysis was performed with porous cellulose beads or silica beads with or without 2-22 methylene groups. Aß-removal activity of C16-cellulose beads increased with increasing alkyl chain length. In contrast, with cellulose the amount of Aß removed by the silica beads decreased with increasing alkyl chain length. Cellulose beads with 16 or 22 methylene groups were best (over 99 % removal) among all the beads tested (p ≤ 0.01). The adsorbent surfaces were analyzed by near-infrared spectroscopy, which revealed that the optimum beads had a sufficiently hydrophobic surface with an appropriate amount of adsorbed water accessible on the surface. Aß removal efficiency by C16-cellulose beads was investigated for 5 renal failure patients on hemodialysis, resulting in 51.1 ± 6.6 % for Aß1-40 and 43.8 ± 4.5 % for Aß1-42 (p ≤ 0.01). In conclusion, cellulose beads with 16 or 22 methylene groups and an appropriate amount of adsorbed water were the optimum Aß adsorbents. The device with C16-cellulose beads had high Aß removal activity in humans. These adsorbents might be useful for Alzheimer's disease therapy.

Potential therapeutic system for Alzheimer's disease: removal of blood Aßs by hemodialzyers and its effect on the cognitive functions of renal-failure patients.

The pathological changes of Alzheimer's disease include the deposition of amyloid ß protein (Aß) as senile plaques in the brain. We hypothesized that the rapid removal of Aßs from the blood may act as a peripheral Aß drainage sink from the brain. In this study, the plasma Aß concentrations and the cognitive functions were investigated for in 57 patients on hemodailysis (69.4 ± 3.8 years), 26 renal-failure patients without hemodialysis (66.6 ± 14.7 years), and 17 age-matched healthy controls (66.6 ± 4.1 years). The concentrations of plasma Aßs increased along with the decline of renal functions. Moreover, the renal-failure patients without hemodialysis and with poorer renal functions showed lower cognitive functions. The plasma concentrations of Aß(1-42) correlated with serum creatinine (P < 0.001) and Mini-Mental-State Examination scores (P = 0.017). The dialyzers effectively removed Aßs in the blood during hemodialysis sessions. The plasma Aß concentrations showed steady or slightly decreasing along with duration of hemodialysis. The total amount of Aßs removed during a hemodialysis session was calculated to be comparable to the Aßs dissolved in the blood and the cerebrospinal fluid. The MMSE scores of the hemodialysis patients showed no clear decrease in longer hemodialysis duration. Therefore, the therapeutic approach for Alzheimer's disease by removing Aßs from the blood is worthy of further investigation, including whether or not Aßs in the brain decrease.

Accumulation of bisphenol A in hemodialysis patients.

Bisphenol A [BPA, 2,2-bis(4-hydoxyphenyl)propane], an industrial chemical used in the production of polycarbonate, epoxide resin, and polyarylate, is considered to be an endocrine-disrupting chemical. BPA may be present in some hollow-fiber dialyzers used in hemodialysis. In this study, we tested the amounts of BPA eluted from various hollow fibers. Furthermore, we measured the BPA concentration in the sera of 22 renal disease predialysis patients, as well as 15 patients who were receiving hemodialysis, to see if there is BPA accumulation in these patients. The elution test of BPA showed that a much larger amount of BPA was eluted from polysulfone (PS), and polyester-polymeralloy hollow fibers. Among renal disease patients who had not undergone hemodialysis, the serum BPA concentration increased as the renal function deteriorated, showing a significant negative association. In a crossover test between PS and cellulose (Ce) dialyzers, the predialysis serum BPA concentration of PS dialyzer users decreased after changing to a Ce dialyzer, and the serum BPA increased again after switching back to PS dialyzers. In patients who were using PS dialyzers, the BPA level significantly increased after a dialysis session. However, in the Ce dialyzer users, the BPA level decreased. Since accumulation of BPA could affect the endocrine or metabolic system of the human body, it is important to perform further investigations on dialysis patients.

Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay.

Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERalpha). Our results revealed that, while DEHP has some binding affinity for ERalpha, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERalpha. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERalpha was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERalpha, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERalpha.