Phenyl sulfate, indoxyl sulfate and p-cresyl sulfate decrease glutathione level to render cells vulnerable to oxidative stress in renal tubular cells.

In chronic kidney disease patients, oxidative stress is generally associated with disease progression and pathogenesis of its comorbidities. Phenyl sulfate is a protein-bound uremic solute, which accumulates in chronic kidney disease patients, but little is known about its nature. Although many reports revealed that protein-bound uremic solutes induce reactive oxygen species production, the effects of these solutes on anti-oxidant level have not been well studied. Therefore, we examined the effects of protein-bound uremic solutes on glutathione levels. As a result, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decreased glutathione levels in porcine renal tubular cells. Next we examined whether phenyl sulfate-treated cells becomes vulnerable to oxidative stress. In phenyl sulfate-treated cells, hydrogen peroxide induced higher rates of cell death than in control cells. Buthionine sulfoximine, which is known to decrease glutathione level, well mimicked the effect of phenyl sulfate. Finally, we evaluated a mixture of indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate at concentrations comparable to the serum concentrations of hemodialysis patients, and we confirmed its decreasing effect on glutathione level. In conclusion, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decrease glutathione levels, rendering the cells vulnerable to oxidative stress.

Anti-oxidative effect of AST-120 on kidney injury after myocardial infarction.

BACKGROUND AND PURPOSE: Chronic kidney disease (CKD) is a crucial risk factor for cardiovascular disease (CVD), and combined CKD and CVD further increases morbidity and mortality. Here, we investigated effects of AST-120 on oxidative stress and kidney injury using a model of myocardial infarction (MI) in rats. EXPERIMENTAL APPROACH: At 10 weeks, male spontaneously hypertensive rats (SHR) were divided into three groups: SHR (n = 6), MI (n = 8) and MI + AST-120 (n = 8). AST-120 administration was started at 11 weeks after MI. At 18 weeks, the rats were killed, and blood and urine, mRNA expression and renal histological analyses were performed. Echocardiography was performed before and after MI. KEY RESULTS: At 18 weeks, the BP was significantly lower in the MI and MI+AST-120 groups than in the SHR group. Elevated levels of indoxyl sulfate (IS), one of the uremic toxins, in serum and urine were reduced by AST-120 treatment, compared with the MI group. Markers of oxidative stress in urine and serum biomarkers of kidney injury were decreased in the MI+AST-120 group compared with the other two groups. Renal expression of mRNAs for kidney injury related-markers were decreased in the MI+AST-120 group, compared with the MI group. In vitro data also supported the influence of IS on kidney injury. Immunohistological analysis showed that intrarenal oxidative stress was reduced by AST-120 administration. CONCLUSIONS AND IMPLICATIONS: Serum IS was increased after MI and treatment with AST-120 may have protective effects on kidney injury after MI by suppressing oxidative stress.

Classification of Five Uremic Solutes according to Their Effects on Renal Tubular Cells.

Background/Aims. Uremic solutes, which are known to be retained in patients with chronic kidney disease, are considered to have deleterious effects on disease progression. Among these uremic solutes, indoxyl sulfate (IS) has been extensively studied, while other solutes have been studied less to state. We conducted a comparative study to examine the similarities and differences between IS, p-cresyl sulfate (PCS), phenyl sulfate (PhS), hippuric acid (HA), and indoleacetic acid (IAA). Methods. We used LLC-PK1 cells to evaluate the effects of these solutes on viable cell number, cell cycle progression, and cell death. Results. All the solutes reduced viable cell number after 48-hour incubation. N-Acetyl-L-cysteine inhibited this effect induced by all solutes except HA. At the concentration that reduced the cell number to almost 50% of vehicle control, IAA induced apoptosis but not cell cycle delay, whereas other solutes induced delay in cell cycle progression with marginal impact on apoptosis. Phosphorylation of p53 and Chk1 and expression of ATF4 and CHOP genes were detected in IS-, PCS-, and PhS-treated cells, but not in IAA-treated cells. Conclusions. Taken together, the adverse effects of PCS and PhS on renal tubular cells are similar to those of IS, while those of HA and IAA differ.

Protein-bound polysaccharide-K augments the anticancer effect of fluoropyrimidine derivatives possibly by lowering dihydropyrimidine dehydrogenase expression in gastrointestinal cancers.

Protein-bound polysaccharide-K (PSK) enhances the antitumor effect of anticancer drug when used clinically in combination with such drugs. PSK is known to act by immune-mediated mechanisms; however, the relationship between PSK and metabolic enzymes of anticancer drugs is unknown. We used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) clinically to evaluate the sensitivity of anticancer drugs. In the present study, we modified the CD-DST by adding peripheral blood mononuclear cells (PBMCs) (immuno-CD-DST) and examined the antitumor effect of PSK in combination with anticancer drugs. First, HCT116 human colon cancer cells were cultured with PSK and 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouridine (5'-DFUR) in the presence or absence of PBMCs, and the antiproliferative effects were compared. In the presence of PBMCs, PSK augmented the inhibitory effects of 5-FU and 5'-DFUR on HCT116 cell proliferation. Next, using human gastric cancer and colon cancer cell lines, the effects of PSK on mRNA expression of various metabolic enzymes of fluoropyrimidines: dihydropyrimidine dehydrogenase (DPD), thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase, were examined by real-time PCR. PSK significantly enhanced DPD mRNA expression in all of the cancer cell lines tested, but not those of the other enzymes. Addition of IFN-α and TRAIL, cytokines known to inhibit DPD expression, to the cultures reduced DPD mRNA expression in the cancer cells. When PBMC samples collected from healthy volunteers were cultured with PSK, IFN-α mRNA expression increased in 3 of the 5 PBMC samples, while TRAIL mRNA expression was unchanged. The present results propose the possibility that PSK induces PBMCs to express IFN-α which inhibits DPD expression, and consequently augments the antitumor effect of 5-FU or 5'-DFUR. Immuno-CD-DST is useful for evaluating drugs with immunological mechanisms of action.

Protein-bound polysaccharide-K induces apoptosis via mitochondria and p38 mitogen-activated protein kinase-dependent pathways in HL-60 promyelomonocytic leukemia cells.

Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101). PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated by modulating host immune systems; however, recent studies showed antiproliferative activity of PSK. Therefore, we examined the mechanism underlying the antiproliferative activity of PSK using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL-60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to elucidate the mechanism of the antiproliferative activity. Western blotting was performed to detect phosphorylated p38 mitogen-activated protein kinase (MAPK). A p38 MAPK inhibitor, SB203580, was used to examine the roles in PSK-induced apoptosis and growth inhibition. Flow cytometry was performed for mitochondrial membrane potential detection. PSK activated caspase-3 and induced p38 MAPK phosphorylation. Co-treatment with SB203580 blocked PSK-induced apoptosis, caspase-3 activation and growth inhibition. PSK induced apoptosis via the mitochondrial pathway. The depolarization of mitochondria induced by PSK was reversed by co-treatment with SB203580. The present study revealed that PSK induced apoptosis in HL-60 cells via a mitochondrial and p38 MAPK-dependent pathway.

Protein-bound polysaccharide-K (PSK) induces apoptosis via p38 mitogen-activated protein kinase pathway in promyelomonocytic leukemia HL-60 cells.

BACKGROUND/AIM: Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101) and is clinically used in combination therapy for gastrointestinal cancer and small-cell lung carcinoma. We have previously demonstrated that PSK induces apoptosis and inhibites proliferation of promyelomonocytic leukemia HL-60 cells, but the signaling pathway for this action remains to be elucidated. In HL-60 cells, the mitogen-activated protein kinase (MAPK) pathway has been reported to be involved in stimuli-induced apoptosis. Therefore, involvement of the p38 MAPK pathway in PSK-induced apoptosis was herein investigated. MATERIALS AND METHODS: HL-60 cells were used in this study. Western blotting was performed to detect phosphorylated p38 MAPK. A p38 MAPK inhibitor, SB203580, was used to examine the roles of p38 MAPK in PSK-induced apoptosis and growth inhibition. RESULTS: PSK induced p38 MAPK phosphorylation. Co-treatment with SB203580 blocked PSK-induced apoptosis, caspase-3 activation and growth inhibition. CONCLUSION: The p38 MAPK pathway plays an important role in PSK-induced apoptosis.

[Analysis of the mechanism of apoptosis induction by PSK].

Previously, we reported that PSK induces apoptosis and growth inhibition in HL60 cells. In this study, we tried to clarify the mechanism of how PSK induces apoptosis. Because several reports suggested that apoptosis of HL60 cells is mediated by activation of p38MAPK, we examined whether p38MAPK is involved in PSK-induced apoptosis. First, we found that PSK induced p38MAPK phosphorylation, which is considered as its activation. Next, we demonstrated that SB203580, inhibitor of p38MAPK, inhibited PSK-induced apoptosis. These results suggest that p38MAPK plays an important role in PSK-induced apoptosis.

Protein-bound polysaccharide-K (PSK) induces apoptosis and inhibits proliferation of promyelomonocytic leukemia HL-60 cells.

Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101), and is clinically used in combination therapy for gastrointestinal cancer and small cell lung carcinoma. PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated, by modulating host immune systems, such as the activation of immune effector cells and the neutralization of transforming growth factor-beta (TGFß) activity. Direct inhibition of tumor cell proliferation has been reported as another mechanism, but how PSK induces such an effect remains to be elucidated. Here, the anti-proliferative activity of PSK was examined using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to clarify the mechanism of anti-proliferative activity. Caspase-3 activation followed by apoptosis are involved at least in part in the PSK-induced anti-proliferative activity against HL-60 cells.

[Analysis of the mechanism of apoptosis induction by PSK].

Although PSK is an antitumor drug with immunomodulating effects, it has also been shown to have a direct action on cancer cells. This study analyzed the mechanism of the direct action of PSK on cancer cells, focused on the apoptosis-inducing effect. First, the cell growth inhibitory effect of PSK was examined using seven cancer cell lines, and HL60 cells were found to be strongly inhibited. Next, using HL60 cells, the apoptosis-inducing effect of PSK was examined using Annexin-V/Propidium iodide immunostaining and DNA fragmentation. The results indicated that PSK induced the apoptosis of HL60 cells. When the effect of PSK on protein expression of apoptosis-related factors was analyzed using an apoptosis array, over-expression of pro-caspase-3 and under-expression of factors such as cIAP-1, and cIAP-2 were observed. Furthermore, FACS analysis confirmed an increase in percentage of cells expressing activated caspase-3. These findings suggest that PSK induces apoptosis of HL60 cells and inhibits cell proliferation.

Screening of drugs that suppress Ste11 MAPKKK activation in yeast identified a c-Abl tyrosine kinase inhibitor.

The yeast MAPKKK Ste11 activates three MAP kinase pathways, including pheromone signaling, osmosensing, and pseudohyphal/invasive growth pathways. We identified two chemical compounds, BTB03006 and GK03225, that suppress growth defects induced by Ste11 activation in diploid yeast cells. BTB03006, but not GK03225, was found to suppress growth defects induced by both alpha-factor and Ste4 G(beta) overexpression in the pheromone signaling pathway, suggesting that GK03225 is an osmosensing pathway-specific inhibitor. We also performed genome-wide suppressor analysis for Ste11 activation, using a yeast deletion strains collection, and identified PBS2 and HOG1, and several genes associated with chaperone functions, which represent potential target proteins of the drugs screened from Ste11 activation. GK03225 possesses an Iressa-like quinazoline ring structure, and its chemical analog, 11N-078, suppresses c-Abl human tyrosine kinase activity. These results suggest that drug screening in yeast can identify human tyrosine kinase inhibitors and other drugs for human diseases.

Administration of oral charcoal adsorbent (AST-120) suppresses low-turnover bone progression in uraemic rats.

BACKGROUND: Using a rat model of renal failure with normal parathyroid hormone levels, we had demonstrated previously that bone formation decreased depending on the degree of renal dysfunction, and hypothesized that uraemic toxins (UTx) are associated with the development of low-turnover bone development, complicating renal failure. In this study, focusing on indoxyl sulphate (IS) as a representative UTx, we analysed the effect of an oral charcoal adsorbent AST-120, which removes uraemic toxins and their precursors from the gastrointestinal tract, on bone turnover. METHODS: AST-120 or vehicle was administered orally to model rats with uraemia and low turnover bone. Bone turnover was analysed by histomorphometry. Expression of osteoblast-related genes and oat-3 gene was analysed by reverse transcription polymerase chain reaction. RESULTS: In rats treated with vehicle, serum IS level increased with time after renal dysfunction, while bone formation decreased accompanied by down-regulation of the parathyroid/parathyroid-related peptide hormone receptor, alkaline phosphatase and osteocalcin genes. Administration of AST-120 inhibited the accumulation of IS in blood and ameliorated bone formation. Bone formation rate was 2.4 +/- 1.7 microm(3)/m(2)/year in controls given vehicle and was 11.7 +/- 2.4 microm(3)/m(2)/year in rats administered with AST-120 (P < 0.05). AST-120 treatment also reversed the down-regulation of osteoblast-related genes. Gene expression of oat-3 was detected in the tibia of rats. CONCLUSION: Administration of the oral charcoal adsorbent AST-120 decreases the osteoblast cytotoxicity of UTx including IS, and suppresses progression of low bone turnover in uraemic rats.

Uremic toxin and bone metabolism.

Patients with end-stage renal disease (ESRD) develop various kinds of abnormalities in bone and mineral metabolism, widely known as renal osteodystrophy (ROD). Although the pathogenesis of ESRD may be similar in many patients, the response of the bone varies widely, ranging from high to low turnover. ROD is classified into several types, depending on the status of bone turnover, by histomorphometric analysis using bone biopsy samples [1,2]. In the mild type, bone metabolism is closest to that of persons with normal renal function. In osteitis fibrosa, bone turnover is abnormally activated. This is a condition of high-turnover bone. A portion of the calcified bone loses its lamellar structure and appears as woven bone. In the cortical bone also, bone resorption by osteoclasts is active, and a general picture of bone marrow tissue infiltration and the formation of cancellous bone can be observed. In osteomalacia, the bone surface is covered with uncalcified osteoid. This condition is induced by aluminum accumulation or vitamin D deficiency. The mixed type possesses characteristics of both osteitis fibrosa and osteomalacia. The bone turnover is so markedly accelerated that calcification of the osteoid cannot keep pace. In the adynamic bone type, bone resorption and bone formation are both lowered. While bone turnover is decreased, there is little osteoid. The existence of these various types probably accounts for the diversity in degree of renal impairment, serum parathyroid hormone (PTH) level, and serum vitamin D level in patients with ROD. However, all patients share a common factor, i.e., the presence of a uremic condition.