Satohiro Masuda

Human recombinant erythropoietin directly stimulates B cell immunoglobulin production and proliferation in serum‐free medium

The effect of human recombinant erythropoietin (Epo) on B cell responses was studied in a serum‐free medium. Epo enhanced IgM production and thymidine uptake by a human IgM‐producing lymphoblasloid cell line, CBL. This effect was specific to Epo since enhancement was blocked by anti‐Epo antibody but not by control antibody. Among the various cytokines, interleukin‐4 (IL‐4) enhanced IgM production and thymidine uptake while IL‐6 enhanced IgM production without affecting thymidine uptake. In contrast, other cytokines including IL‐1β, IL‐2, IL‐5, interferon‐alpha (IFN‐α), interferon‐gamma (IFN‐γ), or granulocyte/macrophage colony‐stimulating factor (GM‐CSF) were without effect. However, the enhancing effect of Epo is different from that of IL‐4 or IL‐6, since Epo effect was not blocked by anti‐IL‐4 antibody or anti‐IL‐6 antibody. Moreover, specific binding of Epo was detected on CBL cells. Epo also enhanced immunoglobulin (IgG, IgM and IgA) production and thymidine uptake by purified tonsil small resting B cells stimulated by Staphylococcus aureus Cowan strain I (SAC) or by large activated B cells. In contrast, Epo had no effect on unstimulated smalt resting B cells. These results indicate that Epo could directly stimulate activated and differentiated B cells and could enhance B cell immunoglobulin production and proliferation.

Therapeutic Drug Monitoring of Everolimus: A Consensus Report

Abstract: In 2014, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology called a meeting of international experts to provide recommendations to guide therapeutic drug monitoring (TDM) of everolimus (EVR) and its optimal use in clinical practice. EVR is a potent inhibitor of the mammalian target of rapamycin, approved for the prevention of organ transplant rejection and for the treatment of various types of cancer and tuberous sclerosis complex. EVR fulfills the prerequisites for TDM, having a narrow therapeutic range, high interindividual pharmacokinetic variability, and established drug exposure–response relationships. EVR trough concentrations (C0) demonstrate a good relationship with overall exposure, providing a simple and reliable index for TDM. Whole-blood samples should be used for measurement of EVR C0, and sampling times should be standardized to occur within 1 hour before the next dose, which should be taken at the same time everyday and preferably without food. In transplantation settings, EVR should be generally targeted to a C0 of 3–8 ng/mL when used in combination with other immunosuppressive drugs (calcineurin inhibitors and glucocorticoids); in calcineurin inhibitor-free regimens, the EVR target C0 range should be 6–10 ng/mL. Further studies are required to determine the clinical utility of TDM in nontransplantation settings. The choice of analytical method and differences between methods should be carefully considered when determining EVR concentrations, and when comparing and interpreting clinical trial outcomes. At present, a fully validated liquid chromatography tandem mass spectrometry assay is the preferred method for determination of EVR C0, with a lower limit of quantification close to 1 ng/mL. Use of certified commercially available whole-blood calibrators to avoid calibration bias and participation in external proficiency-testing programs to allow continuous cross-validation and proof of analytical quality are highly recommended. Development of alternative assays to facilitate on-site measurement of EVR C0 is encouraged.

Conformational Change in Transfer RNA Is an Early Indicator of Acute Cellular Damage

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

Precise comparison of protein localization among OCT, OAT, and MATE in human kidney

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.

Efficacy of aprepitant for the prevention of chemotherapy-induced nausea and vomiting with a moderately emetogenic chemotherapy regimen: a multicenter, placebo-controlled, double-blind, randomized study in patients with gynecologic cancer receiving paclitaxel and carboplatin

BackgroundSubstance P contributes to the hypersensitivity reaction (HSR) to paclitaxel in a rat model. Aprepitant acts as an inhibitor of the binding of substance P to the neurokinin-1 receptor and, consequently, may reduce the frequency of paclitaxel-induced HSR. While aprepitant has a prophylactic effect against vomiting caused by high-dose cisplatin, the benefits of aprepitant have not been clearly demonstrated in patients receiving paclitaxel and carboplatin (TC) combination chemotherapy.MethodsWe conducted a multicenter, placebo-controlled, double-blind, randomized study in Japanese patients with gynecologic cancer who received TC combination chemotherapy. Patients received aprepitant or placebo together with both a 5-HT3 receptor antagonist and dexamethasone prior to chemotherapy. The primary endpoint was the proportion of patients with HSR, and the secondary endpoints were the proportion of patients with “no vomiting”, “no significant nausea”, and complete response, respectively.ResultsOf the 324 randomized patients, 297 (151 in the aprepitant group; 146 in the placebo group) were evaluated. The percentage of patients with HSR (9.2 vs. 7.5xa0%, respectively; Pxa0=xa00.339) was not significantly different between the groups. The percentage of “no vomiting” patients (78.2 vs. 54.8xa0%; Pxa0<xa00.0001), “no significant nausea” patients (85.4 vs. 74.7xa0%; Pxa0=xa00.014), and patients showing complete response (61.6 vs. 47.3xa0%, Pxa0=xa00.0073) was significantly higher in the aprepitant group than in the placebo group.ConclusionThe administration of aprepitant did not have a prophylactic effect on the HSR but was effective in reducing nausea and vomiting in gynecologic cancer patients receiving TC combination chemotherapy.

Molecular Markers of Tubulointerstitial Fibrosis and Tubular Cell Damage in Patients with Chronic Kidney Disease.

In chronic kidney disease (CKD), progressive nephron loss causes glomerular sclerosis, as well as tubulointerstitial fibrosis and progressive tubular injury. In this study, we aimed to identify molecular changes that reflected the histopathological progression of renal tubulointerstitial fibrosis and tubular cell damage. A discovery set of renal biopsies were obtained from 48 patients with histopathologically confirmed CKD, and gene expression profiles were determined by microarray analysis. The results indicated that hepatitis A virus cellular receptor 1 (also known as Kidney Injury Molecule-1, KIM-1), lipocalin 2 (also known as neutrophil gelatinase-associated lipocalin, NGAL), SRY-box 9, WAP four-disulfide core domain 2, and NK6 homeobox 2 were differentially expressed in CKD. Their expression levels correlated with the extent of tubulointerstitial fibrosis and tubular cell injury, determined by histopathological examination. The expression of these 5 genes was also increased as kidney damage progressed in a rodent unilateral ureteral obstruction model of CKD. We calculated a molecular score using the microarray gene expression profiles of the biopsy specimens. The composite area under the receiver operating characteristics curve plotted using this molecular score showed a high accuracy for diagnosing tubulointerstitial fibrosis and tubular cell damage. The robust sensitivity of this score was confirmed in a validation set of 5 individuals with CKD. These findings identified novel molecular markers with the potential to contribute to the detection of tubular cell damage and tubulointerstitial fibrosis in the kidney.

Urinary kidney injury molecule-1 and monocyte chemotactic protein-1 are noninvasive biomarkers of cisplatin-induced nephrotoxicity in lung cancer patients

PurposeAcute kidney injury (AKI) is a common and serious adverse effect of cisplatin-based chemotherapy. However, traditional markers of kidney function, such as serum creatinine, are suboptimal, because they are not sensitive measures of proximal tubular injury. We aimed to determine whether the new urinary biomarkers such as kidney injury molecule-1 (KIM-1), monocyte chemotactic protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL) could detect cisplatin-induced AKI in lung cancer patients in comparison with the conventional urinary proteins such as N-acetyl-β-d-glucosaminidase (NAG) and β2-microglobulin.MethodsWe measured KIM-1, MCP-1, NGAL, NAG, and β2-microglobulin concentrations in urine samples from 11 lung cancer patients, which were collected the day before cisplatin administration and on days 3, 7, and 14. Subsequently, we evaluated these biomarkers by comparing their concentrations in 30 AKI positive (+) and 12 AKI negative (−) samples and performing receiver operating characteristic (ROC) curve analyses.ResultsThe urinary levels normalized with urine creatinine of KIM-1 and MCP-1, but not NGAL, NAG, and β2-microglobulin in AKI (+) samples were significantly higher than those in AKI (−) samples. In addition, ROC curve analyses revealed that KIM-1 and MCP-1, but not NGAL, could detect AKI with high accuracy (area under the curve [AUC]xa0=xa00.858, 0.850, and 0.608, respectively). The combination of KIM-1 and MCP-1 outperformed either biomarker alone (AUCxa0=xa00.871).ConclusionsUrinary KIM-1 and MCP-1, either alone or in combination, may represent biomarkers of cisplatin-induced AKI in lung cancer patients.

Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity

Because of the difficulty in detecting segment-specific response in the kidney, we investigated the molecular events underlying acute kidney injury in the proximal tubules of rats with cisplatin (cis-diamminedichloroplatinum II)-induced nephrotoxicity. Microarray analysis revealed that mRNA levels of several cytokines and chemokines, such as interleukin-1beta, chemokine (C-C motif) ligand (CCL) 2, CCL20, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10 were significantly increased after cisplatin treatment in both isolated proximal tubules and whole kidney. Interestingly, tubular CCL2 mRNA levels increased soon after cisplatin administration, whereas CCL2 mRNA levels in whole kidney first decreased and then increased. Levels of both CCL2 and kidney injury molecule-1 (KIM-1) in the whole kidney increased after cisplatin administration. Immunofluorescence analysis revealed CCL2 changes in the proximal tubular cells initially and then in the medullary interstitium. Urinary CCL2 excretion significantly increased approximately 3-fold compared with controls the day after cisplatin administration (5mg/kg), when no changes were observed plasma creatinine and blood urea nitrogen levels. Urinary levels of KIM-1 also increased 3-fold after cisplatin administration. In addition, urinary CCL2 rather than KIM-1 increased in chronic renal failure rats after administration of low-dose cisplatin (2mg/kg), suggesting that urinary CCL2 was selective for cisplatin-induced nephrotoxicity in renal impairment. These results indicated that the increase in cytokine and chemokine expression in renal epithelial cells might be responsible for kidney deterioration in cisplatin-induced nephrotoxicity, and that urinary CCL2 is associated with tubular injury and serves as a sensitive and noninvasive marker for the early detection of cisplatin-induced tubular injury.

Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning.

Urinary neutrophil gelatinase-associated lipocalin: A useful biomarker for tacrolimus-induced acute kidney injury in liver transplant patients

Tacrolimus is widely used as an immunosuppressant in liver transplantation, and tacrolimus-induced acute kidney injury (AKI) is a serious complication of liver transplantation. For early detection of AKI, various urinary biomarkers such as monocyte chemotactic protein-1, liver-type fatty acid-binding protein, interleukin-18, osteopontin, cystatin C, clusterin and neutrophil gelatinase-associated lipocalin (NGAL) have been identified. Here, we attempt to identify urinary biomarkers for the early detection of tacrolimus-induced AKI in liver transplant patients. Urine samples were collected from 31 patients after living-donor liver transplantation (LDLT). Twenty recipients developed tacrolimus-induced AKI. After the initiation of tacrolimus therapy, urine samples were collected on postoperative days 7, 14, and 21. In patients who experienced AKI during postoperative day 21, additional spot urine samples were collected on postoperative days 28, 35, 42, 49, and 58. The 8 healthy volunteers, whose renal and liver functions were normal, were asked to collect their blood and spot urine samples. The urinary levels of NGAL, monocyte chemotactic protein-1 and liver-type fatty acid-binding protein were significantly higher in patients with AKI than in those without, while those of interleukin-18, osteopontin, cystatin C and clusterin did not differ between the 2 groups. The area under the receiver operating characteristics curve of urinary NGAL was 0.876 (95% confidence interval, 0.800–0.951; P<0.0001), which was better than those of the other six urinary biomarkers. In addition, the urinary levels of NGAL at postoperative day 1 (pu200a=u200a0.0446) and day 7 (pu200a=u200a0.0006) can be a good predictive marker for tacrolimus-induced AKI within next 6 days, respectively. In conclusion, urinary NGAL is a sensitive biomarker for tacrolimus-induced AKI, and may help predict renal event caused by tacrolimus therapy in liver transplant patients.