Yutaka Tonomura

Evaluation of the usefulness of urinary biomarkers for nephrotoxicity in rats.

Since nephrotoxicity affects the development of drug candidates, it is important to detect their toxicity at an early stage of drug development. In this study, we measured twelve urinary nephrotoxic biomarkers [total protein, albumin, kidney injury molecule-1 (KIM-1), clusterin, beta2-microglobulin, cystatin-c, alpha-glutathione S-transferase, mu-glutathione S-transferase, N-acetyl-beta-d-glucosaminidase, lactate dehydrogenase (LDH), aspartate aminotransferase and neutrophil gelatinase-associated lipocalin (NGAL)] and two conventional blood nephrotoxic biomarkers (creatinine and blood urea nitrogen) in rat models treated intravenously with puromycin aminonucleoside (PAN) or cisplatin (CDDP), which are known to induce glomerular injury or proximal tubular injury, respectively, and evaluated their usefulness by receiver operating characteristic analysis. In the PAN-treated rats, urinary albumin and (NGAL) were dramatically increased, which were thought to be caused by the dysfunction of proximal tubule in addition to glomerular injury. Conversely, based on its early and time-dependent increase, its large magnitude of alteration and its high accuracy and sensitivity of detection, (KIM-1) in urine appeared to be the best biomarker for detection of CDDP-induced proximal tubular injury. Moreover, (LDH) was considered useful for broad detection of damaged nephrons, because of its broad distribution along the nephron. Therefore, combinatorial measurement of these biomarkers may be a powerful tool for highly effective screening of nephrotoxicity.

Evaluation of the usefulness of biomarkers for cardiac and skeletal myotoxicity in rats.

Since cardiac and skeletal myotoxicity affect the development of drug candidates, it is important to detect their toxicity at an early stage of drug development. For that purpose, in this study, the usefulness of several cardiac and skeletal myotoxic biomarkers in blood were evaluated using two rat models treated intraperitoneally with an acetylcholinesterase inhibitor carbofuran (CAF) or a synthetic catecholamine isoproterenol (ISO). The biomarkers assayed were fatty acid binding protein 3 (Fabp3), myosin light chain 1 (MLC1), cardiac troponin I (cTnI), cardiac troponin T (cTnT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK). CAF and ISO treatment of rats induced greater increases in the levels of Fabp3, MLC1, cTnI and cTnT than in the levels of AST, LDH and CK. A kinetic analysis indicated that the levels of all of the biomarkers had returned to the basal level by 24h after drug administration. Pathological examination revealed lesions in the heart, mainly at the left ventricle and septum, in both CAF- and ISO-treated rats. CAF-treated rats showed widespread lesions of skeletal muscle that were independent of muscle fiber type, while in ISO-treated rats locoregional lesions were observed only in slow twitch muscle. Receiver operating characteristic curve analysis of the sensitivity of the tested biomarkers indicated that MLC1 and cTnT were the most effective biomarkers of cardiotoxicity. For skeletal myotoxicity, Fabp3 and MLC1 were the most effective biomarkers based on the specific tissue distribution of these proteins. Conversely, the rapid blood clearance of these markers should be taken into account when considering the use of these biomarkers.

Biomarker panel of cardiac and skeletal muscle troponins, fatty acid binding protein 3 and myosin light chain 3 for the accurate diagnosis of cardiotoxicity and musculoskeletal toxicity in rats

Cardiotoxicity and musculoskeletal toxicity can be life-threatening, and thus have strong impact on both the development and marketing of drugs. Because the conventional biomarkers such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) have low detection power, there has been increasing interest in developing biomarkers with higher detection power. The current study examined the usefulness of several promising biomarkers, cardiac and skeletal muscle troponins (cTnI, cTnT and sTnI), fatty acid binding protein 3 (FABP3) and myosin light chain 3 (MYL3), and compared the obtained data to AST, LDH and CK in rat models treated with various myotoxic and non-myotoxic compounds (isoproterenol, metaproterenol, doxorubicin, mitoxantrone, allylamine, cyclosporine A, cyclophosphamide, aminoglutethimide, acetaminophen, methapyrilene, allylalcohol and α-naphthylisothiocyanate). These promising biomarkers were found to be superior to the conventional biomarkers, as they had a specific and abundant distribution within the heart and/or skeletal muscles; exhibited a positive correlation between the amplitude of increases and the degree of pathological alterations; had higher diagnostic accuracy for detecting pathological alterations; and had the additive effect of improving the diagnostic accuracy of conventional biomarkers. However, these promising biomarkers have several drawbacks including a rapid clearance, the fact that they are affected by renal dysfunction, and different reactivity to the mode of action of individual myotoxicants. In conclusion, the promising biomarkers cTnI, cTnT, FABP3, MYL3, and sTnI demonstrated sensitivity and specificity for cardiac and skeletal myotoxicity that was superior to those of conventional biomarkers, while we should pay attention to the drawbacks of these biomarkers when used in toxicity studies.

Plasma miR‐208 as a useful biomarker for drug‐induced cardiotoxicity in rats

Cardiotoxicity is one of the major safety concerns in drug development. Therefore, detecting and monitoring cardiotoxicity throughout preclinical and clinical studies is important for pharmaceutical companies. The present study was conducted in order to explore a plasma miRNA biomarker for cardiotoxicity in rats. As organ specificity is an important factor for a biomarker, we analyzed the miRNA microarray dataset in 55 organs/tissues in normal male rats. Based on this analysis, 5 miRNAs consisting of miR‐208 (heart‐specific), miR‐1, miR‐133a, miR‐133b (heart and skeletal muscle‐specific) and miR‐206 (skeletal muscle‐specific) were selected. Next, we evaluated the usefulness of those 5 miRNAs as circulating biomarkers in rats administered with single‐dose isoproterenol or doxorubicin. Plasma miR‐208 was consistently increased through 24 h after dosing in rats administered with isoproterenol, whereas plasma concentrations of cardiac troponin (cTn) showed transient elevation. In contrast, the plasma levels of miR‐1, miR‐133a, miR‐133a and miR‐206 were elevated after treatment with doxorubicin, probably as a result of skeletal muscle toxicity. Additionally, the plasma miR‐208 level was elevated even after repeat‐dose administration (once daily for 7 days) of isoproterenol under which the pathological condition proceeded to the sub‐chronic phase such as fibrosis. Thus, our data suggest that miR‐208 is a promising plasma biomarker for cardiotoxicity in rats. Monitoring of plasma miR‐208 levels in rats may lead to more accurate evaluation of cardiotoxicity in preclinical studies. Copyright

Identification of potential genomic biomarkers for early detection of chemically induced cardiotoxicity in rats

Cardiotoxicity represents one of the most serious side effects of new drugs. It is essential for pharmaceutical companies to detect potential cardiotoxicity of candidate drugs in non-clinical studies during the early stages of drug development. In this study, we aimed to detect potential genomic biomarkers of rat cardiotoxicity using a toxicogenomics approach. In order to achieve this, we induced cardiac lesions in rats following treatment with the three prototypical cardiotoxic compounds isoproterenol, doxorubicin and carbofuran. We then undertook histopathological examination and microarray analysis at 8 or 24h after single dosing. Using statistical and cluster analysis, we extracted 36 probe sets commonly up-regulated by the three cardiotoxic compounds. GO analysis revealed that these genes were functionally associated with either chemotaxis, tissue regeneration, positive regulation of cell proliferation, cellular organization and morphogenesis events in accordance with the degeneration of myocardium and inflammation observed in the histopathology analysis. Most of selected genes showed transient up-regulation at different time point for each compound. However, among these genes, Spp1, Fhl1, Timp1, Ccl7 and Reg3b revealed a sustained up-regulation with high expression levels at both time points for all three compounds. In conclusion, even though definitive validation studies are required for the establishment of their usefulness and reliability, these identified genes may prove to be the most promising candidate genomic biomarkers of cardiotoxicity in rats.

Identification of metabolomic biomarkers for drug-induced acute kidney injury in rats.

Nephrotoxicity is a common side effect observed during both nonclinical and clinical drug development investigations. The present study aimed to identify metabolomic biomarkers that could provide early and sensitive indication of nephrotoxicity in rats. Metabolomic analyses were performed using capillary electrophoresis–time‐of‐flight mass spectrometry on rat plasma collected at 9 and 24 h after a single dose of 2‐bromoethylamine or n‐phenylanthranilic acid and at 24 h after 7 days of repeated doses of gentamicin, cyclosporine A or cisplatin. Among a total of 169 metabolites identified, 3‐methylhistidine (3‐MH), 3‐indoxyl sulfate (3‐IS) and guanidoacetate (GAA) were selected as candidate biomarkers. The biological significance and reproducibility of the observed changes were monitored over time in acute nephrotoxicity model rats treated with a single dose of cisplatin, with the glomerular filtration rate monitored by determination of creatinine clearance. Increased plasma levels of 3‐MH and 3‐IS were related to a decline in glomerular filtration due to a renal failure. In contrast, the decrease in plasma GAA, which is synthesized from arginine and glycine in the kidneys, was considered to reflect decreased production due to renal malfunction. Although definitive validation studies are required to confirm their usefulness and reliability, 3‐MH, 3‐IS and GAA may prove to be valuable plasma biomarkers for monitoring nephrotoxicity in rats. Copyright

Decrease in urinary creatinine in acute kidney injury influences diagnostic value of urinary biomarker-to-creatinine ratio in rats

Recent research has revealed several useful urinary biomarkers of renal dysfunction such as acute kidney injury (AKI). For adequate evaluation of altered urinary biomarkers, it is necessary to consider the influence of varied urine flow rate (UFR). Calculation of the excretion rate of a urinary biomarker (UFR-correction) is the gold standard for the correction of UFR variation. An alternative method that is widely used is to calculate the ratio of the biomarker level to urinary creatinine (Ucr-correction). To date, the equivalence between these two methods has been examined only in a steady state situation such as diabetic nephropathy, and the urinary biomarkers examined have been limited to proteinuria and albuminuria. Therefore, we comprehensively addressed the relationship between Ucr-correction and UFR-correction of ten urinary biomarkers N-acetyl-β-d-glucosaminidase (NAG), lactate dehydrogenase (LDH), total protein, albumin, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β(2)-microglobulin, cystatin-c and glutathione S-transferase-α in non-steady state situations such as AKI. All ten urinary biomarkers showed larger amplitude increases in AKI by Ucr-correction than by UFR-correction in linear regression analysis. Moreover, receiver operating characteristic curves analysis suggested that, at least for the biomarkers NAG and LDH, Ucr-correction had higher diagnostic power than UFR-correction. We observed a decrease in the Ucr excretion in AKI that was accompanied by a reduction in creatinine clearance and reduced mRNA expression of the renal organic cation transporter-2, which is known to function as a transporter for creatinine. These results may provide a mechanistic explanation for the phenomena obtained in Ucr-correction. In conclusion, while Ucr-correction could overestimate the degree of AKI, it could also provide higher diagnostic power for AKI than UFR-correction. We should take into consideration of these backgrounds when using the Ucr-correction.

Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure.

The kidney has a capacity to recover from ischemic or toxic insults that result in cell death, and timely tissue repair of affected renal tubules may arrest progression of injury, leading to regression of injury and paving the way for recovery. To investigate the roles of neutrophil gelatinase-associated lipocalin (NGAL/lcn2) and osteopontin (OPN/spp1) during renal regeneration, the expression patterns of NGAL and OPN in the cisplatin-induced rat renal failure model were examined. NGAL expression was increased from day 1 after injection; it was seen mainly in the completely regenerating proximal tubules of the cortico-medullary junction on days 3-35; however, the expression was not seen in abnormally dilated or atrophied renal tubules surrounded by fibrotic lesions. On the other hand, OPN expression was increased from day 5 and the increased expression developed exclusively in the abnormal renal tubules. NGAL expression level well correlated with the proliferating activity in the regenerating renal epithelial cells, whereas OPN significantly correlated with the α-smooth muscle actin-positive myofibroblast appearance, expression of transforming growth factor (TGF)-β1, and the number of CD68-positive macrophages. Interestingly, rat renal epithelial cell line (NRK-52E) treated with TGF-β1 decreased NGAL expression, but increased OPN expression in a dose-dependent manner. Because increases of TGF-β1, myofibroblasts and macrophages contribute to progressive interstitial renal fibrosis, OPN may be involved in the pathogenesis of fibrosis; on the contrary, NGAL may play a role in tubular regeneration after injury. Expression analysis of NGAL and OPN would be useful to investigate the tubule damage in renal-toxicity.

Genomic biomarkers for cardiotoxicity in rats as a sensitive tool in preclinical studies

The development of safer drugs is a high priority for pharmaceutical companies. Among the various toxicities caused by drugs, cardiotoxicity is an important issue because of its lethality. In addition, cardiovascular toxicity leads to the attrition of many drug candidates in both preclinical and clinical phases. Although histopathological and blood chemistry examinations are the current gold standards for detecting cardiotoxicity in preclinical studies, the large number of withdrawals from clinical studies owing to safety problems indicate that a more sensitive tool is required. We recently identified 32 genes that were candidate genomic biomarkers for cardiotoxicity in rats. Based on their functions, the present study focused on 8 of these 32 genes (Spp1, Fhl1, Timp1, Serpine1, Bcat1, Lmcd1, Rnd1 and Tgfb2). Diagnostic accuracy for the genes was determined by a receiver‐operating characteristic (ROC) analysis using more cardiotoxic and non‐cardiotoxic compounds. In addition, an optimized support vector machine (SVM) model that was composed of Spp1 and Timp1 was newly constructed. This new multi‐gene model exhibited a much higher diagnostic accuracy than that observed for plasma cardiac troponin I (cTnI), which is one of the most useful plasma biomarkers for cardiotoxicity detection. Furthermore, we determined that this multi‐gene model could predict potential cardiotoxicity in rats in the absence of any cardiac histopathological lesions or elevations of plasma cTnI. Overall, this multi‐gene model exhibited advantages over classic tools commonly used for cardiotoxicity evaluations in rats. Our current results suggest that application of the model could potentially lead to the production of safer drugs. Copyright

Effects of protein transduction with intact myogenic transcription factors tagged with HIV-1 Tat-PTD (T-PTD) on myogenic differentiation of mouse primary cells.

HIV-1 Tat PTD (T-PTD) has been shown to be a useful carrier for delivering transcription factors into living cells. Tissue specific transcription factors tagged with T-PTD sequences could penetrate into cells and induce tissue-specific differentiation. Meanwhile, many eukaryotic transcription factors containing basic helix-loop-helix (bHLH) motifs have been reported to facilitate protein transduction. In the present work, we developed a method to achieve efficient differentiation of primary cells using transcription factors tagged with T-PTD. MyoD and myogenin are myogenic transcription factors having bHLH domains, and we examined the effects of MyoD, T-PTD-fused MyoD (T-PTD-MyoD) and T-PTD-fused myogenin (T-PTD-MyoG) proteins on the differentiation of mouse primary cells. Although mouse primary cells were differentiated to be multi-nucleated cells (myogenic differentiation) by the addition of unmodified MyoD protein, about four times more differentiated cells were observed when we used modified MyoD, whose N-terminal region was tagged with T-PTD. These results indicated that MyoD protein combined with T-PTD could induce effective cell differentiation.