Project description:Clinical use of a major cancer chemotherapeutic agent, cisplatin, is restricted to dose-limiting renal toxicity. A transcriptomic approach enabled us to extract putative molecules including Pleckstrin homology-like domain, family A, member-3 (Phlda3) contributing to the renal injury. This study investigated the expression of Phlda3 and its regulatory role in tubular injury induced by cisplatin in vivo and in vitro. Real-time PCR and immunoblot assays confirmed that Phlda3 was highly up-regulated in the kidney of mice after a single dose of cisplatin treatment, which preceded increases in the blood urea nitrogen or serum creatinine levels. Particularly, the level of Phlda3 transcript was substantially increased at an early time than that of kidney injury molecule-1, and remained elevated. The effect of cisplatin or acetaminophen treatment on Phlda3 expression in the liver was minimal. Consistently, treatment of NRK52E, a renal tubular cell line, with cisplatin caused increases in Phlda3 mRNA and protein. Knockdown of Phlda3 reversed the decrease in cell viability by cisplatin, supporting the role of Phlda3 in cell death. Akt phosphorylation initially increased after cisplatin treatment, but decreased thereafter. Cisplatin treatment elicited p53 accumulation via mdm2 repression, inducing its target genes, p21 and cyclin G1. Phlda3 deficiency attenuated a decrease in Akt phosphorylation by cisplatin, and prevented p53 accumulation, supporting the role of Phlda3 for p53-mediated tubular cell death. Other renal toxicants, cyclosporine A and CdCl2, also enhanced Phlda3-dependent cell death. Overall, nephrotoxicants including cisplatin induce Phlda3, which leads to p53-mediated tubular cell death via mdm2 repression caused by Akt inhibition. Since the datasets reported on the effect of cisplatin on the kidney were obtained from animals treated with multiple and relatively high doses of cisplatin, we did our cDNA array analyses day 3 after a single dose of cisplatin administration to mice (i.p., 15 mg/kg) and compared the profiles of kidney microarray data. The transcriptomic approach enabled us to find the putative genes encoding molecules contributing to renal injury. Among those genes represented on the microarray, 33 genes were found to be differentially expressed by cisplatin treatment when a 2-fold change cutoff was used. The regulation of exemplary mRNAs identified in the array analysis was confirmed by qRT-PCR. C57BL/6 mice (9 weeks old) were intraperitoneally injected with a single dose of cisplatin (15 mg/kg body weight) or vehicle (saline). The cDNA microarray experiments were done for the kidney of mice 3 days after treatment. Each group contained 3 males.

Project description:Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER in order to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fisher 344 rats were intravenously injected with three different doses (150, 300, and 600 mg/kg) of CER, and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and a slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following the exposure to CER, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose-dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. We also found a subset of G2/M phase genes that exhibited hormesis-like (U-Shape) biphasic dose response; namely, downregulation only at the low and/or middle (subtoxic) doses. Furthermore, we could predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understandings of molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity. Keywords: compound treatment, dose response In the present study, we acquired gene expression profiles in the kidney of rats given subtoxic and toxic doses of cephaloridine (CER) using whole-genome oligonucleotide microarrays. Male rats were injected with vehicle alone and three different doses (150, 300, and 600 mg/kg) of CER, and sacrificed after 24 h. Each dose group contained 3 animals.

Project description:Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER in order to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fisher 344 rats were intravenously injected with three different doses (150, 300, and 600 mg/kg) of CER, and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and a slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following the exposure to CER, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose-dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. We also found a subset of G2/M phase genes that exhibited hormesis-like (U-Shape) biphasic dose response; namely, downregulation only at the low and/or middle (subtoxic) doses. Furthermore, we could predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understandings of molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity. Keywords: compound treatment, dose response

Project description:Endoglin (EDG) is a cell surface protein with an important role in the establishment of neo-angiogenesis and vasculogenic mimicry. EDG is part of the transforming growth factor-β (TGF-β) family, acting as an important co-receptor. EDG is shed from the cell surface into the extracellular compartment by matrix metalloproteinase 14 (MMP14), in its soluble form (sEDG). Both transmembrane and soluble forms of EDG exert important signaling functions in the development of new blood vessels and tumour progression. To better understand the role of EDG in Ewing sarcoma (ES), a deadly neoplasm of late childhood and adolescence, we test the efficacy of OMTX703, an endoglin-targeting antibody-drug conjugate in ES8 xenograft. Having determined an optimal dose for OMTX703, an additional experiment was conducted to assess the mechanism(s) of OMTX703 action and its potential mechanism(s) of resistance following a 2-week exposure to OMTX703 at 0, 10, 30, and 60 mg/kg; 246 proteins were assessed by reverse-phase protein array (RPPA). Analysis of variance (ANOVA), Pearson’s correlation as distance metric and Ward’s linkage as the clustering method using a false discovery rate (FDR) of 0.01, identified 60 proteins that discriminated between treatment groups (Matrix#1-Normalized Values). To investigate the proteomic changes associated with the heightened clinical activity of the 60 mg/kg dose, a secondary analysis was performed, which grouped the 10 mg/kg OMTX703 samples and the 10 mg/kg OMTX003 ones with the placebo-treated samples (Matrix#2-Normalized Values). Using a FDR of 0.0001, an absolute log2 fold change of 1.5, Pearson’s correlation as distance metric and Ward’s linkage as the clustering method, 22 proteins were discriminately identified between the 3 treatment groups (Matrix#2-Normalized Values). Notably, a protein regulator of altered metabolism (RPS6) was exclusively upregulated following OMTX703 (60mg/kg), and a second metabolism biomarker (LDHA) was down-expressed in the 30 and 60 mg/kg-treated groups. Conversely, BRD4 was one of about a dozen proteins that were preferentially down-regulated in samples treated only by 60 mg/kg.

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Delineation of renal protein profiles in aristolochic acid I-induced nephrotoxicity in mice by label-free quantitative proteomics

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