The capacities of urinary trefoil factor 3 (TFF3) and urinary albumin to detect acute renal tubular injury have never been evaluated with sufficient statistical rigor to permit their use in regulated drug development instead of the current preclinical biomarkers serum creatinine (SCr) and blood urea nitrogen (BUN). Working with rats, we found that urinary TFF3 protein levels were markedly reduced, and urinary albumin were markedly increased in response to renal tubular injury. Urinary TFF3 levels did not respond to nonrenal toxicants, and urinary albumin faithfully reflected alterations in renal function. In situ hybridization localized TFF3 expression in tubules of the outer stripe of the outer medulla. Albumin outperformed either SCr or BUN for detecting kidney tubule injury and TFF3 augmented the potential of BUN and SCr to detect kidney damage. Use of urinary TFF3 and albumin will enable more sensitive and robust diagnosis of acute renal tubular injury than traditional biomarkers.
of action of lithium in BD, and these have been re-Sethu Sankaranarayanan, 1 Adam J. Simon, 1 viewed (Gould et al., 2004; Gurvich and Klein, 2002). and Edward M. Scolnick 3 Glycogen synthase kinase 3 (GSK3) and inositol mono-1 Merck & Co., Inc. phosphatase (IMPase) are both inhibited by lithium and West Point, Pennsylvania 19486 have been topics of considerable study as potential 2 Rosetta Inpharmatics LLC, a wholly owned therapeutic targets in BD. subsidiary of Merck & Co. Inc. An effect of lithium on brain inositol levels was dem-401 Terry Avenue North onstrated more then 30 years ago (Allison and Stewart, Seattle, Washington 98011 1971). Inositol phosphates and inositol-containing phospholipids (phosphoinositides) constitute an array of cell signaling molecules with diverse functions (Irvine and Summary Schell, 2001; Majerus, 1992). Most notably, activation of cell surface receptors can activate hydrolysis of Lithium inhibits inositol monophosphatase at theraphosphatidylinositol-4,5-bisphosphate (PIP 2 ) by phospeutically effective concentrations, and it has been pholipase C (PLC) to produce inositol-1,4,5-trisphoshypothesized that depletion of brain inositol levels is phate (IP 3 ), which is a second-messenger molecule. IP 3 an important chemical alteration for lithium's therabinds to its cognate receptor to trigger release of Ca 2+ peutic efficacy in bipolar disorder. We have employed from the endoplasmic reticulum (ER) and a host of adult rat cortical slices as a model to investigate the downstream signaling events. Lithium inhibits two engene regulatory consequences of inositol depletion zymes in the inositol pathway, namely inositol polyeffected by lithium using cytidine diphosphoryl-diacylphosphate 1-phosphatase and IMPase, both uncomglycerol as a functionally relevant biochemical marker petitively with respect to substrate (Majerus, 1992). to define treatment conditions. Genes coding for the Indeed, therapeutically relevant doses of lithium cause neuropeptide hormone pituitary adenylate cyclase acaccumulation of inositol phosphates in the brains of tivating polypeptide (PACAP) and the enzyme that rats and mice (Atack et al., 1992; Sherman et al., 1986). processes PACAP's precursor to the mature form, The "inositol depletion" hypothesis of lithium's action in peptidylglycine ␣-amidating monooxygenase, were BD was formulated by Berridge (Berridge et al., 1989). upregulated by inositol depletion. Previous work has Simply put, lithium blocks IMPase and the recycling of shown that PACAP can increase tyrosine hydroxylase inositol into inositol lipids. This leads to a reduction in (TH) activity and dopamine release, and we found that PIP 2 and a reduction in the ability of the cell (neuron) to the gene for GTP cyclohydrolase, which effectively respond to a stimulus by making IP 3 to stimulate an regulates TH through synthesis of tetrahydrobiopincrease in intracellular Ca 2+ concentration. Thus, neuterin, was also upregulated by inositol depletion. We ronal excitability is reduced, and this somehow leads propos...
To test the hypothesis that urokinase-type plasminogen activator (uPA) plays an important role in liver regeneration in vivo, partial hepatectomy was performed on wild-type and uPA-deficient (uPA−/−) mice. Mice were studied at 24, 44, and 96 h and at 8 days and 4 wk post-partial hepatectomy for evidence of regeneration, as measured by mitotic indexes and [3H]thymidine incorporation. In wild-type mice, thymidine incorporation peaked at 44 h and this index was reduced by 47% in uPA−/− mice ( P= 0.02). By 8 days, however, liver mass was comparable in both groups. Histological analysis revealed the presence of focal areas of fibrin deposition and cellular loss by 24 h that were more severe and prevalent in uPA−/− mice than in wild-type mice (62 and 23%, respectively; χ2 = 3.939, P = 0.047). In contrast, regeneration was not impaired in uPA receptor (uPAR)-deficient mice at 24 and 44 h. Taken together, these data indicate that uPA, independent of its interaction with the uPAR, plays an important role in liver regeneration in vivo.
Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here we describe translation of those NRF1/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned co-culture system (HEPATOPAC®) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI positive and negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to de-risk DILI potential from chemically reactive metabolites by guiding structure activity relationship hypotheses and candidate selection.
Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target the BCR-ABL junction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety of BCR-ABL junctions in CML patients, we utilized gene editing of the human ABL gene for clinical applications. Using the ABL gene-edited virus in K562 cells, we detected 41.2% indels in ABL sgRNA_2-infected cells. The ABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced the ABL gene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population in ABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with the ABL gene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using the ABL gene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of the ABL gene in maintaining CML cell survival and tumorigenicity in vitro and in vivo. ABL gene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.
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