Metabonomics evaluations of age-related changes in the urinary compositions of male Sprague Dawley rats and effects of data normalization methods on statistical and quantitative analysis

<p>Abstract</p> <p>Background</p> <p>Urine from male Sprague-Dawley rats 25, 40, and 80 days old was analyzed by NMR and UPLC/MS. The effects of data normalization procedures on principal component analysis (PCA) and quantitative analysis of NMR-based metabonomics data were investigated. Additionally, the effects of age on the metabolic profiles were examined by both NMR and UPLC/MS analyses.</p> <p>Results</p> <p>The data normalization factor was shown to have a great impact on the statistical and quantitative results indicating the need to carefully consider how to best normalize the data within a particular study and when comparing different studies. PCA applied to the data obtained from both NMR and UPLC/MS platforms reveals similar age-related differences. NMR indicated many metabolites associated with the Krebs cycle decrease while citrate and 2-oxoglutarate, also associated with the Krebs cycle, increase in older rats.</p> <p>Conclusion</p> <p>This study compared four different normalization methods for the NMR-based metabonomics spectra from an age-related study. It was shown that each method of normalization has a great effect on both the statistical and quantitative analyses. Each normalization method resulted in altered relative positions of significant PCA loadings for each sample spectra but it did not alter which chemical shifts had the highest loadings. The greater the normalization factor was related to age, the greater the separation between age groups was observed in subsequent PCA analyses. The normalization factor that showed the least age dependence was total NMR intensity, which was consistent with UPLC/MS data. Normalization by total intensity attempts to make corrections due to dietary and water intake of the individual animal, which is especially useful in metabonomics evaluations of urine. Additionally, metabonomics evaluations of age-related effects showed decreased concentrations of many Krebs cycle intermediates along with increased levels of oxidized antioxidants in urine of older rats, which is consistent with current theories on aging and its association with diminishing mitochondrial function and increasing levels of reactive oxygen species. Analysis of urine by both NMR and UPLC/MS provides a comprehensive and complementary means of examining metabolic events in aging rats.</p

Proteomic Candidate Biomarkers of Drug-Induced Nephrotoxicity in the Rat

Improved biomarkers of acute nephrotoxicity are coveted by the drug development industry, regulatory agencies, and clinicians. In an effort to identify such biomarkers, urinary peptide profiles of rats treated with two different nephrotoxins were investigated. 493 marker candidates were defined that showed a significant response to cis-platin comparing a cis-platin treated cohort to controls. Next, urine samples from rats that received three consecutive daily doses of 150 or 300 mg/kg gentamicin were examined. 557 potential biomarkers were initially identified; 108 of these gentamicin-response markers showed a clear temporal response to treatment. 39 of the cisplatin-response markers also displayed a clear response to gentamicin. Of the combined 147 peptides, 101 were similarly regulated by gentamicin or cis-platin and 54 could be identified by tandem mass spectrometry. Most were collagen type I and type III fragments up-regulated in response to gentamicin treatment. Based on these peptides, classification models were generated and validated in a longitudinal study. In agreement with histopathology, the observed changes in classification scores were transient, initiated after the first dose, and generally persistent over a period of 10–20 days before returning to control levels. The data support the hypothesis that gentamicin-induced renal toxicity up-regulates protease activity, resulting in an increase in several specific urinary collagen fragments. Urinary proteomic biomarkers identified here, especially those common to both nephrotoxins, may serve as a valuable tool to investigate potential new drug candidates for the risk of nephrotoxicity

Evaluating the Stability of RNA-Seq Transcriptome Profiles and Drug-Induced Immune-Related Expression Changes in Whole Blood.

Methods were developed to evaluate the stability of rat whole blood expression obtained from RNA sequencing (RNA-seq) and assess changes in whole blood transcriptome profiles in experiments replicated over time. Expression was measured in globin-depleted RNA extracted from the whole blood of Sprague-Dawley rats, given either saline (control) or neurotoxic doses of amphetamine (AMPH). The experiment was repeated four times (paired control and AMPH groups) over a 2-year span. The transcriptome of the control and AMPH-treated groups was evaluated on: 1) transcript levels for ribosomal protein subunits; 2) relative expression of immune-related genes; 3) stability of the control transcriptome over 2 years; and 4) stability of the effects of AMPH on immune-related genes over 2 years. All, except one, of the 70 genes that encode the 80s ribosome had levels that ranked in the top 5% of all mean expression levels. Deviations in sequencing performance led to significant changes in the ribosomal transcripts. The overall expression profile of immune-related genes and genes specific to monocytes, T-cells or B-cells were well represented and consistent within treatment groups. There were no differences between the levels of ribosomal transcripts in time-matched control and AMPH groups but significant differences in the expression of immune-related genes between control and AMPH groups. AMPH significantly increased expression of some genes related to monocytes but down-regulated those specific to T-cells. These changes were partially due to changes in the two types of leukocytes present in blood, which indicate an activation of the innate immune system by AMPH. Thus, the stability of RNA-seq whole blood transcriptome can be verified by assessing ribosomal protein subunits and immune-related gene expression. Such stability enables the pooling of samples from replicate experiments to carry out differential expression analysis with acceptable power

Identification of whole blood mRNA and microRNA biomarkers of tissue damage and immune function resulting from amphetamine exposure or heat stroke in adult male rats.

This work extends the understanding of how toxic exposures to amphetamine (AMPH) adversely affect the immune system and lead to tissue damage. Importantly, it determines which effects of AMPH are and are not due to pronounced hyperthermia. Whole blood messenger RNA (mRNA) and whole blood and serum microRNA (miRNA) transcripts were identified in adult male Sprague-Dawley rats after exposure to toxic AMPH under normothermic conditions, AMPH when it produces pronounced hyperthermia, or environmentally-induced hyperthermia (EIH). mRNA transcripts with large increases in fold-change in treated relative to control rats and very low expression in the control group were a rich source of organ-specific transcripts in blood. When severe hyperthermia was produced by either EIH or AMPH, significant increases in circulating organ-specific transcripts for liver (Alb, Fbg, F2), pancreas (Spink1), bronchi/lungs (F3, Cyp4b1), bone marrow (Np4, RatNP-3b), and kidney (Cesl1, Slc22a8) were observed. Liver damage was suggested also by increased miR-122 levels in the serum. Increases in muscle/heart-enriched transcripts were produced by AMPH even in the absence of hyperthermia. Expression increases in immune-related transcripts, particularly Cd14 and Vcan, indicate that AMPH can activate the innate immune system in the absence of hyperthermia. Most transcripts specific for T-cells decreased 50-70% after AMPH exposure or EIH, with the noted exception of Ccr5 and Chst12. This is probably due to T-cells leaving the circulation and down-regulation of these genes. Transcript changes specific for B-cells or B-lymphoblasts in the AMPH and EIH groups ranged widely from decreasing ≈ 40% (Cd19, Cd180) to increasing 30 to 100% (Tk1, Ahsa1) to increasing ≥500% (Stip1, Ackr3). The marked increases in Ccr2, Ccr5, Pld1, and Ackr3 produced by either AMPH or EIH observed in vivo provide further insight into the initial immune system alterations that result from methamphetamine and AMPH abuse and could modify risk for HIV and other viral infections

Time course of nephrotoxicity classification by the urinary models composed of gentamicin-sensitive polypeptide markers.

<p>Mean classification factors obtained with the linear (left row) and SVM (right row) model variants of the marker panels AKI_rat_lin_101 (upper panel) and AKI_rat_lin_54seq (lower panel) for urine samples from untreated animals (green curve) and animals treated with 150 (blue curve) and 300 mg/kg (red curve) gentamicin once daily for three consecutive days over time.</p

Correlation of urinary peptide biomarkers, as well as the biomarker models with histopathology changes and other investigated kidney injury biomarkers.

<p>Given are short protein name of the UniProt database with the position of the firs and last amino acid in parenthesis, for the sequenced biomarkers and names of the biomarker models, rank correlation coefficients (and p-values) between histopathology, necrosis, apoptosis, regeneration scores, classical clinical chemistry endpoints (BUN, serum creatinine, albumin) and the detection level of the sequenced candidate biomarkers and the biomarker models\ classification scores.</p

Group specific polypeptide profiles for the 101 drug-induced nephrotoxicity markers in the gentamicin-treated rat cohort.

<p>The compiled data sets of urine samples from gentamicin-treated rats at different doses and sampling days are shown. The data sets were divided into four groups according to the days of sampling after initiation of treatment (samples collected at day 1+2; day 3+7+10; day 15+18+22 and day 29+36+44 from left to right) and for the gentamicin dose (0, 150 and 300 mg/kg gentamicin from the top to bottom). Molecular mass of the analyzed polypeptides (kDa) in logarithmic scale is plotted against CE migration time (min). The mean signal intensity is represented on the z-axis of the 3D plot.</p

Study design and rationale for biomarker selection.

<p>Depicted is the size of the sample cohorts used in the biomarker definition step and the number of selected peptides after statistical comparison of the respective treated and control groups. Numbers of peptides identified in both cis-platin and genatmicin treated rats as markers are denominated in the intersection of the diagrams. The significant marker candidates were visually inspected and only those candidates were selected that showed a clear response to gentamicin. In the next step the direction of regulation was controlled for the markers identified in both studies and selected were only those demonstrating similar (up or down) regulation with both cis-platin and gentamicin. In the last step the number of markers for which the amino acid sequence could be resolved is listed.</p

Additional file 1: of Vascular-directed responses of microglia produced by methamphetamine exposure: indirect evidence that microglia are involved in vascular repair?

Figure S1A. DAB-Iba1 labeled microglia activation in the septum at 3 days after METH exposure. Figure S1B. DAB-Iba1 labeled microglia in the septum and hippocampus at 1day after METH exposure. Figure S2A. DAB-Iba1 labeled microglia in the hippocampus at 3 days after METH exposure. Figure S2B. DAB-Iba1 labeled microglia in the cudate/putamen at 1 day after METH exposure. Figure S3A. Coincidence of degenerating (FJc+) neurons and activated microglia in the thalamus at 3 days after METH. Figure S3B. FJc-labeling in the septum at 3 days after METH exposure. Figure S4. FJc-labeling in the medial hippocampus at 3 days after METH exposure. Figure S9. DAB-GFAP labeled astrocytes in the medial hippocampus at 3 days after METH exposure. (PDF 15680 kb