The Toxicologist - Society of Toxicology

577 URANYL NITRATE INHIBITS LACTATE
GLUCONEOGENESIS IN ISOLATED HUMAN AND
MOUSE RENAL PROXIMAL TUBULES: A CELLULAR
METABOLOMIC STUDY.
A. Conjard-Duplany 1, 2 , S. Renault 1 , H. Faiz 1 , R. Gadet 1 , B. Ferrier 1, 2 , G.
Martin 1, 2 and G. Baverel 2 . 1 Metabolomics and Metabolic Diseases, Inserm, Lyon
Cedex 08, France and 2 Metabolys Inc., Lyon, Rhône-Alpes, France.
As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl
nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological
substrate lactate. For this, isolated human and mouse renal proximal
tubules were incubated with variously labelled lactates in the absence and the presence
of uranyl nitrate. In the millimolar range, uranyl nitrate reduced lactate removal
and gluconeogenesis and the cellular ATP level in a dose-dependent fashion.
After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-
13C]-, or L-[2-13C]-, or L-[3-13C]lactate, substrate utilization and product formation
were measured by enzymatic and NMR spectroscopic methods. In the presence
of 3mM uranyl nitrate, glucose production and the intracellular ATP content
were significantly reduced in both human and mouse tubules. Combination of enzymatic
and NMR measurements with a mathematical model of lactate metabolism
revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic
enzymes in the presence of 3 mM uranyl nitrate; in human and mouse
tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and
32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase),
and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that
natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans
and mice. Moreover, they show that the cellular metabolomic approach is a precious
tool to evaluate the effects of nephrotoxic compounds.
578 GENDER SPECIFIC DIFFERENCES IN THE
EXPRESSION OF URINARY MARKERS OF
INFLAMMATION AND OXIDATIVE STRESS.
I. S. Richards and M. M. Bourgeois. EOH, University of South Florida COPH,
Tampa, FL.
Activation of inflammatory and oxidative stress pathways and the onset of renal,
cardiovascular, cerebrovascular and other systemic damage have been shown to be
mechanistically linked. This study compares the expression of inflammatory, stress
and vascular biomarkers in the urine of male and female donors. Specimens were
assayed for aldosterone, total protein, creatine kinase, creatinine, c reactive protein,
heat shock protein, microalbumin, myeloperoxidase, myoglobin, neutrophil gelatinase
associated lipocalin, proatrial natriuretic peptide, vascular endothelial growth
factor and interleukins 1 alpha, 1 beta, 6 and 10 using enzyme-linked immunosorbent
assays. Many of these markers have been assayed exclusively in
serum specimens without reference to gender specific differences. Urine specimens
are non-invasive and may provide for qualitative clinical testing once appropriate
normal ranges have been established. Increased expression of these markers is associated,
in varying degrees of specificity, with vascular damage, oxidative stress and
inflammation. Establishing a link between gender and biomarker expression in
urine is the first step in developing a diagnostic tool that may permit rapid selection
of an appropriate clinical intervention. Our research suggests that significant
gender based differences may exist in the expression of urinary inflammatory and
stress biomarkers.
This work has been supported in part by the Agency for Community Treatment
and Services of Tampa.
579 URINARY KIDNEY INJURY MOLECULE-1 (KIM-1) AS A
RENAL BIOMARKER IN GENTAMICIN (GEN)-
INDUCED RENAL INJURY AND RECOVERY.
R. Rouse 1 , L. Zhang 2 , P. Harlow 3 , J. Zhang 1 , P. Espandiari 4 , S. Stewart 1 , B.
Rosenzweig 1 , K. Thompson 1 and N. Sadrieh 5 . 1 Division of Applied Pharmacology
Research, U.S. FDA, CDER, Silver Spring, MD, 2 Office of Clinical Pharmacology,
U.S. FDA, CDER, Silver Spring, MD, 3 Division of Cardiovascular and Renal
Products, U.S. FDA, CDER, Silver Spring, MD, 4 Division of Metabolism and
Endocrinology Products, U.S. FDA, CDER, Silver Spring, MD and 5 Science and
Research Staff, U.S. FDA, CDER, Silver Spring, MD.
This study was undertaken to determine and characterize the sensitivity of Kim-1
relative to that of existing biomarkers of acute kidney injury. The evolution and recovery
of renal injury was followed in male SD rats given Gen (0, 75, 150, or 300
mg/kg) for up to three consecutive days. Representatives from each group were sacrificed
at 11 time points over 45 days. Necropsy sampling included serum for blood
urea nitrogen (BUN) and creatinine (sCr), urine for Kim-1,kidney for RNA extraction,
PCR, and histopathology evaluation. Urinary Kim-1 (uKim-1) levels showed
a significant dose-dependent increase over days 1 to 7 that peaked at day 7, and
then returned to control levels by day 15. Changes in uKim-1 preceded
histopathology changes and were preceded by changes in Kim1 gene expression.
Increases in BUN and sCr were lower in magnitude than those of uKim-1.
Decreases in elevated BUN and sCr levels preceded decreases in elevated uKim-1
levels during recovery. A completely blinded pathology evaluation using a semiquantative
scale of 0-5 showed a correlation of the severity of kidney injury with all
biomarkers. Discrimination methods including receiver operating
characteristics(ROC) analysis indicated that uKim-1 and renal Kim1 gene expression
significantly outperformed BUN and sCr for the detection of renal injury.
Conclusions: 1) Kim-1 is a more sensitive biomarker for detecting acute renal injury
than BUN and sCr during both evolution and recovery of injury. 2) Kim-1 excretion
into urine diminishes with tissue repair. 3) The correlation of Kim-1 excretion
into urine with tissue injury was better during injury evolution than during
repair and recovery.
580 METABOLOMIC ANALYSIS OF RAT URINE
FOLLOWING ACUTE EXPOSURE TO
PERFLUORINATED CHEMICALS.
W. M. Henderson 1 , T. W. Collette 1 , D. J. Dix 2 and D. R. Ekman 1 . 1 National
Exposure Research Laboratory, U.S. EPA, Athens, GA and 2 National Center for
Computational Toxicology, U.S. EPA, Research Triangle Park, NC.
Perfluorinated chemicals (PFCs), namely perfluorooctanoic acid (PFOA) and perfluorooctane
sulfonate (PFOS), represent an emerging class of persistent and bioaccumulative
compounds. Global occurrence of these fluorochemicals, coupled with
probable human exposure, has prompted investigations of the biochemical impacts
of PFCs that elicit toxicity through modulation of peroxisome proliferator-activated
receptors (PPAR) as well as other modes of action. Genomic studies have
shown that PFOA and PFOS affect genes involved in cholesterol synthesis and fatty
acid metabolism and result in signs of steatosis and hepatomegaly in rats. As a biomarker-based
approach, this study focused on the use of metabolomics for identifying
fluxes in the endogenous metabolome using proton nuclear magnetic resonance
( 1 H-NMR) and both liquid and gas chromatography coupled to mass spectrometry
(LC-MS n and GC-MS). To study this, male SD rats were dosed daily by gavage for
5 days with 20 mg/kg PFOA or 10 mg/kg PFOS. Urine was collected 24 hrs prior
to, and twice daily during, the exposure period at 8 and 16 hr intervals. Urine was
either buffered ( 1 H-NMR), filtered and diluted (LC-MS n ), or extracted with chloroform:methanol,
lyophilized and derivatized (GC-MS) prior to analysis. Spectra
were subjected to principal components (PCA) and partial least squares discriminant
analysis (PLS-DA) to determine the effects of each PFC on the urinary
metabolite profile. For each analytical platform, differences between the control
and exposed rats were observed at the earliest time point. Moreover, PFC-related effects
were temporal and classes sustained distinct separation following three days of
exposure. Components of the spectra responsible for time and PFC-dependent
clustering are being investigated. Identification of significant changes in urinary
metabolites will aid in identifying biomarkers associated with PPAR activation, hepatotoxicity,
and exposure to these and other PFCs.
581 ROADMAP FOR NOVEL BIOMARKER CANDIDATE
NOMINATION FOR PREDICTIVE SAFETY TESTING
CONSORTIUM (PSTC) HEPATOTOXICITY WORKING
GROUP.
W. J. Bailey. Analytical & Systems Toxicology, Merck & Co., Inc., West Point, PA.
Arginase 1 (Arg1) and glutathione S-alkyltransferase Alpha (Gstα) are two novel
biomarkers of liver toxicity. “Fit-for-purpose” ELISA assays have been validated and
performance of these biomarkers has been assessed for specificity and sensitivity relative
to ALT over a number of studies. The data in support of, and strategy used to
evaluate and nominate these biomarkers for potential Voluntary Exploratory Data
Submission (VXDS) will be detailed as a guidance for future efforts in bringing
candidate biomarkers forward. Roadmap milestones should include 1) clearly defined
biological claims described in a Biomarker Research plan submitted to C-Path
and the FDA; 2) mechanistic/biological rationale for the biomarker; 3) assay validation;
4) sample/study assessment based on the biological claims; 4) performance
evaluation relative to ALT and histopathology; 5) nomination within the PSTC
Working Group; 6) independent validation from PSTC members; 7) VXDS submission.
The real world experience of Arg1 and Gstα will be used to illustrate the
process.
124 SOT 2010 ANNUAL MEETING