ABSTRACT
A label-free and convenient strategy for PARP-1 activity assay and inhibitors assessment has been developed based on the fluorescence resonance energy transfer (FRET) between a cationic conjugated polymer (CCP) and supercharged green fluorescent protein (scGFP).
Subject(s)
Enzyme Assays/methods , Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Cations/chemistry , Cations/metabolism , Drug Evaluation, Preclinical/methods , Green Fluorescent Proteins/metabolism , Humans , Models, Molecular , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/analysis , Poly(ADP-ribose) Polymerases/blood , Poly(ADP-ribose) Polymerases/urine , Polymers/chemistry , Polymers/metabolismABSTRACT
The ischemia/reperfusion (I/R) model in rats allows pharmacological investigation of protective renal effects of certain agents to thereby diminish the incidence of delayed graft function (DGF). The aim of this study was to determine the effects of preconditioning with triiodothyronine (T(3)) on renal function and oxidative status in renal I/R injury. Forty male Wistar rats were preconditioned with T(3) (100 microg/kg) or control (normal saline) at 24 hours prior to 45 minutes of renal ischemia, followed by a 4-hour (groups C-4h and T(3)-4h) or 24-hour (groups C-24h and T(3)-24h) reperfusion period. We determined renal function parameters (urea, creatinine, and proteinuria), oxidative stress biomarkers in plasma (malondialdehyde [MDA], glutathione [GSH], and superoxide dismutase [SOD]), urine (hydrogen peroxide [H(2)O(2)]), and renal tissue (GSH and MDA), and poly(ADP-ribose) polymerase (PARP-1) expression. Proteinuria was significantly lower in the T(3)-treated group (4.63 +/- 1.9 vs 9.27 +/- 0.72 mg/mL/100 g body weight). Pretreated rats showed lower levels of plasma and tissue MDA and urine H(2)O(2) (50.57 +/- 1.17 vs 71.16 +/- 1.14 micromol/100 g body weight). The T(3) treatment was associated with lower postischemia GSH concentrations (3.82 +/- 1.16 vs 4.89 +/- 0.68 nmol/mg protein) and higher SOD levels at 24 hours (11.27 +/- 0.86 vs 9.92 +/- 1.77 nmol/mg protein). Preconditioning with the hormone also reduced PARP-1 tissue expression by 18% (P Subject(s)
Ischemic Preconditioning/methods
, Poly(ADP-ribose) Polymerases/genetics
, Reperfusion Injury/physiopathology
, Triiodothyronine/pharmacology
, Animals
, Diuresis
, Glutathione/blood
, Glutathione/metabolism
, Hydrogen Peroxide/blood
, Kidney Cortex/physiopathology
, Kidney Medulla/physiopathology
, Male
, Malondialdehyde/blood
, Malondialdehyde/metabolism
, Oxidative Stress/physiology
, Poly(ADP-ribose) Polymerases/urine
, Proteinuria
, Rats
, Rats, Wistar
, Reperfusion Injury/urine
ABSTRACT
Monitoring level of the metabolites of the coenzyme NAD such as nicotinamide and its oxidized and methylated derivatives is important due to therapeutic applications of these compounds and monitoring of oxidative stress. We evaluated feasibility of using HPLC with electrospray ion-trap mass detection for single run separation and quantitation of all the NAD metabolites. We achieved good separation and retention of all the metabolites of interest using reversed-phase with ion-pairing. Single ion monitoring or tandem MS were used for detection and quantitation of the specific compounds with good linearity. The method was able to detect all the physiological metabolites in plasma samples of rats and humans or in urine. However, full validation is necessary before this method could be routinely applied.
