¹H NMR spectroscopic studies on the characterization of renal cell lines and identification of novel potential markers of in vitro nephrotoxicity

Abstract

Cell cultures are increasingly used in the evaluation of chemically-induced nephrotoxicity. The utili of renal cell culture systems in toxicology would be improved, however, if better characterized and more specific markers of toxicity were available. High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy is well suited to the study of toxicological events and has identified many novel markers of nephrotoxicity in vivo. In this study, (1)H NMR spectroscopy has been used to characterize the biochemical composition of two renal cell lines of different nephronal origin, LLC-PK1 (pig proximal tubule) and Madin-Darby canine kidney (MDCK, distal tubule). The early biochemical responses of these cell lines to the model proximal tubular toxin S-(1,2dichlorovinyl)i-L-cysteine (DCVC) and the renal medullary toxin 2-chloroethanamine (CEA) have also been investigated. For each line, 500 MHz (1)H NMR spectra of protein-free acetone extracts of cells and culture medium gave characteristic and reproducible profiles of low MW constituents, including amino and organic acids, glucose and soluble membrane precursors, such as choline and myo-inositol. Treatment-related changes in several low MW compounds not routinely measured in toxicological studies were revealed by NMR specboscopy before marked cytotoxicity was observed by phase contrast microscopy. For example, LLC-PK1 cells treated with 60 μM DCVC showed a marked decrease in intracellular choline levels within 3 h which suggests an effect on the balance of choline synthesis and utilization. Wrthin 9 h of treatment with DCVC there were decreases in intracellular acetate and alanine concentrations which may be indicative of a decrease in fatty acid oxidation and biglyceride metabolism accompanied by an increase in gluconeogenesis. In MDCK cells, 1 h post treatment with 5 mM CEA, intracellular glycine was decreased. This study indicates the potential power and applicability of (1)H NMR spectroscopy for evaluating the biochemical and metabolic effects of toxins in cell culture systems and provides a novel approach to identifying new markers of tissue damage.

Magic Angle Spinning NMR and 1H−31P Heteronuclear Statistical Total Correlation Spectroscopy of Intact Human Gut Biopsies

“…NMR spectroscopy has been widely and successfully used to measure multiple endogenous metabolites simultaneously in a variety of tissues and biofluids in a range of physiological and pathological states. ,− When combined with multivariate statistical techniques, such studies have efficiently enabled the discovery of novel biomarker combinations . A unique feature of NMR spectroscopy as a structural analytical tool is its nondestructive nature that allows biofluids, , cells, and intact tissues, and even the dynamic biochemical reactions in those systems, to be measured and monitored through time noninvasively …”

High resolution magic angle spinning 1H nuclear magnetic resonance analysis of intact prostatic hyperplastic and tumour tissues

“…Virtually no lipid is seen in the acetonitrile-water extracts in either case (this having been precipitated together with the proteins) and this is also true of perchloric acid or trichloroacetic acid extracts in other tissues. 10 As lipid is an important diagnostic biomarker for the tumour cell type, it is unfortunate that this is lost in the cell extraction procedure. The relative weakness of the aromatic amino acid signals (from tyrosine and phenylalanine) could be due to the increased lipid binding in the cancer tissue, which shortens their T 2 s and hence increases their line widths (T 2 contributions to line broadening are not reduced by MAS).…”

Characterization of Metabolites in IntactStreptomyces citricolorCulture Supernatants Using High-Resolution Nuclear Magnetic Resonance and Directly Coupled High-Pressure Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy

“…There is therefore a need for new techniques that can provide rapid multiparametric information about the microbial fermentation process. NMR spectroscopy has been shown to be a useful tool in the analysis of complex biological mixtures such as mammalian biofluids (1), cell cultures (2,3), and microbial cultures (4 -6). High-resolution 1 H NMR spectroscopy has the potential to detect and identify a range of low-molecular-weight biological compounds at low concentration in untreated broth supernatants.…”