Linking imaging to omics utilizing image-guided tissue extraction.

Phenotypic heterogeneity is commonly observed in diseased tissue, specifically in tumors. Multimodal imaging technologies can reveal tissue heterogeneity noninvasively in vivo, enabling imaging-based profiling of receptors, metabolism, morphology, or function on a macroscopic scale. In contrast, in vitro multiomics, immunohistochemistry, or histology techniques accurately characterize these heterogeneities in the cellular and subcellular scales in a more comprehensive but ex vivo manner. The complementary in vivo and ex vivo information would provide an enormous potential to better characterize a disease. However, this requires spatially accurate coregistration of these data by image-driven sampling as well as fast sample-preparation methods. Here, a unique image-guided milling machine and workflow for precise extraction of tissue samples from small laboratory animals or excised organs has been developed and evaluated. The samples can be delineated on tomographic images as volumes of interest and can be extracted with a spatial accuracy better than 0.25 mm. The samples remain cooled throughout the procedure to ensure metabolic stability, a precondition for accurate in vitro analysis.

The Immunosuppressant Mycophenolic Acid Alters Nucleotide and Lipid Metabolism in an Intestinal Cell Model.

The study objective was to elucidate the molecular mechanisms underlying the negative effects of mycophenolic acid (MPA) on human intestinal cells. Effects of MPA exposure and guanosine supplementation on nucleotide concentrations in LS180 cells were assessed using liquid chromatography-mass spectrometry. Proteomics analysis was carried out using stable isotope labeling by amino acids in cell culture combined with gel-based liquid chromatography-mass spectrometry and lipidome analysis using 1H nuclear magnetic resonance spectroscopy. Despite supplementation, depletion of guanosine nucleotides (p < 0.001 at 24 and 72 h; 5, 100, and 250 µM MPA) and upregulation of uridine and cytidine nucleotides (p < 0.001 at 24 h; 5 µM MPA) occurred after exposure to MPA. MPA significantly altered 35 proteins mainly related to nucleotide-dependent processes and lipid metabolism. Cross-reference with previous studies of MPA-associated protein changes widely corroborated these results, but showed differences that may be model- and/or method-dependent. MPA exposure increased intracellular concentrations of fatty acids, cholesterol, and phosphatidylcholine (p < 0.01 at 72 h; 100 µM MPA) which corresponded to the changes in lipid-metabolizing proteins. MPA affected intracellular nucleotide levels, nucleotide-dependent processes, expression of structural proteins, fatty acid and lipid metabolism in LS180 cells. These changes may compromise intestinal membrane integrity and contribute to gastrointestinal toxicity.

Collagen fibers mediate MRI-detected water diffusion and anisotropy in breast cancers.

Collagen 1 (Col1) fibers play an important role in tumor interstitial macromolecular transport and cancer cell dissemination. Our goal was to understand the influence of Col1 fibers on water diffusion, and to examine the potential of using noninvasive diffusion tensor imaging (DTI) to indirectly detect Col1 fibers in breast lesions. We previously observed, in human MDA-MB-231 breast cancer xenografts engineered to fluoresce under hypoxia, relatively low amounts of Col1 fibers in fluorescent hypoxic regions. These xenograft tumors together with human breast cancer samples were used here to investigate the relationship between Col1 fibers, water diffusion and anisotropy, and hypoxia. Hypoxic low Col1 fiber containing regions showed decreased apparent diffusion coefficient (ADC) and fractional anisotropy (FA) compared to normoxic high Col1 fiber containing regions. Necrotic high Col1 fiber containing regions showed increased ADC with decreased FA values compared to normoxic viable high Col1 fiber regions that had increased ADC with increased FA values. A good agreement of ADC and FA patterns was observed between in vivo and ex vivo images. In human breast cancer specimens, ADC and FA decreased in low Col1 containing regions. Our data suggest that a decrease in ADC and FA values observed within a lesion could predict hypoxia, and a pattern of high ADC with low FA values could predict necrosis. Collectively the data identify the role of Col1 fibers in directed water movement and support expanding the evaluation of DTI parameters as surrogates for Col1 fiber patterns associated with specific tumor microenvironments as companion diagnostics and for staging.

Molecular MRI in the Earth's Magnetic Field Using Continuous Hyperpolarization of a Biomolecule in Water.

In this work, we illustrate a method to continuously hyperpolarize a biomolecule, nicotinamide, in water using parahydrogen and signal amplification by reversible exchange (SABRE). Building on the preparation procedure described recently by Truong et al. [ J. Phys. Chem. B , 2014 , 118 , 13882 - 13889 ], aqueous solutions of nicotinamide and an Ir-IMes catalyst were prepared for low-field NMR and MRI. The (1)H-polarization was continuously renewed and monitored by NMR experiments at 5.9 mT for more than 1000 s. The polarization achieved corresponds to that induced by a 46 T magnet (P = 1.6 × 10(-4)) or an enhancement of 10(4). The polarization persisted, although reduced, if cell culture medium (DPBS with Ca(2+) and Mg(2+)) or human cells (HL-60) were added, but was no longer observable after the addition of human blood. Using a portable MRI unit, fast (1)H-MRI was enabled by cycling the magnetic field between 5 mT and the Earth's field for hyperpolarization and imaging, respectively. A model describing the underlying spin physics was developed that revealed a polarization pattern depending on both contact time and magnetic field. Furthermore, the model predicts an opposite phase of the dihydrogen and substrate signal after one exchange, which is likely to result in the cancelation of some signal at low field.

Hypoxic tumor environments exhibit disrupted collagen I fibers and low macromolecular transport.

Hypoxic tumor microenvironments result in an aggressive phenotype and resistance to therapy that lead to tumor progression, recurrence, and metastasis. While poor vascularization and the resultant inadequate drug delivery are known to contribute to drug resistance, the effect of hypoxia on molecular transport through the interstitium, and the role of the extracellular matrix (ECM) in mediating this transport are unexplored. The dense mesh of fibers present in the ECM can especially influence the movement of macromolecules. Collagen 1 (Col1) fibers form a key component of the ECM in breast cancers. Here we characterized the influence of hypoxia on macromolecular transport in tumors, and the role of Col1 fibers in mediating this transport using an MDA-MB-231 breast cancer xenograft model engineered to express red fluorescent protein under hypoxia. Magnetic resonance imaging of macromolecular transport was combined with second harmonic generation microscopy of Col1 fibers. Hypoxic tumor regions displayed significantly decreased Col1 fiber density and volume, as well as significantly lower macromolecular draining and pooling rates, than normoxic regions. Regions adjacent to severely hypoxic areas revealed higher deposition of Col1 fibers and increased macromolecular transport. These data suggest that Col1 fibers may facilitate macromolecular transport in tumors, and their reduction in hypoxic regions may reduce this transport. Decreased macromolecular transport in hypoxic regions may also contribute to poor drug delivery and tumor recurrence in hypoxic regions. High Col1 fiber density observed around hypoxic regions may facilitate the escape of aggressive cancer cells from hypoxic regions.

A hyperpolarized equilibrium for magnetic resonance.

Nuclear magnetic resonance spectroscopy and imaging (MRI) play an indispensable role in science and healthcare but use only a tiny fraction of their potential. No more than ≈10 p.p.m. of all ¹H nuclei are effectively detected in a 3-Tesla clinical MRI system. Thus, a vast array of new applications lays dormant, awaiting improved sensitivity. Here we demonstrate the continuous polarization of small molecules in solution to a level that cannot be achieved in a viable magnet. The magnetization does not decay and is effectively reinitialized within seconds after being measured. This effect depends on the long-lived, entangled spin-order of parahydrogen and an exchange reaction in a low magnetic field of 10⁻³ Tesla. We demonstrate the potential of this method by fast MRI and envision the catalysis of new applications such as cancer screening or indeed low-field MRI for routine use and remote application.

A continuous-flow, high-throughput, high-pressure parahydrogen converter for hyperpolarization in a clinical setting.

Pure parahydrogen (pH(2) ) is the prerequisite for optimal pH(2) -based hyperpolarization experiments, promising approaches to access the hidden orders of magnitude of MR signals. pH(2) production on-site in medical research centers is vital for the proliferation of these technologies in the life sciences. However, previously suggested designs do not meet our requirements for safety or production performance (flow rate, pressure or enrichment). In this article, we present the safety concept, design and installation of a pH(2) converter, operated in a clinical setting. The apparatus produces a continuous flow of four standard liters per minute of ≈98% enriched pH(2) at a pressure maximum of 50 bar. The entire production cycle, including cleaning and cooling to 25 K, takes less than 5 h, only ≈45 min of which are required for actual pH(2) conversion. A fast and simple quantification procedure is described. The lifetimes of pH(2) in a glass vial and aluminum storage cylinder are measured to be T(1C) (glass vial) =822 ± 29 min and T(1C) (Al cylinder) =129 ± 36 days, thus providing sufficiently long storage intervals and allowing the application of pH(2) on demand. A dependence of line width on pH(2) enrichment is observed. As examples, (1) H hyperpolarization of pyridine and (13) C hyperpolarization of hydroxyethylpropionate are presented.

Association of DJ-1/PTEN/AKT- and ASK1/p38-mediated cell signalling with ischaemic cardiomyopathy.

AIMS: Dilated cardiomyopathies from chronic ischaemia (ISCM) or idiopathic (IDCM) pathological mechanisms are accompanied by similar clinical symptoms but may differ in protein expression, cell metabolism, and signalling processes at the cellular level. Using a combination of proteomic and metabolomic profiling, we sought to decipher the relationships between the metabolism and cellular signalling pathways in human heart tissues collected from patients with ISCM, IDCM, and those without heart disease and dilation. METHODS AND RESULTS: The comparative analysis suggested a decrease in glycolysis, Krebs cycle, and malate-aspartate shuttle activities in both types of cardiomyopathies and an increase in ketone body oxidation only in ISCM. Chronic ischaemic injury was associated with increased DJ-1 and decreased phosphatase and tensin homolog (PTEN) protein expression. The reduced PTEN expression was accompanied by increased phosphorylation of cell-protective AKT. Phosphorylation at T845 of apoptosis signal-regulating kinase 1 and p38 mitogen-activated protein kinase proteins, with no change in the phosphorylation of extracellular signal-regulated kinases, was also observed. The downregulation of peptidyl-prolyl cis/trans isomerase and NF-κB essential modulator potentially inhibits NF-κB-initiated processes. CONCLUSION: The present study characterized differences in the molecular mechanisms, metabolism, and pathological cell signalling associated with ISCM and IDCM, which may provide novel targets for intervention at the cellular level.

Collagen I fiber density increases in lymph node positive breast cancers: pilot study.

Collagen I (Col1) fibers are a major structural component in the extracellular matrix of human breast cancers. In a preliminary pilot study, we explored the link between Col1 fiber density in primary human breast cancers and the occurrence of lymph node metastasis. Col1 fibers were detected by second harmonic generation (SHG) microscopy in primary human breast cancers from patients presenting with lymph node metastasis (LN+) versus those without lymph node metastasis (LN-). Col1 fiber density, which was quantified using our in-house SHG image analysis software, was significantly higher in the primary human breast cancers of LN+ (fiber volume=29.22%±4.72%, inter-fiber distance=2.25±0.45 µm) versus LN- (fiber volume=20.33%±5.56%, inter-fiber distance=2.88±1.07 µm) patients. Texture analysis by evaluating the co-occurrence matrix and the Fourier transform of the Col1 fibers proved to be significantly different for the parameters of co-relation and energy, as well as aspect ratio and eccentricity, for LN+ versus LN- cases. We also demonstrated that tissue fixation and paraffin embedding had negligible effect on SHG Col1 fiber detection and quantification. High Col1 fiber density in primary breast tumors is associated with breast cancer metastasis and may serve as an imaging biomarker of metastasis.

Low-salt diet and cyclosporine nephrotoxicity: changes in kidney cell metabolism.

Cyclosporine (CsA) is a highly effective immunosuppressant used in patients after transplantation; however, its use is limited by nephrotoxicity. Salt depletion is known to enhance CsA-induced nephrotoxicity in the rat, but the underlying molecular mechanisms are not completely understood. The goal of our study was to identify the molecular effects of salt depletion alone and in combination with CsA on the kidney using a proteo-metabolomic strategy. Rats (n = 6) were assigned to four study groups: (1) normal controls, (2) low-salt fed controls, (3) 10 mg/kg/d CsA for 28 days on a normal diet, (4) 10 mg/kg/d CsA for 28 days on low-salt diet. Low-salt diet redirected kidney energy metabolism toward mitochondria as indicated by a higher energy charge than in normal-fed controls. Low-salt diet alone reduced phospho-AKT and phospho-STAT3 levels and changed the expression of ion transporters PDZK1 and CLIC1. CsA induced macro- and microvesicular tubular epithelial vacuolization and reduced energy charge, changes that were more significant in low-salt fed animals, probably because of their more pronounced dependence on mitochondria. Here, CsA increased phospho-JAK2 and phospho-STAT3 levels and reduced the phospho-IKKγ and p65 proteins, thus activating NF-κB signaling. Decreased expression of lactate transport regulator CD147 and phospho-AKT was also observed after CsA exposure in low-salt rats, indicating a decrease in glycolysis. In summary, our study suggests a key role for PDZK1, CD147, JAK/STAT, and AKT signaling in CsA-induced nephrotoxicity and proposes mechanistic explanations on why rats fed a low-salt diet have higher sensitivity to CsA.

On the spin order transfer from parahydrogen to another nucleus.

The hyperpolarization of nuclear spins holds great potential e.g. for biomedical research. Strong signal enhancements have been demonstrated e.g. by transforming the spin order of parahydrogen (pH(2)) to net polarization of a third nucleus (e.g. (13)C) by means of a spin-order-transfer (SOT) sequence. The polarization achieved is vitally dependent on the sequence intervals, which are a function of the J-coupling constants of the molecule to be polarized. How to derive the SOT sequence intervals, the actual values for molecules as well as the (theoretical) polarization yield and robustness, however, are not fully described. In this paper, (a) we provide the methods to obtain the SOT intervals for a given set of J-coupling constants (i.e. of a new hyperpolarization agent); (b) exemplify these methods on molecules from literature, providing the hitherto missing intervals and simulated polarization yield; and (c) assess the robustness of the sequences towards B(1) and J-coupling errors. Close to unity polarization is obtained for all molecules and sequences. Furthermore, the loss of polarization caused by erroneous B(1) and J-coupling constants is reduced by choosing the channel and phase of some pulses in the SOT sequences appropriately.

Fast three-dimensional proton spectroscopic imaging of the human brain at 3 T by combining spectroscopic missing pulse steady-state free precession and echo planar spectroscopic imaging.

The combination of the principles of two fast spectroscopic imaging (SI) methods, spectroscopic missing pulse steady-state free precession and echo planar SI (EPSI) is described as an approach toward fast 3D SI. This method, termed missing pulse steady-state free precession echo planar SI, exhibits a considerably reduced minimum total measurement time T(min), allowing a higher temporal resolution, a larger spatial matrix size, and the use of k-space weighted averaging and phase cycling, while maintaining all advantages of the original spectroscopic missing pulse steady-state free precession sequence. The minor signal-to-noise ratio loss caused by using oscillating read gradients can be compensated by applying k-space weighted averaging. The missing pulse steady-state free precession echo planar SI sequence was implemented on a 3 T head scanner, tested on phantoms and applied to healthy volunteers.

Combined reversed phase HPLC, mass spectrometry, and NMR spectroscopy for a fast separation and efficient identification of phosphatidylcholines.

In respect of the manifold involvement of lipids in biochemical processes, the analysis of intact and underivatized lipids of body fluids as well as cell and tissue extracts is still a challenging task, if detailed molecular information is required. Therefore, the advantage of combined use of high-pressure liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy will be shown analyzing three different types of extracts of the ubiquitous membrane component phosphatidylcholine. At first, different reversed phase modifications were tested on phosphatidylcholines (PC) with the same effective carbon number (ECN) for their applicability in lipid analysis. The results were taken to improve the separation of three natural PC extract types and a new reversed phase (RP)-HPLC method was developed. The individual species were characterized by one- and two-dimensional NMR and positive or negative ion mode quadrupole time of flight (q-TOF)-MS as well as MS/MS techniques. Furthermore, ion suppression effects during electrospray ionisation (ESI), difficulties, limits, and advantages of the individual analytical techniques are addressed.

Age and sex differences in the effects of the immunosuppressants cyclosporine, sirolimus and everolimus on rat brain metabolism.

Application of the widely used immunosuppressant (ISS) cyclosporine (CsA) is severely limited by a number of serious side-effects such as kidney and neurotoxicity. As we have shown before, CsA exhibits metabolic toxicity in brain-models. The macrolide ISSs sirolimus (SRL) and everolimus (RAD) are capable of modulating these CsA-induced effects. It was our aim to study the age-dependent metabolic changes in the rat brain after ISS-treatment and the possible role of the blood-brain-barrier in modulation of CsA metabolic toxicity. Young and adult rats were treated orally with one ISS alone or in combination with CsA for six days. Metabolic changes were assessed by nuclear magnetic resonance (NMR) spectroscopy of brain extracts as toxicodynamic endpoints. Brain P-glycoprotein (P-gp) and ISS concentrations were determined as pharmacokinetic endpoints. Young rats were more susceptible to CsA-induced inhibition of the Krebs cycle (glutamate: 78% of controls, glutamine: 82%, GABA: 71% in young vs. 85%, 89%, 92% in adult rats). Increased glycolysis after CsA-treatment was sufficient to maintain the energy state at control levels in adult brains, but not in the young rat brains (phosphocreatine: 35%). Tissue concentrations of CsA and SRL within the brain of young rats were three-fold higher, while concentrations of P-gp were three-fold higher in adult rat brains. Our results suggest that age-dependent differences in the blood-brain barrier led to increased ISS brain concentrations and hence inhibition of brain energy metabolism.

Metabolism of [U-(13)C]aspartate by astroglial cultures: nuclear magnetic resonance analysis of the culture media.

In brain the amino acid L-aspartate serves roles as: (1) putative transmitter, (2) protein precursor, (3) donor of atoms for the biosynthesis of pyrimidine and purine bases, and (4) fuel for energy metabolism. Astrocytes dominate aspartate clearance in brain, and in culture they take up aspartate and quickly metabolize it. In brain, only astrocytes were shown to express the enzymes for de novo pyrimidine biosynthesis. To gain more details about the spectrum of metabolites generated from aspartate and subsequently released by cultured astrocytes a (13)C-nuclear magnetic resonance analysis was performed of [U-(13)C]aspartate supplemented incubation media exposed to astroglial cultures. The results show that astrocytes readily metabolize aspartate and release into their culture media (13)C-isotopomers of lactate, glutamine, citrate and alanine. Despite the presence in astroglial cells of two tandem enzymes of pyrimidine biosynthesis and their mRNAs, pyrimidine nucleotide-related heterocyclic compounds such as dihydroorotate and orotate could not be detected in the culture media.

Toxicodynamic effects of ciclosporin are reflected by metabolite profiles in the urine of healthy individuals after a single dose.

WHAT IS ALREADY KNOWN ABOUT THE SUBJECT * Ciclosporin's nephrotoxicity initially targets the proximal tubule and is, at least in part, driven by increased formation of oxygen radicals. * (1)H-nuclear magnetic resonance spectroscopy (NMR)- and mass spectrometry (MS)-based biochemical profiling (metabolomics) allows for the sensitive detection of metabolite pattern changes in urine. * In systematic studies in rats we showed that ciclosporin caused urine metabolite pattern changes typical for proximal tubule damage and that these pattern changes seemed to be more sensitive than established clinical kidney function markers such as serum creatinine concentrations. WHAT THIS PAPER ADDS * This study showed that urine metabolite pattern changes as assessed by (1)H-NMR and HPLC-MS are sensitive enough to detect the effect of ciclosporin as early as 4 h after a single oral dose. * In our previous rat studies, changes in urine metabolite pattern in response to ciclosporin translated into healthy humans, indicating the involvement of the same toxicodynamic mechanisms. * The results provide proof of concept for further development of this combination molecular marker strategy into diagnostic tools for the detection and monitoring of drug nephrotoxicity. AIMS The immunosuppressant ciclosporin is an efficient prophylaxis against transplant organ rejection but its clinical use is limited by its nephrotoxicity. Our previous systematic studies in the rat indicated urine metabolite pattern changes to be sensitive indicators of the negative effects of ciclosporin on the kidney. To translate these results, we conducted an open label, placebo-controlled, crossover study assessing the time-dependent toxicodynamic effects of a single oral ciclosporin dose (5 mg kg(-1)) on the kidney in 13 healthy individuals. METHODS In plasma and urine samples, ciclosporin and 15-F(2t)-isoprostane concentrations were assessed using HPLC-MS and metabolite profiles using (1)H-NMR spectroscopy. RESULTS The maximum ciclosporin concentrations were 1489 +/- 425 ng ml(-1) (blood) and 2629 +/- 1308 ng ml(-1) (urine). The increase in urinary 15-F(2t)-isoprostane observed 4 h after administration of ciclosporin indicated an increase in oxidative stress. 15-F(2t)-isoprostane concentrations were on average 2.9-fold higher after ciclosporin than after placebo (59.8 +/- 31.2 vs. 20.9 +/- 19.9 pg mg(-1) creatinine, P < 0.02). While there were no conclusive changes in plasma 15-F(2t)-isoprostane concentrations or metabolite patterns, non-targeted metabolome analysis using principal components analysis and partial least square fit analysis revealed significant changes in urine metabolites typically associated with negative effects on proximal tubule cells. The major metabolites that differed between the 4 h urine samples after ciclosporin and placebo were citrate, hippurate, lactate, TMAO, creatinine and phenylalanine. CONCLUSION Changes in urine metabolite patterns as a molecular marker are sufficiently sensitive for the detection of the negative effects of ciclosporin on the kidney after a single oral dose.

Separation of phospholipid classes by hydrophilic interaction chromatography detected by electrospray ionization mass spectrometry.

A new isocratic separation method was developed for separation of phospholipid (PL) classes based on a silica hydrophilic interaction liquid chromatography (HILIC) column with electrospray ionization (ESI) mass spectrometric detection. Although HILIC is typically used for polar compounds, also amphiphilic molecules like phospholipids can be separated very well. Compared to normal-phase (NP) chromatography, which is usually used for PL class separation, HILIC has the advantage to use on-line ESI-MS detection because its eluents are ESI compatible. Furthermore, this HILIC method is isocratic and hence less time consuming than most (gradient) NP HPLC methods. A chromatographic baseline separation of a standard mixture containing phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC) was achieved within a total run time of 17 min using a mobile phase consisting of acetonitrile, methanol and ammonium acetate 10 mM. The new method was subsequently tested on phospholipid fractions of a body fluid (human blood plasma) and a tissue extract (swine brain) whereby it achieved nearly the same baseline separation of the PL classes. The detected classes in both cases were PE, PC, SM and LPC.

Immunosuppressant neurotoxicity in rat brain models: oxidative stress and cellular metabolism.

Coadministration of the calcineurin inhibitor cyclosporine (CsA) and the mTOR inhibitors sirolimus (SRL) or everolimus (RAD) increases the efficacy of immunosuppression after organ transplantation. Neurotoxicity of CsA is a major clinical problem. Our goal was to assess the effects of CsA, SRL, and RAD on brain cell metabolism. The studies included the comparison of immunosuppressant-mediated effects on glucose metabolism, energy production, and reactive oxygen species (ROS) formation in perfused rat brain slices, primary rat astrocytes, and C6 glioma cells. In brain slices and astrocytes, CsA inhibited Krebs cycle metabolism, while activating anaerobic glycolysis, most likely to compensate for the inhibition of mitochondrial energy production. SRL and RAD inhibited cytosolic glycolysis but did not cause changes in mitochondrial energy production. CsA + SRL inhibited Krebs cycle and glycolysis, thus reducing the ability of the cell to compensate for the negative effects of CsA on mitochondrial nucleoside triphosphate synthesis. In contrast to SRL at the concentrations tested, RAD reduced the CsA-induced ROS formation and antagonized CsA-induced effects on glucose and energy metabolism. Surprisingly, in C6 cells, SRL and RAD exposure resulted in high ROS concentrations without significant impairment of cell metabolism. Our results suggested that SRL enhances CsA-induced ROS formation and negative metabolic effects in brain cells, while RAD seems to antagonize the CsA effects. However, the three models showed different metabolic responses when challenged with the study drugs. In contrast to SRL, RAD enhances ROS formation in C6 glioma cells but has only minor effects on normal rat brain tissue.

Association of immunosuppressant-induced protein changes in the rat kidney with changes in urine metabolite patterns: a proteo-metabonomic study.

The basic mechanisms underlying calcineurin inhibitor (CI) nephrotoxicity and its enhancement by sirolimus are still largely unknown. We investigated the effects of CIs alone and in combination with sirolimus on the renal proteome and correlated these effects with urine metabolite pattern changes. Thirty-six male Wistar rats were assigned to six treatment groups (n = 4/group for proteome analysis and n = 6/group for urine (1)H NMR metabolite pattern analysis): vehicle controls, sirolimus 1 mg/kg/day, cyclosporine 10 mg/kg/day, cyclosporine 10 mg/kg/day + sirolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day, tacrolimus 1 mg/kg/day + sirolimus 1 mg/kg/day. After 28 days, 24 h-urine was collected for (1)H NMR-based metabolic analysis and kidneys were harvested for 2D-gel electrophoresis and histology. Cyclosporine affected the following groups of proteins: calcium homeostasis (regucalcin, calbindin), cytoskeleton (vimentin, caldesmon), response to hypoxia and mitochondrial function (prolyl 4-hydroxylase, proteasome, NADH dehydrogenase), and cell metabolism (kidney aminoacylase, pyruvate dehydrogenase, fructose-1,6-bis phosphate). Several of the changes in protein expression, confirmed by Western blot, were associated with and explained changes in metabolite concentrations in urine. Representative examples are an increase in kidney aminoacylase expression (decrease of hippurate concentrations in urine), up regulation of pyruvate dehydrogenase and fructose-1,6-bisphosphatase, (increased glucose metabolism), and down regulation of arginine/glycine-amidino transferase (most likely due to an increase in creatinine concentrations). Protein changes explained and qualified immunosuppressant-induced metabolite pattern changes in urine.