Urinary Metabolomics in Monitoring Acute Tubular Injury of Renal Allografts: A Preliminary Report

doi:10.1016/j.transproceed.2011.08.109

Transplantation Proceedings

Volume 43, Issue 10, December 2011, Pages 3738-3742

https://doi.org/10.1016/j.transproceed.2011.08.109 Get rights and content

Abstract

Acute tubular injury (ATI) is very common in biopsy specimens from renal allografts that suffer from delayed graft function (DGF) or dysfunction. Currently there are few reports on investigating small molecule metabolites in urine samples from transplant recipients as a noninvasive method to predict the ATI of renal allografts instead of an allograft biopsy. In our study matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) was used to analyze small molecule metabolites in urine samples from renal transplant recipients with biopsy-proven slight ATI or moderate ATI or acute tubular necrosis (ATN). To evaluate the ATI-specific value of those small molecules, we applied the Principal Component Analysis (PCA) program. Mass spectra data were imported into the PCA, where loading graphs were constructed to express the constituents of the urine samples. Slight ATI, moderate ATI, or ATN of renal allografts were separated obviously in the loading graph. The position of urine samples in the graph may reflect the tubular injury status of allografts. A farther apart point from the original site may mean the allograft suffered from more severe ATI (even ATN), and vice versa. Detection of small molecule metabolites in urine samples of recipients through MALDI-FTMS may offer a promising noninvasive, high throughput, rapid tool to predict ATI/ATN of renal allografts.

Section snippets

Selection and Description of Participants

We collected 53 urine samples from 5 transplant recipients whose biopsy specimens showed slight or moderate ATI or ATN, and 10 urine samples from their donors as normal controls. No prisoners or organs from prisoners were used in our study. Informed consent was obtained from the patients.

Each specimen was collected in 2 10-mL sterile polypropylene centrifuge tubes, 1 for the following analysis and another for comparison of doubtful samples. Urine aliquots taken with a disposable 1.5-mL

Results

Our primary study classified all 5 recipients into 3 groups based on renal allograft biopsy and clinical manifestations: 24-hour urine output, serum creatinine (Scr), delayed graft function (DGF), and so on. First-week dialyzed patients formed the DGF group. Nondialyzed patients were divided into slow graft function (SGF) or immediate graft function (IGF) cohorts according to whether the Scr at day 7 was higher or lower than 221 μmol/L (25 mg/mL). The detailed clinical information and

Discussion

ATI is a common finding among early renal allograft biopsies that are performed due to dysfunction or DGF, which show a aggregate prevalence of 32%–65%.2, 12 It has been viewed mostly as a nonspecific, relatively harmless result of early graft insults, without adverse effects on long-term graft function or survival, unless complicated by rejection episodes.13, 14 But ATI has been linked to inferior long-term graft function.¹ ATI is a common pathological course during the early recovery process.

Acknowledgments

We are grateful to Dr Yue Su of Shanghai University of Traditional Chinese Medicine for her assistance in the data handling of PCA.

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In the last decade, the progress in genomics, proteomics and metabolomics (more specifically, lipidomics) approaches has allowed finding robust, predictive and useful biomarkers in RT such as peptides and proteins [7]. Although metabolomics has been the least assessed in renal transplantation as compared to proteomics, urinary metabolomic assays were successfully applied to assess kidney transplant function in patients and to predict acute renal allograft rejection [11,12]. Similarly, the level of OS metabolites increased as chronic kidney disease progressed, significantly correlating with the level of renal function [13].
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Urine metabolomics has also been used to monitor renal allograft function in five Asian transplant recipients whose biopsy specimens showed slight or moderate ATI or acute tubular necrosis (ATN) [107]. Changes among urine small molecule metabolites were capable to distinguish between ATI and ATN status after transplantation [107]. Data from metabolomics GWAS demonstrate that there are differences in individual metabolic profiles in blood and/or urine that may be linked to underlying genetic variation but we are still not at a point where an integrated “metabologenomic” profile can be provided for a person with kidney disease.
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This review article presents an overview of the recent developments in the fields of metabolomics and proteomics in relation to CKD. Among the many different proteomic biomarkers proposed, there is particular interest in the CKD273 classifier, a urinary proteome biomarker reported to predict CKD progression and with implementation potential. Other individual non-invasive peptidomic biomarkers that are potentially relevant for CKD detection include type 1 collagen, uromodulin and mucin-1. Despite the limited sample sizes and variability of the metabolomics studies, some metabolites such as trimethylamine N-oxide, kynurenine and citrulline stand out as potential biomarkers in CKD.
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This study demonstrated how rapidly the kidney responds following a dose of calcineurin inhibitor (CNI) and how urine metabolite patterns can reflect biochemical changes in the kidney [2,5]. These studies in combination with other previous studies [4,9] suggested that, in addition to established kidney function markers such as creatinine and uric acid, it will be of clinical value to also monitor changes in Krebs cycle intermediates (citrate, succinate, oxoglutarate, and lactate), markers of oxidative stress (trimethylamine oxide (TMAO), reabsorption (glucose, sorbitol), as well as active secretion and kidney amino acylase activity (hippurate) [3,5,9,10]. Although various combinations of these markers have previously been examined using nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), to our knowledge no high-performance liquid chromatography- tandem mass spectrometry-based (LC-MS/MS) assay to simultaneously analyze all of these metabolites in human urine [3,5,9,11,12] has been published yet.
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A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of ten metabolites associated with the Krebs cycle, purine degradation, and oxidative stress in human urine was developed and validated. Normal values were assessed in healthy adult (n = 120) and pediatric (n = 36) individuals. In addition, 9 pediatric renal transplant recipients patients were evaluated before and after initial dosing of the immunosuppressant tacrolimus in a proof-of-concept study.
The assay met all predefined acceptance criteria. The lower limit of quantification ranged from 0.1 to 1000 μmol/l. Inter-day trueness and imprecisions ranged from 91.4–112.9% and 1.5–12.4%, respectively. The total assay run time was 5.5 minutes.
Concentrations of glucose, sorbitol, and trimethylamine oxide (TMAO) were elevated in pediatric renal transplant patients (n = 9) prior to transplantation as well as before and immediately after initial dosing of tacrolimus. One month post-transplant urine metabolite patterns matched those of healthy children (n = 36).
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Another LC-MS metabolomic analysis identified HVA-SO4 as a biomarker in urine from ARI children [95]. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry has been used to analyze small molecule metabolites in urine from renal transplant recipients with varying levels of acute tubular injury [96]. 1H NMR or MS metabolomics is a powerful and reliable analytical approach for nephrotoxicity studies.
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: Grants provided by the National Natural Science Foundation of China (no. 81070595), Science and Technology Commission of Shanghai Municipality (no. 08410701300, 08DZ2293100, 09DZ2260300, 09411952000, and 10DZ2212000).

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Kidney transplantationComplication: MetabolicUrinary Metabolomics in Monitoring Acute Tubular Injury of Renal Allografts: A Preliminary Report

Abstract

Section snippets

Selection and Description of Participants

Results

Discussion

Acknowledgments

Am J Transplant

Transplant Proc

J Chromatogr A

J Chromatogr A

J Am Soc Mass Spectrom

Am J Kidney Dis

Enhanced MCP-1 expression during ischemia/reperfusion injury is mediated by oxidative stress and NF-kappaB

Kidney Int

Kidney transplantation
Complication: Metabolic
Urinary Metabolomics in Monitoring Acute Tubular Injury of Renal Allografts: A Preliminary Report