MSACL 2019 EU Abstract

Podocyturia Evaluation in Women with Preeclampsia and Fabry Disease Patients Using a Tandem Mass Spectrometry Approach

Christiane Auray-Blais (1), Tristan Martineau (1), Michel Boutin (1), Anne-Marie Côté (2), Bruno Maranda (1), Daniel Bichet (3)
(1) Division of Medical Genetics, Université de Sherbrooke, Sherbrooke, Quebec, Canada (2) Division of Nephrology, Université de Sherbrooke, Quebec, Canada (3) Université de Montréal, Quebec, Canada

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Christiane Auray-Blais (Presenter) Université de Sherbrooke

Presenter Bio: Christiane Auray-Blais is the Director of the Neonatal Urine Screening Program for hereditary metabolic disorders in Sherbrooke, QC. She holds a Ph.D. in radiobiology from the Faculty of Medicine and Health Sciences (FMHS) at the Université de Sherbrooke and postdoctoral studies from Duke University Medical Center in North Carolina, US. She has a master’s degree in Health Law from the Faculty of Law at the Université de Sherbrooke and a bachelor’s degree in biochemistry. She is the author of more than 275 publications, book chapters, abstracts and articles. She is a full professor in the Medical Genetics Division in the Department of Pediatrics at the FMHS and a researcher at the Clinical Research Centre in Sherbrooke, and in the Mother-Child Axis. She is the principal investigator and co-investigator in numerous research grants.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction. Podocyturia is a possible early sign of kidney abnormalities in patients. Currently, kidney damages are assessed using proteinuria measurements and the estimated glomerular filtration rate. Unfortunately, these parameters might not always be efficient in early detection of all patients. Most analytical techniques for the analysis of podocyturia are tedious, time-consuming, and may lead to results variability. We opted to develop a reliable methodology for podocyturia evaluation in patients with kidney involvement.
Objectives. The main objective of this project was to devise an efficient analytical tool for the analysis of peptides characteristic of podocyte proteins (podocin and podocalyxin) using a tandem mass spectrometry (MS/MS) method for early diagnosis of various kidney abnormalities. Random urine samples from women with preeclampsia, Fabry disease patients and controls were analyzed and correlations with other kidney disease biochemical parameters evaluated.
Method. A reversed-phase ultra-performance liquid chromatography coupled to a tandem mass spectrometry (UPLC-MS/MS) method was developed/validated to quantify peptides of podocalyxin and podocin in urine supernatant by using specific cleavable peptides and standards. One mL was pipetted from the supernatant of centrifuged random urine samples. A heavy cleavable peptide standard was added for each protein. Samples were treated with sodium deoxycholate, dithiothreitol and iodoacetamide prepared in an ammonium bicarbonate buffer to ensure an optimal trypsin digestion (2 h at 37oC). Tryptic peptides were purified by solid-phase extraction and evaporated. Samples were resuspended, filtered and analyzed using an Acquity-I Class Xevo UPLC TQ-S MS/MS (Waters Corp.).
Results. Peptides [ATFNPAQDK+2H]2+ (m/z 496.25->558.29 (y5)) and [APAATVVDVDEVR+2H]2+ (m/z 671.35->831.42 (y7)) were selected to quantify podocalyxin and podocin, respectively. The validation of the method for intraday- and interday assays showed biases below 15%. The molecules were stable at -20oC and -80oC. Our results show that a severe albuminuria content in urine samples did not unfavorably impact on the results. Podocalyxin levels were higher than podocin levels in patients, especially in pregnant women. Women with preeclampsia had abnormal levels of both proteins with a higher sensitivity for podocalyxin. Slightly increased levels of podocin were observed in Fabry males, while both protein levels were increased in untreated Fabry females compared to controls. Positive correlations were found in the preeclampsia groups: podocalyxin and podocin levels correlated with blood pressure, albuminuria and proteinuria levels. In the Fabry groups, podocalyxin levels correlated with urine glycosphingolipids, such as globotriaosylceramide and globotriaosylsphingosine levels.
Conclusion. This multiplex quantitative podocyturia methodology is reliable. It is a valuable investigative tool for patients with kidney involvement.