MSACL 2016 EU Abstract

Proteome Analysis to Discovery of Biomarkers of Kidney Disease

Pamella Malagrino (Presenter)
University of São Paulo Medical School, São Paulo

Bio: I graduated in biology and finished my master degree in science in 2014. Currently, I am a PhD student in medical school at USP, and I am developing my project at Heart Institute under the supervision of Dr. Alexandre da Costa Pereira. I have been working with metabolomics analysis by nuclear magnetic resonance and GC-MS / MS for 6 years. Add I have been working with proteomic analysis developed on Q-TOF, MALDI-TOF and ESI-Q-Orbitrap. Currently, we are also beginning the analysis of imaging in kidney tissue. My PhD project is a continuation of my master degree and it aims the discovery of new biomarkers and a better understanding of acute kidney injury (AKI) for a better diagnosis and treatment of AKI and others kidney disease. We described a panel of metabolites that characterized ischemia and 55 proteins candidates to kidney biomarker in urine and serum.

Authorship: Pamella Araujo Malagrino1, Gabriela Venturini1, Patrícia Schneider Yogi1, Rafael Dariolli1, Kallyandra Padilha1, Bianca Kiers1, Tamiris Carneiro Gois1, Karina Helena Morais Cardozo2, Valdemir Malecho Carvalho2, Jéssica Silva Salgueiro2, Adriana Castello Costa Girardi1, Silvia M. O. Titan3, José Ed
1Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo Medical School, São Paulo, SP, Brazil; 2Grupo Fleury, São Paulo, SP, Brazil; 3Renal Division, Department of Clinical Medicine, Faculty of Medicine, University of São Paulo Medical School, São Paulo, SP, Brazil

Short Abstract We performed the proteomic of different tissues of ischemia/reperfusion (I/R) swine model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60 minutes) and 4, 11 and 16 hours post-reperfusion and renal cortex after 24hours of reperfusion. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 55 systemic proteins, four of them predominantly renal, candidates for biomarkers of renal disease. We validated one of the identified biomarkers, DPPIV, in a set of patients with diabetic nephropathy.

Long Abstract

Current diagnosis acute kidney injury (AKI) based on acute alterations in serum creatinine or urine output is late and unspecific. The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are predominantly renal. In fact, putative biomarkers for this condition have also been identified in a number of other clinical scenarios, such as cardiovascular diseases, chronic kidney failure or in patients being treated in intensive care units from a number of conditions. So, suitable animal models, such as pigs, can help in the identification of specific biomarkers for kidney disease. Here, we have used different tissues form a controlled, unilateral, percutaneous porcine renal ischemia/reperfusion (I/R) model to identify new, predominantly renal biomarker candidates for AKI.

Methods. Animal model experiments were conducted according to Malagrino and collaborators [1]. Briefly, five female pigs (Sus scrofa domesticus 15-20 kg) were submitted to unilateral renal I/R. Initially, a 6F multipurpose guide-catheter was introduced in the right femoral artery until the right renal artery. After an angiography, a balloon-catheter was introduced together with guide-catheter and inflated for 120 min (ischemia), followed by deflation (reperfusion) for 24 h. After reperfusion, the ischemic and contralateral kidneys were collected. Fifty milliliters of urine were collected directly from the bladder and serum was sampled from the inferior vena cava above the renal veins. In our model, urine and serum samples came from both the ischemic and contralateral kidneys. This was done, as opposed to the selectively collection from the ischemic kidney, in order to detect subtle biochemical changes generated by renal I/R that could be used as an early, and systemic, biomarker for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60 minutes) and 4, 11 and 16 hours post-reperfusion. In addition, contralateral and ischemic kidneys were analyzed after 24hours of reperfusion. The proteins were digested with trypsin and analyzed on the Q-ExactiveTM. To process proteomics date, Maxquant was used. Paired Student’s t-test was used to compare means between ischemic and contralateral kidney. The two-tailed test with p<0.05 and false discovery rate (FDR < 0.05) was considered significant. Pathway and network analysis were performed in Ingenuity Pathway Analysis and Gene Onthology. The validation of one candidate to biomarker, DPPIV, was performed by urine DPP IV activity assay in 56 patients with diabetic nephropathy.

Results. In renal cortex proteome, we observed an increase in the synthesis of proteins in the ischemic kidney compared to the contralateral, highlighting by pathway analysis the transcription factors and epithelial adherens junction proteins important for kidney injury recovery after 24 hours. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 6 proteins in the serum that probably came from the ischemic kidney and may provide a set of targets for AKI screening. Additionally, we identified also 49, being 4 predominantly renal, proteins in urine based on the TiGER database. Aiming at showing the biological and clinical significance of our finds, we selected one protein (DPPIV) among the four candidates to biomarker for biological validation. The main reason to choose DPPIV was due to the high expression of this endopeptidase in the kidney tubule. This protein was also described as changed in other kidney diseases and, nowadays, DPPIV inhibitors are widely used in diabetic treatment. DPPIV is an 88KDa protein, which suggest the presence of this protein in urine may be due to secretion by tubular epithelial cells or due to tubular injury. In our swine model, we demonstrate an increase in urinary DPPIV activity 4 hours after reperfusion, followed by its recovery after 11 hours. In addition to validation of DPPIV in urine in our model, we also describe the relation between this potential biomarker and measures associated with kidney function in a small cohort of humans with kidney disease. These new results add to the overall description of biomarker identification pointing towards the usability of these newly identified proteins as clinical tools for disease stratification. We believe new studies are also important for validation of the other biomarkers that, in addition to DPPIV, can contribute to a better diagnosis and prognosis of kidney injury.

Conclusion. We describe four urinary proteins with predominantly renal gene expression that are altered in response to controlled kidney I/R and can be biomarkers of kidney dysfunction. Three of these proteins were related to AKI for the first time in our work. Altogether, this integrative proteome analysis can provide a panel of potential and predominantly renal biomarkers in many levels, considering changes that occur in the tissue and echo in serum and urine protein profiles. Therefore, through proteome analysis we could identify new candidates to kidney disease that can serve as non-invasive biomarkers for kidney dysfunction. Future studies with target analytical tools should be developed to validate these findings and identify which of the described proteins have clinical potential as non-invasive biomarkers.

References & Acknowledgements:

[1] P.A. Malagrino, G. Venturini, P.S. Yogi, R. Dariolli, K. Padilha, B. Kiers, et al., Catheter-based induction of renal ischemia/reperfusion in swine: description of an experimental model, Physiol. Rep. 2 (2014) 1–13. doi:10.14814/phy2.12150.

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