Proteins & Peptides
Proteome analysis of acute renal ischemia
Pamella Araujo Malagrino1, Gabriela Venturini da Silva1, Rafael Dariolli1, Patrícia
Schneider Yogi1, Joaquim Maurício da Motta Leal Filho1, Tamiris Carneiro Gois1,
Kallyandra Padilha1, Bianca Kiers1, Denise Maria Avancini Costa Malheiros2, Ana
Carolina Zeri3, Rafael Canevarolo3, Alexandre da Costa Pereira1.
pamalagrino@gmail.com
Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of Sao
Paulo Medical School, Sao Paulo, Brazil;
2
Laboratory of Cellular, Genetic, and Molecular Nephrology, University of São Paulo,
São Paulo, Brazil.
3
Nacional Laboratory of Bioscience, CNPEM, Sao Paulo, Brazil.
1
Background: Acute kidney injury (AKI) is a serious complication in hospitalized patients mainly
caused by ischemia/reperfusion (I/R). I/R increase reactive oxygen species and nitrogen leading to
cell damage and protein modifications such as nitration[1]. Thus, our purpose was analyzed
nitrated proteins in response to renal I/R in a porcine model. Methods. Pigs (n=5) were subjected
to 120 minutes of renal ischemia by catheter-balloon into the right renal artery followed by 24
hours reperfusion. Serial serum was collected of cava vein over renal veins during all experiment
and both kidneys were collected after reperfusion. Analysis of renal structure, function and
oxidative stress were performed by histological analysis, serum analysis (creatinine, NGAL, total
nitrated proteins) and urine analyses (proteins, fractional excretion, glucose). Proteins of kidney
tissue (ischemic and control) were enriched with anti-nitrotyrosine immunoprecipitation, digested
and analyzed by nanoLC/MS-Q-Exactive. The spectra were analyzed by Label Free MaxQuant
analysis and differential proteins were subjected to analysis of systems biology by software
MetaCore and Ingenuity. Results. Level of serum creatinine, NGAL and nitrated protein were
increased as well as proteins, electrolytes and glucose in urine. Acute tubular necrosis (ATN) was
identified in every animal, but only two animals showed levels of serum creatinine positive to AKI
(above 150% of baseline values). These results confirm our early model of renal I/R, proving the
decrease of renal function and increase of oxidative stress. Kidney nitroproteome showed 17
proteins exclusive in control group and 48 of ischemic group. These proteins were representatives
of cardiovascular and renal disease. In renal disease these proteins were associated with kidney
injury (necrosis, proliferation, inflammation, nephritis, glomerular injury) and it is related to
reorganization of cytoskeleton and cell adhesion. Through proteome analysis, was observed that
ischemic kidney lost transporters proteins of ionic channel and glucose, consistent with ATN
diagnostic and urinary biochemistry analysis. Add these proteins showed an increase in
metabolism of protein (expression and transport) and carbohydrate, with catabolism reduction of
short fatty acid and sulfur aminoacids. Conclusions. Nitroproteome is a powerful analysis to
explore physiological changes in kidney caused by ischemia and reperfusion. Ischemia/Reperfusion
in kidney causes changes in protein profile that are related to alterations in glucose and ions
output. This study supports the development of new diagnostics and early treatment of acute
kidney injury.
[1] Li, C. & Jackson, R. M. American journal of physiology: Cell physiology. 2002, 282, C227–41.