Evaluation of Tryptic Podocin Peptide in Urine Sediment Using LC-MS-MRM Method as a Potential Biomarker of Glomerular Injury in Dogs with Clinical Signs of Renal and Cardiac Disorders.

Barbara Szczepankiewicz,Remigiusz Bąchor,Robert Pasławski,Andrzej Konieczny,Urszula Pasławska,Zbigniew Szewczuk,Natalia Siwińska

doi:10.3390/molecules24173088

Abstract

The early asymptomatic stage of glomerular injury is a diagnostic challenge in the course of renal and extra-renal disease, e.g., heart insufficiency. It was found that podocin, a podocyte-specific protein present in the urine, may serve as a biomarker in the diagnosis of glomerular disease in humans and animals including glomerulonephritis, glomerulosclerosis, amyloidosis, or nephropathy. Therefore, there is a need of development of the sensitive and straightforward method of urinary podocin identification. In this work, we report our extended research under the glomerular injury investigation in dogs by application of clinical examination and LC-MS-MRM method in the identification of canine podocin in urine samples. The LC-MS-MRM method is based on the identification of podocin tryptic peptide with the 218H-AAEILAATPAAVQLR-OH232 sequence. The model peptide was characterized by the highest ionization efficiency of all the proposed model podocin tryptic peptides in a canine urine sediment according to the LC-MS/MS analysis. The obtained results revealed the presence of the model peptide in 40.9% of dogs with MMVD (active glomerular injury secondary to heart disease = cardiorenal syndrome-CRS) and 33.3% dogs with chronic kidney disease. The potential applicability of the developed methodology in the analysis of podocin in canine urine sediments was confirmed.

Highlights

Podocytes are glomerular epithelial cells that line the urinary side of the capillary loops
The aim of this work was to analyze the applicability of liquid chromatography coupled with mass spectrometry (LC-MS)-Multiple Reaction Monitoring (MRM) method in the detection of obtained withurine the clinical, andkidney biochemical data and urine test podocin in results the canine sampleshematological, with diagnosed dysfunction andwith to correlate theresults
We present our investigation on the application of LC-MS-MRM method in the analysis of podocin tryptic peptides in canine urine samples as a potent podocyte biomarker and correlation of the obtained results with the clinical, hematological, and biochemical data and urine test results

Summary

Introduction

Podocytes are glomerular epithelial cells that line the urinary side of the capillary loops This highly specialized cells completely enwrap the glomerular capillaries with their primary and secondary processes. These processes combine with the neighboring foot processes forming a so-called slit diaphragm. Because of their anatomic localization at the outer aspect of the capillary loops, podocytes are constantly challenged by oxygen radicals, cytokines, immune complexes, and inflammatory processes and by mechanical forces (i.e., glomerular hypertension and filtration pressure) [1,2,3,4]. Podocyturia enables the differentiation between active and inactive processes [7] It seems that the podocytes may be more useful in the diagnosis of kidney diseases in dogs than in cats. Most of dogs and humans suffer from glomerular injury [11]

Objectives

Methods

Results

Discussion

Conclusion