Validation of an enzyme-linked immunosorbent assay (ELISA) for quantification of endostatin levels in mice as a biomarker of developing glomerulonephritis.

Gregory Shearer,Jacqueline Wallwitz,Dagmar Stoiber,Petra Aigner,Emilio Casanova,Anton Bauer,Eva Bauer,Elisabeth Gadermaier

doi:10.1371/journal.pone.0220935

Abstract

Endostatin, the C-terminal fragment of type XVIII collagen, was shown to be one of the most potent endothelial cell-specific inhibitors of angiogenesis. As altered circulating endostatin concentration is associated with impaired kidney function, new tools for measuring endostatin in rodents may be helpful to further investigate and understand its role within kidney disease progression. A novel and commercially available ELISA for the quantification of mouse and rat endostatin was developed and validated according to international quality guidelines including the parameters specificity, robustness, accuracy, dilution linearity, precision, limit of detection (LOD) and lower limit of quantification (LLOQ). Endostatin and blood urea nitrogen (BUN) concentration were measured in mice with a glomerulonephritis phenotype. The validation revealed that within the range of 0.5–32 nmol/L the immunoassay is robust and highly specific for the measurement of rodent endostatin with high sensitivity (LOD 0.24 nmol/L, LLOQ 0.5 nmol/L) and good reproducibility (intra- and inter-assay CV <10%). Also accuracy and dilution linearity were within the range of acceptance. BCL2 transgenic and ETV6/RUNX1;BCL2 double transgenic mice develop a glomerulonephritis phenotype over time, which was displayed by staining of kidney sections. Even before full manifestation of disease serum endostatin concentration rises significantly, whereas BUN levels just slightly increase. This newly developed and commercially available ELISA provides a reliable and accurate tool for the quantification of mouse and rat endostatin and may give new perspectives in the investigation of the role of endostatin as an important and early biomarker for reduced kidney function. Measurement of endostatin concentration is recommended to be used as a superior biomarker for chronic kidney disease compared to BUN.

Highlights

Decreased kidney function may have a wide range of reasons like infections, toxins, genetic disorders, metabolic dysfunction like type 2 diabetes or autoimmune diseases
acute kidney injury (AKI) is defined as a sudden reduction in the glomerular filtration rate (GFR) and renal output, which results in the accumulation of nitrogenous waste [1], whereas chronic kidney disease (CKD) is characterized by structural or functional abnormalities of the kidney with implications for health over a time period for at least three months [2]
As there is no high-quality quantification tool available, this study presents the development, validation and application of an enzyme-linked immunosorbent assay (ELISA) for measuring rodent endostatin levels, which is available commercially

Summary

Introduction

Decreased kidney function may have a wide range of reasons like infections, toxins, genetic disorders, metabolic dysfunction like type 2 diabetes or autoimmune diseases. AKI is defined as a sudden reduction in the glomerular filtration rate (GFR) and renal output, which results in the accumulation of nitrogenous waste [1], whereas CKD is characterized by structural or functional abnormalities of the kidney with implications for health over a time period for at least three months [2]. Endostatin was already discovered 1997 by O’Reilly and colleagues and is a 20 kDa inhibitor of endothelial cell proliferation in vitro and a potent inhibitor of angiogenesis and tumor growth in vivo [4] It is the C-terminal fragment of type XVIII collagen and emerges mainly during extracellular matrix remodeling [5]. Endostatin could be used as a prediction marker for AKI in critically ill patients [11]

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