Journal of Molecular Biomarkers and Clinical Trials

Automated Image Analysis of Podocyte Desmin Immunostaining in a Rat Model of Sub-Acute Doxorubicin-Induced Glomerulopathy

*James Eric McDuffie
Investigative Toxicology Department, Janssen Research & Development, United States

*Corresponding Author:
James Eric McDuffie
Investigative Toxicology Department, Janssen Research & Development, United States
Email:jmcduffi@its.jnj.com

Published on: 2016-11-15

Abstract

Remarkable histopathology findings first identified by light microscopic examination of hematoxylin and eosin stained tissues often warrant further characterization. Recent advancements in the field of molecular diagnostic pathology include fully automated whole-slide imaging. Such tools support best practices for digital imaging and quantification of positive immunohistochemical staining. Consequently, we developed a novel computational method to automatically detect total glomeruli in whole-tissue sections and to quantify areas and intensities of desmin immunolabeling in podocytes. We propose that our method represents a feasible, accurate and efficient alternative to semi-automated quantitative methods which require tedious manual tracing of glomerular borders and allow operator bias (e.g., random selection of glomeruli with enhanced staining) when evaluating glomerular alterations and associated changes in marker localization. Real-time quantitative polymerase chain reaction analysis of adjacent tissues may reveal simultaneous changes in nephron-specific genomic biomarkers. Indeed, samples from the same rats with evidence of doxorubicin-induced primary glomerular toxicity, revealed increased gene expression changes in podocyte markers (desmin and podocin) concurrent to upregulated microRNA-34c3p in macrodissected flash-frozen kidney cortices (anatomic site for podocytes). We demonstrated innovative approaches to consider when monitoring for invasive changes in glomerular-specific kidney safety biomarkers following nephrotoxicant exposure

Keywords

Doxorubicin, Podocyte, Glomeruli, Automated Image Analysis, Rat

Introduction

Drug-induced kidney injury (DIKI) may occur in the absence of remarkable clinical signs of toxicity or clinically relevant increases in routine kidney safety biomarkers [1]. Both blood urea nitrogen (BUN) and serum creatinine are routine biomarkers which lack sensitivity and specificity for nephrotoxicity [1-3]. Remarkable alterations in glomerular filtration rate (GFR) are the best indicator of reduced kidney function. Selected novel, protein-based rat kidney safety biomarkers in urine have been qualified for use in conjunction with BUN, serum creatinine and histopathology to enable monitoring for compound induced tubular and/or glomerular toxicity [1]. In the rat, drug-induced tubular lesions may be predicted by increases in urinary protein levels of kidney injury molecule 1 (KIM-1), albumin, trefoil factor 3 and clusterin. Impairment of tubular reabsorption and alterations or damage of glomeruli may be identified by changes in urinary levels of total protein, cystatin C and B2-microglobulin. MicroRNAs (miRNAs) function at the post-transcriptional level by modifying translation, or promote the cleavage of their target mRNAs [4]. Because miRNAs play a key role in gene regulation and demonstrate high inter-species conservation, these species have the potential to serve as novel indicators of DIKI that may outperform traditional biomarkers; when elevated, miRNAs have the potential to serve as novel genomic indicators associated with renal injury that may outperform BUN and serum creatinine when monitoring for translatable DIKI liabilities [5].