InVitro Selection of Cell-Internalizing DNA Aptamers in a Model System of Inflammatory Kidney Disease.

Glory Ranches,Alexander Hüttenhofer,Taras Stasyk,Herbert Schramek,Günter Mayer,Andreas Ploner,Gert Mayer,Melanie Lukasser

doi:10.1016/j.omtn.2017.06.018

Abstract

Chronic kidney disease (CKD) is a progressive pathological condition marked by a gradual loss of kidney function. Treatment of CKD is most effective when diagnosed at an early stage and patients are still asymptomatic. However, current diagnostic biomarkers (e.g., serum creatinine and urine albumin) are insufficient for prediction of the pathogenesis of the disease. To address this need, we applied a cell-SELEX (systematic evolution of ligands by exponential enrichment) approach and identified a series of DNA aptamers, which exhibit high affinity and selectivity for cytokine-stimulated cells, resembling some aspects of a CKD phenotype. The cell-SELEX approach was driven toward the enrichment of aptamers that internalize via the endosomal pathway by isolating the endosomal fractions in each selection cycle. Indeed, we demonstrated co-localization of selected aptamers with lysosomal-associated membrane protein 1 (LAMP-1), a late endosomal and lysosomal marker protein, by fluorescence in situ hybridization. These findings are consistent with binding and subsequent internalization of the aptamers into cytokine-stimulated cells. Thus, our study sets the stage for applying selected DNA aptamers as theragnostic reagents for the development of targeted therapies to combat CKD.

Highlights

The diagnosis of chronic kidney disease (CKD) is currently based on the estimated glomerular filtration rate and urinary albumin excretion
In Vitro Selection of DNA Aptamers for Chronic kidney disease (CKD) For in vitro selection, we employed a single-stranded DNA library consisting of approximately 1015 different DNA molecules and containing a 43-nucleotide randomized region, flanked by 18-nt and 19-nt primer sequences at the 50 and 30 ends, respectively (Figure 1)
renal proximal tubular epithelial cell (RPTEC)/TERT1 cells were stimulated with a mixture of pro-inflammatory and Molecular Therapy: Nucleic Acids are present on unstimulated cells and whose expression level is increased upon stimulation

Summary

Introduction

The diagnosis of chronic kidney disease (CKD) is currently based on the estimated glomerular filtration rate (eGFR) and urinary albumin excretion. A progressive decrease in the former results in stages G1 (preserved eGFR) to G5 (severely reduced eGFR, often necessitating initiation of renal replacement therapy), and the latter is defined as stages A1 (normal urinary albumin excretion) to A3 (albuminuria in excess of 300 mg/day).[1] A recent report on the global prevalence of CKD based on stages of glomerular filtration rate was published by Mills et al.[2] in 2015. The histopathology is characterized by an infiltration of inflammatory cells, tubular cell loss, (myo) fibroblast accumulation, and rarefaction of the peritubular vasculature, accompanied by deposition of the interstitial matrix,[7] and most studies suggest that progression to renal failure correlates more closely with tubular than with glomerular damage.[7,8,9] As a proof of concept, Grgic et al.[10] recently developed a unique mouse model of kidney injury and presented evidence that acute injury to the proximal renal tubules is sufficient to produce the full spectrum of pathological changes associated with progressive CKD

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