In vitrostudy of putative genomic biomarkers of nephrotoxicity through differential gene expression using gentamicin

Abstract

Drug-induced nephrotoxicity is one of the most frequently observed effects in long-term pharmacotherapy. The effects of nephrotoxicity are commonly discovered later due to a lack of sensitivity in in vivo methods. Therefore, researchers have tried to develop in vitro alternative methods for early identification of toxicity. In this study, LLC-PK1 cells were exposed to gentamicin through MTT and trypan blue assay. Concentrations of 4 (low), 8 (medium) and 12 (high) mM, were used to evaluate differential gene expression. A panel of genes was selected based on gene expression changes. The search for sequences of mRNA encoding proteins previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information (USA). RNA was extracted from the cells, and RT-qPCR was performed to evaluate differential expression profiles of the selected genes. Among the 11 analyzed genes, four proved to be differentially up-regulated in cells exposed to gentamicin: HAVcr1, caspase 3, ICAM-1 and EXOC6. According to this study's results, we suggest that these genes play an important role in the mechanism of in vitro nephrotoxicity caused by gentamicin and can be used as early in vitro biomarkers to identify nephrotoxicity when developing safer drugs.

Glomerular filtration drug injury: In vitro evaluation of functional and morphological podocyte perturbations

“…The apparent permeability of the podocytes was evaluated at the end of the incubation time. The two concentrations were chosen in agreement with the literature [11][12][13] and after preliminary assays were done to evaluate the highest nontoxic dose for each compound in our podocyte model. Untreated and PAN-treated cells served as negative and positive control, respectively, for podocyte dysfunction.…”

Nephrotoxicity Biomarkers: Role and Significance in the Diagnosis of Drug-Induced Kidney Injury

“…Так, в исследовании, проведенном в США в 2017 г., было показано, что воздействие гентамицина вызывает повышение экспрессии четырех генов, кодирующих HAVcr1, ICAM-1, EXOC6 и каспазу-3. Сделан вывод о том, что эти гены играют важную роль в механизмах нефротоксичности, вызванной гентамицином, и могут быть использованы в качестве ранних биомаркеров нефротоксичности в исследованиях in vitro при разработке безопасных ЛП [84].…”

Biomarkers and in vitro strategies for nephrotoxicity and renal disease assessment

“…Although KIM-1 is a sensitive and specific marker for kidney tubular injury, it is difficult to measure it in acute settings [53]. By contrast, Ichimura, et al [54] demonstrated that human KIM-1 exhibits homology to HAVCR1, and studies using gentamicin [55,56], amphotericin B [57], and cisplatin (no publishing data) have shown that the gene HAVCR1 (KIM1) might be used as in vitro biomarkers of nephrotoxicity.…”

“…Hence, it is paramount to use new tools in order to develop renal AOPs. Some studies have been performed utilizing biochemistry [154][155][156] and genomics [55][56][57]. However, it is important to make these data public through database, like the Comparative Toxicogenomics Database (CTD), in an attempt to achieve faster and more reliable results.…”