Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron

Abstract

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Keywords: biomarkers; kidney injury; microRNAs; safety assessment.

Figure 1.

Strategy to identify and characterize the localization of nephron segment specific miRNAs. Abbreviations: G, glomerulus; PT, proximal tubule; TAL, thick ascending limb of the loop of Henle; CD, collecting duct.

Figure 2.

Compound structures for Sanofi compounds (A) X, (B) Y, and (C) Z. The pharmacological targets of compounds X, Y, and Z are Factor Xa, adenosine A3 receptor, and GPR119, respectively.

Figure 3.

Urinary microRNAs measured in rat studies using TaqMan low-density array A cards.

Figure 4.

Venn diagram of urinary microRNAs (miRNAs) in rats treated with segment selective nephrotoxicants. miRNAs highlighted in the diagram correspond to miRNAs candidates further characterized in nephron segments isolated by laser capture microdissection.

Figure 5.

Digital droplet PCR profiles of selected urinary microRNA top candidates in nephron segments isolated by laser capture microdissection (LCM) in Sprague Dawley rats. Candidates for (A) glomerulus (G), proximal tube (PT), and thick ascending limb (TAL) of the loop of Henle and collecting duct (CD) were measured in their respective nephron segments. Ratios calculated by using miR-30d-5p as normalizer per region and rat, multiplied by 100 to obtain more easily comparable numbers. miR-34c-3p was measured in LCM samples from only 1 rat.

Figure 6.

Principal component analysis of sRNA-seq normalized microRNA counts in nephron segments isolated by laser capture microdissection (LCM) in Sprague Dawley rats. A, Principal component 1 (PC1; x-axis) and PC2 (y-axis) view. B, PC3 (z-axis) view. First 3 PCs shown, representing 73% of the total variance. Each data point represents different sequencing lanes. Shapes designate individual rat samples. Colors represent different nephron regions (see key). Colored shaded regions represent groupings of each nephron segment sampled via LCM.

Figure 7.

Small RNA-sequencing (sRNA-seq) profiles of selected miRNA top candidates in nephron segments isolated by laser capture microdissection in Sprague Dawley rats. A, Hierarchical clustering of region-specific or -enriched miRNAs based on sRNA-seq. Top (B) glomerulus (G), (C) proximal tube (PT), (D) collecting duct (CD), and (E) thick ascending limb (TAL) candidates of medium to high expression based on normalized counts (>500 in target regions) are shown with the exception of miR-210-3p and -221-3p, which had lower expression but which were identified as potential candidates in the urinary miRNA analysis.

Figure 8.

FirePlex profiles of selected microRNA top candidates in nephron segments isolated by laser capture microdissection in Sprague Dawley rats. (A) glomerulus (G), (B) proximal tubule (PT), (C) thick ascending limb (TAL) of the loop of Henle and collecting duct (CD).

Figure 9.

Correlation coefficient comparison between digital droplet PCR, FirePlex, and small RNA-sequencing measurements of nephrotoxicity microRNA (miRNA) candidates across nephron segments isolated by laser capture microdissection in Sprague Dawley rats. Color gradient was adjusted per miRNA per method across 4 nephron segments. Correlation gradient was based on all comparisons. *, raw sequencing count for these miRNA were below cutoff (<100 total counts for all samples).