Identification of urinary microRNA biomarkers for in vivo gentamicin-induced nephrotoxicity models

Abstract

Background: Although previous in vivo studies explored urinary microRNA (miRNA), there is no agreement on nephrotoxicity-specific miRNA biomarkers.

Objectives: In this study, we assessed whether urinary miRNAs could be employed as biomarkers for nephrotoxicity.

Methods: For this, literature-based candidate miRNAs were identified by reviewing the previous studies. Female Sprague-Dawley rats received subcutaneous injections of a single dose or repeated doses (3 consecutive days) of gentamicin (GEN; 137 or 412 mg/kg). The expression of miRNAs was analyzed by real-time reverse transcription-polymerase chain reaction in 16 h pooled urine from GEN-treated rats.

Results: GEN-induced acute kidney injury was confirmed by the presence of tubular necrosis. We identified let-7g-5p, miR-21-3p, 26b-3p, 192-5p, and 378a-3p significantly upregulated in the urine of GEN-treated rats with the appearance of the necrosis in proximal tubules. Specifically, miR-26-3p, 192-5p, and 378a-3p with highly expressed levels in urine of rats with GEN-induced acute tubular injury were considered to have sensitivities comparable to clinical biomarkers, such as blood urea nitrogen, serum creatinine, and urinary kidney injury molecule protein.

Conclusions: These results indicated the potential involvement of urinary miRNAs in chemical-induced nephrotoxicity, suggesting that certain miRNAs could serve as biomarkers for acute nephrotoxicity.

Keywords: Acute kidney injury; biomarker; gentamicin; microRNAs; nephrotoxicity.

Fig. 1. Levels of clinical biomarkers after single and repeated administration of GEN. CD rats were subcutaneously administrated with single dose or repeated doses (3 consecutive days) of GEN (137 or 412 mg/kg) or vehicle (sterile saline), and sacrificed 24 h after the final treatment. Urine samples were individually collected for 16 h prior to necropsy. (A) Clinical parameters in rat serum: BUN and sCr. (B) Relative expression of KIM-1 in rat urine, normalized to urinary creatinine content (ng/mg) and then expressed as treatment-to-vehicle ratios. Values indicate the mean ± SE (n = 4).

BUN, blood urea nitrogen; sCr, serum creatinine; KIM-1, kidney injury molecule-1; GEN, gentamicin. ^*p < 0.05 and ^**p < 0.01 compared with the vehicle control.

Fig. 2. Histopathological changes in kidney tissues after single and repeated administration of GEN. CD rats were subcutaneously administrated with single dose or repeated doses (3 consecutive days) of GEN (137 or 412 mg/kg) or vehicle (sterile saline), and sacrificed 24 h after the final treatment. (A) Microscopic images (×200) of hematoxylin and eosin-stained formalin-fixed kidney sections (approximately 5 μm in thickness). Degeneration/necrosis (square areas) with basophilic tubules(arrowheads) were noted in the repeated treatment of GEN (412 mg/kg). Hyaline casts (arrowhead blank) in the tubules were observed in all GEN-treated groups. (B) Microscopic findings were graded on a scale of 0 (absent), 1 (minimal), 2 (mild), 3 (moderate), and 4 (severe). Data are shown as number of animals observed.

GEN, gentamicin.

Fig. 3. Comparison of miR-26b-3p, miR-192-5p, and miR-378a-3p highly expressed in GEN-induced acute tubular injury. Female Sprague-Dawley were subcutaneously administrated with single dose or repeated doses (3 consecutive days) of GEN (137 or 412 mg/kg) or vehicle (sterile saline). Urine samples were individually collected for 16 h prior to necropsy at 24 h post-dose and used for quantitative real-time reverse transcription-polymerase chain reaction analysis. The treatment-to-vehicle ratios (fold changes) were calculated from the C_t value of each miRNA normalized to that of a synthetic spike‐in control RNA, miR‐39, and transformed to log₂. Values indicate the mean ± SE (n = 4).

MiRNA, microRNA; GEN, gentamicin. ^*p < 0.05 and ^**p < 0.01 compared with the vehicle control.