MiRNA profiling of urinary exosomes to assess the progression of acute kidney injury

Hiroko Sonoda,Sang-Ho Kwon,Deepak Nihalani,Byung Rho Lee,Je-Hyun Yoon,Masahiro Ikeda,Ki-Hoon Park

doi:10.1038/s41598-019-40747-8

Abstract

Because exosomes have gained attention as a source of biomarkers, we investigated if miRNAs in exosomes (exo-miRs) can report the disease progression of organ injury. Using rat renal ischemia-reperfusion injury (IRI) as a model of acute kidney injury (AKI), we determined temporally-released exo-miRs in urine during IRI and found that these exo-miRs could reliably mirror the progression of AKI. From the longitudinal measurements of miRNA expression in kidney and urine, we found that release of exo- miRs was a regulated sorting process. In the injury state, miR-16, miR-24, and miR-200c were increased in the urine. Interestingly, expression of target mRNAs of these exo-miRs was significantly altered in renal medulla. Next, in the early recovery state, exo-miRs (miR-9a, miR-141, miR-200a, miR-200c, miR-429), which share Zeb1/2 as a common target mRNA, were upregulated together, indicating that they reflect TGF-β-associated renal fibrosis. Finally, release of exo-miRs (miR-125a, miR-351) was regulated by TGF-β1 and was able to differentiate the sham and IRI even after the injured kidneys were recovered. Altogether, these data indicate that exo-miRs released in renal IRI are associated with TGF-β signaling. Temporal release of exo-miRs which share targets might be a regulatory mechanism to control the progression of AKI.

Highlights

Acute kidney injury (AKI) is a condition that results in an abrupt decrease in renal function and is known to be a risk factor for progression of chronic kidney disease (CKD)[1]
Male rats (Sprague-Dawley, SD, 10 wks) were purchased from Kyudo (Saga, Japan) and the rats were randomly divided into two groups: a sham operation group and a renal ischemia/reperfusion injury (IRI) group subjected to an IR operation
Histological sections were stained with periodic acid-Schiff (PAS) or Masson’s trichrome (MT) reagents (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), and microscopic observation of the stained specimen was performed as previously described[4]

Summary

Materials and Methods

For urinary exosome analysis for RT-PCR, urine samples were subjected to the total exosome isolation reagent from urine (Thermo Fisher) and RNAs were extracted using miRNeasy kit (Qiagen). 12.5 ng of RNAs isolated from urinary exosomes were used to generate poly(T) primed cDNA and miRNA expression analyses were performed according to miRCURY Locked Nucleic Acid (LNA) miRNA PCR assays (Qiagen) or custom primers for U6 RNA Forward : 5′-gcttcggcagcacatatacta-3′; Reverse, 5′-cgaatttgcgtgtcatccttg-3′). 15 ng of total RNAs were used to generate poly(T) primed cDNA and miRNA expression analyses were performed according to miRCURY LNA miRNA PCR assays (Qiagen). Unsaturated signals on the blots were captured with Odyssey Fc Imaging System (Li-Cor)

Injury vs Recovery

Results and Discussion

Additional Information