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Abstract: SA-PO1005

The miR-143/145 Cluster Induced by TGF-β1 Suppresses Wilms Tumor 1 Expression in Cultured Human Podocytes

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Tabei, Akifumi, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Sakairi, Toru, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Ohishi, Yuko, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Watanabe, Mitsuharu, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Nakasatomi, Masao, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Hamatani, Hiroko, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Ikeuchi, Hidekazu, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Kaneko, Yoriaki, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
  • Kopp, Jeffrey B., National Institutes of Health, Bethesda, Maryland, United States
  • Hiromura, Keiju, Gunma Daigaku Igakubu Fuzoku Byoin, Meabashi, Gunma, Japan
Background

Transforming growth factor (TGF)-β1 contributes to podocyte injury in various glomerular diseases, including diabetic kidney disease (DKD), probably in part by attenuating expression of Wilms’ tumor 1 (WT1). We aimed to identify microRNAs associated with TGF-β1-induced podocyte injury.

Methods

miR-microarray studies were performed using total RNA extracted from differentiated human podocytes exposed to 5 ng/mL TGF-β1 for 24 h and control podocytes. db/db mice were used as animal models of type-2 diabetic nephropathy.

Results

Microarray analysis identified miR-143 as the miR with the greatest increase following exposure to TGF-β1 (2.3 fold increase as 2ΔLMR vs. control, p=0.0000077). We confirmed by reverse transcription-quantitative polymerase chain reaction that the miR-143/145 cluster expression significantly increased in a time-dependent manner for up to 48 hours after exposure to of TGF-β1 in cultured human podocytes (13.3 ± 3.7/ 17.4 ± 0.4 fold at 48 h vs. 0 h). We also observed upregulation of miR-143 in glomeruli of 16-week-old type 2 diabetic db/db obese mice compared to control mice, although miR-145 levels were comparable. Lentiviral ectopic expression of miR-143 and miR-145 in cultured human podocytes suppressed mRNA and protein expression of WT1 compared to ectopic expression of scramble allele as control, both with TGF-β1 exposure and non-exposure (0.37 ±0.057/ 0.49 ±0.084 fold vs. control for mRNA, 0.44 ± 0.093/ 0.38 ± 0.066 fold vs control for protein). Further, inhibition of SMAD signaling by shRNA targeting SMAD4 and inhibition of mammalian target of rapamycin (mTOR) signaling by exposure to rapamycin partially and significantly reversed the TGF-β1-induced increase in miR-143/145 and decrease in mRNA and protein expression of WT1.

Conclusion

In summary, in cultured human podocytes, TGF-β1 induces expression of the miR-143/145 cluster, in part through the classic SMAD and mTOR pathway, and miR-143/145 reduces expression of WT1. Thus, miR-143/145 may contribute to the TGF-β1-induced podocyte injury.

Funding

  • Commercial Support – Bayer Yakuhin, Ltd