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<p>The ease with which microRNA inhibitors (antimiRs) can be delivered varies with the intended target cell type and tissue. AntimiRs are of interest as potential therapeutics for kidney conditions, including ischaemia–reperfusion injury in transplantation. During ex-situ human kidney perfusion, antimiRs are delivered to the proximal tubule epithelium without the use of transfection reagents by an endocytic process. Here we investigate whether antimiR uptake by proximal tubule epithelial cells (PTEC) occurs <i>in vitro</i>, without transfection reagents, and which endocytic entry mechanism is responsible. PTEC were isolated from human kidneys declined for transplantation and maintained in culture. PTEC were shown to take up antimiR through detection of increased fluorescent signal associated with the antimiR label. A vesicular pattern of uptake was demonstrated on fluorescence microscopy, in keeping with endocytic uptake. Endocytosis was confirmed by co-occurrence of the antimiR with endocytic markers and temperature dependence of uptake. Megalin inhibition with receptor associated protein to target receptor-mediated endocytosis had no effect on antimiR uptake, whereas the macropinocytosis inhibitor 5-(<i>N</i>-ethyl-<i>N</i>-isopropyl)-amiloride reduced antimiR uptake. Our results demonstrate antimiR can be delivered to primary human PTEC <i>in vitro</i> without the use of transfection reagents and support macropinocytosis as the dominant pathway of antimiR entry.</p>

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AntimiR uptake by human proximal tubule epithelial cells is predominantly by macropinocytosis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月29日

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  • Emily K. Glover,
  • Rolando Berlinguer-Palmini,
  • Emily R. Thompson,
  • Colin Wilson,
  • Laura Denby,
  • Gary Reynolds,
  • Simi Ali,
  • Martin Lowe,
  • Rachel Lennon,
  • Neil S. Sheerin

摘要

The ease with which microRNA inhibitors (antimiRs) can be delivered varies with the intended target cell type and tissue. AntimiRs are of interest as potential therapeutics for kidney conditions, including ischaemia–reperfusion injury in transplantation. During ex-situ human kidney perfusion, antimiRs are delivered to the proximal tubule epithelium without the use of transfection reagents by an endocytic process. Here we investigate whether antimiR uptake by proximal tubule epithelial cells (PTEC) occurs in vitro, without transfection reagents, and which endocytic entry mechanism is responsible. PTEC were isolated from human kidneys declined for transplantation and maintained in culture. PTEC were shown to take up antimiR through detection of increased fluorescent signal associated with the antimiR label. A vesicular pattern of uptake was demonstrated on fluorescence microscopy, in keeping with endocytic uptake. Endocytosis was confirmed by co-occurrence of the antimiR with endocytic markers and temperature dependence of uptake. Megalin inhibition with receptor associated protein to target receptor-mediated endocytosis had no effect on antimiR uptake, whereas the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)-amiloride reduced antimiR uptake. Our results demonstrate antimiR can be delivered to primary human PTEC in vitro without the use of transfection reagents and support macropinocytosis as the dominant pathway of antimiR entry.