Abstract: TH-OR46
Downregulation of Syndecan-1 and Alternative Complement in Renal Proximal Tubular Epithelial Cells by Crotamine/Sirna Complexes
Session Information
- Technology-Based Approaches in Nephrology
November 03, 2022 | Location: W308, Orange County Convention Center‚ West Building
Abstract Time: 05:42 PM - 05:51 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- van den Born, Jacob, Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Campeiro, Joana D., Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Dam, Wendy, Universitair Medisch Centrum Groningen, Groningen, Groningen, Netherlands
- Hayashii, Miriian A F, Universidade Federal de Sao Paulo, Sao Paulo, São Paulo, Brazil
Background
In proteinuria, syndecan-1, an epithelial heparan sulfate proteoglycan, serves as a docking platform for filtrated urinary properdin in the apical membranes of proximal tubular epithelial cells (PTEC) activating the complement system via alternative pathway. Targeting PTEC aiming to reduce syndecan-1 expression might be useful to slow down the alternative complement activation during proteinuria. Crotamine is a non-viral cell-penetrating peptide which after ip injection accumulates in PTEC via apical endocytosis. We now tested crotamine-siRNA complexes for in vitro and in vivo targeting of PTEC.
Methods
The complexes formed by crotamine and syndecan-1 siRNA were characterized by biophysical methods. After the in vitro transfection of HK2 cells with crotamine-siRNA complexes, the efficiency to downregulate the syndecan-1 expression, properdin binding, and subsequently, complement deposition was assessed by FACS and qRT-PCR. The targeted internalization into PTEC in vivo was evaluated by confocal microscopy of kidney sections from mice injected with fluorescently-labeled crotamine-siRNA complexes.
Results
We demonstrated that the efficient complex formation is time- and crotamine-siRNA ratio-dependent and that crotamine is able to protect siRNA against degradation by endonucleases. After 48 h, the transfection with the complex reduced ~50% of syndecan-1 expression at both mRNA and protein levels (both p<0.01) in vitro. Subsequently, properdin binding was also comparably reduced (p<0.001) and the alternative pathway activation declined ~60% (p<0.001). Moreover, ip injection of the fluorescently-labeled crotamine-siRNA complexes in mice showed siRNA presence in the cell membranes of proximal tubular cells, followed by internalization into these tubular cells.
Conclusion
We show for the first time the use of crotamine as a non-viral nanocarrier for PTEC-specific delivery of siRNA both in vitro and in vivo. Successful reduction of the expression of syndecan-1 was accompanied by down modulation of alternative complement activation by PTECs in vitro. We suggest crotamine as a prototypic next generation kidney-specific non-viral vector to modulate aberrant gene expression in kidney PTECs, for instance, in proteinuric renal diseases.
Funding
- Government Support – Non-U.S.