Vasopressin V2R-targeting peptide carrier mediates siRNA delivery into collecting duct cells

Abstract

Internalization of receptor proteins after interacting with specific ligands has been proposed to facilitate siRNA delivery into the target cells via receptor-mediated siRNA transduction. In this study, we demonstrated a novel method of vasopressin V2 receptor (V2R)-mediated siRNA delivery against AQP2 in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. We synthesized the dDAVP conjugated with nine D-arginines (dDAVP-9r) as a peptide carrier for siRNA delivery. The structure of synthetic peptide carrier showed two regions (i.e., ligand domain to V2R (dDAVP) and siRNA carrying domain (nine D-arginine)) bisected with a spacer of four glycines. The results revealed that 1) synthesized dDAVP-9r peptides formed a stable polyplex with siRNA; 2) siRNA/dDAVP-9r polyplex could bind to the V2R of IMCD cells and induced AQP2 phosphorylation (Ser 256); 3) siRNA/dDAVP-9r polyplex was stable in response to the wide range of different osmolalities, pH levels, or to the RNases; 4) fluorescein-labeled siRNA was delivered into V2R-expressing MDCK and LLC-PK1 cells by siRNA/dDAVP-9r polyplex, but not into the V2R-negative Cos-7 cells; and 5) AQP2-siRNA/dDAVP-9r polyplex effectively delivered siRNA into the IMCD cells, resulting in the significant decrease of protein abundance of AQP2, but not AQP4. Therefore, for the first time to our knowledge, we demonstrated that V2R-mediated siRNA delivery could be exploited to deliver specific siRNA to regulate abnormal expression of target proteins in V2R-expressing kidney cells. The methods could be potentially used in vivo to regulate abnormal expression of proteins associated with disease conditions in the V2R-expressing kidney cells.

Figure 1. Structure of dDAVP-9r peptide and formation of siRNA/dDAVP-9r polyplex.

A) PEP-FOLD simulations give similar structure folding of dDAVP region and vasopressin peptide. The lowest energy structure of dDAVP-4 Gly-9 Arg peptide (dDAVP, light blue; 4 Gly, gray blue; and 9 Arg, blue) was aligned with vasopressin peptide (CYFQNC, 1jk4-b, yellow). Disulfide bond of dDAVP was highlighted by red and mercaptopropionyl N-terminal modification is indicated by orange circle. B) Formation of siRNA/dDAVP-9r polyplex was examined at the various mole ratios by electrophoretic mobility shift assay.

Figure 2. Internalization of siRNA/dDAVP-9r polyplex in V2R-expressing cells and V2R-negative cells.

A) Cellular uptake of fluorescein-labeled siRNA in V2R-expressing MDCK cells and LLC-PK1 cells, and V2R-negative Cos-7 cells was examined by flow cytometry. Black line: FITC-siRNA alone; green line: FITC-siRNA/9r polyplex (1∶10 mole ratio, siRNA:peptide); red line: FITC-siRNA/dDAVP-9r polyplex (1∶10 mole ratio, siRNA:peptide); and blue line: FITC-siRNA/dDAVP-9r (1∶40 mole ratio, siRNA:peptide). B) Laser scanning confocal microscopic examination of the MDCK cells treated by FITC-labeled siRNA only or FITC-labeled siRNA complexed with 9r peptides (1∶10 mole ratio) or with dDAVP-9r peptides (1∶10 or 1∶40 mole ratio). Nuclei of the MDCK cells were stained with DAPI. All fluorescence imaging studies were performed at least three times and images demonstrated were representative of the majority of analyzed cells.

Figure 3. Stability of siRNA/dDAVP-9r polyplex against RNase A, osmolality, and pH.

A) siRNA (100 pmol) or siRNA/dDAVP-9r polyplex at the various mole ratios were incubated in the absence or the presence of RNase A for 1 h or 6 h. B) siRNA/dDAVP-9r polyplex (1∶10 or 1∶40 mole ratio) were incubated under different osmolalities generated by NaCl and urea or different pH levels for 60 min after formation of the polyplex. The polyplex was incubated in the absence or the presence of 0.2% SDS, and siRNAs were electrophoresed on 2% agarose gel containing EtBr.

Figure 4. Phosphorylation of AQP2 at S256 in response to dDAVP, dDAVP-9r, and siRNA/dDAVP-9r polyplex treatment in IMCD cells of rat kidney.

A) IMCD suspension was treated by dDAVP (10⁻⁸ M), dDAVP-9r peptide (10⁻⁸ M), or siRNA/dDAVP-9r polyplex (1∶10) for 3 min. B) Changes of the phosphorylated AQP2 (S256) expression relative to total AQP2 were examined by semiquantitative immunoblotting (n = 4 in each group). n, number of samples which were prepared from each well.

Figure 5. Semiquantitative immunoblotting of AQP2 and AQP4 expression in primary cultured IMCD cells of rat kidney treated by AQP2-siRNA/dDAVP-9r polyplex.

A) The siRNA/dDAVP-9r polyplex (1∶10) containing 200 pmol of AQP2-siRNA was incubated in the IMCD tubule suspension for 2 h. After incubation, the cells were cultured at 12-well plate for 72 h, and then AQP protein expression was analyzed by semiquantitative immunoblotting. B) Protein abundance of AQP2 and AQP4 in IMCD cells treated with the polyplex containing either non-target-siRNA or AQP2-siRNA was evaluated by semiquantitative immunoblotting (n = 8 in each group). *P<0.05 compared with the treatment with non-target-siRNA; n, number of samples which were prepared from each well.