Kim-1 Targeted Extracellular Vesicles: A New Therapeutic Platform for RNAi to Treat AKI

Abstract

Background: AKI is a significant public health problem with high morbidity and mortality. Unfortunately, no definitive treatment is available for AKI. RNA interference (RNAi) provides a new and potent method for gene therapy to tackle this issue.

Methods: We engineered red blood cell-derived extracellular vesicles (REVs) with targeting peptides and therapeutic siRNAs to treat experimental AKI in a mouse model after renal ischemia/reperfusion (I/R) injury and unilateral ureteral obstruction (UUO). Phage display identified peptides that bind to the kidney injury molecule-1 (Kim-1). RNA-sequencing (RNA-seq) characterized the transcriptome of ischemic kidney to explore potential therapeutic targets.

Results: REVs targeted with Kim-1-binding LTH peptide (REV_LTH) efficiently homed to and accumulated at the injured tubules in kidney after I/R injury. We identified transcription factors P65 and Snai1 that drive inflammation and fibrosis as potential therapeutic targets. Taking advantage of the established REV_LTH, siRNAs targeting P65 and Snai1 were efficiently delivered to ischemic kidney and consequently blocked the expression of P-p65 and Snai1 in tubules. Moreover, dual suppression of P65 and Snai1 significantly improved I/R- and UUO-induced kidney injury by alleviating tubulointerstitial inflammation and fibrosis, and potently abrogated the transition to CKD.

Conclusions: A red blood cell-derived extracellular vesicle platform targeted Kim-1 in acutely injured mouse kidney and delivered siRNAs for transcription factors P65 and Snai1, alleviating inflammation and fibrosis in the tubules.

Keywords: P65; RNAi; Snai1; acute kidney injury; extracellular vesicles; kidney injury molecule-1.

Graphical abstract

Figure 1.

Identification of Kim-1–targeting peptides by phage display. (A) Schematic illustration of the preparation of REV-based kidney-targeted RNAi therapy against AKI. (B) Table of the five candidate Kim-1–binding peptides identified by in vitro phage display. (C) His-tagged peptides were coated on nickel beads, then incubated with kidney lysates from I/R mice. The captured Kim-1 protein was analyzed with Western blot to examine the binding affinity of candidate peptides to Kim-1. n=3 independent experiments. (D, E) For in vivo biodistribution, mice subjected to unilateral renal I/R were administered intravenously with FITC-labeled peptides. n=3 mice per group. (D) Ex vivo imaging of kidneys (left: sham, right: I/R) and quantitative analysis of the I/R/sham ratio at 6 hours after injection. (E) Correlation analysis between the average radiance of FITC-labeled peptides and Kim-1 protein in I/R kidney. (F) Different concentrations of FITC-labeled peptides were incubated with Kim-1–coated plates, and the bound peptides were imaged with IVIS spectrum. n=3 independent experiments. (G) Representative confocal images showing the colocalization of FITC-labeled LTH or Scrbl with Kim-1 in sections from I/R kidney. Scale bar, 10 μm. Quantification on the basis of 24 sections from three mice. (H) Representative confocal images showing the binding of FITC-labeled LTH or Scrbl to Kim-1-overexpressed TECs. Scale bar, 20 μm. Quantification on the basis of 30 fields from three independent experiments. The specificity of LTH in recognizing Kim-1 is determined by the ratio of LTH⁺Kim-1⁺/LTH⁺ tubules or cells. Data are presented as mean±SD, *P<0.05, **P<0.01 versus LTH, one-way ANOVA.

Figure 2.

LTH efficiently directs REVs to the injured kidney. (A) Schematic diagram of conjugating LTH to REV surface by a two-step reaction. (B) Size distribution of REV and REV_LTH. Representative TEM image of REV_LTH. (C) Western blot analysis of EV markers (Alix, CD63, CD81) and RBC marker (Hemoglobin A) in REV and REV_LTH. (D–H) For imaging analysis, the scrambled and LTH peptide were labeled with FITC. (D) Representative confocal image of REV_LTH. Yellow shows the colocalization of FITC-labeled LTH with PKH26-labeled REV. Scale bar, 1 μm. Quantification on the basis of 30 fields from three independent experiments. (E) Ex vivo imaging of kidneys from unilateral I/R mice injected intravenously with REV_Scrbl and REV_LTH. n=4 mice (REV_Scrbl); n=6 mice (REV_LTH). (F) Representative confocal images showing the colocalization of REV_LTH with Kim-1–positive tubules in sections from I/R kidney. Scale bar, 30 μm. Quantification on the basis of 30 sections from three mice per group. (G) Live-cell imaging of the cellular uptake of REV_LTH in HEK293 cells expressing Kim-1-RFP at indicated times. Scale bar, 5 μm. See Supplemental Video 1. Zoomed image indicates the binding of REV_LTH to Kim-1. (H) Representative confocal images of REV_Scrbl and REV_LTH in Kim-1⁺ HEK293 cells after 30 minutes of incubation. Scale bar, 20 μm. Quantification on the basis of 150 cells from three independent experiments. Data are presented as mean±SD *P<0.05, ***P<0.001 versus REV_Scrbl, two-tailed t test.

Figure 3.

P65 and Snai1 are potential therapeutic targets involved in I/R pathogenesis. (A) Heat map of the upregulated transcription factors in I/R kidney compared with sham kidney. n=3 mice (sham); n=4 mice (I/R). (B) RT-qPCR analysis of P65 and Snai1 mRNA levels in kidney tissues. n=8 mice (sham); n=18 mice (I/R). (C) Immunohistochemical analysis of P-p65 and Snai1 expression in kidney sections. Scale bar, 50 μm. Zoom panels show the nuclear localization of P-p65 and Snai1 in tubules of I/R kidney. (D) Representative confocal images of P-p65/Kim-1 and Snai1/Kim-1-immunostained I/R kidney. Scale bar, 20 μm. Data are presented as mean±SD **P<0.01, ***P<0.001 versus sham, two-tailed t test.

Figure 4.

Targeting of P65 and Snai1 in tubules using siRNA-loaded REV_LTH. (A) Size distribution and representative TEM images of REV_LTH-siP65 and REV_LTH-siSnai1. (B) The loading efficiency of Cy3-labeled siRNA with or without cholesterol modification or cholesterol-modified siRNA without REV_LTH. n=3 independent experiments. ***P<0.001 versus REV_LTH+chol-siRNA. (C) Representative confocal image of REV_LTH-siP65. White shows the colocalization of FITC-labeled LTH and Cy3-labeled siP65 with DID-labeled REVs. Scale bar, 1 μm. (D) Gel retention assay of siP65 in REV_LTH-siP65 treated with RNase or RNase combined with Triton. n=3 independent experiments. ***P<0.001 versus untreated control. (E) Ex vivo imaging of I/R kidney from unilateral I/R mice administered intravenously with REV_Scrbl-siP65 and REV_LTH-siP65. n=3 mice (REV_Scrbl-siP65); n=6 mice (REV_LTH-siP65). **P<0.01 versus REV_Scrbl-siP65. (F, G) Mice were subjected to 35 minutes of bilateral renal I/R injury and received treatment every 24 hours upon reperfusion, five times in total. All of the mice were sacrificed at day 5 post-I/R injury. (F) Western blot analysis of P-p65 and Snai1 protein levels in tubules. n=4 mice per group. *P<0.05, **P<0.01, ***P<0.001 versus REV_LTH-siRNA. (G) Immunohistochemical analysis of P-p65 and Snai1 expression in I/R kidney treated with REV_LTH-siScrbl or REV_LTH-siRNA. Scale bar, 50 μm. Quantification on the basis of four mice, with at least five sections counted in each. ***P<0.001 versus REV_LTH siScrbl. Data are presented as mean±SD one-way ANOVA (B, D, F), two-tailed t test (E, G).

Figure 5.

REV_LTH-siP65/siSnai1 combination therapy alleviates I/R-induced ischemic AKI. (A) Schematic diagram of the experimental design. In brief, mice were subjected to bilateral renal I/R and received REV_LTH-siP65/siSnai1 or REV_LTH-siScrbl every 24 hours upon reperfusion, five times in total. (B) Serum creatinine and (C) blood urea nitrogen at day 0, 1, and 5 post-I/R. n=6 mice (sham, I/R, siScrbl); n=8 mice (siP65/siSnai1). (D) Quantification of kidney injury on the basis of PAS staining. n=6 mice (sham, I/R, siScrbl); n=8 mice (siP65/siSnai1). (E) Representative images of PAS staining. Scale bar, 100 μm. (F) Immunostaining of macrophages (CD68) and T cells (CD3) in the tubulointerstitial region. Scale bar, 20 μm. Quantification on the basis of six mice with at least five sections counted in each. (G) RT-qPCR analysis of proinflammatory cytokine mRNA levels in kidney tissues. n=6 mice per group. (H) RT-qPCR analysis of fibrotic factor mRNA levels in kidney tissues. n=6 mice per group. (I, J) Mice were subjected to 40 minutes of unilateral renal I/R, and REV_LTH-siP65/siSnai1 or REV_LTH-siScrbl was administered upon reperfusion and continued every 24 hours for 5 consecutive days. Mice were euthanized at day 28 after reperfusion. n=6 mice per group. (I) Representative images of PAS staining of I/R kidneys treated with siP65/siSnai1 or siScrbl. Scale bar, 100 μm. (J) Representative images of Masson trichrome staining and quantification of fibrotic area. Scale bar, 100 μm. Data are presented as mean±SD *P<0.05, **P<0.01, ***P<0.001 versus I/R or siScrbl, one-way ANOVA (B–D), two-tailed t test (F–J).

Figure 6.

REV_LTH-siP65/siSnai1 combination therapy attenuates UUO-induced obstructive AKI. (A) Schematic diagram of the experimental design. In brief, mice were treated daily starting at day 1 after UUO surgery with REV_LTH-siP65/siSnai1 or REV_LTH-siScrbl, and were sacrificed on day 14 after surgery. (B, D) Representative images of PAS staining and quantification of kidney injury. Scale bar, 50 μm. n=6 mice (sham, UUO, siScrbl); n=8 mice (siP65/siSnai1). (C, E) Representative images of Masson trichrome staining and quantification of fibrotic area. Scale bar, 100 μm. n=6 mice (Sham, UUO, siScrbl); n=8 mice (siP65/siSnai1). (F) Immunostaining of macrophages (CD68) and T cells (CD3) in the tubulointerstitial region. Scale bar, 20 μm. Quantification on the basis of six mice with at least five sections counted in each. (G) RT-qPCR analysis of proinflammatory cytokine mRNA levels in kidney tissues. n=6 mice per group; (H) RT-qPCR analysis of fibrotic factor mRNA levels in kidney tissues. n=6 mice per group. Data are presented as mean±SD *P<0.05, ***P<0.001 versus UUO or siScrbl, one-way ANOVA (B, C), two-tailed t test (F–H).