A Novel Approach to Deliver Therapeutic Extracellular Vesicles Directly into the Mouse Kidney via Its Arterial Blood Supply

Abstract

Diseases of the kidney contribute a significant morbidity and mortality burden on society. Localized delivery of therapeutics directly into the kidney, via its arterial blood supply, has the potential to enhance their therapeutic efficacy while limiting side effects associated with conventional systemic delivery. Targeted delivery in humans is feasible given that we can access the renal arterial blood supply using minimally invasive endovascular techniques and imaging guidance. However, there is currently no described way to reproduce or mimic this approach in a small animal model. Here, we develop in mice a reproducible microsurgical technique for the delivery of therapeutics directly into each kidney, via its arterial blood supply. Using our technique, intra-arterially (IA) injected tattoo dye homogenously stained both kidneys, without staining any other organ. Survival studies showed no resulting mortality or iatrogenic kidney injury. We demonstrate the therapeutic potential of our technique in a mouse model of cisplatin-induced acute kidney injury (AKI). IA injection of mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) successfully reversed AKI, with reduced physiological and molecular markers of kidney injury, attenuated inflammation, and restoration of proliferation and regeneration markers. This reproducible delivery technique will allow for further pre-clinical translational studies investigating other therapies for the treatment of renal pathologies.

Keywords: acute kidney injury; extracellular vesicles; intra-arterial delivery; locoregional delivery; mesenchymal stromal cells; microsurgery; targeted therapy.

Figure 1

Selective ligation allows for selective kidney labeling. (A) Normal anatomy of the mouse abdominal aorta, showing origin of the celiac trunk (CT), superior mesenteric artery (SMA), renal arteries, and kidneys. (B) Suture ligation sites, including the proximal aorta between the origins of the CT and SMA, the SMA, and the distal aorta. A metal clip is placed temporarily on the left renal artery to allow delivery of therapeutics first to the right renal artery. (C) Selective ligation and clip placement shown inside the mouse abdomen. (D) Result of tattoo dye injection into the distal aorta without described ligation technique. (E,F) Result of tattoo dye injection with our ligation and clipping technique.

Figure 2

Physiological and molecular markers of kidney injury. (A) Study protocol. (B) Gross appearance of kidney. (C) Percent survival, animal body weight, and kidney weight. (D) Serum concentration of blood urea nitrogen (BUN), creatinine, and NGAL, as measured by serum ELISA. (E) mRNA expression of KIM-1, TIMP-1, and NGAL in kidney lysates, as measured by qRT-PCR. (F) Urine concentrations of KIM-1, TIMP-1, and NGAL, as measured by urine ELISA. Measurements were taken at day 12. Each group has n = 8 mice, except for survival data for which n = 10 for untreated controls and n = 20 mice for other groups. Significant difference ^a p < 0.05: relative to untreated control group; ^b p < 0.05: relative to AKI + IA saline group.

Figure 3

Inflammatory cytokines. (A) Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) of kidney tissue, stained for TNF-α and NF-κB, and quantification of tubular casts. (B) Serum concentrations of TNF-α and IL-6, as measured by serum ELISA. (C) Western blot and quantification of NF-κB and β-actin from kidney lysate. (D) mRNA expression of NF-κB, as measured by qRT-PCR. Measurements were taken at day 12. Each group has n = 5 mice for TNF-α and IL-6 measurements and n = 3 mice for NF-κB measurements. Significant difference ^a p < 0.05: relative to untreated control group; ^b p < 0.05: relative to AKI + IA saline group.

Figure 4

Proliferation and regeneration markers. (A) Immunohistochemistry (IHC) of FGF2 and Ki67 in kidney tissue, and quantification of Ki67+ cells. (B) Western blot and quantification of Ki67, FGF2, FGF23, and β-actin from kidney lysate. (C) Western blot and quantification of pAMPK, pERK, and β-actin from kidney lysate. Measurements were taken at day 12. Each group has n = 3 pooled mice. Significant difference ^a p < 0.05: relative to untreated control group; ^b p < 0.05: relative to AKI + IA saline group.