Mitochondrial transplantation by intra-arterial injection for acute kidney injury

Abstract

Acute kidney injury is a common clinical disorder and one of the major causes of morbidity and mortality in the postoperative period. In this study, the safety and efficacy of autologous mitochondrial transplantation by intra-arterial injection for renal protection in a swine model of bilateral renal ischemia-reperfusion injury were investigated. Female Yorkshire pigs underwent percutaneous bilateral temporary occlusion of the renal arteries with balloon catheters. Following 60 min of ischemia, the balloon catheters were deflated and animals received either autologous mitochondria suspended in vehicle or vehicle alone, delivered as a single bolus to the renal arteries. The injected mitochondria were rapidly taken up by the kidney and were distributed throughout the tubular epithelium of the cortex and medulla. There were no safety-related issues detected with mitochondrial transplantation. Following 24 h of reperfusion, estimated glomerular filtration rate and urine output were significantly increased while serum creatinine and blood urea nitrogen were significantly decreased in swine that received mitochondria compared with those that received vehicle. Gross anatomy, histopathological analysis, acute tubular necrosis scoring, and transmission electron microscopy showed that the renal cortex of the vehicle-treated group had extensive coagulative necrosis of primarily proximal tubules, while the mitochondrial transplanted kidney showed only patchy mild acute tubular injury. Renal cortex IL-6 expression was significantly increased in vehicle-treated kidneys compared with the kidneys that received mitochondrial transplantation. These results demonstrate that mitochondrial transplantation by intra-arterial injection provides renal protection from ischemia-reperfusion injury, significantly enhancing renal function and reducing renal damage.

Keywords: acute kidney injury; ischemia-reperfusion injury; mitochondrial transplantation; renal.

Conflict of interest statement

P. J. del Nido and J. D. McCully have patents pending for the isolation and usage of mitochondria and are consultants with CellVita Sciences, Inc. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

Figures

Fig. 1.

Description of experimental model: intra-arterial injection of autologous mitochondria in the nonischemic kidney. Female Yorkshire pigs (40–60 kg) were sedated and intubated. The left femoral artery and right carotid artery were cannulated with a 6-F angiography sheath. Femoral or jugular venous lines and a Foley urinary catheter were also placed. Once the renal arteries were angiographically identified, animals were divided into different experimental groups. A small piece of muscle was surgically extracted from the sternocleidomastoid muscle using a 6-mm biopsy punch (~0.01 g) and used for mitochondrial isolation, which was performed 30 min before the injection of mitochondria. Single injections were delivered as a bolus antegrade into each renal artery (1 × 109 in 6 mL respiration buffer, n = 10; A). Serial injections of respiration buffer containing mitochondria (3 injections of 1 × 109 in 6 mL respiration buffer for each injection, n = 10) were delivered every 20 min (B). Following each injection, 5 mL of saline flush were injected as a flush. Before the injections, a 15-cm midline laparotomy incision was made, and bilateral renal arteries were identified and bluntly dissected. An ultrasonic transit time flow was placed circumferentially around each renal artery. Sixty minutes after the first injection of autologous mitochondria, the animal was subsequently euthanized. Blood and urine samples were collected every 20 min.

Fig. 2.

Description of experimental model: intra-arterial injection of autologous mitochondria or vehicle after renal ischemia-reperfusion injury. A: description of the experimental model. B: female Yorkshire pigs (40–60 kg) were sedated and intubated. The left or right femoral artery and the carotid artery were cannulated with a 6-F angiography sheath. Femoral or jugular venous lines and a Foley urinary catheter were also placed. Selective catheterization of the renal arteries was performed using a 6-F multipurpose guide catheter. Occlusion of the renal arteries was performed by a 6-F balloon catheter inflated in the proximal portion of the renal artery, totally occluding the blood flow to the kidneys for 60 min. C: confirmation of the occlusion was acquired by injection of iodinated contrast medium in the aorta and by checking for any opacification of the vessel of the kidneys. A small piece of muscle was surgically extracted from the sternocleidomastoid muscle using a 6-mm biopsy punch (~0.01 g) and used for mitochondrial isolation, which was performed 30 min before the injection of mitochondria. Following 60 min of occlusion, the balloons were deflated and carefully removed. D: angiography was performed to confirm renal artery patency and establishment of renal reperfusion. To evaluate efficacy of intra-arterial delivery of mitochondria after ischemia-reperfusion injury, a group of animals (n = 12) received either a single injection of vehicle (10 mL, n = 6) or vehicle solution containing mitochondria (1 × 109 in 10 mL, n = 6) immediately at reperfusion after a period of 60 min of bilateral warm ischemia. Following each injection, 5 mL of saline flush were injected as a flush. Animals were then allowed to reperfuse the kidneys under physiological conditions for the next 24 h and were subsequently euthanized. Blood and urine samples were collected right before and after bilateral renal ischemia at 2, 6, and 24 h after occlusion.

Fig. 3.

Renal function during intra-arterial injection of autologous mitochondria in the nonischemic kidney. A: right renal artery flow. B: left renal artery flow. C: mean arterial pressure (MAP). D: central vein pressure (CVP). E: urine output. F: glomerular filtration rate (GFR). All results are means ± SE for each group. Blue arrows show the time of injection of mitochondria in the serial injection group; the red arrow represents the time of injection of mitochondria in the single injection group. In A–D, data are shown for the time of injection and 1 min after each injection or the 20-min interval. Data were analyzed by two-way repeated-measures ANOVA with the Benjamini and Hochberg's false discovery rate (n = 10 in both groups). *P < 0.05, designated time point vs. baseline for serial injections; #P < 0.05, designated time point vs. baseline for single injection.

Fig. 4.

Biodistribution of autologous mitochondria by intra-arterial injection in the nonischemic kidney. 18F-rhodamine-6G-labeled mitochondria were administered through a single injection of 1 × 109 for each kidney. A whole body positron emission tomography and computerized tomography were then performed after euthanization of the animal 10 min following mitochondrial transplantation to obtain coronal (A), sagittal (B), and transverse (C) views.

Fig. 5.

Renal uptake of autologous mitochondria by intra-arterial injection in the nonischemic kidney. Representative immunofluorescent and immunohistochemical images demonstrating mitochondrial uptake in the kidneys are shown. A: injected kidney sections were fluorescently stained for nuclei using the DNA stain DAPI (blue) and for both human and swine mitochondria with Mito-Tracker Green FM (green) and for human mitochondria only with human mitochondrial MTCO2 antibody (red). Original magnification: ×40. B: representative immunohistochemistry photomicrographs of kidneys that received either vehicle or human mitochondria. Mitochondria were labeled with MTCO2 (red) antibody, which labels human mitochondria. Original magnification: ×20. White arrows indicate examples of transplanted human mitochondria. n = 1.

Fig. 6.

Renal function during intra-arterial injection of autologous mitochondria or vehicle after renal ischemia-reperfusion injury. A: plasma creatinine. B: estimated glomerular filtration rate (eGFR). C: blood urea nitrogen. D: urine output. All results are means ± SE for each group. Injection is denoted by the dotted vertical line. Data were analyzed by two-way repeated-measures ANOVA with the Benjamini and Hochberg's false discovery rate (n = 6 in both groups). *P < 0.05, mitochondria vs. vehicle.

Fig. 7.

Safety assessment of mitochondrial transplantation in renal ischemia-reperfusion injury. A: potassium. B: sodium. C: phosphorus. D: calcium. E: lactates. F: bicarbonates. G: pH. All results are means ± SE for each group. Injection is denoted by the dotted vertical line. Data were analyzed by two-way repeated measures ANOVA with the Benjamini and Hochberg's false discovery rate (n = 6 in both groups).

Fig. 8.

Representative light and electron microscopy images after intra-arterial injection of autologous mitochondria or vehicle and ischemia-reperfusion injury. A and B: the renal cortex of the vehicle-treated kidney showed extensive coagulative necrosis of primarily proximal tubules following 60 min of ischemia and 24 h of reperfusion, while the kidney that received mitochondrial transplantation showed only patchy mild acute tubular injury (Masson trichrome). Original magnification: ×20. C and D: these 1-μm plastic (araldite-epon)-embedded sections demonstrate in greater detail the marked difference between the confluent tubular necrosis seen in the vehicle-treated group (C) versus the more limited and likely sublethal proximal tubular injury in the mitochondria-treated group (D), consisting primarily of matrix with swelling and degenerative changes in organelles (toluidine blue). Original magnification: ×40. E and F: transmission electron microscopy of mitochondrial morphology from the same animals shown in C and D revealing dysmorphic mitochondria in the vehicle-treated group (E), with swelling, loss or distortion of cristae, and other alterations in the matrix; in contrast, the mitochondria-treated group (F) displayed mitochondria with normal morphology that were oriented along the basolateral infoldings of an intact proximal tubular cell. Original magnification: ×25,000. White arrows in E and F indicate the mitochondria.

Fig. 9.

Gross kidney anatomy and renal tissue injury at 24 h of reperfusion after intra-arterial injection of autologous mitochondria or vehicle and ischemia-reperfusion injury. A: gross anatomy of the left kidney of the vehicle-treated group, showing pale areas indicative of infarction and red hemorrhagic areas, while the left kidney of the mitochondria-treated group had normal gross morphology following 60 min of bilateral renal artery occlusion and 24 h of reperfusion. Acute tubular necrosis (ATN) score assessment of renal injury showed increased injury in the cortex of the vehicle-treated group compared with the mitochondria-treated group (B), while no difference was noted in the medulla (C). D: IL-6 expression in the renal cortex in the vehicle-treated group compared with the mitochondria-treated group. All results are means ± SE for each group. Data were analyzed by a Mann-Whitney U test (n = 12 in both groups; 2 kidneys/animal were analyzed). *P < 0.05, mitochondria vs. vehicle.