Irisin-Encapsulated Mitochondria-Targeted Biomimetic Nanotherapeutics for Alleviating Acute Kidney Injury

Abstract

Acute kidney injury (AKI) is the sudden decrease in renal function that can be attributed to dysregulated reactive oxygen species (ROS) production and impaired mitochondrial function. Irisin, a type I membrane protein secreted by skeletal muscles in response to physical activity, has been reported to alleviate kidney damage through regulation of mitochondrial biogenesis and oxidative metabolism. In this study, a macrophage membrane-coated metal-organic framework (MCM@MOF) is developed as a nanocarrier for encapsulating irisin to overcome the inherent characteristics of irisin, including a short circulation time, limited kidney-targeting ability, and low membrane permeability. The engineered irisin-mediated biomimetic nanotherapeutics have extended circulation time and enhanced targeting capability toward injured kidneys due to the preservation of macrophage membrane proteins. The irisin-encapsulated biomimetic nanotherapeutics effectively mitigate acute ischemia-reperfusion injury by protecting mitochondrial function and modulating SOD2 levels in renal tubular epithelial cells. The present study provides novel insights to advance the development of irisin as a potential therapeutic approach for AKI.

Keywords: acute kidney injury; biomimetic nanocarriers; irisin; mitochondria.

Scheme 1

Schematic illustration of MCM@MOF@irisin biomimetic nanotherapeutics designed for targeting mitochondria in the kidney to ameliorate the acute renal injury induced by ischemia‐reperfusion.

Figure 1

Preparation and characterization of MCM@MOF@irisin biomimetic nanotherapeutics. a) Schema of the preparation of MCM@MOF@irisin biomimetic nanotherapeutics. b) The SEM (b1) and TEM (b2) images of MOF. Scale bar = 100 nm. c) The TEM image of MCM@MOF nanocarrier. Scale bar = 50 nm. d) Zeta potentials of MOF, MCM, MCM@MOF, and MCM@MOF@irisin measured by dynamic light scattering (DLS) (n = 3 per group). e) Confocal laser scanning microscopy (CLSM) images show the colocalization of DiD‐labeled MOF nanoparticles and NBD‐labeled macrophage membrane after MCM@MOF biomimetic nanocarrier internalization in RAW264.7 cells. The colors are nucleus (blue), MCM‐NBD “shell” (green), and MOF‐DiD “core” (red). Scale bar = 10 µm. f) Heat map of proteins related to inflammation and chemotaxis detected in RAW‐M, MCM, and MCM‐MOF (n = 2 per group). Group 1 represents proteins related to inflammation. Group 2 represents proteins related to chemotaxis. Group 3 represents proteins related to both inflammation and chemotaxis. g) Western blots of CCR2 and cluster of differentiation (CD) proteins (CD18, CD40, and CD47) in RAW‐M, MCM, MCM@MOF, and MOF (n = 3 per group). (h) The release characteristics of irisin from MCM@MOF@irisin in buffers with different pH values (n = 3 per group). Data are presented as mean ± SD.

Figure 2

Immune‐evasive and kidney‐targeting ability of MCM@MOF@irisin nanotherapeutics. a) Confocal fluorescence images of RAW264.7 macrophages incubated with DiD‐labeled MOF or DiD‐labeled MCM@MOF for 8 h (n = 6 per group). Scale bar = 50 µm. b) Pharmacokinetics of MOF and MCM@MOF over a span of 24 h (n = 3 per group). The relative zinc content of MOF and MCM@MOF nanocarrier remaining in the blood at different time points after intravenously injection into mice via ICP‐AES measurement. c) Confocal fluorescence images of HUVECs incubated at 4 °C for 2 h in PBS (Control) or MCM@MOF nanocarrier after pretreatment with or without TNF‐α (50 ng mL ⁻¹) for 6 h, MCM@MOF (red), HUVECs nucleus (blue), and ICAM‐1 (green) (n = 3 per group). Scale bar = 20 µm. d) Overview of the animal experimental design for the in vivo imaging in the unilateral renal ischemia/reperfusion injury model. e) Fluorescence images and their quantification by IVIS imaging system in kidneys from mice treated with Cy5.5 labeled‐irisin or Cy5.5 labeled‐MCM@MOF@irisin at 2 h and 24 h after their injection (n = 3 per group). Color scale, Min = 5.0 × 10⁷, Max = 1.2 × 10⁸. f) Biodistribution and quantification of irisin‐Cy5.5 and MCM@MOF@irisin‐Cy5.5 that accumulated in major organs, including the brain, heart, liver, spleen, and lung, 2 h and 24 h after their intravenous administration (n = 3 per group). Color scale, Min = 5.0 × 10⁷, Max = 3.0 × 10⁸. All images acquired with the same detection conditions, exposure time (t = 0.2 s), and excitation light power. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, ns, not significant.

Figure 3

MCM@MOF@irisin nanotherapeutics against renal I/R injury in mice. a) Representative pictures of kidneys from mice with saline, irisin, MCM@MOF, and MCM@MOF@irisin treatments after renal I/R injury; sham‐operated mice treated with saline served as negative control. b,c) The levels of Scr and BUN in mice with the different treatments after 24 h (n = 6 per group). (d1) PAS staining of kidney slices of renal I/R‐injured mice with different treatments; sham‐operated mice treated with saline served as negative control (n = 6 per group). Scale bar = 100 µm. (d2) Quantification of tubular injury based on PAS staining. (e1) Renal tubular apoptosis in renal I/R‐injured mice with different treatments evaluated by TUNEL (n = 6 per group). Scale bar = 50 µm. (e2) The quantification of tubular apoptosis based on TUNEL staining. (f1) The expression of KIM‐1 in mice receiving the different treatments was assessed by immunofluorescence staining (n = 6 per group). Scale bar = 20 µm. (f2) Quantification of KIM‐1 fluorescence intensity. g) Western blot showed protein levels of cytochrome c and cleaved‐caspase 3 after different treatments in I/R injured kidneys. β‐actin was used as an internal control. h) The relative renal mRNA levels of inflammatory cytokine genes were measured by real‐time quantitative PCR (n = 6 per group). i) The levels of inflammatory factors TNF‐α, IL‐6, IL‐1β and CCL‐2 in I/R injury kidneys under different treatments. The data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4

MCM@MOF@irisin protects renal tubular epithelial cells subjected to hypoxia‐reoxygenation (H/R) by ameliorating mitochondrial function. a) The mitochondrial targeting capability of irisin and MCM@MOF@irisin in HK‐2 cells under normoxia and H/R conditions observed by CLSM (n = 6 per group). The colors are nuclei (blue), MitoTracker (green), and irisin‐Cy5.5 (red). HK‐2 cells were incubated with irisin for 6 h after H/R treatment for 24 h. Scale bar = 10 µm. b) Confocal fluorescence images and quantitative analysis of mitochondrial membrane potential in HK‐2 cells stained with JC‐1 after the different treatments (n = 6 per group). Scale bar = 50 µm. c) Intracellular ROS levels were measured with DCFH‐DA fluorescence probe under different treatments. Scale bar = 50 µm. The data are presented as mean ± SD. * P < 0.05, **** P < 0.0001. ns, not significant.

Figure 5

MCM@MOF@irisin protected mitochondria from damage in renal I/R injured mice. a) Bio‐TEM images of mitochondrial injury and percentage of damaged mitochondria in mice kidneys (n = 3 per group). Scale bar = 1 µm. b) Fluorescence images and quantification of oxidative stress, using DHE staining, in kidney slices of I/R injured mice with different treatments (n = 6 per group). Scale bar = 20 µm. c,d) Mitochondrial respiratory chain complex I and II enzymatic activity in I/R injury kidneys (n = 6). The data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Figure 6

MCM@MOF@irisin mitigated fibrogenesis and the progression of CKD in kidneys of mice after unilateral ischemia/reperfusion injury. a) Treatment protocol of the renal I/R injury mice model. b) PAS staining of kidney slices of renal I/R‐injured mice with different treatments; sham‐operated mice treated with saline served as negative control (n = 6 per group). Scale bar = 100 µm. (c1) Representative images of Sirius red staining, in kidney slices of renal I/R‐injured mice with different treatments (n = 6 per group). Scale bar = 100 µm. (c2) Quantification of fibrotic area based on Sirius red staining. d,e) Expression of the fibrotic marker α‐SMA, COL‐1 in kidney slices of renal I/R‐injured mice (n = 6 per group). Scale bar = 20 µm. The data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 7

The biosafety evaluation of MCM@MOF nanocarrier. a–d) ALT, AST, BUN, and Scr levels in blood of mice after the intravenous injection of MOF or MCM@MOF (n = 3 per group). e) H&E‐stained histological sections from major organs, 21 days after the intravenous administration of saline, MOF, or MCM@MOF into healthy mice (n = 3 per group). Scale bar = 100 µm. The data presented as mean ± SD. ns, not significant.

Figure 8

The protection of MCM@MOF@irisin on mitochondria via modulation of SOD2. a) SOD activities of H/R injured HK‐2 cells after incubation in PBS, irisin, MCM@MOF, or MCM@MOF@irisin; HK‐2 cells incubated with PBS under normoxia served as control (n = 6 per group). b) SOD activities of kidneys after treatment with saline, irisin, MCM@MOF, or MCM@MOF@irisin (n = 6 per group). c) Western blot analysis of SOD2 expression in the kidneys of AKI mice following treatment with saline, irisin, MCM@MOF, or MCM@MOF@irisin (n = 6 per group). β‐actin served as a loading control.