Intravital imaging of real-time endogenous actin dysregulation in proximal and distal tubules at the onset of severe ischemia-reperfusion injury

Abstract

Severe renal ischemia-reperfusion injury (IRI) can lead to acute and chronic kidney dysfunction. Cytoskeletal modifications are among the main effects of this condition. The majority of studies that have contributed to the current understanding of IRI have relied on histological analyses using exogenous probes after the fact. Here we report the successful real-time visualization of actin cytoskeletal alterations in live proximal and distal tubules that arise at the onset of severe IRI. To achieve this, we induced fluorescent actin expression in these segments in rats with hydrodynamic gene delivery (HGD). Using intravital two-photon microscopy we then tracked and quantified endogenous actin dysregulation that occurred by subjecting these animals to 60 min of bilateral renal ischemia. Rapid (by 1-h post-reperfusion) and significant (up to 50%) declines in actin content were observed. The decline in fluorescence within proximal tubules was significantly greater than that observed in distal tubules. Actin-based fluorescence was not recovered during the measurement period extending 24 h post-reperfusion. Such injury decimated the renal architecture, in particular, actin brush borders, and hampered the reabsorptive and filtrative capacities of these tubular compartments. Thus, for the first time, we show that the combination of HGD and intravital microscopy can serve as an experimental tool to better understand how IRI modifies the cytoskeleton in vivo and provide an extension to current histopathological techniques.

Organ Scaffold Characterizations Using Intravital Microscopy

“…To this end, a variety of macroscopic and microscopic approaches have been used to evaluate whole kidney scaffold structure and function [8][9][10][11][12][13][14][15] . In several animal models, such techniques have shown that the decellularization process successfully removes native cellular/tissue components and decellularizing agents while retaining the vascular and ECM structures.…”

“…Qualitative and quantitative assessments of tissue autofluorescence, velocity within the microcirculation, and microarchitecture porosity of scaffolds were acquired in live rats on day 0, day 1, day 2, and day 7 after transplantation and compared to the native (non-transplanted) organs. This sophisticated imaging technology's ability to evaluate renal dynamics in vivo is documented extensively [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . Our manuscript aims to highlight methods of monitoring cellular/subcellular processes in real-time within transplanted kidney scaffolds, which can be extended to other organ scaffold systems.…”

“…Static micrographs and time-series videos were obtained using the multi-photon microscope system. Imaging data was collected on day 0, day 1, day 2, and day 7 time points to examine cell nuclear patterns in native kidneys and scaffolds, changes in innate/decellularized tissue fluorescence levels, and microvascular integrity/function, using well-established techniques 13,15,[18][19][20][21][22][23][24]26,[29][30][31] . To conduct this analysis, we randomly selected 4 regions within a microscopic field and measured the blue and green pseudocolor fluorescence levels in nuclear and epithelial compartments, respectively.…”

Digital technologies: advancing individualized treatments through gene and cell therapies, pharmacogenetics, and disease detection and diagnostics

“…Gene therapies are emerging to restore or counter malfunctioning genes in conditions adversely influencing a patient's quality of life without pharmacological intervention, radiotherapy, or surgery (Goswami et al, 2019). This form of therapy has made significant progress since this concept arose in the 1960s and 1970s (Friedmann, 1992;Niidome and Huang, 2002;Collins and Thrasher, 2015), but tragic failures in clinical settings (Uddin et al, 2020) along with pervasive obstacles related to nucleic acid delivery (Corridon et al, 2013;Collett et al, 2017;Shi et al, 2017;Kolb et al, 2018;van Haasteren et al, 2020;Corridon et al, 2021), have limited its progression. However, a recent and significant achievement in this field has come from the CRISPR-Cas system.…”

Analysis of vascular architecture and parenchymal damage generated by reduced blood perfusion in decellularized porcine kidneys using a gray level co-occurrence matrix

“…The incidence of kidney failure, otherwise known as end-stage renal disease (ESRD) is rising globally (Arikan et al, 2021;Kari et al, 2021). Unfortunately, there is no cure for this condition, which can develop form the progression of acute and chronic injuries (Hsu and Hsu, 2016;Kolb et al, 2018;Corridon et al, 2021). Currently, transplantation is the best option to treat ESRD.…”