A novel method for successful induction of interdigitating process formation in conditionally immortalized podocytes from mice, rats, and humans

Doi, Kotaro; Kimurâ, Hiroshi; Wada, Takehiko; Tanaka, Tetsuhiro; Hiromura, Keiju; Saleem, Moin A.; Inagi, Reiko; Nangaku, Masaomi; Fujii, Teruo

doi:10.1016/j.bbrc.2021.07.029

Cited by 2 publications

(2 citation statements)

References 29 publications

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“…In morphology of foot process-like structures, lamellipodia and filopodia of podocytes were not much investigated in culture, and the nomenclature is sometimes inconsistent. Even though podocytes in culture usually only express rudimentary foot processes, laminin coating of culture dishes has been shown to promote cell process formation [29][30][31][32]. Therefore, we coated the substrate podocytes that were seeded on with laminin.…”

Section: Discussionmentioning

confidence: 99%

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Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

Kraus¹,

Rose

Krüger

et al. 2023

Cells

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Podocytes are critical components of the glomerular filtration barrier, sitting on the outside of the glomerular basement membrane. Primary and secondary foot processes are characteristic for podocytes, but cell processes that develop in culture were not studied much in the past. Moreover, protocols for diverse visualization methods mostly can only be used for one technique, due to differences in fixation, drying and handling. However, we detected by single-cell RNA sequencing (scRNAseq) analysis that cells reveal high variability in genes involved in cell type-specific morphology, even within one cell culture dish, highlighting the need for a compatible protocol that allows measuring the same cell with different methods. Here, we developed a new serial and correlative approach by using a combination of a wide variety of microscopic and spectroscopic techniques in the same cell for a better understanding of podocyte morphology. In detail, the protocol allowed for the sequential analysis of identical cells with light microscopy (LM), Raman spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Skipping the fixation and drying process, the protocol was also compatible with scanning ion-conductance microscopy (SICM), allowing the determination of podocyte surface topography of nanometer-range in living cells. With the help of nanoGPS Oxyo®, tracking concordant regions of interest of untreated podocytes and podocytes stressed with TGF-β were analyzed with LM, SEM, Raman spectroscopy, AFM and SICM, and revealed significant morphological alterations, including retraction of podocyte process, changes in cell surface morphology and loss of cell-cell contacts, as well as variations in lipid and protein content in TGF-β treated cells. The combination of these consecutive techniques on the same cells provides a comprehensive understanding of podocyte morphology. Additionally, the results can also be used to train automated intelligence networks to predict various outcomes related to podocyte injury in the future.

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Section: Discussionmentioning

confidence: 99%

“…Cell protrusions of ciPodocytes were quite long. It has been shown before in podocytes, including ciPodocytes, that laminin accelerates podocyte process formations that were often mutually interlocked [29][30][31][32]. Promotion of filopodia formation, due to laminin coating, has been described in other cells before [45].…”

Section: Discussionmentioning

confidence: 99%

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

Kraus¹,

Rose

Krüger

et al. 2023

Cells

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Glomerulus-on-a-Chip: Current Insights and Future Potential Towards Recapitulating Selectively Permeable Filtration Systems

Doi¹,

Kimura²,

Matsunaga³

et al. 2022

IJNRD

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Glomerulopathy, characterized by a dysfunctional glomerular capillary wall, results in proteinuria, leading to end-stage renal failure and poor clinical outcomes, including renal death and increased overall mortality. Conventional glomerulopathy research, including drug discovery, has mostly relied on animal experiments because in-vitro glomerulus models, capable of evaluating functional selective permeability, was unavailable in conventional in-vitro cell culture systems. However, animal experiments have limitations, including time- and cost-consuming, multi-organ effects, unstable reproducibility, inter-species reliability, and the social situation in the EU and US, where animal experiments have been discouraged. Glomerulus-on-a-chip, a new in-vitro organ model, has recently been developed in the field of organ-on-a-chip research based on microfluidic device technology. In the glomerulus-on-a-chip, the podocytes and endothelial cells are co-cultured in a microfluidic device with physical stimuli that mimic the physiological environment to enhance cell function to construct a functional filtration barrier, which can be assessed by permeability assays using fluorescently labeled molecules including inulin and albumin. A combination of this glomerulus-on-a chip technology with the culture technology to induce podocytes and endothelial cells from the human pluripotent stem cells could provide an alternative organ model and solve the issue of animal experiments. Additionally, previous experiments have verified the difference in the leakage of albumin using differentiated podocytes derived from patients with Alport syndrome, such that it could be applied to intractable hereditary glomerulopathy models. In this review, we provide an overview of the features of the existing glomerulus-on-a-chip systems, focusing on how they can address selective permeability verification tests, and the challenges they involved. We finally discuss the future approaches that should be developed for solving those challenges and allow further improvement of glomerulus-on-a-chip technologies.

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A novel method for successful induction of interdigitating process formation in conditionally immortalized podocytes from mice, rats, and humans

Cited by 2 publications

References 29 publications

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

Glomerulus-on-a-Chip: Current Insights and Future Potential Towards Recapitulating Selectively Permeable Filtration Systems

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