Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury

Namwanje, Maria; Bisunke, Bijay; Rousselle, Thomas; Lamanilao, Gene G; Sunder, Venkatadri S; Patterson, Elizabeth C; Kuscu, Canan; Kuscu, Cem; Maluf, Daniel G.; Kiran, Manjari; Mas, Valeria R.; Eason, James D.; Bajwa, Amandeep

doi:10.3390/ijms22105386

Cited by 9 publications

(6 citation statements)

References 68 publications

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“…Seahorse Assay, Mitochondrial DNA Quantification, and Transmission Electron Microscopy HK-2 cells were transferred to a Seahorse 24-well tissue culture plate, oxygen consumption rate was measured, and parameters were calculated as described in our previous work. 33,34 Total DNA was extracted from Boston University mouse proximal tubular cells by incubating cells in sodium dodecyl sulfate lysis buffer with proteinase K overnight at 55°C followed by ethanol precipitation. The abundance of mitochondrial DNA was quantified using RT-PCR by assessing the ratio of mouse mitochondrially encoded nicotinamide adenine dinucleotide N1hydrogen dehydrogenase, subunit 1 (ND1) relative to mouse nuclear DNA HK-2.…”

Section: Primary Tubular Cell Culture and Transductionmentioning

confidence: 99%

Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31

Silvaroli,

Bisunke,

Kim

et al. 2024

JASN

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Background: The synthetic tetra-peptide SS-31 shows promise in alleviating mitochondrial dysfunction associated with common diseases. However, the precise pharmacological basis of its mitoprotective effects remains unknown. Methods: To uncover the biological targets of SS-31, we performed a genome-scale CRISPR screen in HK-2 cells, a cell culture model where SS-31 mitigates cisplatin-associated cell death and mitochondrial dysfunction. The identified hit candidate gene was functionally validated using knockout cell lines, siRNA mediated downregulation, and tubular epithelial specific conditional knockout mice. Biochemical interaction studies were also performed to examine the interaction of SS-31 with the identified target protein. Results: Our primary screen and validation studies in HK-2 and primary murine tubular epithelial cells showed that phospholipid scramblase 3 (PLSCR3), an understudied inner mitochondrial membrane protein, is essential for the protective effects of SS-31. For in vivo validation, we generated tubular epithelial-specific knockout mice and found that Plscr3 gene ablation did not influence kidney function under normal conditions or affect the severity of cisplatin and rhabdomyolysis-associated AKI. However, Plscr3 gene deletion completely abrogated the protective effects of SS-31 during cisplatin and rhabdomyolysis-associated AKI. Biochemical studies showed that SS-31 directly binds to a previously uncharacterized N-terminal domain and stimulates PLSCR3 scramblase activity. Finally, PLSCR3 protein expression was found to be increased in the kidneys of AKI patients. Conclusions: PLSCR3 is identified as the essential biological target that facilitates the mitoprotective effects of SS-31 in vitro and in vivo. PLSCR3 agonists can alleviate mitochondrial dysfunction linked to AKI.

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Section: Primary Tubular Cell Culture and Transductionmentioning

confidence: 99%

Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31

Silvaroli,

Bisunke,

Kim

et al. 2024

JASN

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“…In this study, the mechanism of MCU-mediated mitochondrial damage remains a mystery. A large number of studies reported in recent years that mitochondrial quality control is an important mechanism involved in the pathogenesis, injury, and recovery of AKI [17][18][19][20][21]. Mitochondrial dynamics has been considered to be a crucial component of mitochondrial quality control and may be associated with MCU.…”

Section: Discussionmentioning

confidence: 99%

“…There is evidence from animal studies that other acute kidney injury (AKI) induced by multiple pathogenic factors such as I/R, cisplatin, sepsis, folic acid, and rhabdomyolysis are associated with mitochondrial dynamics, including ssion and fusion [17][18][19][20][21]. Mitochondrial dynamics is an important part of mitochondrial quality control, which is responsible for maintaining the relative stability of mitochondrial quantity, morphology and function, that is, mitochondrial homeostasis.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial calcium uniporter-mediated mitochondrial dynamics imbalance contributes to contrast medium-induced renal tubular cell injury

Huang,

Lv,

Chen

et al. 2024

Preprint

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Background Contrast-induced acute kidney injury (CI-AKI) is the acute onset of renal failure occurring 24–48 hours after intravascular injection of contrast medium (CM), which is a common cause of hospital-acquired acute kidney injury. Previous researches on CI-AKI have demonstrated that cytoplasmic Ca2+ overload and mitochondrial damage were strongly associated with CI-AKI, but the precise pathogenesis remains elusive. Therefore, we aimed to identify the role of mitochondrial calcium uniporter (MCU), the most important Ca2+ unidirectional channel of mitochondria, in CM-induced tubular epithelial cell injury and explore the molecular conformation of MCU interacting with iohexol. Methods Human renal proximal tubular epithelial (HK-2) cells were incubated with 100 mg I/ml iohexol. Cell injury and apoptosis were detected by Cell Counting Kit-8 and flow cytometry. The mitochondrial Ca2+ level was evaluated by Rhod-2 fluorescence. Mitochondrial damage was assessed by transmission electron microscopy, fluorescence of mitotracker, and JC-1. Protein expression of dynamin-related protein 1 (DRP1) and optic atrophy 1 (OPA1) was assessed by Western Blot. Results Iohexol administration successfully induced HK-2 cell injury and apoptosis. Moreover, there is an interaction between Iohexol and MCU. We also demonstrated that iohexol could lead to increase of mitochondrial Ca2+ concentration, upregulation of MCU expression, mitochondrial injury, and mitochondrial dynamics imbalance (excessive mitochondrial fission and loss of mitochondrial fusion) in HK-2 cells. Of note, inhibiting MCU by Ru360 efficiently maintaining mitochondrial function by reducing mitochondrial Ca2+ influx and improving impaired mitochondrial dynamics, thereby protecting HK-2 cells from CM-induced injury and apoptosis. On the contrary, the activation of MCU by spermine aggravated cell injury under the same mechanisms. Conclusions The present study illustrated a novel molecular mechanism of CI-AKI involving MCU-mediated mitochondrial dynamics imbalance, and suppression of MCU exhibited a cytoprotective effect on CM-treated renal tubular cells.

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“…Following this, cells were washed (2×) and cocultured with iMSC or cultured as a monoculture. Cocultured THP-1 cells were transferred to a Seahorse 24-well tissue culture plates and oxygen consumption rate (OCR) was measured, and parameters were calculated as previously described [ 24 ]. Briefly, prior to the assay, the media was changed to unbuffered DMEM (Gibco #12800-017, pH 7.4, 37 °C), and cells were equilibrated for 1 h at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Mesenchymal Stem Cell Induced Foxp3(+) Tregs Suppress Effector T Cells and Protect against Retinal Ischemic Injury

Agrawal

Rasiah

Bajwa

et al. 2021

Cells

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Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. This study tested the hypothesis that MSC induces upregulation of transcription factor forkhead box protein P3 (Foxp3) in T cells to elicit immune modulation, and thus, protect against retinal damage. Induced MSCs (iMSCs) were generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a noninsertional reprogramming approach. In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes significantly increased oxygen consumption rate (OCR) for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ T regulatory cells (Tregs) in coculture with mouse splenocytes. In in-vivo studies, iMSCs transplanted in ischemia-reperfusion (I/R) injured eye significantly increased Foxp3+ Tregs in the retina compared to that of saline-injected I/R eyes. Furthermore, iMSC injected I/R eyes significantly decreased retinal inflammation as evidenced by reduced gene expression of IL1β, VCAM1, LAMA5, and CCL2 and improved b-wave amplitudes compared to that of saline-injected I/R eyes. Our study demonstrates that iMSCs can transfer mitochondria to immune cells to suppress the effector T cell population. Additionally, our current data indicate that iMSC can enhance differentiation of T cells into Foxp3 Tregs in vitro and therapeutically improve the retina’s immune function by upregulation of Tregs to decrease inflammation and reduce I/R injury-induced retinal degeneration in vivo.

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Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury

Cited by 9 publications

References 68 publications

Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31

Genome-Wide CRISPR Screen Identifies Phospholipid Scramblase 3 as the Biological Target of Mitoprotective Drug SS-31

Mitochondrial calcium uniporter-mediated mitochondrial dynamics imbalance contributes to contrast medium-induced renal tubular cell injury

Mesenchymal Stem Cell Induced Foxp3(+) Tregs Suppress Effector T Cells and Protect against Retinal Ischemic Injury

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