scholarly journals Overexpression of MnSOD Protects against Cold Storage-Induced Mitochondrial Injury but Not against OMA1-Dependent OPA1 Proteolytic Processing in Rat Renal Proximal Tubular Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1272
Author(s):  
Julia Tobacyk ◽  
Grishma KC ◽  
Lee Ann MacMillan-Crow
Keyword(s):  
Cell Viability ◽  
Cold Storage ◽  
Renal Injury ◽  
Rat Kidney ◽  
Proteolytic Processing ◽  
Independent Manner ◽  
Western Blots ◽  
Proximal Tubular ◽  
Dependent Cell

Kidneys from deceased donors undergo cold storage (CS) preservation before transplantation. Although CS is a clinical necessity for extending organ quality preservation, CS causes mitochondrial and renal injury. Specifically, many studies, including our own, have shown that the triggering event of CS-induced renal injury is mitochondrial reactive oxygen species (mROS). Here, we explored the role of OMA1-depedent OPA1 proteolytic processing in rat kidney proximal tubular epithelial (NRK) cells in an in vitro model of renal CS (18 h), followed by rewarming (6 h) (CS + RW). The involvement of mROS was evaluated by stably overexpressing manganese superoxide dismutase (MnSOD), an essential mitochondrial antioxidant enzyme, in NRK cells. Western blots detected rapid OPA1 proteolytic processing and a decrease in ATP-dependent cell viability in NRK cells subjected to CS + RW compared to control cells. Small interfering RNA (siRNA) knockdown of OMA1 reduced proteolytic processing of OPA1, suggesting that OMA1 is responsible for OPA1 proteolytic processing during CS + RW-induced renal injury. Overexpression of MnSOD during CS + RW reduced cell death, mitochondrial respiratory dysfunction, and ATP-dependent cell viability, but it did not prevent OMA1-dependent OPA1 processing. These data show for the first time that OMA1 is responsible for proteolytically cleaving OPA1 in a redox-independent manner during renal cell CS.

scholarly journals Specific BK Channel Activator NS11021 Protects Rat Renal Proximal Tubular Cells from Cold Storage—Induced Mitochondrial Injury In Vitro

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 825 ◽  
Author(s):  
Stephen Shrum ◽  
Nancy J. Rusch ◽  
Lee Ann MacMillan-Crow
Keyword(s):  
Cold Storage ◽  
Rat Kidney ◽  
Bk Channel ◽  
K Uptake ◽  
Western Blots ◽  
Α Subunit ◽  
Mitochondrial Injury ◽  
Mitochondrial Fractions ◽  
Proximal Tubular

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS causes mitochondrial injury, which may exacerbate renal graft dysfunction. Here, we explored whether adding NS11021, an activator of the mitochondrial big-conductance calcium-activated K+ (mitoBK) channel, to CS solution can mitigate CS-induced mitochondrial injury. We used normal rat kidney proximal tubular epithelial (NRK) cells as an in vitro model of renal cold storage (18 h) and rewarming (2 h) (CS + RW). Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from NRK cells. The fluorescent K+-binding probe, PBFI-AM, revealed that isolated mitochondria from NRK cells exhibited mitoBK-mediated K+ uptake, which was impaired ~70% in NRK cells subjected to CS + RW compared to control NRK cells maintained at 37 °C. Importantly, the addition of 1 μM NS11021 to CS solution prevented CS + RW-induced impairment of mitoBK-mediated K+ uptake. The NS11021–treated NRK cells also exhibited less cell death and mitochondrial injury after CS + RW, including mitigated mitochondrial respiratory dysfunction, depolarization, and superoxide production. In summary, these new data show for the first time that mitoBK channels may represent a therapeutic target to prevent renal CS-induced injury.

scholarly journals MiR-144-3p Aggravated Cartilage Injury in Rheumatoid Arthritis by Regulating BMP2/PI3K/Akt Axis

2021 ◽  
Author(s):  
Mei-Li Mo ◽  
Jin-Mei Jiang ◽  
Xiao-Ping Long ◽  
Li-Hu Xie
Keyword(s):  
Extracellular Matrix ◽  
Cell Viability ◽  
Cell Injury ◽  
Tissue Injury ◽  
Cartilage Injury ◽  
Luciferase Assay ◽  
Tunel Staining ◽  
Western Blots

Abstract Objectives Present study aimed to illustrate the role of miR-144-3p in RA. Methods N1511 chondrocytes were stimulated by IL-1β to mimic RA injury model in vitro. Rats were subjected to injection of type II collagen to establish an in vivo RA model and the arthritis index score was calculated. Cell viability was determined by CCK-8. The expression of cartilage extracellular matrix proteins (Collagen II and Aggrecan) and matrix metalloproteinases protein (MMP-13) were determined by qRT-PCR and western blots. Cell apoptosis was measured by Flow cytometry. ELISA was applied to test the secretion of pro-inflammatory cytokines (IL-1β and TNF-α). Tissue injury and apoptosis were detected by HE staining and TUNEL staining. Interaction of miR-144-3p and BMP2 was verified by dual luciferase assay. Results MiR-144-3p was dramatically increased in IL-1β induced N1511 cells. MiR-144-3p depletion elevated cell viability, suppressed apoptosis, pro-inflammatory cytokine releasing, and extracellular matrix loss in IL-1β induced N1511 cells. Moreover, miR-144-3p targeted BMP2 to modulate its expression negatively. Activation of PI3K/Akt signaling compromised inhibition of BMP2 induced aggravated N1511 cell injury with IL-1β stimulation. Inhibition of miR-144-3p alleviated cartilage injury and inflammatory in RA rats. Conclusion Collectively, miR-144-3p could aggravate chondrocytes injury inflammatory response in RA via BMP2/PI3K/Akt axis.

In vitro Phosphorylation of Rat Kidney Proximal Tubular Brush Border Membranes

1985 ◽  
Vol 8 (1) ◽  
pp. 19-29
Author(s):  
Jürg Biber ◽  
Vito Sealera ◽  
Heini Murer
Keyword(s):  
Brush Border ◽  
Rat Kidney ◽  
Brush Border Membranes ◽  
Proximal Tubular

GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange

2002 ◽  
Vol 115 (12) ◽  
pp. 2529-2539
Author(s):  
Xuedong Kang ◽  
Alexander Szallies ◽  
Marc Rawer ◽  
Hartmut Echner ◽  
Michael Duszenko
Keyword(s):  
Trypanosoma Brucei ◽  
Signal Sequence ◽  
Intracellular Localization ◽  
Glycosylation Site ◽  
Proteolytic Processing ◽  
Gpi Anchor ◽  
Hydrophobic Region ◽  
Western Blots ◽  
Vitro Assay

GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies. The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.

Cold storage of peripheral nerves: An in vitro assay of cell viability and function

Glia ◽  
1994 ◽  
Vol 10 (2) ◽  
pp. 121-131 ◽  
Author(s):  
Allan D. O. Levi ◽  
Peter J. Evans ◽  
Susan E. Mackinnon ◽  
Richard P. Bunge
Keyword(s):  
Cell Viability ◽  
Cold Storage ◽  
Peripheral Nerves ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
And Function

scholarly journals Modulation of cholestan-3β,5α,6β-triol toxicity by butylated hydroxytoluene, α-tocopherol and β-carotene in newborn rat kidney cells in vitro

1997 ◽  
Vol 78 (3) ◽  
pp. 479-492 ◽  
Author(s):  
Alison M. Wilson ◽  
Ruth M. Sisk ◽  
Nora M. O'Brien
Keyword(s):  
Lipid Peroxidation ◽  
Cell Viability ◽  
Rat Kidney ◽  
Oxidation Products ◽  
Butylated Hydroxytoluene ◽  
Cholesterol Oxidation ◽  
Newborn Rat ◽  
Membrane Function ◽  
Β Carotene

Cholesterol oxidation products (COP) have been reported to influence vital cellular processes such as cell growth, cell proliferation, membrane function and de novo sterol biosynthesis. The objectives of the present study were: (1) to develop an in vitro model using newborn rat kidney (NRK) cells to investigate the actions of COP; (2) to investigate the effect of COP on cell viability, endogenous antioxidant enzymes activities, i.e. superoxide dismutase (EC 1.15.1.1; SOD) and catalase (EC 1.11.1.6; CAT), and the extent of lipid peroxidation in this model; (3) to determine whether the addition of 100–1000 nm-α-tocopherol, β-carotene or butylated hydroxytoluene (BHT) could protect against COP-induced cytotoxicity. NRK cells were cultured in the presence of various concentrations (5–50 μM) of cholesterol or cholestan-3β,5α,6β-triol (cholestantriol) for a period of 24 h. Cholesterol over the range 5–50 μM did not induce cytotoxicity as indicated by the neutral-red-uptake assay or the lactate dehydrogenase (EC 1.1.1.27)-release assay. However, cell viability was compromised by the addition of > 10 μM-cholestantriol (P < 0.05). The addition of β-carotene (100–1000 nM) did not increase cell viability significantly in cholestantriol-supplemented cells. However, the addition of α-tocopherol (1000 nM) and BHT (1000 nM) significantly increased percentage cell viability above that of the cholestantriol-supplemented cells but not back to control levels. SOD and CAT activities in NRK cells significantly decreased (P < 0.05) following incubation with cholestantriol. The addition of > 750 nM-α-tocopherol, β-carotene or BHT returned SOD and CAT activities to that of the control. Lipid peroxidation was significantly induced (P < 0.05) in the presence of cholestantriol. Supplementation of the cells with α-tocopherol (250, 500 or 1000 nM) or BHT (750 or 1000 nM) resulted in a reduction in the extent of lipid peroxidation (P < 0.05). The addition of β-carotene over the concentration range of 250–1000 nM did not reduce lipid peroxidation significantly compared with cells exposed to cholestantriol alone. These findings suggest that addition of exogenous antioxidants may be beneficial in the prevention of COP-induced toxicity in vitro.

Different effects of arsenate and phosphonoformate on Pi transport adaptation in opossum kidney cells

2009 ◽  
Vol 297 (3) ◽  
pp. C516-C525 ◽  
Author(s):  
Ricardo Villa-Bellosta ◽  
Víctor Sorribas
Keyword(s):  
Rat Kidney ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Competitive Inhibitors ◽  
Renal Adaptation ◽  
Proximal Tubular ◽  
Dietary Phosphate ◽  
Definition Of

The main nonhormonal mechanism for controlling inorganic phosphate (Pi) homeostasis is renal adaptation of the proximal tubular Pi transport rate to changes in dietary phosphate content. Opossum kidney (OK) cell line is an in vitro renal model that maintains the ability of renal adaptation to the extracellular Pi concentration. We have studied how two competitive inhibitors of Pi transport, arsenate [As(V)] and phosphonoformate (PFA), affect adaptation to low and high Pi concentrations. OK cells show very high affinity for As(V) (inhibitory constant, Ki 0.12 mM) when compared with the rat kidney. As(V) very efficiently reversed the adaptation of OK cells to low Pi (0.1 mM), whereas PFA induced adaptation similar to 0.1 mM Pi. Adaptation with 2 mM Pi or As(V) was characterized by decreases in the maximal velociy ( Vmax) of Pi transport and an abundance of the NaPi-IIa Pi transporter in the plasma membrane, shown by the protein biotinylation. Conversely, PFA and 0.1 mM Pi increased the Vmax and transporter abundance. Changes in the Vmax were limited to a 50% variation, which was not paralleled by changes in the concentration of Pi or of the inhibitor. OK cells are very sensitive to As(V), but the effects are reversible and noncytotoxic. These effects can be interpreted as As(V) being transported into the cell, thereby mimicking a high Pi concentration. PFA blocks the uptake of Pi but is not transported, and it therefore simulates a low Pi concentration inside the cell. To conclude, a mathematical definition of the adaptation process is reported, thereby explaining the limited changes in Pi transport Vmax.

scholarly journals Protective Effect of Shikimic Acid against Cisplatin-Induced Renal Injury: In Vitro and In Vivo Studies

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1681
Author(s):  
Jinkyung Lee ◽  
Quynh Nhu Nguyen ◽  
Jun Yeon Park ◽  
Sullim Lee ◽  
Gwi Seo Hwang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protective Effect ◽  
Cell Viability ◽  
Renal Injury ◽  
Shikimic Acid ◽  
Kidney Injury ◽  
In Vivo Studies ◽  
Artemisia Absinthium ◽  
Vitro Assay

Nephrotoxicity is a serious side effect of cisplatin, which is one of the most frequently used drugs for cancer treatment. This study aimed to assess the renoprotective effect of Artemisia absinthium extract and its bioactive compound (shikimic acid) against cisplatin-induced renal injury. An in vitro assay was performed in kidney tubular epithelial cells (LLC-PK1) with 50, 100, and 200 µg/mL A. absinthium extract and 25 and 50 µM shikimic acid, and cytotoxicity was induced by 25 µM cisplatin. BALB/c mice (6 weeks old) were injected with 16 mg/kg cisplatin once and orally administered 25 and 50 mg/kg shikimic acid daily for 4 days. The results showed that the A. absinthium extract reversed the decrease in renal cell viability induced by cisplatin, whereas it decreased the reactive oxidative stress accumulation and apoptosis in LLC-PK1 cells. Shikimic acid also reversed the effect on cell viability but decreased oxidative stress and apoptosis in renal cells compared with the levels in the cisplatin-treated group. Furthermore, shikimic acid protected against kidney injury in cisplatin-treated mice by reducing serum creatinine levels. The protective effect of shikimic acid against cisplatin-mediated kidney injury was confirmed by the recovery of histological kidney injury in cisplatin-treated mice. To the best of our knowledge, this study is the first report on the nephroprotective effect of A. absinthium extract and its mechanism of action against cisplatin-induced renal injury.

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