scholarly journals Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

2019 ◽  
Vol 20 (10) ◽  
pp. 2379 ◽  
Author(s):  
Johannes Fels ◽  
Bettina Scharner ◽  
Ralf Zarbock ◽  
Itzel Pamela Zavala Guevara ◽  
Wing-Kee Lee ◽  
...  
Keyword(s):  
Cell Viability ◽  
Proximal Tubule ◽  
Protein Complexes ◽  
Binding Protein ◽  
Proximal Tubule Cell ◽  
Lipocalin 2 ◽  
High Passage ◽  
Β2 Microglobulin ◽  
Low Passage ◽  
Passage Cells

Cadmium (Cd2+) in the environment is a significant health hazard. Chronic low Cd2+ exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd2+ binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, β2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd2+ nephrotoxicity claims that Cd2+-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd2+-metallothionein concentrations; (ii) the KD of megalin for metallothionein is ~105-times higher than (Cd2+)-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd2+-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, β2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd2+-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd2+-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd2+-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd2+-β2-microglobulin, Cd2+-albumin and Cd2+-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd2+-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.

PLAQUE FORMATION BY POLIO AND MEASLES VIRUSES IN BS-C-1 (HOPPS) CELLS DERIVED FROM THE AFRICAN GREEN MONKEY

1965 ◽  
Vol 11 (3) ◽  
pp. 435-439 ◽  
Author(s):  
John Furesz ◽  
Pierre Moreau
Keyword(s):  
Cell Line ◽  
Measles Virus ◽  
Plaque Assay ◽  
Plaque Formation ◽  
African Green Monkey ◽  
High Passage ◽  
Green Monkey ◽  
Simple Medium ◽  
Low Passage ◽  
Passage Cells

BS-C-1 cells, cultivated in a simple medium designated M-E, were found to be suitable for a plaque assay of polio and measles viruses. In the plaque assay both low- and high-passage levels of the BS-C-1 cell line were highly susceptible to measles virus but titers of polioviruses were lower in the high-passage cells. Low-passage cells, preserved and stored at −196 °C for prolonged periods, are recommended for the titration of polio and measles viruses.

scholarly journals Puerarin blocks the aging phenotype in human dermal fibroblasts

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249367
Author(s):  
Yuki Kamiya ◽  
Mao Odama ◽  
Aki Mizuguti ◽  
Shigeru Murakami ◽  
Takashi Ito
Keyword(s):  
Dermal Fibroblast ◽  
Smooth Muscle Actin ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
High Passage ◽  
Normal Human ◽  
Low Passage ◽  
Passage Cells ◽  
Beta Galactosidase ◽  
Functional Defects

Dermal fibroblast aging contributes to aging-associated functional defects in the skin since dermal fibroblasts maintain skin homeostasis by interacting with the epidermis and extracellular matrix. Here, we found that puerarin, an isoflavone present in Pueraria lobata (Kudzu), can prevent the development of the aging-phenotype in human dermal fibroblasts. Normal human dermal fibroblasts (NHDFs) were subcultivated and high-passage cells were selected as senescent cells, whereas low-passage cells were selected as a young cell control. Puerarin treatment increased cell proliferation and decreased the proportion of senescence-associated beta-galactosidase-positive cells in a high-passage culture of NHDFs. Moreover, puerarin treatment reduced the number of smooth muscle actin (SMA)-positive myofibroblasts and the expression of a reticular fibroblast marker, calponin 1 (CNN1), which were induced in high-passage NHDFs. Fulvestrant, an estrogen receptor antagonist, blocked the puerarin-mediated downregulation of SMA and CNN1. Our results suggest that puerarin may be a useful functional food that alleviates aging-related functional defects in dermal fibroblasts.

scholarly journals Renal Proximal Tubule Cell Cannabinoid-1 Receptor Regulates Bone Remodeling and Mass via a Kidney-to-Bone Axis

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 414
Author(s):  
Saja Baraghithy ◽  
Yael Soae ◽  
Dekel Assaf ◽  
Liad Hinden ◽  
Shiran Udi ◽  
...  
Keyword(s):  
Bone Mass ◽  
Proximal Tubule ◽  
Bone Remodeling ◽  
Kidney Function ◽  
Bone Cells ◽  
Critical Role ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Protective Effects ◽  
Cannabinoid 1 Receptor

The renal proximal tubule cells (RPTCs), well-known for maintaining glucose and mineral homeostasis, play a critical role in the regulation of kidney function and bone remodeling. Deterioration in RPTC function may therefore lead to the development of diabetic kidney disease (DKD) and osteoporosis. Previously, we have shown that the cannabinoid-1 receptor (CB1R) modulates both kidney function as well as bone remodeling and mass via its direct role in RPTCs and bone cells, respectively. Here we employed genetic and pharmacological approaches that target CB1R, and found that its specific nullification in RPTCs preserves bone mass and remodeling both under normo- and hyper-glycemic conditions, and that its chronic blockade prevents the development of diabetes-induced bone loss. These protective effects of negatively targeting CB1R specifically in RPTCs were associated with its ability to modulate erythropoietin (EPO) synthesis, a hormone known to affect bone mass and remodeling. Our findings highlight a novel molecular mechanism by which CB1R in RPTCs remotely regulates skeletal homeostasis via a kidney-to-bone axis that involves EPO.

scholarly journals Silencing of lipocalin-2 and its receptor improved cardiomyocytes viability via decreasing iron uptake, mitochondrial fission, mitophagy and apoptosis under iron overload condition

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S Kumfu ◽  
S.C Chattipakorn ◽  
N Chattipakorn
Keyword(s):  
Calcium Channels ◽  
Iron Overload ◽  
Cell Viability ◽  
Iron Uptake ◽  
Mitochondrial Fission ◽  
Iron Accumulation ◽  
Funding Source ◽  
Lipocalin 2 ◽  
Intracellular Iron ◽  
Beneficial Effects

Abstract Background Iron overload cardiomyopathy is a common cause of death in iron overload patients. L-type calcium channels (LTCC) and T-type calcium channels (TTCC) have been shown to play important roles for iron uptake into the heart under iron overload condition. Recently, cardiomyocytes which exposed to lipocalin-2 (LCN-2) have been shown to increase apoptosis due to excessive intracellular iron accumulation. However, the mechanistic roles of LCN-2 and LCN-2 receptor (LCN-2R) as iron transporters in cardiomyocytes under iron overload condition have never been investigated. Purpose We hypothesized that the LCN-2 and LCN-2R are alternate iron uptake pathways into cardiomyocytes under iron overload condition. Methods H9c2 cardiomyocytes were treated with either LCN-2 siRNA or LCN-2R siRNA for 72 hr or LTCC blocker (verapamil), TTCC blocker (TTA-P2), or iron chelator deferiprone (DFP) for 1 hr. After treatment, cells were exposed to ferric ammonium citrate (FAC, Fe3+) or FAC + 1mM ascorbic acid (Fe2+) at 200 μM for 48 hr. Intracellular iron level, cell viability, mitochondrial dynamics, mitophagy and apoptosis were determined. Results Both Fe2+ and Fe3+ treated groups showed significantly increased intracellular iron uptake, decreased cell viability, increased mitochondrial fission, mitophagy and apoptotic protein expression in cardiomyocytes. Under Fe2+ overload condition, treatments with LTCC blocker, TTCC blocker, and DFP could significantly decrease intracellular iron accumulation and increase cell viability via decreasing mitochondrial fission, mitophagy and cleaved caspase-3 (Figure), whereas both LCN-2 and LCN-2R siRNA treatment had no beneficial effects on these parameters. Under Fe3+ overload condition, treatment with LCN-2 siRNA, LCN-2R siRNA, and DFP showed beneficial effects on those parameters, whereas neither LTCC nor TTCC blocker provided these benefits (Figure 1). Conclusion Silencing of LCN-2 and LCN-2R increased cardiomyocyte viability via decreasing iron uptake, mitochondrial fission, mitophagy and apoptosis under Fe3+ iron overload condition. Meanwhile, treatment with calcium channel blockers improved cardiomyocytes viability via decreasing iron uptake, mitochondrial fission, mitophagy and apoptosis under Fe2+ iron overload condition. All of these findings suggested that LTCC and TTCC played important roles for Fe2+ uptake, whereas LCN-2 and LCN-2R were essential for Fe3+ uptake into the cardiomyocytes under iron overload conditions. Figure 1. Cell viability and apoptosis Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Thailand Research Fund and NSTDA Research Chair Grant (NC)

scholarly journals The major histocompatibility complex class II promoter-binding protein RFX (NF-X) is a methylated DNA-binding protein.

1993 ◽  
Vol 13 (11) ◽  
pp. 6810-6818 ◽  
Author(s):  
X Y Zhang ◽  
N Jabrane-Ferrat ◽  
C K Asiedu ◽  
S Samac ◽  
B M Peterlin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Dna Sequences ◽  
Protein Complexes ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Class Ii ◽  
Major Histocompatibility ◽  
Methylated Dna ◽  
Mammalian Protein

A mammalian protein called RFX or NF-X binds to the X box (or X1 box) in the promoters of a number of major histocompatibility (MHC) class II genes. In this study, RFX was shown to have the same DNA-binding specificity as methylated DNA-binding protein (MDBP), and its own cDNA was found to contain a binding site for MDBP in the leader region. MDBP is a ubiquitous mammalian protein that binds to certain DNA sequences preferentially when they are CpG methylated and to other related sequences, like the X box, irrespective of DNA methylation. MDBP from HeLa and Raji cells formed DNA-protein complexes with X-box oligonucleotides that coelectrophoresed with those containing standard MDBP sites. Furthermore, MDBP and X-box oligonucleotides cross-competed for the formation of these DNA-protein complexes. DNA-protein complexes obtained with MDBP sites displayed the same partial supershifting with an antiserum directed to the N terminus of RFX seen for complexes containing an X-box oligonucleotide. Also, the in vitro-transcribed-translated product of a recombinant RFX cDNA bound specifically to MDBP ligands and displayed the DNA methylation-dependent binding of MDBP. RFX therefore contains MDBP activity and thereby also EF-C, EP, and MIF activities that are indistinguishable from MDBP and that bind to methylation-independent sites in the transcriptional enhancers of polyomavirus and hepatitis B virus and to an intron of c-myc.

Proximal tubule microvilli remodeling and albuminuria in the Ren2 transgenic rat

2007 ◽  
Vol 292 (2) ◽  
pp. F861-F867 ◽  
Author(s):  
Melvin R. Hayden ◽  
Nazif A. Chowdhury ◽  
Shawna A. Cooper ◽  
Adam Whaley-Connell ◽  
Javad Habibi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Proximal Tubule ◽  
Structural Damage ◽  
Kidney Tissue ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Sprague Dawley Rats

TG(mRen2)27 (Ren2) transgenic rats overexpress the mouse renin gene, with subsequent elevated tissue ANG II, hypertension, and nephropathy. The proximal tubule cell (PTC) is responsible for the reabsorption of 5–8 g of glomerular filtered albumin each day. Excess filtered albumin may contribute to PTC damage and tubulointerstitial disease. This investigation examined the role of ANG II-induced oxidative stress in PTC structural remodeling: whether such changes could be modified with in vivo treatment with ANG type 1 receptor (AT1R) blockade (valsartan) or SOD/catalase mimetic (tempol). Male Ren2 (6–7 wk old) and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Systolic blood pressure, albuminuria, N-acetyl-β-d-glucosaminidase, and kidney tissue malondialdehyde (MDA) were measured, and ×60,000 transmission electron microscopy images were used to assess PTC microvilli structure. There were significant differences in systolic blood pressure, albuminuria, lipid peroxidation (MDA and nitrotyrosine staining), and PTC structure in Ren2 vs. Sprague-Dawley rats (each P < 0.05). Increased mean diameter of PTC microvilli in the placebo-treated Ren2 rats ( P < 0.05) correlated strongly with albuminuria ( r2 = 0.83) and moderately with MDA ( r2 = 0.49), and there was an increase in the ratio of abnormal forms of microvilli in placebo-treated Ren2 rats compared with Sprague-Dawley control rats ( P < 0.05). AT1R blockade, but not tempol treatment, abrogated albuminuria and N-acetyl-β-d-glucosaminidase; both therapies corrected abnormalities in oxidative stress and PTC microvilli remodeling. These data indicate that PTC structural damage in the Ren2 rat is related to the oxidative stress response to ANG II and/or albuminuria.

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

2012 ◽  
Vol 303 (3) ◽  
pp. F458-F466 ◽  
Author(s):  
Sara M. Molinas ◽  
Laura Trumper ◽  
Raúl A. Marinelli
Keyword(s):  
Metabolic Acidosis ◽  
Protein Expression ◽  
Proximal Tubule ◽  
Ammonia Excretion ◽  
Renal Proximal Tubule ◽  
In Vivo Studies ◽  
Proximal Tubule Cell ◽  
Inner Mitochondrial Membrane ◽  
Ammonia Transport ◽  
In Cells

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% ( P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (−31%, P < 0.05) as well as in cells exposed to acid (−90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH4Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.

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