Considerations in Comparing Proximal Tubule Cell Culture Models

The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides in the proximal tubule; however, knowledge of regulation of PEPT2 by growth factors and hormones is limited. In the present study, we examined the effects of epidermal growth factor (EGF) on PEPT2 transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 expression by lowering transcription of the rat PepT2 gene or by decreasing rat PepT2 mRNA stability. Previous investigators routinely used SKPT cell culture media with a high (10 ng/ml) EGF concentration. Our study suggests that this might be disadvantageous when studying PEPT2-mediated transport phenomena. These findings demonstrate for the first time EGF-mediated regulation of PEPT2 expression in a kidney cell line. The relevance for kidney regulation of peptide transport activity in physiological and/or pathophysiological situations, where EGF and EGF receptor levels change drastically, remains to be established.

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Development of a human proximal tubule cell culture acidotic/WIT model

Transplantation Proceedings ◽

10.1016/s0041-1345(00)02366-6 ◽

2001 ◽

Vol 33 (1-2) ◽

pp. 895-897

Author(s):

D.R Owen ◽

M Bakeer ◽

K.G.M Brockbank

Keyword(s):

Cell Culture ◽

Proximal Tubule ◽

Proximal Tubule Cell ◽

Tubule Cell ◽

Human Proximal Tubule Cell ◽

Human Proximal Tubule

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Treatment approaches combining cytostatic drugs with the oncolytic parvovirus H-1 increase cytotoxic effects in cell culture models of highly malignant pediatric brain tumors

Klinische Pädiatrie ◽

10.1055/s-0034-1393947 ◽

2014 ◽

Vol 226 (06) ◽

Author(s):

C Westphal ◽

S Öztürk ◽

R Josupeit ◽

B Leuchs ◽

O Witt ◽

...

Keyword(s):

Cell Culture ◽

Brain Tumors ◽

Pediatric Brain Tumors ◽

Cytostatic Drugs ◽

Cytotoxic Effects ◽

Treatment Approaches ◽

Culture Models ◽

Pediatric Brain ◽

Cell Culture Models

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Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/s11626-020-00537-3 ◽

2021 ◽

Author(s):

Terry Riss ◽

O. Joseph Trask

Keyword(s):

Cell Culture ◽

Three Dimensional ◽

3D Culture ◽

Fluorescent Imaging ◽

Culture Models ◽

Assay Methods ◽

Diffusion Rates ◽

Cell Culture Models ◽

Dimensional Cell ◽

Cells Cultured

AbstractAlong with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

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Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication

Toxicological Sciences ◽

10.1093/toxsci/kfv136 ◽

2015 ◽

Vol 147 (2) ◽

pp. 412-424 ◽

Cited By ~ 49

Author(s):

Rowena L. C. Sison-Young ◽

Dimitra Mitsa ◽

Rosalind E. Jenkins ◽

David Mottram ◽

Eliane Alexandre ◽

...

Keyword(s):

Cell Culture ◽

Single Cell ◽

Significant Variation ◽

Human Liver ◽

Drug Disposition ◽

Culture Models ◽

Single Cell Culture ◽

Cell Culture Models

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Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

Molecular and Cellular Biology ◽

10.1128/mcb.01254-05 ◽

2006 ◽

Vol 26 (17) ◽

pp. 6425-6434 ◽

Cited By ~ 119

Author(s):

O. Jameel Shah ◽

Tony Hunter

Keyword(s):

Cell Culture ◽

Tuberous Sclerosis ◽

High Speed ◽

Serine Phosphorylation ◽

Feedback Signal ◽

Insulin Receptor Substrates ◽

Igf I ◽

Culture Models ◽

Cell Culture Models

ABSTRACT The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

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Cell culture models of the ocular barriers

European Journal of Pharmaceutics and Biopharmaceutics ◽

10.1016/j.ejpb.2005.01.009 ◽

2005 ◽

Vol 60 (2) ◽

pp. 207-225 ◽

Cited By ~ 167

Author(s):

Margit Hornof ◽

Elisa Toropainen ◽

Arto Urtti

Keyword(s):

Cell Culture ◽

Culture Models ◽

Cell Culture Models

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Abstract 15951: Palmitate, Not Oleate, is the Preferred Fatty Acid for Cardiac Triacylglycerol Synthesis

Circulation ◽

10.1161/circ.130.suppl_2.15951 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Stephen C Kolwicz ◽

Rong Tian

Keyword(s):

Cell Culture ◽

13C Nmr Spectroscopy ◽

Culture Studies ◽

Triacylglycerol Synthesis ◽

Culture Models ◽

Contracting Heart ◽

And Control ◽

The Relationship ◽

Cell Culture Models ◽

Insight Into

Introduction: Previous studies using cell culture models identified cyto-toxic effects of palmitate and that supplementation with oleate was protective by redirecting palmitate into triacylglycerol (TAG) stores. However, other cull culture studies reported that diacylglycerol transferase 1 (DGAT1), the last enzyme in TAG synthesis, demonstrated a preference for oleate. At present, it is not clear whether the supply of exogenous fatty acids (FA) to the heart is differentially allocated into the endogenous TAG pool. Therefore, the purpose of the present study is to examine the influence of palmitate and/or oleate on cardiac TAG incorporation. METHODS/RESULTS: Hearts were isolated from DGAT1-transgenic (DGAT) and control littermates (CON) and perfused in Langendorff mode with a mixed substrate buffer consisting of glucose, lactate, insulin, and FAs. The FA supply was varied with 0.2mM of both labeled (13C) and unlabeled (12C) FAs in 4 different experiments: 1) 13C/12C palmitate; 2) 13C/12C oleate; 3) 13C palmitate/12C oleate; 4) 13C oleate/12C palmitate. The incorporation of 13C palmitate or 13C oleate into the TAG pool was monitored by 13C NMR spectroscopy. In CON hearts (n=3), the incorporation of palmitate was ~65% higher than oleate when the perfusate contained a homogenous supply of FA. This was also observed in DGAT hearts (n=4) although the incorporation of both palmitate and oleate was ~75% higher compared to CON (P <0.05). In the presence of oleate, palmitate incorporation decreased 25-30% in both CON and DGAT hearts. In contrast, oleate incorporation was diminished by ~50% and ~100% in CON and DGAT hearts, respectively, in the presence of palmitate. CONCLUSIONS: These data suggest that when palmitate and oleate are provided in equal concentrations, palmitate is more readily utilized in the synthesis of endogenous TAG stores in the heart. Furthermore, although overexpression of DGAT increases both oleate and palmitate incorporation, the DGAT1 enzyme demonstrates a preference for palmitate. These findings provide insight into the relationship between exogenous FA supply and endogenous TAG dynamics in the contracting heart.

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