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Kidney proximal human tubule HK-2 cell line as a tool for the investigation of P-glycoprotein modulation by natural compounds
No full textABCB1 (MDR1/P-glycoprotein), the best characterized multidrug transporter belonging to the ABC transporter superfamily, is nowadays
acknowledged to have a major impact on drug resistance to chemotherapy and drug bioavailability and disposition. A number of natural
compounds, dietary phytochemicals and herbal remedies have the ability to modulate P-glycoprotein function and /or expression and
therefore the potentiality to cause food-drug, or herb-drug interactions. The elucidation of these interactions may be important not only to
predict possible undesirable effects deriving from the concomitant intake of herbal constituents and conventional drugs, but also for further
studies of positive uses of these interactions as a way to increase the bioavailability of drugs that are P-gp substrates. In particular, the study
of P-gp inhibition by herbal constituents may provide an approach for the identification of lead compounds for the design of news chemosensitizers
to reverse multidrug resistance in tumor cells. This review describes our recent investigations on the potential herb-drug
interactions involving P-glycoprotein, using as a model the HK-2 cells, an immortalized line of proximal tubule cell derived from human
normal kidney
Albumin influences expression and function of the membrane transporter P-glycoprotein in HK-2 human proximal tubular cells
No full textBackground: In proximal tubular cells exposed to albumin
genes encoding membrane transporters were
found to be up-regulated or down-regulated. P-glycoprotein
(Pgp) is an efflux pump which transports a variety
of compounds outside the cell. In the kidney, Pgp
is located mainly on the luminal side of proximal tubular
cells. The aim of this study was to assess whether
albumin overload influences the expression and function
of Pgp in HK-2 cells.
Methods: Tubular cells were cultured in the presence
of albumin (20 mg/mL) for 24 and 72 hours. Pgp expression
was evaluated by Western blot (WB). ABCB1
gene expression was assessed by RT-PCR. Pgp-mediated
transport was measured by the rhodamine-123
(R-123) test.
Results: WB showed decreased protein expression
(-7% after 24 hours and -65% after 72 hours, vs. controls).
RT-PCR showed that gene expression decreased
to 66% after 72 hours of treatment. The fluorescence
of HK-2 cells was 2.4-fold higher compared with controls
(R-123) test. TNF-α restored Pgp expression and
function.
Conclusions: Tubular cells exposed to albumin present
a decrease in both protein and gene expression of Pgp
with impairment in transport function. The overexposure
of tubular cells to toxic substrates due to Pgp
transport failure represents a novel mechanism of tubular
damage linked to proteinuria
Up-regulation of MDR1 P-glycoprotein by 1,25(OH)2D3 in the proximal tubular cell line HK-2
No full textAlbumin influences expression and function of the membrane transporter P-glycoprotein in HK-2 cells
No full textBACKGROUND:
In proximal tubular cells exposed to albumin genes encoding membrane transporters were found to be up-regulated or down-regulated. P-glyco-protein (Pgp) is an efflux pump which transports a variety of compounds outside the cell. In the kidney, Pgp is located mainly on the luminal side of proximal tubular cells. The aim of this study was to assess whether albumin overload influences the expression and function of Pgp in HK-2 cells.
METHODS:
Tubular cells were cultured in the presence of albumin (20 mg/mL) for 24 and 72 hours. Pgp expression was evaluated by Western blot (WB). ABCB1 gene expression was assessed by RT-PCR. Pgp-mediated transport was measured by the rhodamine-123 (R-123) test.
RESULTS:
WB showed decreased protein expression (-7% after 24 hours and -65% after 72 hours, vs. controls). RT-PCR showed that gene expression decreased to 66% after 72 hours of treatment. The fluorescence of HK-22 cells was 2.4-fold higher compared with controls (R-123) test. TNF-alpha restored Pgp expression and function.
CONCLUSIONS:
Tubular cells exposed to albumin present a decrease in both protein and gene expression of Pgp with impairment in transport function. The overexposure of tubular cells to toxic substrates due to Pgp transport failure represents a novel mechanism of tubular damage linked to proteinuria
Influence of drugs on pgp-mediated transport in the human proximal tubule cell line HK-2
No full textA method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma
Full text linkAntibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization
In vitro effects of Mangifera indica and polyphenols derived on ABCB1/P-glycoprotein activity
No full textMany plant-derived compounds, including polyphenols, are able to affect the function of MDR-1/P-glycoprotein (P-gp ABCB1) multidrug transporter, leading to potential herb-drug interactions. This study evaluated the effects of mango (Mangifera indica L.) stem bark extract, MSBE, and related phenols on P-gp activity in both the HK-2 proximal tubule cell line, constitutively expressing P-gp, and in a Caco-2 cell sub-line selected by resistance to vincristine (Caco-2/VCR) and overexpressing P-gp. The effects of MSBE, mangiferin, norathyriol, catechin, quercetin and gallic acid on P-gp activity were tested by the rhodamine-123 accumulation as well as by the Calcein-AM assays. Effects on esterase activity, which could influence the results of Calcein-AM test, were also assessed. All investigated compounds except for catechin and gallic acid inhibited P-gp activity in HK-2 cells, in the order of mangiferin<norathyriol<quercetin<MSBE. MSBE, quercetin and norathyriol also inhibited significantly esterase activity. Similar effects were obtained in resistant Caco-2/VCR cells, but were negligible in the wild-type ones, expressing low amounts of P-gp. Our results demonstrate, for the first time, that M. indica and polyphenols derived may affect the activity of the multidrug transporter P-gp ABCB1, suggesting the possibility of herb-drug interactions to be explored in depth
P-glycoprotein in HK-2 proximal tubule cell line
No full textP-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell fine from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150 kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP
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