Albumin influences expression and function of the membrane transporter P-glycoprotein in HK-2 human proximal tubular cells.
BACKGROUND
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.
Tramonti
Gianfranco
G
Department of Internal Medicine, University of Pisa, Pisa, Italy. g.tramonti@med.unipi.it
Romiti
Nadia
N
Chieli
Elisabetta
E
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D000328:Adult; D000418:Albumins / Q000009:adverse effects*; D000419:Albuminuria / Q000209:etiology / Q000235:genetics* / Q000652:urine; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media / Q000009:adverse effects; D015870:Gene Expression; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000418:Albumins; D003470:Culture Media; D012313:RNA