Expression of glucose transporters in human peritoneal mesothelial cells.
Glucose containing solutions, the basis of peritoneal dialysis fluids, affect the proliferation and regeneration of peritoneal mesothelial cells (MsC). The aim of this study was to examine mechanisms of glucose transport into MsC, that is, the expression of facilitative glucose transporters (GLUT) and the Na(+)-dependent glucose transporter (SGLT1) in human primary MsC and a transfected MsC line. Since expression of both transporters is differentiation dependent, we investigated the effects of cell differentiation induced by culturing MsC on membranes or by addition of hexamethylene bisacetamide (HMBA; 6 mM), which enhances SGLT1 expression in LLC-PK1 cells. Levels of mRNA for GLUT1 through GLUT4 and SGLT1 were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of the corresponding proteins was examined by Western blotting and localized by immunofluorescence. Active, Na(+)-dependent glucose transport was assessed by alpha-methyl-D-[14C]glucopyranoside (AMG) with and without the SGLT1-specific inhibitor phlorizin and by patch clamp experiments in NaCl or choline-chloride, For Na(+) dependent glucose uptake choline chloride instead of NaCl served as negative control. Facilitative transport was assessed using 2-fluoro-2-deoxy-[14C]-D-glucose (FDG) with and without the inhibitors cytochalasin B or phloretin. Primary and transfected MsC express GLUT1 and GLUT3 mRNA while no transcripts were found for GLUT2 and GLUT4. No SGLT1 transcript was detectable in subconfluent cells. Semiquantitative RT-PCR analysis documented that the addition of the differentiation inducer HMBA to confluent cultures or growth of MsC on membranes for seven days produced a down-regulation of mRNA for GLUT1, no change for GLUT3, and a substantial increase for SGLT1 mRNA. Under these conditions MsC express SGLT1 protein and possess a Na(+)-dependent glucose uptake as assessed by AMG. Phlorizin (1 mM) inhibits AMG uptake by 30 to 40%. In patch clamp experiments the addition of extracellular glucose depolarized the membrane potential only in the presence of sodium. These results indicate that differentiated MsC express GLUT1, GLUT3, and SGLT1. Further characterization of these transport mechanisms and their regulation may help to understand the cellular effects of glucose on MsC in peritoneal dialysis.
Schröppel
B
B
Medizinische Poliklinik, Ludwig-Maximilians-Universität, Munich, Germany.
Fischereder
M
M
Wiese
P
P
Segerer
S
S
Huber
S
S
Kretzler
M
M
Heiss
P
P
Sitter
T
T
Schlöndorff
D
D
D001483:Base Sequence; D001693:Biological Transport, Active; D002455:Cell Division; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D010537:Peritoneum / Q000166:cytology* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014162:Transfection
D017931:DNA Primers; D051272:Glucose Transporter Type 1; D051273:Glucose Transporter Type 2; D051274:Glucose Transporter Type 3; D051275:Glucose Transporter Type 4; D008562:Membrane Glycoproteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C495677:SLC2A1 protein, human; C495687:SLC2A3 protein, human; C495692:SLC2A4 protein, human; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; C027020:methylglucoside; D012964:Sodium; D005947:Glucose