Amino Acid Transporter LAT3 Is Required for Podocyte Development and Function

Abstract

LAT3 is a Naϩ-independent neutral L-amino acid transporter recently isolated from a human hepatocellular carcinoma cell line. Although liver, skeletal muscle, and pancreas are known to express LAT3, the tissue distribution and physiologic function of this transporter are not completely understood. Here, we observed that glomeruli express LAT3. Immunofluorescence, confocal microscopy, and immunoelectron microscopy revealed that LAT3 localizes to the apical plasma membrane of podocyte foot processes. In mice, starvation upregulated glomerular LAT3, phosphorylated AKT1, reconstituted the actin network, and elongated foot processes. In the fetal kidney, we observed intense LAT3 expression at the capillary loops stage of renal development. Finally, zebrafish morphants lacking lat3 function showed collapsed glomeruli with thickened glomerular basement membranes. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of selective glomerular permeability. Our data suggest that LAT3 may play a crucial role in the development and maintenance of podocyte structure and function by regulating protein synthesis and the actin cytoskeleton.

Role of amino acid transporter LAT2 in the activation of mTORC1 pathway and the pathogenesis of crescentic glomerulonephritis

“…Anti-mouse LAT3 polyclonal antibody was previously described. 38 The following antibodies were purchased from the suppliers as indicated: anti-phospho-S6 ribosomal protein rabbit monoclonal (91B2) (Ser235/236), anti-p70S6 kinase rabbit polyclonal, anti-phospho-p70S6 kinase rabbit polyclonal (Thr389), anti-4E-BP1 rabbit polyclonal and anti-phospho-4E-BP1 rabbit polyclonal (Tyr37/46) (Cell Signaling Technology, Beverly, MA, USA); anti-vascular endothelial growth factor (VEGF) monoclonal (Novus Biologicals, Littleton, CO, USA); anti-AKT1 phospho-specific (pS473) polyclonal (Rockland, Gilbertsville, PA, USA); anti-b-actin monoclonal (SigmaAldrich, St Louis, MO, USA); goat anti-mouse and goat antirabbit horseradish peroxidase (Dako, Kyoto, Japan) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). Nephritogenic anti-GBM monoclonal antibody (clone b35) was purchased from Chondrex (Redmond, Wash, USA).…”

“…Pure glomeruli were isolated by subjecting the rats to magnetic beads perfusion method as previously described. 38 To isolate pure glomeruli with intact Bowman's capsule, collagenase treatment was excluded. Isolated glomeruli were used for western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR).…”

“…32 Expression of LAT2 has been shown in the epithelium of kidney proximal tubules and the digestive tract. [33][34][35][36][37] LAT3 and LAT4 have been localized to the apical plasma membrane of podocytes 38 and the distal tubules and collecting ducts, 39 respectively. In the present study, we have used a rat model of CGN to investigate the activation of mTORC1 pathway as a possible underlying factor in the crescent formation of glomerular epithelial cells.…”

mTORC1 activation triggers the unfolded protein response in podocytes and leads to nephrotic syndrome

“…22 Cells were cultured in the absence or presence of PAN (5 mg/ml) in a time-dependent manner in RPMI-1640 medium that contained 100 U/ml penicillin/streptomycin supplemented with 5% fetal bovine serum at 37 1C. For mTORC1 inhibition experiment, cells were cultured in the presence of everolimus at the concentration of 5 ng/ml for 30 min before PAN incubation.…”

“…For mTORC1 inhibition experiment, cells were cultured in the presence of everolimus at the concentration of 5 ng/ml for 30 min before PAN incubation. Cells were lysed on ice in lysis buffer, 22 and then protein samples were subjected to western blot analysis as described below.…”