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

Abstract

Molecular mechanisms and signaling pathways leading to cellular proliferation and lesion formation in the crescentic glomerulonephritis (CGN) remain elusive. In the present study we have explored a potential role of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway and amino acid transporter (LAT) in the pathogenesis of CGN. Immunohistochemistry and western blot analysis of glomeruli isolated from a rat model of CGN revealed that activation of mTORC1 preceded crescent formation in glomerular parietal epithelial cells (PECs) and podocytes. Daily treatment of rats with the mTOR inhibitor everolimus just after induction of CGN was not beneficial and instead led to increased cellular necrosis of PECs. However, daily treatment starting 7 days after the onset of CGN was beneficial and maintained intact glomeruli. Out of three forms of L-type neutral amino acid transporters (LAT1-LAT3) studied here, only LAT2 was found to be upregulated in the PECs and podocytes in advance of the crescent formation as well as in the crescent lesion itself. Cell culture study revealed that plasma membrane expression of LAT2 markedly stimulated mTORC1 signaling pathway, which was significantly abrogated by coexistence of LAT inhibitor. Finally, LAT inhibitor significantly abrogated development of crescent formation of CGN on day 7. Our data suggest that LAT2 may have a pivotal role in the pathogenesis of CGN by activating the mTORC1 pathway in the glomerular epithelial cells. Crescentic glomerulonephritis (CGN) is the most severe form of glomerulonephritis, and if untreated, progresses to endstage renal failure within days or weeks of diagnosis. Despite different etiologies and clinical manifestations among patients with CGN, there is a common glomerular pathology characterized by the disruption of glomerular basement membrane (GBM), followed by the flow of plasma proteins and inflammatory cells into the Bowman's space. 1 Several studies suggest that proliferating glomerular epithelial cells and accumulation of infiltrated macrophages are the main components of the cellular crescents. 2-6 Recent reports have further revealed that the cellular crescent lesions in CGN consist of podocytes in addition to glomerular parietal epithelial cells (PECs) and macrophages. [7][8][9] It is increasingly evident that proinflammatory cytokines and growth hormones released by proliferating cells in the glomerulus are involved in the pathogenesis of crescent formation. [10][11][12][13] These factors stimulate the p38 mitogen-activated protein kinase (MAPK) pathway, resulting in the production of inflammatory mediators. [14][15][16] The involvement of the MAPK pathway in the pathogenesis of CGN was first reported by Bokemeyer et al, 17 who demonstrated a rapid and sustained activation of extracellular signal-regulated kinase in the glomeruli isolated from rat model of anti-GBM nephritis. A further study demonstrated that both podocytes and the crescent lesion are the main source of p38MAPK activation, although additional signaling path...

Glomerular parietal epithelial cells in kidney physiology, pathology, and repair

“…Indeed, CXCL12 blockade increased renal mRNA expression levels of nephrin and podocin, two podocyte-slit membrane-related proteins that serve as markers of podocyte differentiation [47], further suggesting that pharmacological treatments can be used to modulate PEC growth and/or differentiation into podocytes. Other potential therapeutic targets for PECs in disease might include epidermal growth factor [48] and its receptor [49], as well as the amino acid transporter LAT2 [50]. More recently, Ueno et al [51] showed that inhibiting Notch in vivo in experimental collapsing FSGS reduced the expression of mesenchymal-like markers including alpha smooth muscle actin, vimentin, and snail.…”

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

“…23 Isolated glomeruli were homogenized and lysed on ice in lysis buffer as described above. Protein samples from the glomeruli and cultured podocytes were separated on 7.5% and 4-20% gradient gels under reducing conditions.…”

“…Levels of everolimus in the serum samples were measured using the liquid chromatography/mass spectrometry (LC-MS) system (LCMS-2020, Shimadzu) as recently described 23 but with minor modifications. Briefly, serum samples from the rats treated with everolimus or vehicle on day 5 were taken 2 h after gavage.…”

Mammalian target of rapamycin complex 1 activation in podocytes promotes cellular crescent formation

“…mTORC1 is negatively regulated by Tsc1/Tsc2 through inhibiting Rheb. Abnormal activation of mTORC1 participates in the pathogenesis of many types of kidney disease, including polycystic kidney disease, diabetic nephropathy (12,18,22,25), podocyte dysfunction, glomerular sclerosis, and kidney fibrosis (4,10,11,14,15,27,30). However, whether mTORC1 signaling activation in podocytes contributes to crescent formation remains obscure.…”