Megalin is downregulated via LPS-TNF-α-ERK1/2 signaling pathway in proximal tubule cells
Highlights
► Low-concentration lipopolysaccharide downregulates megalin in proximal tubule cells. ► Lipopolysaccharide-induced TNF-α-ERK1/2 signaling is involved in the pathway. ► Chronic metabolic endotoxemia in diabetes may thus cause reduced megalin functions. ► It is likely to result in urinary loss of proteins and nutrients including vitamins.
Introduction
Diabetic nephropathy (DN) is a worldwide leading cause of end-stage renal disease. Proteinuria/albuminuria is an important clinical sign for the initiation and progression of DN [1], [2] as well as a risk marker of cardiovascular disease (CVD) [3], [4]. Although proteinuria has been generally assumed to be a result of increased permeability of serum proteins (mostly albumin) through glomeruli, it is also attributed to impaired reabsorption of the proteins by proximal tubule cells (PTCs) [5].
Megalin is a large (∼600 kDa) glycoprotein member of the low-density lipoprotein receptor family [6] that is expressed abundantly at the apical membranes of PTCs [7]. Megalin plays a critical role in the reabsorption (endocytosis) of glomerular-filtered proteins including albumin and low-molecular-weight proteins [7]. Vitamin D binding protein is one of megalin’s endocytic ligands, and megalin knockout mice display decreased utilization of vitamin D for its activation in PTCs [8], [9]. Selenoprotein P, a major carrier of selenium, is also endocytosed by megalin and provides selenium required for PTCs to synthesize glutathione peroxidase 3 (GPx3), a circulating antioxidant [10].
Decreased megalin expression in PTCs is found in the early diabetic stages in experimental animal [11], [12]. It is also suggested that the function of megalin is impaired in patients at the early stages of DN, since low-molecular-weight proteinuria are frequently observed in those patients [13], [14]. Impairment of megalin function in patients with DN is also supported by the facts that the patients are often complicated with deficiencies of vitamin D and GPx3 [15], [16], which are associated with the development of CVD [17], [18]. Thus, regulation of megalin expression and its function in PTCs is a key determinant for the early prevention of proteinuria/albuminuria and the risk of CVD in diabetic patients [19]. However, the mechanisms of the regulation are not fully understood.
Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and acts as endotoxin by being released from lysed bacteria. In sepsis, the serum LPS level is increased, which induces severe inflammatory responses in multiple organs including kidney [20]. LPS is filtered by glomeruli and mainly targeted to PTCs via Toll-like receptor 4 located at the apical cell membrane [20], [21]. The serum LPS level is also chronically elevated in subjects with obesity and type 2 diabetes; although it is lower than that in septic patients [22]. Such elavation in serum LPS in those subjects is named “metabolic endotoxemia” [23], which is considered to be consequences of changes in gut microbiota associated with high-fat diet and increased intestinal permeability of bacteria to circulation [24]. Chronic infectious complications such as periodontitis may be another cause of LPS increase in those subjects [25]. However, the effects of low-level LPS on the initiation and progression of DN, in particular, on the endocytic function of PTCs and the mechanisms of proteinuria/albuminuria in DN are not known.
In this study, we investigated low-level LPS-mediated effects on megalin expression and function in immortalized rat PTCs (IRPTCs), in order to elucidate the mechanism of megalin dysfunction that is involved in the development of proteinuria/albuminuria and the risk of CVD in diabetes.
Section snippets
Cell culture
IRPTCs were kindly gifted by Dr. Julie R. Ingelfinger. The cells were maintained in DMEM (low-glucose) supplemented with 5% FCS, 1× non-essential amino acids and 25 mM HEPES at 37 °C and 5% CO2 [26]. Cell culture reagents were obtained from Invitrogen (Carlsbad, CA) unless indicated. The cells were grown to confluence on 6- or 12-well tissue culture plates, washed twice with the culture medium without FCS and serum-starved for 24 h. The cells were then incubated with LPS (10–100 ng/ml)
Incubation of IRPTCs with low-concentration LPS for 48 h reduced megalin protein expression and its endocytic function
In previous studies with cultured renal epithelial cells, LPS was used at high concentrations (1–10 μg/ml) to study its effects on the cells, emulating the condition of septic shock [29], [30]. In this study, however, we used LPS at a lower concentration of 10 (or 100 at most) ng/ml to investigate more chronic effects of LPS on PTCs, which simulated the condition associated with metabolic endotoxemia.
By incubating IRPTCs with LPS at the low concentration for 48 h, megalin protein expression was
Acknowledgments
The authors thank Dr. Julie R. Ingelfinger for providing IRPTCs. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (19590941 and 21591023), and a Grant for Promotion of Niigata University Research Projects.
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Selenium nanoparticles involve HSP-70 and SIRT1 in preventing the progression of type 1 diabetic nephropathy
2014, Chemico-Biological InteractionsCitation Excerpt :Selenium is an essential requirement for effective GPx activity [11]. Major selenium carrying protein, megalin, is also reported to be down regulated in diabetic nephropathy, which further leads to altered antioxidant status [12]. High levels of selenium in blood plasma have been correlated with reducing several types of cancers, cardiovascular diseases, muscle disorders and to a certain extent diabetes mellitus [13].
Acute endotoxemia in mice induces downregulation of megalin and cubilin in the kidney
2012, Kidney InternationalCitation Excerpt :This finding may indicate that the downregulation of megalin and cubilin in response to LPS is independent of systemic and renal hemodynamic or tubular alterations, such as hypotension, reduced renal blood flow, and reduced glomerular filtration, which we and others observed in this in vivo model.26 In subsequent in vitro studies in primary PTCs, endotoxin was clearly shown to decrease the cellular uptake of FITC-labeled albumin, which is in line with previous observations.21,27 The decrease in the cellular uptake of FITC-labeled albumin was paralleled by a decrease in cubilin and megalin mRNA expression.
- 1
Present address: Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Japan.