Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin

Abstract

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as “albumin-induced albumin endocytosis”. Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells

“…LLC-PK1 cells were transfected with megalin construct lacking the whole extracellular domain tagged with HA epitope (MegT0-HA) [7] . This approach is well known to study the traffic and half-life of megalin [20] , [24] . EEA1 and LAMP1 were used as markers of early endosomes and lysosomes, respectively.…”

“…LLC-PK1 cells, a well-characterized porcine PT cell line, were cultured in low-glucose DMEM supplemented with 10 % heat-inactivated FBS and 1 % antibiotic-antimycotic at 37 °C in 5 % CO 2 [20] . HEK-293A cells, a parental human embryonic kidney cell line, were cultured in high-glucose DMEM supplemented with 10 % heat-inactivated FBS and 1 % antibiotic-antimycotic at 37 °C in 5 % CO 2 [21] .…”

“…LLC-PK1 cells were stably transfected with MegT0-HA plasmid, which contains a truncated form of megalin that lacks the extracellular domain and is tagged with hemagglutinin (HA), as previously described [20] . Porcine Megalin siRNA (BIAGB-000005) and siGENOME control pool non-targeting (D-001206-13-05) were purchased from Dharmacon (Lafayette, CO, USA).…”

“…BSA-FITC, dextran-FITC or DQ-albumin were used as markers to assess receptor-mediated albumin endocytosis, fluid-phase albumin endocytosis and lysosomal albumin degradation, respectively [20] . Cells were incubated with 100 μg/mL of these compounds for 30 min (BSA-FITC, dextran-FITC) or 60 min (DQ-albumin) at 37 °C.…”

Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway

“…Therein, phosphorylated forms of AKT were detected after HSA stimulation and prevention of HSA protection was achieved with PI3K inhibitors [ 23 ]. Albumin endocytosis has been shown to activate the PI3K/AKT pathway, which in a feed-forward mechanism, stimulated albumin endocytosis [ 24 ]. Precisely, physiological albumin concentrations (0.01 mg/mL BSA) promoted AKT phosphorylation, which peaked at 15 min [ 24 ].…”