AKT/PKB‐Megalin Positive Feedback Loop Expands Albumin Endocytosis in Proximal Tubule Cells

Abstract

Albuminuria is an independent risk factor for mortality in cardiovascular diseases and is a marker of progression to end-stage renal disease (ESRD). It is accepted that proximal tubule epithelial cells (PTECs) play a central role in the genesis of albuminuria. PTECs reabsorb 3-7g of filtered albumin daily through a receptor-mediated endocytosis being megalin one of the main receptors. Nowadays, one of the main challenges is to understand the regulatory mechanisms involved in this process. In this context, PI3K and AKT have been pointed out as a central mechanism to control albumin endocytosis in PTECs but the mechanism involved is poorly known. Here, we investigated how PI3K/AKT pathway and receptor-mediated albumin endocytosis are interconnected. The incubation of LLC-PK1 cells, a model of PTECs, with albumin 0.01 mg/mL promoted a transient AKT activation measured by its phosphorylation (pAKT) at Ser473 (S473) and by phosphorylation of GSK-3β at Ser9 (S9). Pre-incubation with Pit-Stop-2, an endocytosis inhibitor, prevented AKT phosphorylation (S473) induced by albumin. Using confocal microscopy, the increase of pAKT S473 in subapical vesicles was observed indicating that albumin endocytosis promotes endosomal AKT activation. Acute inhibition of PI3K and AKT by wortmannin (10-6M) and MK-2206 (10-5M) reduced albumin endocytosis capacity and lysosomal delivery by 50%, accessed by BSA-FITC and DQ-BSA uptake, respectively. To study megalin trafficking, LLC-PK1 cells were transfected with a truncated megalin tagged with HA, named MegT0. The inhibition of PI3K/AKT pathway reduced MegT0 surface expression what was followed by an increase in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to the reduction of megalin recycling. Conversely, we studied the possible feedback between PI3K/AKT pathway and receptor-mediated albumin endocytosis. LLC-PK1 cells were pre-incubated with albumin 0.01 mg/mL, washed, and BSA-FITC endocytosis was accessed. Albumin pre-incubation induced receptor-mediated albumin endocytosis what was abolished when PI3K/AKT pathway was inhibited. This result shows that albumin expands albumin endocytosis capacity at physiological concentration. To test this mechanism in vivo, BALB/c micewere i.v. injected with L-lysine, a known inhibitor of megalin-mediated albumin endocytosis in PTECs. L-lysine reduced pAKT S473 and albumin endocytosis, promoting albuminuria. Additionally, wortmannin and MK-2206 incubation reduced albumin endocytosis ex vivo in renal cortex obtained from BALB/c mice. In conclusion, our results suggest a positive feedback between megalin and PI3K/AKT pathway what could be correlated with the capacity of albumin reabsorption in PTECs and the genesis of albuminuria.