Renal proximal tubule cells have a remarkable ability to reabsorb large quantities of albumin through megalin-mediated endocytosis. This is an essential process for overall body homeostasis. Overstressing this endocytic system with a prolonged excess of albumin is injurious to proximal tubule cells. How these cells function and protect themselves from injury is unknown. Here, we show that megalin is the sensor that determines whether cells will be protected or injured by albumin. Megalin, through a novel mechanism, binds PKB in a D-3-phosphorylated phospholipid-insensitive manner, anchoring PKB in the luminal plasma membrane. Whereas low doses of albumin are protective, an overload of albumin decreases megalin expression followed by a reduction of plasma membrane PKB, PKB activity, and Bad phosphorylation induced by PKB. The result is albumin-induced apoptosis. These results reveal a model for PKB distribution in the plasma membrane and elucidate mechanisms involved in both the protective and toxic effects of albumin on proximal tubule cells. In addition, our findings suggest a mechanism for the progression of chronic kidney disease to end-stage renal disease.

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.

The molecular mechanisms involved in the Ang-(1–7) [angiotensin-(1–7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1–7) has a biphasic effect on the proximal tubule Na+-ATPase activity, with the stimulatory effect mediated by the AT1 receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na+-ATPase by Ang-(1–7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT1 receptor-mediated stimulation. In this condition, Ang-(1–7) at 0.1 nM inhibited the Na+-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT2 receptor, and by 1 μM GDP[β-S] (guanosine 5′-[β-thio]diphosphate), an inhibitor of G protein. Ang-(1–7) at 0.1 M induced [35S]GTP[S] (guanosine 5′-[γ-[35S]thio]triphosphate) binding and 1 μg/ml pertussis toxin, an inhibitor of Gi/o protein, reversed the Ang-(1–7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1–7) on the Na+-ATPase activity was completely reversed by 0.1 μM LY83583, an inhibitor of guanylate cyclase, and by 2 μM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1–7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 μM LY83583. Taken together, these data indicate that Ang-(1–7) inhibits the proximal tubule Na+-ATPase by interaction with the AT2 receptor that subsequently activates the Gi/o protein/cGMP/PKG pathway.

The role of albumin reabsorption in proximal tubule (PT) cells has emerged as an important factor in the genesis of albuminuria observed in the early stages of diabetes. Evidence has shown that a decrease in megalin expression could be the key mechanism in this process. In the present work, we investigated the molecular mechanism underlying the modulation of albumin endocytosis in LLC-PK1 cells, a model of PT cells. High glucose concentrations (HG) inhibited megalin expression and albumin endocytosis after 48 h of incubation. This inhibitory effect involves the entrance of glucose into PT cells through SGLT located at the luminal membrane. Once inside PT cells, glucose is diverted to the hexosamine biosynthetic pathway (HBP) increasing O-GlcNAcylation of several intracellular proteins, including PKB. This process promotes the inhibition of PKB activity measured by its phosphorylation at T308 and S473 and phosphorylation of specific substrates, GSK3β and TSC2. The decrease in PKB activity led to a decrease in megalin expression and, consequently, reducing albumin endocytosis in LLC-PK1 cells. HG did not change mTORC2 activity, responsible for phosphorylated PKB at S473. In addition, HG activated the mTORC1/S6K pathway, but this effect was not correlated to the decrease in megalin expression or albumin endocytosis. Taken together, our data help to clarify the current understanding underlying the genesis of tubular albuminuria induced by hyperglycemia in the early stage of diabetes pathogenesis.