PKB and megalin determine the survival or death of renal proximal tubule cells

Abstract

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.

Fig. 1.

PKB interacts with megalin in the luminal membrane. LLC-PK1 cells were grown on Transwell cell culture inserts and transfected with either WT GFP-PKB (a–c) or its mutants GFP-PKB (T308A) (d–f) or GFP-PKB (S473A) (g–i). Blue (anti-ZO-1 antibody) represents tight junctions, green represents GFP, red represents megalin, and yellow (white arrows) represents the overlay of megalin and PKB imagines. ZO-1 staining is shown in all merged pictures, indicating that the pictures are captured at apical level. GFP-PKB and megalin display a punctuated pattern, appearing both near the plasma membrane and in the cytoplasm. [Scale bar, 10 μm (n = 3).]

Fig. 2.

Short-term effect of physiological albumin concentrations on PKB and megalin interactions and PKB activity. LLC-PK1 cells were grown on six-well plates, kept overnight in medium depleted of serum, and incubated with different concentrations of albumin for 5 min. Ctr, control; Wort, 0.5 μM wortmannin; and P-GSK-3, phosphorylated glycogen synthase kinase-3. Albumin concentrations are expressed in mg/ml. PKB was immunoprecipitated with mouse monoclonal PKB antibody where indicated. (A) Effect of albumin on PKB and megalin interactions (n = 4). (B) Effect of albumin on PKB phosphorylation (n = 6). (C) PKB activity measured by phosphorylation of GSK-3, as described in Materials and Methods. (Lower) Densitometry of phospho-GSK-3 (P-GSK-3) (n = 4). (D) Wortmannin and LY29004 reverse the stimulatory effect of albumin on PKB phosphorylation (n = 5). The megalin and phospho-residues bands were quantified and normalized by total PKB.

Fig. 3.

Chronic effects of pathophysiological albumin concentrations on the PKB–megalin interaction. LLC-PK1 cells were grown on a six-well plate, kept overnight in medium depleted of serum in the absence or presence of albumin ranging from 0.01 to 20 mg/ml. Ctr, control. Albumin concentrations are expressed in mg/ml. (A) Effect of albumin on PKB–megalin interactions. PKB was immunoprecipitated with mouse monoclonal PKB antibody followed by immunoblotting for megalin and total PKB (n = 6). (B) Effect of albumin on PKB and megalin levels. Megalin and PKB bands were quantified and normalized by β-actin (n = 6). (C) Effect of albumin on megalin mRNA. Real-time PCR was used to quantify megalin. β-Actin was used as a control (n = 4).

Fig. 4.

Albumin modulates PKB in the plasma membrane. Albumin concentrations are expressed in mg/ml. (A) LL-CPK cells were grown on Transwell cell culture inserts and transiently transfected with GFP-PKB WT. Cells were kept overnight in medium depleted of serum without (a–c) or with (d–f) albumin (20 mg/ml). Blue (anti-ZO-1 antibody) represents tight junctions, green represents GFP, red represents megalin, and yellow (white arrows) represents the overlay of megalin and PKB images. [Scale bar, 10 μm (n = 3).] (B) LLC-PK1 cells were grown on a six-well plate, kept overnight in medium depleted of serum, and incubated for 3 h with 0.5 μM wortmannin (Wort) or for 24 h with 0.01 and 20 mg/ml albumin. The membrane fraction was isolated and immunoblotting for PKB and β-actin performed. Bands of PKB were quantified and normalized to β-actin (n = 4). (C) Chronic effect of albumin on PKB phosphorylation. PKB was immunoprecipitated with mouse monoclonal PKB antibody followed by immunoblotting with anti-phosphoThr-308, anti-phosphoSer-473, or total PKB antibodies. Phosphoresidues were quantified and normalized by total PKB (n = 6).

Fig. 5.

Albumin-induced apoptosis. LLC-PK1 cells were grown on six-well plates, kept for 24 h in medium depleted of serum in the absence or presence of albumin or in the presence of FBS, as indicated. Albumin concentrations are expressed in mg/ml. (A) Chronic effect of albumin on Bad phosphorylation. Phospho-Bad was detected by immunoblotting and normalized by total Bad (n = 4). (B and C) Cells were kept in medium with different concentrations of serum for 24 h in the absence or presence of albumin, as indicated in the figure legends. Apoptosis was measured by using annexin V-FITC as a marker of the early phase of apoptosis. Propidium iodide was used as a marker of cell viability. The stain was quantified by FACS (n = 4). (D) Cells were kept in medium without or with serum for 24 h. Protein extracted and immunoblotted with anti-phosphoThr-308, anti-phosphoSer-473, or total PKB antibodies was performed (n = 3).