Distinct functions of megalin and cubilin receptors in recovery of normal and nephrotic levels of filtered albumin

Abstract

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

Keywords: amnionless; cubilin; disabled-2; endocytosis; megalin; proteinuria; proximal tubule.

Fig. 1.

Three pathways mediate dose-dependent albumin uptake by opossum kidney (OK) cells. A: OK cells incubated with 50 µg/mL albumin or 2 mg/mL albumin were fixed and visualized by confocal microscopy. Images were acquired and processed identically, and a maximum projection of a representative field is shown. Scale bar = 10 µm. B: OK cells were incubated with the indicated concentrations of Alexa Fluor 647-albumin at 37°C for 15 min and then rapidly washed and solubilized, and cell-associated albumin was assessed by spectrofluorimetry. Global fitting of >30 experiments was used to resolve the curve into three components: saturable high-affinity (C1; red) and low-affinity (C2; green) uptake components and an underlying nonsaturable component (C3; blue) that we attribute to fluid phase uptake. The half-maximal uptake values for the high-affinity and low-affinity components were 44 and 295 µg/mL albumin, respectively, and the respective maximal uptake capacities in this experiment were 4,883 and 2,494. C: curves shown in B are replotted on a semi-log scale to more easily visualize the three uptake components. The total curve is color coded to show the regions where albumin uptake is dominated by the high-affinity (red), low-affinity (green), or nonsaturable (blue) component. AU, arbitrary units.

Fig. 2.

Efficient knockdown of megalin, cubilin, and disabled-2 (Dab2) impairs uptake of physiological concentrations of filtered albumin. Cells were transfected with the indicated siRNAs and cultured on Transwells as described in materials and methods. Samples were solubilized, and equal protein loads were run on SDS-PAGE and Western blotted to detect cubilin (A), megalin (B), or Dab2 (C). Similar results were observed in 2 independent experiments. Opossum kidney cells transfected with the indicated siRNAs were incubated with 50 µg/mL Alexa Fluor 647-albumin for 15 min, fixed, and then processed to detect actin and cubilin (D), megalin (E), or Dab2 (F). Scale bar = 10 µm. ctrl, control.

Fig. 3.

Knockdown of cubilin, megalin, and disabled-2 (Dab2) differentially affect albumin uptake via high- and low-affinity pathways. The average (black) curves and individual components (C1, red; C2, green; and C3, blue) obtained by fitting the average kinetic profiles of albumin uptake in control (A), cubilin siRNA (B), megalin siRNA (C), and Dab2 siRNA (D) data sets are plotted. AU, arbitrary units.

Fig. 4.

Effect of cubilin, amnionless (AMN), megalin, and disabled-2 (Dab2) knockdown on high- and low-affinity albumin uptake capacity. The albumin uptake profile from each experiment was deconvolved, and high-affinity (V₁; A) and low-affinity (V₂; B) uptake capacities calculated for each condition are plotted as individual points with the mean and SD noted. Statistical significance was assessed by a Kruskal-Wallis test (one-way ANOVA with Dunn’s multiple comparisons test. *P ≤ 0.01, **P < 0.05, and ****P < 0.0001 vs. control (Ctrl). C: ratio of V₂ to V₁ obtained from the mean V₂ and V₁ values from each group is plotted (means ± SE).

Fig. 5.

Megalin takes up albumin independently of cubilin in nephrotic podocin knockout (KO) mice. A−F: immunostaining for cubilin (red), megalin (blue), and albumin (green) in kidney sections from control mice (A), inducible cubilin KO mice (B), inducible megalin KO mice (C), inducible podocin KO mice (D), inducible podocin/cubilin KO mice (E), and inducible podocin/megalin KO mice (F). Confocal images were taken at ×20 magnification. Scale bars = 40 μm. Only megalin- and cubilin-positive cells take up albumin in normal mice; under proteinuria, cubilin expression is dispensable for albumin uptake (but megalin is not).

Fig. 6.

Megalin [low-density lipoprotein-related protein 2 (LRP2)] to cubilin (CUBN) mRNA transcript ratios in opossum kidney (OK) cells and the mouse kidney. Digital droplet PCR was performed using validated primers for LRP2 and CUBN to enable direct quantitation of mRNA transcript ratios in OK cells and in the mouse kidney cortex. Data from RNA isolated from 3 independent experiments are plotted. A: absolute mRNA transcript counts for LRP2 and CUBN in the mouse kidney and OK cells are plotted. B: plot of the LRP2-to-CUBN transcript ratio in the mouse kidney and OK cells. The mean fold difference in LRP2 over CUBN expression was 3.5 ± 0.12 and 1.3 ± 0.06 for the mouse kidney and OK cells, respectively.

Fig. 7.

siRNA knockdown of cubilin (CUBN), megalin [low-density lipoprotein-related protein 2 (LRP2)], or disabled-2 (Dab2) inhibits uptake of a fluid phase marker. Opossum kidney (OK) cells transfected with the indicated siRNAs were incubated with the 250–1000 µg/mL Alexa Fluor 647-dextran for 15 min at 37°C and then rapidly washed and solubilized, and cell-associated fluorescence was quantified. Mean uptake ± SD from 3 independent experiments are plotted, with maximal uptake in control cells normalized to 100%. Uptake at each time point for every condition was significantly different from control by two-way ANOVA (**P > 0.002; ****P < 0.0001).

Fig. 8.

Model for uptake of normal and nephrotic levels of albumin along the proximal tubule. Based on our data and published results by others, we hypothesize that normally filtered albumin is retrieved exclusively in the S1 segment by high-affinity binding to cubilin in association with amnionless (AMN) and/or megalin. Under nephrotic conditions where cubilin-mediated uptake is saturated, the concentration of albumin will increase progressively along the tubule as fluid is reabsorbed via paracellular and transcellular transport mechanisms. We hypothesize that passive uptake of albumin via its incorporation into endocytic vesicles whose formation requires the expression of megalin [and disabled-2 (Dab2)] accounts for the enormous reserve capacity for albumin uptake under nephrotic conditions. See the discussion for more details.