Differential kidney proximal tubule cell responses to protein overload by albumin and its ligands

Abstract

Albuminuria is frequently associated with proximal tubule (PT) cytotoxicity that can feed back to cause glomerular damage and exacerbate kidney disease. PT cells express megalin and cubilin receptors that bind to and internalize albumin over a broad concentration range. How the exposure to high concentrations of albumin leads to PT cytotoxicity remains unclear. Fatty acids and other ligands bound to albumin are known to trigger production of reactive oxygen species (ROS) that impair PT function. Alternatively or in addition, uptake of high concentrations of albumin may overload the endocytic pathway and elicit downstream responses. Here, we used a well-differentiated PT cell culture model with high endocytic capacity to dissect the effects of albumin versus its ligands on endocytic uptake and degradation of albumin, production of ROS, and cell viability. Cellular responses differed dramatically, depending on the preparation of albumin tested. Knockdown of megalin or cubilin failed to prevent ROS production mediated by albumin ligands, suggesting that receptor-mediated internalization of albumin was not necessary to trigger cellular responses to albumin ligands. Moreover, albumin induced cytotoxic responses when added to the basolateral surface of PT cells. Whereas overnight incubation with high concentrations of fatty acid-free albumin had no overt effects on cell function or viability, lysosomal degradation kinetics were slowed upon longer exposure, consistent with overload of the PT endocytic/degradative pathway. Together, the results of our study demonstrate that the PT responds independently to albumin and to its ligands and suggest that the consequences of albumin overload in vivo may be dependent on metabolic state.

Keywords: endocytosis; lipotoxicity; palmitate; proteinuria; proximal tubule; reactive oxygen species.

Fig. 1.

Differential effect of albumin ligands with proximal tubule (PT) endocytic capacity. Opossum kidney (OK) cells were incubated for 3 h (A) or 17–24 h (B) in serum-free medium without (control) or with apically added BSA [20 mg/mL fraction V BSA (FrV-BSA), fatty acid-free BSA (FAF-BSA), or palmitate-loaded BSA (Palm-BSA)] or 50% human serum (HuSer), as noted. Cells were then rinsed several times and incubated for 30 min with Alexa Fluor-647 albumin, and cell-associated fluorescence was quantified by spectrofluorimetry. Data were normalized to the protein recovered in each sample. The mean control uptake of all experiments was set to 100, and each point represents data from an independent experiment. *P < 0.02 and **P ≤ 0.01 by one-way ANOVA (Dunnett’s multiple-comparisons test). C: recovered protein concentrations (means ± SD, normalized to control) from cells treated for 3–48 h with albumin or HuSer as above. ***P ≤ 0.0005 and ****P ≤ 0.0001 by one-way ANOVA (Dunnett’s multiple-comparisons test): 3 h, n = 3; 17–24 h (combined), n = 9; 48 h, n = 5. D: control cells or cells treated overnight with albumin or HuSer and incubated for 30 min with Alexa Fluor-647 albumin (blue) were fixed and processed to detect actin (green). Maximum projections sections of confocal stacks are shown to confirm that exposure to FrxV-BSA or Palm-BSA reduces cellular albumin uptake and alters cell morphology but does not affect the integrity of the monolayer. Scale bar = 10 µm.

Fig. 2.

Albumin ligands differentially trigger mitochondrial oxidative stress. Opossum kidney (OK) cells were incubated for 17–24 h in serum-free medium without (control) or with apically added BSA [20 mg/mL fraction V BSA (FrV-BSA), fatty acid-free BSA (FAF-BSA), or palmitate-loaded BSA (Palm-BSA)] or 50% human serum (HuSer) and then lysed, and aconitase activity was measured and quantified as described in materials and methods. Data from 3 independent experiments performed in duplicate were normalized to untreated controls. Means and SDs are plotted, and each point represents data from a single experiment. *P ≤ 0.05, **P ≤ 0.02, and ****P < 0.0001 by one-way ANOVA (Dunnett’s multiple-comparisons test).

Fig. 3.

Megalin and cubilin receptor knockdown impair albumin uptake but not reactive oxygen species (ROS) production in response to albumin ligands. A: equal amounts of lysate from cells transfected with control, megalin, or cubilin siRNA were blotted with antibodies against megalin or cubilin. B: albumin uptake (1-h incubation, corrected for protein recovery) was quantified in cells transfected with control, megalin, or cubilin siRNA uptake. Means ± SD of triplicate samples are plotted. ****P ≤ 0.0001 vs. control siRNA by one-way ANOVA (Dunnett’s multiple-comparisons test). C: aconitase activity was measured in cells transfected with the indicated siRNA and incubated for 18 h. Means and SDs are plotted; each point represents the mean of duplicate samples from 4 independent experiments. D: protein recovery was quantified in cells transfected with the indicated siRNA and incubated for 18 h in serum-free medium without (control) or with 20 mg/mL apically added fatty acid-free (FAF-BSA) or palmitate-loaded BSA (Palm-BSA). Means and SDs are plotted; each point represents the mean of duplicate samples from 4 independent experiments. ***P ≤ 0.0005 and ****P ≤ 0.0001 by two-way ANOVA (Dunnett’s multiple-comparisons test).

Fig. 4.

Basolateral exposure to BSA impairs apical endocytosis. Opossum kidney (OK) cells were incubated for 3 h (A) or 17–20 h (B) in serum-free medium without (control) or with apically (Ap) or basolaterally (Bl) added BSA [20 mg/mL fatty acid-free BSA (FAF-BSA) or palmitate-loaded BSA (Palm-BSA) or oleic acid-loaded BSA (Olea-BSA)]. Cells were then rinsed several times and incubated for 60 min with apically added Alexa Fluor-647 albumin, and uptake was quantified as described in materials and methods. Data were normalized for protein recovery. Each point represents the average uptake of duplicate samples (normalized to control), and the mean ± SD of 2–3 experiments for each condition is indicated by the bar. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0005, and ****P ≤ 0.0001 by one-way ANOVA (Dunnett’s multiple-comparisons test).

Fig. 5.

Chronic albumin overload slows degradation kinetics. Opossum kidney (OK) cells were pretreated with 20 mg/mL apical fatty acid-free BSA (FAF-BSA) for 72 h or 20 mg/mL Fraction V BSA (FrxV-BSA) or palmitate-loaded BSA (Palm-BSA) was added basolaterally for 3 h. After being washed, cells were incubated with ¹²⁵I-labeled albumin (¹²⁵I-albumin) for 15 min. A: total ¹²⁵I-albumin internalized under each condition (normalized to control; mean ± SD of 5 experiments). *P ≤ 0.05 and ****P ≤ 0.0001 by Kruskal-Wallis test (Dunnett’s multiple-comparisons test). B: cells incubated with FAF-BSA, FrxV-BSA, or Palm-BSA as in A were solubilized, and equal protein loads were Western blotted with anti-megalin antibody. A representative blot is shown, and normalized intensities (means ± range) from 2 independent experiments are plotted. C: cells treated as above were returned to culture after internalization of ¹²⁵I-albumin. Apical and basolateral medium from individual filters was collected, and cells were solubilized after 10, 20, or 30 min. Trichloroacetic acid-soluble ¹²⁵I-dpm are plotted as the percent total for each condition. The mean ± SD of 3 experiments is plotted. *P ≤ 0.05 and **P ≤ 0.01 by two-way ANOVA (Dunnett’s multiple-comparisons test).