Figure 1.
Endocytosed albumin co-localized with lysosomes and was degraded in podocytes.
Confocal microscopy image demonstrating co-localization of albumin and lysosomes in a podocyte treated with 1.5 mg/mL FITC-albumin and Cathepsin B Magic Red for 1 hour. FITC- albumin was excited with a 488 nm laser and Magic Red with a 633 nm laser. FITC-Albumin is labeled green (A) and Magic Red, which selectively localizes in lysosomes, is labeled red (B). Yellow fluorescence represents albumin and Magic Red co-localization (C). Scale bar is 20 µm. D) Representative Western blot demonstrating the time course of albumin degradation. Podocytes were treated with 1.5 mg/mL FITC-albumin for 1 h then washed. Cells were harvested at time zero, 15, 30, 45, and 60 min. Albumin abundance in cell lysates was quantified by Western blot analysis using an antibody to FITC. E) Densitometry demonstrating an exponential decrease in abundance of FITC-albumin over 60 minutes normalized to GAPDH and abundance at time zero (n = 4).
Figure 2.
Albumin degradation in podocytes decreased with lysosome inhibition, but not proteasome inhibition.
Densitometry and representative Western blot demonstrating albumin degradation. Podocytes were pretreated with standard media (controls, n = 7), 50 µM chloroquine (lysosome inhibitor, n = 5), or 10 µM MG-132 (proteasome inhibitor, n = 4) for 24 h. At 24 h, podocytes were treated with 1.5 mg/mL FITC-albumin for 1 h then washed. Cells were harvested at time zero, 30 min, and 60 min after washing. A) Albumin abundance in cell lysates was quantified by Western blot analysis using an antibody to FITC. B) Albumin abundance in chloroquine treated cells was significantly increased compared to control and MG-132 treated cells at 30 min (*p<0.05) and 60 min (**p<0.05). Densitometry was normalized to GAPDH and time zero.
Figure 3.
Exposure of podocytes to albumin and chloroquine increased lysosome fluorescence.
Representative individual podocytes are shown for A) negative control B) albumin (5 mg/mL) exposure for 24 h C) chloroquine (50 µM) exposure for 24 h D) albumin (5 mg/mL) + chloroquine (50 µM) exposure for 24 h. After treatment with Magic Red Cathepsin B, individual lysosomes fluoresce red in the presence of active cathepsin B. E),Exposure to albumin and chloroquine increased the density of lysosome fluorescence. The effect was potentiated in podocytes exposed to both albumin and chloroquine. Fluorescence is expressed in arbitrary units. Scale bar is 20 µm. (n = 8 for all treatment groups, * p<0.05 vs control. **p<0.05 vs albumin or chloroquine alone).
Figure 4.
Prolonged exposure of podocytes to albumin and chloroquine decreased lysosome activity.
Lysosome activity in living podocytes was determined by fluorescence recovery after photobleaching (FRAP). Using confocal microscopy, regions of interest containing individual lysosomes were isolated and photobleached. Exponential curves of the recovery of fluorescence intensity, which represents cathepsin B activity, were obtained. A) Representative recovery curves for control (black), 24 h albumin exposed (red), 24 h chloroquine exposed (green), and podocytes exposed to both albumin and chloroquine for 24 h (blue). B) Bar graph of recovery half times (mean +/− SEM), which are inversely related to cathepsin B activity. Cathepsin B activity was decreased in 24 h chloroquine (n = 10) and 24 h albumin (n = 10) exposed podocytes compared to control (n = 10), *p<0.05. Cathepsin B activity decreased further in podocytes exposed to both albumin and chloroquine for 24 h (n = 10) compared to each exposure alone, **p<0.05.
Figure 5.
Chloroquine potentiated the effect of albumin on increased cytokine release in podocytes.
Amount of A) IL-6, B) TNF, and C) IL-1β released into the medium after exposure to 5 mg/mL albumin (white bars), 50 µM chloroquine (grey bars) and 5 mg/mL albumin plus 50 µM chloroquine (black bars). IL-6 (n = 4), TNF (n = 4), and IL-1β (n = 3)were increased in podocytes exposed to both albumin and chloroquine compared to either exposure alone at 18 hrs, *p<0.0001.
Figure 6.
Lysosome inhibition increased podocyte cell death.
Shown are the percentages of dead podocyte cells after 24(5 mg/mL), chloroquine (50 µM), and albumin (5 mg/mL) plus chloroquine (50 µM). Compared to control podocytes (n = 5), cell death was increased in albumin (n = 5) and chloroquine (n = 5) exposed podocytes,* p<0.05. Cell death was greater in podocytes exposed to both albumin and chloroquine (n = 5) compared to either albumin or chloroquine and compared to the control, **p<0.05.
Figure 7.
Albuminuria, glomerulosclerosis, and lysosomal associated membrane protein 1 (LAMP-1) staining was increased in Denys-Drash mice compared to control mice.
Representative periodic acid-Shiff (PAS) staining of glomeruli from A) wild type and B) Denys-Drash mice. Glomerulosclerosis was qualitatively increased in Denys-Drash mice compared to controls. Representative fluorescence staining for LAMP-1 (green) in glomeruli from C) Denys-Drash and wild type mice. LAMP-1 staining was qualitatively increased in Denys-Drash mice compared to controls. LAMP1 staining appeared to be most prominent in podocytes as demonstrated by the increased intensity at the periphery of the glomerulus. The round bodies within the glomeruli that stain non-specifically for actin and LAMP1 are erythrocytes. E) Urine albumin to creatinine ratios were increased at 4 weeks in Denys-Drash (n = 3) compared to wild type (n = 5) mice. (Shown here on a logarithmic scale as mean ±SEM, p = 0.04).