In dendritic cells, FcRn is required to traffic immune complexes to the lysosome for processing [9]. To examine whether FcRn is required to direct ICs to the lysosome in podocytes, we treated WT and FcRn KO podocytes with immune complexes for 4 hours and examined colocalization between ICs and the lysosomal compartment using confocal microscopy. We found that in FcRn KO podocytes, there was significantly less colocalization between ICs and LAMP1-positive (lysosomal) compartments (Fig. 1A), suggesting decreased trafficking of ICs to the lysosomes in the FcRn KO podocytes. We hypothesized that in FcRn KO podocytes, ICs might preferentially be diverted to recycling endosomes. We used Rab11 as a recycling endosome marker and found increased colocalization of ICs and Rab11-positive compartments in FcRn KO podocytes compared to wild type (Fig. 1B), suggesting preferential trafficking of ICs to recycling endosomes in the KO cells.
Lysosomal size and cellular position differ in WT and FcRn KO podocytes
Given the differences in IC trafficking to the lysosome in WT and FcRn KO podocytes, we examined lysosomal size and position in these cells. Previous studies have shown that lysosomal function is modulated by the size and position of lysosomes within the cell, with larger more mature and proteolytically active lysosomes being found closer to the cell nucleus [12, 15, 16]. We examined lysosomal area in WT and FcRn KO podocytes at baseline and after treatment with ICs. We found that at baseline, lysosomal area was significantly decreased in FcRn KO podocytes compared to WT (Fig. 2A), suggesting that in the FcRn KO cells, lysosomes are less active. After treatment with ICs for 4 hours, WT podocytes had a non-significant increase in surface area compared to baseline conditions. In contrast, after IC treatment FcRn KO podocytes had a significant increase in lysosomal area compared to baseline although the average lysosomal area was still significantly decreased compared to WT podocytes treated with ICs (Fig. 2A).
Lysosomal activation results in migration of lysosomes from more peripheral cellular locations to the perinuclear region [17–19]. Given that lysosomes in FcRn KO podocytes have decreased surface area compared to WT suggesting altered lysosomal function in FcRn KO podocytes, we hypothesized that their cellular location in response to an immune challenge would be different. To test this hypothesis, we examined lysosomal distribution in WT and FcRn KO podocytes after an immune challenge. After treatment with ICs for 4 hours, lysosomes in WT podocytes clustered around the nucleus (arrows) whereas in FcRn KO cells, lysosomes remained distributed throughout the cell (Fig. 2B), suggesting that treatment with ICs did not result in lysosomal activation in the knockout.
Colocalization of lysosomes with late endosomal compartments is altered in FcRn KO podocytes
Since trafficking of exogenous proteins to the lysosome involves transit through the late endosome [20], we sought to determine if colocalization of lysosomes and late endosomes was altered in KO podocytes. We examined lysosomal colocalization with late endosomal (Rab7 positive) compartments. After treating podocytes with ICs for 4 hours, we found that there was significantly less colocalization between late endosomal (Rab7+) compartments and lysosomal (LAMP1+) compartments in the KO (Fig. 3).
FcRn KO results in fewer acidic compartments in podocytes and alterations in LAMP1 and cathepsin B activity in podocytes
We next examined lysosomal acidity and expression of the lysosomal membrane protein LAMP1 as well as one of the key lysosomal enzymes, cathepsin B. Lysotracker Red is a cell permeant dye that preferentially accumulates in acidic compartments in living cells [21]. To examine acidic compartments in podocytes in real time, we stained WT and FcRn KO podocytes at baseline and after treatment with ICs with Lysotracker Red. We found that at baseline Lysotracker Red staining was significantly decreased in KO podocytes compared to WT (Fig. 4A). Treatment with ICs increased Lysotracker red staining in KO podocytes but staining was still significantly decreased compared to that in WT podocytes treated with ICs, suggesting decreased lysosomal activation in KO cells.
Given the differences in lysosomal appearance and cellular location, we examined the expression of LAMP1, a lysosomal marker, and cathepsin B, a cysteine protease specific to lysosomes, in WT and FcRn KO podocytes. We treated podocytes with ICs for 4 hours and 48 hours to mimic acute and chronic exposure to an immune challenge. We found that in WT podocytes, LAMP 1 expression was unchanged across any treatment condition at 4 hours (Fig. 4B). Treatment with NP-ovalbumin for 4 hours, resulted in a significant decrease in LAMP1 in KO podocytes compared to WT (Fig. 4B). Treatment with ICs for 4 hours also led to a significant decrease in LAMP1 expression in the KO compared to untreated WT cells. After 48 hours of treatment, there was no difference in LAMP1 expression for WT versus KO for any of the treatment conditions (Fig. 4B), suggesting that KO podocytes upregulated expression of LAMP1 protein after sustained treatment with NP-ovalbumin or immune complexes.
In contrast, treatment of WT and KO podocytes with IgG, NP-ovalbumin or immune complexes did not result in any significant change in cathepsin B expression after 4 hours (Fig. 4B). However, by 48 hours, treatment with IgG, NP-ovalbumin or immune complexes resulted in a significant decrease in cathepsin B expression in WT podocytes compared to KO (Fig. 4B), suggesting that sustained treatment led to downregulation of cathepsin B expression at the protein level in WT podocytes.
Cathepsin B activity is significantly decreased in FcRn KO podocytes after an immune challenge.
Based on the differences in lysosomal size, location and cathepsin B expression at the protein level, we hypothesized that there would also be a difference in cathepsin B activity in WT versus FcRn KO podocytes. We used Cathepsin B Magic Red as this reagent allows determination of the activity of this enzyme in lysosomes in living cells in real time [14]. To examine cathepsin B activity after treatment with immune complexes, WT or FcRn KO podocytes were treated with ICs for 4 or 48 hours. Cathepsin B activity was measured in live cells using fluorescence recovery after photobleaching (FRAP) as previously described [14, 22]. Cathepsin B Magic Red is a fluorophore coupled to a substrate specific for cathepsin B. When the substrate is cleaved by cathepsin B, the fluorophore is released and fluoresces after being excited by the appropriate wavelength. FRAP was performed by bleaching a region of interest containing a lysosome and then the time to recovery of the Magic Red signal was measured (Fig. 5A). The intensity of the Magic Red signal over time relative to the initial intensity was graphed. Representative curves for each condition are shown in Fig. 5B. The half-time (t1/2) of fluorescence recovery is indirectly proportional to cathepsin B activity – i.e. shorter t1/2 to recovery indicates increased cathepsin B activity. After treatment with ICs, FcRn KO podocytes had significantly longer t1/2 to recovery compared to WT podocytes at both 4 hours and 48 hours, indicating decreased cathepsin B activity in the FcRn KO cells (Fig. 5C). Interestingly, while t1/2 recovery was significantly increased in the KO podocytes at both 4 and 48 hours, overall t1/2 recovery was increased in both WT and KO podocytes at 48 hours compared to 4 hours, suggesting an overall decrease in cathepsin B activity with prolonged exposure to ICs.
Signaling pathways differ between WT and FcRn KO podocytes after treatment with ICs
To examine cell survival signaling pathways after treatment with immune complexes, we studied Akt and phospho-Akt expression at baseline and after treatment with IgG, NP-ova or immune complexes. Phosphorylation of Akt at serine 473 (S473) upregulates cell metabolism and growth [23]. There was no increase in phospho-Akt expression compared to baseline after treatment with ICs in KO podocytes whereas WT podocytes showed increased pAkt expression compared to baseline starting 10 minutes after treatment (Fig. 6). Similarly, KO podocytes treated with NP-ovalbumin did not show an increase in p-Akt expression compared to baseline whereas WT podocytes had increased p-Akt expression compared to baseline at 10 minutes (Fig. 6). In contrast, both WT and KO podocytes had increased pAkt expression compared to baseline after treatment with IgG.
We also examined the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway after treatment of WT or FcRn KO podocytes with IgG, NP-ovalbumin or ICs. Phosphorylation of ERK1/2 is a critical step in promoting G1 to S transition and upregulating cell proliferation [24]. We found that phospho-ERK1/2 was significantly upregulated in WT podocytes compared to KO podocytes 10 minutes after treatment with ICs. In contrast to IC treatment, however, we found that after treatment with NP-ovalbumin, phospho-ERK1/2 was significantly upregulated in KO podocytes at 10 minutes compared to WT (Fig. 6). The different signaling responses to IgG alone versus immune complexes suggests that podocytes have the ability to distinguish monomeric IgG from ICs.
To further investigate cell proliferation we performed an EdU assay. As expected, we found that growing cells in phosphate buffered saline (PBS) caused a significant reduction in cell proliferation (Fig. 7). We found that at baseline, KO cells grown in regular media with 10% FBS (standard conditions) had a reduction in cell proliferation compared to WT cells grown in 10% FBS. Interestingly, treatment with immune complexes caused a significant reduction in proliferation in both WT and KO podocytes compared to WT podocytes grown in standard conditions. In KO podocytes, treatment with IgG alone also caused a significant reduction in proliferation compared to WT podocytes grown in standard conditions.