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. 2013 Jul;169(6):1239-51.
doi: 10.1111/bph.12214.

γ-Secretase inhibition promotes fibrotic effects of albumin in proximal tubular epithelial cells

C Slattery  1 , Y Jang, W A Kruger, D H Hryciw, A Lee, P Poronnik
Affiliations

γ-Secretase inhibition promotes fibrotic effects of albumin in proximal tubular epithelial cells

C Slattery et al. Br J Pharmacol. 2013 Jul.

Abstract

Background and purpose: Albuminuria is an important biomarker of renal dysfunction and is a major mediator of renal damage and fibrosis during kidney disease. The mechanisms underlying albumin-induced renal fibrosis remain unclear. There has been significant interest in γ-secretase activity in tubular epithelial cells in recent times; however, its potential role in albumin-induced fibrosis has not been investigated.

Experimental approach: The primary aim of this study was to examine the role of γ-secretase in albumin-induced fibrotic effects in proximal tubular cells. The effects of increasing albumin concentrations on fibrosis indicators and mediators in the human HK-2 cell line were examined in the presence and absence of a γ-secretase inhibitor, compound E.

Key results: Treatment with albumin resulted in a number of pro-fibrotic effects, including up-regulation of fibronectin, TGF-β1 and the EGF-R. Interestingly, similar effects were observed in response to treatment with the γ-secretase inhibitor, compound E. Co-treatment of cells with albumin and an EGF-R inhibitor, AG-1478, resulted in significant inhibition of the observed pro-fibrotic effects, suggesting a major role for the EGF-R in albumin-induced fibrotic events. Albumin-induced effects on the EGF-R appeared to be mediated through inhibition of γ-secretase activity and were dependent on ERK-MAPK signalling.

Conclusions and implications: These results provide novel insights into the mechanisms of albumin-induced fibrotic effects in tubular epithelial cells, suggesting important roles for the γ-secretase and the EGF-R. These results suggest that the proposed use of γ-secretase inhibitors as anti-fibrotic agents requires further investigation.

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Figures

Figure 1
Figure 1
Albumin endocytosis in HK-2 tubular epithelial cells. (A) HK-2 cells were grown to confluence on 48-well plates. Cells were exposed to TR-albumin (50 μg·mL−1) at 24, 48, 72 and 96 h after confluence, for 2 h at which time TR-albumin fluorescence in cell lysates was measured. Data are expressed as % of maximal albumin uptake observed over the time-course. (B) OK cells and HK-2 cells cultured on 48-well plates were exposed to TR-albumin (50 μg·mL−1) in the absence and presence of latrunculin A (2 μM) for 2 h at which time TR-albumin fluorescence in cell lysates was measured. Error bars represent the mean ± SEM of at least three independent experiments. **P < 0.01 and ***P < 0.001 indicate statistically significant difference compared with the control. (C) Equal amounts of whole cell lysates (OK cells and HK-2 cells) were subjected to SDS-PAGE and probed with antibodies for megalin and cubilin. Blots shown are representative of at least three independent experiments. (D) HK-2 cells cultured on coverslip cells were incubated with TR-albumin (50 μg·mL−1) for 2 h at 37°C and then fixed. Fluorescent albumin distribution was assessed by confocal microscopy.
Figure 2
Figure 2
Determination of CpdE working concentration in HK-2 tubular epithelial cells. (A) HK-2 cells grown on 6-well culture plates were exposed to increasing concentrations of CpdE or DAPT for 6 h. Whole cell lysates were prepared in CHAPSO and γ-secretase activity in whole cell lysates was determined using fluorescence-based assays as described in the Methods section. (B) HK-2 cells grown on 96-well culture plates were exposed to increasing concentrations of CpdE for 72 h. Cell viability was assessed using the MTT assay and LDH assays.
Figure 3
Figure 3
Effects of albumin on TGF-β and fibronectin in HK-2 tubular epithelial cells. HK-2 cells grown on 6-well culture plates were treated with control medium, albumin (0.1, 1 or 5 mg·mL−1) or CpdE (100 nM) for 72 h. Cell supernatants were collected and stored at −20°C. (A) The levels of TGF-β1 in cell supernatants obtained were determined by specific elisa. Each value represents the mean ± SEM of three independent experiments performed in duplicate. (B) HK-2 cells on 24 well plates were co-transfected with CAGA-luciferase and pCMV-hRL for 18 h. Cells were treated with control medium, TGF-β1 (5 ng·mL−1), albumin (5 mg·mL−1) or CpdE (100 nM) for 24 or 72 h, and harvested for assay of luciferase activity. Each value represents the mean ± SEM of three independent experiments performed in triplicate after normalization to Renilla luciferase activity. (C) After concentration, cell supernatants were subjected to SDS-PAGE and probed with an antibody for fibronectin. Band intensities were quantified densitometrically and are shown as mean ± SEM of three independent experiments. *Indicates statistically different to control: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4
Effects of albumin on HES1 expression in HK-2 tubular epithelial cells. HK-2 were grown on 6-well culture plates. Cells were treated with control medium, albumin (5 mg·mL−1), CpdE (100 nM) or TGF-β1 (5 ng·mL−1) for 72 h, and to EDTA or EDTA + CpdE for 1 h. Whole cell lysates were subjected to SDS-PAGE and probed with an antibody for HES1. β-Actin levels were determined for normalization. Band intensities were quantified densitometrically and are shown as mean ± SEM of three independent experiments. *Indicates statistically different to control: ***P < 0.001.
Figure 5
Figure 5
Immediate effects of albumin on EGF-R and ERK-MAPK in HK-2 tubular epithelial cells. HK-2 were grown on 6-well culture plates. Cells were treated with control medium or albumin (5 mg·mL−1) for 3 h. Whole cell lysates prepared at 5, 10, 30 and 60 min were subjected to SDS-PAGE and probed with an antibody for (A) p-EGF-R, wc-EGF-R and (B) p-ERK, wc-ERK. β-Actin levels were determined for normalization. Band intensities were quantified densitometrically and are shown as mean ± SEM of three independent experiments (phospho levels were normalized against appropriate whole cell protein levels). *Indicates statistically different to control: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
Sustained effects of albumin on EGF-R and ERK-MAPK in HK-2 tubular epithelial cells. (A) HK-2 were grown on 6-well culture plates. Cells were treated with control medium, albumin (0.1, 1 or 5 mg·mL−1) or CpdE (100 nM) for 72 h. Whole cell lysates were subjected to SDS-PAGE and probed with an antibody for (A) p-EGF-R, wc-EGF-R and (B) p-ERK, wc-ERK. β-Actin levels were determined for normalization. Band intensities were quantified densitometrically and are shown as mean ± SEM of three independent experiments (phospho levels were normalized against appropriate whole cell protein levels). *Indicates statistically different from the control: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7
Figure 7
Effects of EGF-R inhibition on albumin-induced TGF-β1 and fibronectin secretion from HK-2 tubular epithelial cells. HK-2 cells grown on 6-well culture plates were treated with control medium, albumin (0.1, 1 or 5 mg·mL−1) or CpdE (100 nM), in the presence or absence of the EGF-R inhibitor, AG-1784 (250 nM), for 72 h. Cell supernatants were collected and stored at −20°C. (A) The levels of TGF-β1 in cell supernatants obtained were determined by specific elisa. Each value represents the mean ± SEM of three independent experiments performed in duplicate. (B) After concentration, cell supernatants were subjected to SDS-PAGE and probed with an antibody for fibronectin. Band intensities were quantified densitometrically and are shown as mean ± SEM of three independent experiments. *Indicates statistically different from the control: *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 8
Figure 8
Effects of ERK-MAPK inhibition on albumin-induced effects on EGF-R expression and γ-secretase activity. (A) HK-2 cells grown on 6-well culture plates were treated with control medium or albumin (5 mg·mL−1) in the presence or absence of an ERK-MAPK inhibitor, U0126 (10 μM), for 72 h. Whole cell lysates were subjected to SDS-PAGE and probed with an antibody for wc-EGF-R. β-Actin levels were determined for normalization. Band intensities were quantified densitometrically and are shown as mean ± SEM of three independent experiments (normalized for β-actin levels). (B) HK-2 cells grown on 6-well culture plates were treated with control medium, albumin (5 mg·mL−1) in the presence or absence of U0126 (10 μM), or CpdE, for 72 h. Whole cell lysates were prepared in CHAPSO and γ-secretase activity in whole cell lysates was determined using a fluorescence-based assays as described in the Methods section. *Indicates statistically different from the control: *P < 0.05, **P < 0.01, ***P < 0.001.
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