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. 2019 Dec;30(12):2370-2383.
doi: 10.1681/ASN.2019030321. Epub 2019 Nov 1.

Proximal Tubule-Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis

Eirini Kefaloyianni  1 , Manikanda Raja Keerthi Raja  1 , Julian Schumacher  1 , Muthu Lakshmi Muthu  1 , Vaishali Krishnadoss  1 , Sushrut S Waikar  2 , Andreas Herrlich  3
Affiliations

Proximal Tubule-Derived Amphiregulin Amplifies and Integrates Profibrotic EGF Receptor Signals in Kidney Fibrosis

Eirini Kefaloyianni et al. J Am Soc Nephrol. 2019 Dec.

Abstract

Background: Sustained activation of EGF receptor (EGFR) in proximal tubule cells is a hallmark of progressive kidney fibrosis after AKI and in CKD. However, the molecular mechanisms and particular EGFR ligands involved are unknown.

Methods: We studied EGFR activation in proximal tubule cells and primary tubular cells isolated from injured kidneys in vitro. To determine in vivo the role of amphiregulin, a low-affinity EGFR ligand that is highly upregulated with injury, we used ischemia-reperfusion injury or unilateral ureteral obstruction in mice with proximal tubule cell-specific knockout of amphiregulin. We also injected soluble amphiregulin into knockout mice with proximal tubule cell-specific deletion of amphiregulin's releasing enzyme, the transmembrane cell-surface metalloprotease, a disintegrin and metalloprotease-17 (ADAM17), and into ADAM17 hypomorphic mice.

Results: Yes-associated protein 1 (YAP1)-dependent upregulation of amphiregulin transcript and protein amplifies amphiregulin signaling in a positive feedback loop. YAP1 also integrates signals of other moderately injury-upregulated, low-affinity EGFR ligands (epiregulin, epigen, TGFα), which also require soluble amphiregulin and YAP1 to induce sustained EGFR activation in proximal tubule cells in vitro. In vivo, soluble amphiregulin injection sufficed to reverse protection from fibrosis after ischemia-reperfusion injury in ADAM17 hypomorphic mice; injected soluble amphiregulin also reversed the corresponding protective proximal tubule cell phenotype in injured proximal tubule cell-specific ADAM17 knockout mice. Moreover, the finding that proximal tubule cell-specific amphiregulin knockout mice were protected from fibrosis after ischemia-reperfusion injury or unilateral ureteral obstruction demonstrates that amphiregulin was necessary for the development of fibrosis.

Conclusions: Our results identify amphiregulin as a key player in injury-induced kidney fibrosis and suggest therapeutic or diagnostic applications of soluble amphiregulin in kidney disease.

Keywords: chronic kidney disease; epidermal growth factor; fibrosis; proximal tubule.

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Figures

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Graphical abstract
Figure 1.
Figure 1.
Low-affinity EGFR ligands induce sustained EGFR phosphorylation in HPTCs. Equimolar amounts (17 pM) of each ligand were used to stimulate HPTCs for the time points indicated. EGFR phosphorylation was examined by (A) Western blot and (B) densitometric analysis was performed after normalization to nonstimulated cells. Tubulin was used for normalization (loading control). Western blots and quantifications shown are representative of three independent experiments.
Figure 2.
Figure 2.
Sustained EGFR reactivation requires transcription and release of endogenous AREG. (A and B) HPTCs were pretreated with DMSO or BB94 (10 ∝M) for 30 minutes and then treated with different low-affinity EGFR ligands for 24 hours. (A) EGFR phosphorylation and downstream ERK1/2 phosphorylation was quantified by Western blot (left panel) followed by densitometric analysis (graph) after normalization to control-stimulated cells (% Control). (B) Endogenous sAREG released in cell culture medium was measured by ELISA and presented as percentile of sAREG concentration in control-stimulated cell medium (% Control). (C) HPTCs were treated with different EGFR ligands (shown in the legend) for 24 hours and mRNA expression of endogenous EGFR ligands (shown in x-axis) was tested by quantitative PCR. Results are presented after normalization to non-stimulated cells (fold control). (D) HPTCs were treated with different EGFR ligands for 4 hours and then culture medium was changed to fresh medium containing AREG-neutralization antibody or vehicle for 20 hours. EGFR phosphorylation was quantified by Western blot (left panel) and by densitometric analysis (right column graph) after normalization to control-stimulated/vehicle-treated cells (% Control). Tubulin was used as loading control in (A and D). For all experiments, n=3–6. *P<0.05; **P<0.01.
Figure 3.
Figure 3.
AREG transcriptional upregulation and sustained EGFR reactivation require YAP1. (A) Cells were control-treated or treated with sAREG for 24 hours and total YAP1 or phospho-YAP1 (pYAP1) levels were examined by Western blot in whole cell lysates (left panel). Quantification of phospho-YAP1 and total YAP1 levels, as well as the ratio of phospho-to-total YAP1, is presented in the graphs after densitometric analysis (right panels). Tubulin was used as loading control. (B) HPTCs were control-treated or treated with sAREG for 24 hours and preparations of nuclear and cytoplasmic fractions were analyzed by Western blot (left panels). Tubulin was used as loading control for cytoplasmic fractions and histone 2A was used as loading control for nuclear fractions. Quantification of total YAP1 levels is presented after densitometric analysis (right panel graph). (C and D) HPTCs were transfected with control siRNA (siControl) or siRNA against YAP1 (siYAP1) and at 48 hours post-transfection, serum-starved cells were control-treated or treated with sAREG for 24 hours. Endogenous AREG and YAP1 expression was tested by quantitative PCR ([C], results presented as fold of siControl/control-treated cells) and release of endogenous sAREG to the cell culture medium was tested by ELISA ([D], presented as percent siControl/control-treated cells). Results are presented after normalization for siControl transfected/control-stimulated cells. (E) Control or siYAP1 transfected cells were treated at 48 hours post-transfection with different EGFR ligands for 24 hours and EGFR phosphorylation was tested by Western blot (left panel). Densitometric analysis is presented after normalization for siControl transfected/control-stimulated cells (right panel graph, stars denote significant difference from the respective ligand-treated siControl sample). (F and G) ADAM17 PTC-KO mice or their ADAM17 WT littermates were subjected to IRI or sham surgery and after 5 days their kidneys were collected and tdTomato+ PTCs were sorted by FACS for subsequent mRNA extraction and quantitative PCR analysis of (F) AREG and (G) YAP1 expression. Results are presented after normalization to PTCs from ADAM17 WT/sham surgery mice (% Control). n=3–4; *P<0.05, **P<0.01.
Figure 4.
Figure 4.
sAREG injection reverses protection from fibrosis in ADAM17 PTC-KO and ADAM17ex/ex hypomorph mice. ADAM17 PTC-KO mice or their ADAM17 WT littermates, or ADAM17 hypomorphic mice (ex/ex) or their wt littermates (wt/wt), were subjected to IRI and received daily i.p. injection of saline or sAREG (18.3 ng/gr body wt) for 4 days, as indicated. Kidneys were collected at day 5. (A) TdTomato-positive PTCs were isolated by FACS and the expression of AREG, YAP1, MCP1, MIP1A, RANTES, and TGFβ was examined by quantitative PCR. Results are presented after normalization to ADAM17 WT mice. (B–E) The profibrotic markers fibronectin and αSMA were examined by IF staining (left panels) and stained area for each protein (% area covered) was quantified (right panels, graphs). White stars in (E) denote αSMA-positive blood vessels that were excluded from the quantification. n=3–4; *P<0.05; **P<0.01.
Figure 5.
Figure 5.
AREG PTC-KO reduces kidney proinflammatory and profibrotic markers after ischemic injury. (A) The efficiency of AREG PTC-KO was tested in KO mice and control littermates (AREG WT) by quantitative PCR for AREG after mRNA isolation from cortical samples. (B) Mice were subjected to IRI and kidneys were collected 5 days postinjury. The profibrotic markers fibronectin and αSMA were examined by immunofluorescence staining (left panel) and stained area for each (% area covered) were quantified (right column graph). (C) The levels of fibronectin are examined in whole kidney lysates by western blot (left panels, tubulin is used as loading control) and quantified by densitometric analysis (right panel graph). (D) The expression levels of TNFα, MCP1, and αSMA were examined by quantitative PCR using whole kidney mRNA extracts. (E) Macrophage infiltration was examined by F4/80 staining at day 5 post IRI ([E], left panel) and stained area (% area covered) was quantified ([E], right panel graph). n=3–4; *P<0.05; ***P<0.001.
Figure 6.
Figure 6.
AREG PTC-KO reduces kidney fibrosis after UUO. AREG PTC-KO mice or AREG WT littermates were subjected to UUO and kidneys were collected after 7 days. (A and B) The profibrotic markers fibronectin and αSMA were examined by immunofluorescence staining (left panels) and the levels of the stained area per field for each marker (% area covered, right panel graphs) were quantified. (C) The levels of fibronectin are examined in whole kidney lysates by Western blot (left panels, tubulin is used as loading control) and quantified by densitometric analysis (right panel graph). (D) Macrophage infiltration was examined by F4/80 staining (left panels) and the levels of the stained area per field (% area covered, right panel graphs). (E) Collagen deposition was quantified after Sirius red staining. (F) Whole kidney mRNA was extracted and the expression of AREG, HB-EGF, TGFA, epiregulin, epigen, and CCN2 was tested by quantitative PCR and presented after normalization to AREG WT mice subjected to UUO (fold control). (G) Tubular injury was scored in kidney sections after periodic acid–Schiff staining as described under Methods. Representative images ([G], left panel) and scoring results ([G], right panel) are shown. n=4–7; *P<0.05; **P<0.01.
Figure 7.
Figure 7.
PTC-derived AREG sustains profibrotic EGFR signals in PTC by amplification of its own signaling and integration of other EGFR ligand signals via a YAP1-dependent positive feedback loop.
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