EGF Receptor–Dependent YAP Activation Is Important for Renal Recovery from AKI

Abstract

Background

Increasing evidence indicates that renal recovery from AKI stems from dedifferentiation and proliferation of surviving tubule epithelial cells. Both EGF receptor (EGFR) and the Hippo signaling pathway are implicated in cell proliferation and differentiation, and previous studies showed that activation of EGFR in renal proximal tubule epithelial cells (RPTCs) plays a critical role in recovery from ischemia-reperfusion injury (IRI). In this study, we explored RPTC activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ), two key downstream effectors of the Hippo pathway, and their potential involvement in recovery from AKI.

Methods

We used immunofluorescence to examine YAP expression in kidney biopsy samples from patients with clinical AKI and controls (patients with minimal change disease). Studies of RPTC activation of YAP and TAZ used cultured human RPTCs that were exposed to hypoxia-reoxygenation as well as knockout mice (with inducible deletions of Yap, Taz, or both occurring specifically in RPTCs) that were subjected to bilateral IRI.

Results

YAP was activated in RPTCs in kidneys from post-AKI patients and post-IRI mouse kidneys. Inhibition of the interaction of YAP and the TEA domain (TEAD) transcription factor complex by verteporfin or conditional deletion of YAP in RPTCs delayed renal functional and structural recovery from IRI, whereas TAZ deletion had no effect. Activation of the EGFR-PI3K-Akt pathway in response to IRI signaled YAP activation, which promoted cell cycle progression.

Conclusions

This study shows that EGFR-PI3K-Akt–dependent YAP activation plays an essential role in mediating epithelial cell regeneration during kidney recovery from AKI.

The incidence of AKI is estimated to be 15%–20% in hospitalized patients and 50%–70% in patients in the intensive care unit,¹ but few preventive and therapeutic options are available, except for supportive management.² The mortality of patients with severe AKI who need dialysis remains around 40%–70%.^3,4 A common cause of clinically severe AKI is ischemia.⁵ Acute tubular necrosis and apoptosis, glomerular injury, and inflammation are the main pathogenic consequences of ischemia-reperfusion injury (IRI).⁶ The proximal tubular epithelial cells, especially the S3 segment, are the most vulnerable to injury.^7,8 Although some publications have suggested that bone marrow–derived stem cells or an interstitial, nonepithelial stem cell population might serve as a precursor for regenerating tubule epithelial cells, increasing evidence indicates that intrinsic viable epithelial cell dedifferentiation, migration, and proliferation are predominantly responsible for tubular regeneration after AKI.^9–15

Locally produced growth factor–mediated renal epithelial cell dedifferentiation, proliferation, and migration play essential roles in the regeneration of renal epithelial cells after AKI.¹⁶ EGF receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. Binding of the ligands, such as EGF, amphiregulin, or HB-EGF, to EGFR leads to activation of the intrinsic kinase domain and subsequent phosphorylation on specific tyrosine residues within the cytoplasmic tail. EGFR is widely expressed in the mammalian kidney, with high levels of expression in renal proximal tubule epithelial cells (RPTCs).^17,18 Studies by us and others have shown that EGFR expression and activation play a critical role in renal functional and structural recovery from AKI.^19–24

The Hippo signaling pathway is a serine/threonine kinase cascade that controls the balance of cell proliferation, cell differentiation, and cell death, thereby defining organ size and preventing tumorigenesis by phosphorylating and inactivating the downstream effectors Yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ).^25,26 YAP and TAZ share 45% amino acid identity and serve as important transcriptional coactivators in cell nuclei for many transcription factors, with TEA domains (TEADs) being the major YAP-interacting transcription factors.^27–32

Our recent study showed that EGFR activation serves as an upstream cue for YAP expression and activation in diabetic RPTCs.³³ These studies examined the potential role of the YAP activation in EGFR-mediated recovery from acute ischemic injury.

Methods

Materials and Reagents

Erlotinib was purchased from LC Laboratories (Woburn, MA). Antibodies against EGFR (4060S), p-EGFR (3777S), YAP (14074S), TAZ (4883S), pan-TEAD (13295S), Laminin B2 (13823S), Cyclin D (2922S), p-Rb (9307S), Ki67 (9129S), phosphoinositide-dependent kinase 1 (PDK1; 3062S), p-Akt (4060S), Akt (2920S), and β-actin (4970S) were from Cell Signaling Technology (Beverly, MA). LY294002, wortmannin, and antibodies against amphiregulin (AB3152) were from EMD Millipore (Billerica, MA). Antibody against Kidney injury molecule 1 (Kim-1; MAB1817) was from R&D Systems, Inc. (Minneapolis, MN). All secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies were from Life Technologies (Grand Island, NY). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Deidentified Human Kidney Specimens

All sections of human kidney biopsy specimens used in the study were obtained with approval from Department of Nephrology of Huashan Hospital (Shanghai, China). Biopsy specimens of five controls (three women and two men with minimal change disease; ages 22–59 years old; serum creatinine ranged from 45 to 82 μmol/L) and 12 patients with AKI (seven men and five women; ages 29–69 years old; AKI was caused by ischemia, nephrotoxin, or complication of nephrotic syndrome or occurred postkidney transplant with no obvious rejection; the serum creatinine ranged from 240 to 1100 μmol/L) were fixed in paraffin and sectioned at 5 μm. The sections were analyzed by immunofluorescence with antibodies against YAP, and images were captured by using a Leica Confocal Laser Microscope with LAS AF lite software.

Animal Studies

The proximal tubule–specific epithelial cell EGFR deletion mice (EGFR^ptKO) were generated as we have previously described.^18,21 Yap^flox/floxSLC34a1.CreERT2(+), Taz^flox/flox SLC34a1.CreERT2(+), or (Yap/Taz)^flox/flox SLC34a1.CreERT2(+) mice were generated by crossing SLC34a1.CreERT2(+) mice (on mixed C57/Bl6/129 background)³⁴ with the Yap, Taz, or Yap/Taz gene floxed mice (all were on mixed C57/Bl6/Balb/c background).³⁵ At 10 weeks of age, male mice were subjected to peritoneal injection of 120 mg/kg per day of tamoxifen or corn oil for 5 consecutive days to generate inducible RPTC-specific YAP, TAZ, or YAP/TAZ deletion mice (Yap^PTiKO, Taz^PTiKO, or Yap/Taz^PTiDKO) and their respective wild-type control mice. Two weeks after tamoxifen induction, male Yap^PTiKO, Taz^PTiKO, Yap/Taz^PTiDKO, EGFR^ptKO, or wild-type BALB/c mice (Jackson Laboratory, Bar Harbor, ME) treated with or without erlotinib (80 mg/kg per day) were subjected to bilateral renal pedicle clamp for 35 minutes followed by reperfusion as previously described.^21,36 Ten- to 12-weeks-old male Yap^PTWT and Yap^PTiKO mice were subjected to a single dose of cisplatin (25 mg/kg in PBS) or vehicle intraperitoneal injection. The mice were euthanized at 4 days after injection, and kidney tissues were collected for analysis.

Kidney Histology Analyses

Mouse kidneys were harvested and embedded in paraffin at different time points after IRI surgery, and 5-μm tissue sections were stained with hematoxylin and eosin by standard methods. Histopathologic scoring was performed in a blinded fashion in ten consecutive 100× fields per section from different five mice per group, and tubular damage was scored by calculation of the percentage of tubules in the cortical-medullary junction that displayed tubular dilation, tubular epithelium flattening, tubular cast formation, fragments of cells or necrotic epithelium in the tubular lumen, loss of brush border, loss of nuclei, and denudation of the basement membrane. Specifically, zero represents no lesion, one indicates <25%, two indicates 25%–50%, three indicates 50%–75%, and four indicates >75%.

Cell Culture

Human renal proximal tubule epithelial cells (hRPTCs) were purchased from ATCC (Manassas, VA). The cells were maintained in complete growth medium as indicated in the culture methods from ATCC. After 48 hours of culture of the confluent or subconfluent cells, the cells were made quiescent in DMEM/F12 medium with 0.5% FBS for 24 hours. Quiescent cells were incubated in a hypoxic (1% oxygen) chamber for 3 hours followed by reoxygenation for different times to simulate in vivo ischemia-reperfusion conditions.

In Vitro Scratch Wound Assay

Forty-eight hours after control siRNA or YAP1 siRNA transfection, hRPTCs were made quiescent for 24 hours before the assay. A sterile pipette tip was used to make a uniform scratch, and 100 nM EGF was added to the medium immediately after the scratch. Cell images were captured at 24 hours after injury with a Leica Dmirbe Inverted Widefield Microscope (Vanderbilt Medical Center Cell Image Shared Resource Core).

Mouse Proximal Tubule Isolation

The procedure was performed as previously described.^18,37 Specifically, three or four mice per group were euthanized; kidneys were immediately harvested, and mouse cortexes were minced and digested for 30 minutes with 0.03% collagenase (type I; Sigma-Aldrich) in the presence of 0.01% soybean trypsin inhibitor (Sigma-Aldrich) prepared in an isotonic buffer containing 105 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 1.5 mM CaCl₂, 1 mM MgSO₄, 2.0 mM NaH₂PO₄, 10 mM HEPES, 8.3 mM glucose, and 1 mM alanine as well as 0.2% BSA, which has been oxygenated with 95% O₂ and 5% CO₂ at 37°C for 30 minutes. After digestion, the tissue solution was filtered through a 212-μm mesh sieve (Fisher Scientific, Houston, TX). After spinning the pass through at 100×g for 1 minute on a bench top centrifuge, the pellet was resuspended in 30 ml of a 45% Percoll (Sigma Chemical Co., St. Louis, MO) solution in the oxygenated isotonic buffer followed by centrifugation at 20,000×g for 30 minutes at 4°C in a Sorvall centrifuge (Sorvall rotor model: ss-34; Sorvall Products, Newtown, CT). Centrifugation resulted in the separation of four bands. The solution within the third layer containing the proximal tubules was aspirated and centrifuged at 1500 rpm at room temperature for 2 minutes to remove the Percoll solution.

Renal Function Measurement

Mouse blood samples were collected at 1, 2, 4, 7, 10, and 14 days after surgery, and BUN was measured as previously described,³⁸ Serum creatinine was measured by Isotope Dilution LC-MSMS (UAB/UCSD O’Brien Center Core, Birmingham, AL).

Quantitative Real-Time PCR

Total RNA was extracted from isolated renal proximal tubules from wild-type BALB/c mice that were subjected to sham or IRI surgery for 24 hours or 7 days using the miRNeasy Mini Kit (QIAGEN). cDNA was synthesized using the SuperScript III First-Strand Synthesis System (Invitrogen), and quantitative PCR was performed using Power SYBR Green PCR Master Mix (Invitrogen) on the BioRad CFX96 Touch Real-Time PCR Detection System; β-actin was used as an internal control, and relative mRNA levels were normalized to the control sample using the ΔCq method. The sequences of primers used for real-time PCR were mouse Yap1: forward 5′-ACCCTCGTTTTGCCATGAAC-3′, reverse 5′-TTCAACCGCAGTCTCTCCTT-3′ and Actb: forward 5′-CCTCTATGCCAACACAGTGC-3′, reverse 5′-CCTGCTTGCTGATCCACATC-3′.

Transfection of siRNA in hRPTCs

The control and ON-TARGETplus human siRNA SMARTpool: nontargeting Pool (D-001810–10–05), EGFR (L-003114–00–0005), YAP1 (L-012200–00–0005), PDK1 (L-003017–00–0005), and AKT1 (L-003000–00–0005) were purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO). Forty-eight hours after transfection using Effectene Transfection Reagent (QIAGEN), the cells were made quiescent in DMEM/F12 medium with 0.5% FBS for 24 hours. Quiescent cells were incubated in a hypoxic (1% oxygen) chamber for 3 hours followed by reoxygenation for different times to simulate in vivo ischemia-reperfusion conditions. The cells were lysed in RIPA buffer for immunoblotting analysis or fixed for immunofluorescence staining.

Preparation of Cell Nuclear Protein and Cytosol Protein

The procedure was performed as we previously described, with minor modification.³³ Specifically, the isolated renal proximal tubules were further digested in the perfusion buffer (105 mM NaCl, 24 mM NaHCO₃, 5 mM KCl, 2 mM Na₂HPO₄, 1 mM MgSO₄, 1.5 mM CaCl₂, 5 mM d-Glucose, 1 mM l-Alanine, and 10 mM HEPES, pH 7.4) with 1 mg/ml collagenase (type I) for another 15 minutes followed by spinning at 1500 rpm at room temperature for 2 minutes to remove the digestion solution, and then, the isolated mouse RPTCs or cultured hRPTCs after indicated treatments were lysed on ice for 10 minutes in appropriate volumes of lysis buffer A (0.05% Nonidet P-40, 10 mM KCl, 1.5 mM MgCl₂, 10 Mm HEPES, 0.5 mM DTT, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and phosphatase inhibitor cocktail 2, pH 7.4). The cells were gently sonicated followed by centrifugation at 1000×g for 10 minutes at 4°C; then, the supernatant was carefully removed and kept (cytosol protein fraction). The pellets were resuspended in 200–400 μl of buffer B (0.2 mM EDTA, 1.5 mM MgCl₂, 5 mM HEPES, 0.5 mM DTT, 10% Glycerol, 300 mM NaCl, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and phosphatase inhibitor cocktail 2, pH 7.4), and they were sonicated again and kept on ice for 30 minutes for further lysis. Then, the lysates were centrifuged at 14,000×g for 10 minutes at 4°C, and the supernatant was retained (nuclear protein fraction).

Immunoprecipitation and Immunoblotting

Procedures were performed as we previously described.^18,38

Immunofluorescence Analysis

Immunofluorescence was performed on paraffin-embedded tissues or cultured hRPTCs fixed by 4% paraformaldehyde using standard techniques as previously described,^18,33 and images were captured using an Olympus FV1000 Invert Confocal Microscope (Vanderbilt Medical Center Cell Image Shared Resource Core).

Statistical Analyses

Data are presented as means±SEM for at least three separate experiments (each in triplicate). An unpaired t test was used for statistical analysis, and for multiple group comparisons, ANOVA with Bonferroni corrections using GraphPad Prism version 7 was used. A P value of <0.05 compared with control was considered statistically significant.

Results

YAP Was Activated in Ischemic RPTCs

YAP activation by suppression of the Hippo signaling pathway by growth factors or mechanical stress has been shown to play an important role in both cancer pathogenesis and tissue repair after injury.^39–50 To explore the potential role of YAP activation in kidney recovery from AKI, we used immunofluorescence to examine YAP expression in kidney biopsy samples from patients with clinical AKI. YAP expression was minimally detected in the RPTC nucleus in the kidneys of patients with minimal change disease (used as controls), but nuclear distribution of YAP in the RPTC was enhanced in the kidneys of patients with AKI, which is indicated by costaining with the proximal tubule marker LTA (Figure 1A, Supplemental Figure 1).

Figure 1.

YAP was activated in RPTC of post-AKI patient and post-IRI mouse kidneys. Increased Yes-associated protein (YAP) expression and nuclear distribution in post-AKI kidneys from patients and ischemia-reperfusion–injured mouse kidneys. (A) Representative immunofluorescence staining images of a control biopsy kidney specimen from a woman with minimal change nephropathy (32 years old; serum creatinine was 82 μmol/L) and a kidney specimen from an man with AKI (53 years old with ischemia-induced AKI; serum creatinine was 230 μmol/L, which was down to 60 μmol/L after 3 weeks of hemodialysis). Arrows indicate nuclear YAP-positive renal proximal tubule epithelial cells (RPTCs). Blue, DAPI; green, LTA; red, YAP. (B–E) Nine- to 10-week-old wild-type BALB/c mice (n=7–10 mice per group) were subjected to bilateral ischemia for 35 minutes followed by reperfusion for 24 hours or 7 days. (B) YAP protein and (C) mRNA increased after ischemia-reperfusion injury (IRI), and (D) immunohistochemistry and (E) immunofluorescence confirmed increased tubular expression and nuclear localization. Values are mean±SEM (n=5–10 for each group). Original magnification, ×100 in D, upper panel; ×400 in D, lower panel; ×600 in E. *P<0.05; ^#P<0.01.

We determined that YAP protein expression increased in mouse renal cortical tissue lysates at 24 hours post-IRI and was still elevated at 7 days after injury (Figure 1B). YAP mRNA in isolated RPTC was also upregulated at both 24 hours and 7 days post-IRI (Figure 1C). YAP protein expression upregulation in RPTCs was also detected in another model of AKI, secondary to cisplatin injection (Supplemental Figure 2). The YAP upregulation in response to IRI was primarily located in the renal tubule epithelial cell nuclei, although there was moderate positive staining of YAP in some peritubular cell nuclei (Figure 1D). In nonstressed mouse kidney, there was low expression of YAP in RPTC nuclei, although noticeable YAP could be detected in the distal convoluted tubules, indicated by colocalization with calbindin (Supplemental Figure 3A), and thick ascending limb tubules, indicated by colocalization Tamm–Horsfall protein (Supplemental Figure 3B) in control mice. After IRI, YAP expression in proximal tubules was dramatically enhanced, with increased nuclear distribution (Figure 1E).

Activation of YAP in RPTCs Promoted Renal Recovery from AKI

To define the pathophysiologic function of YAP activation in the RPTC in response to IRI, mice received intraperitoneal administration of a well characterized inhibitor of YAP-TEAD interactions, verteporfin (100 mg/kg every other day).^33,51,52 Verteporfin effectively inhibited YAP-TEAD association in response to IRI (Figure 2A) and delayed functional recovery compared with vehicle-treated mice (BUN: 21.3±2.4 versus 55.9±6.9 mg/dl; serum creatinine: 0.18±0.015 versus 0.26±0.023 mg/dl; n=5 at 7 days post-IRI) (Figure 2, B and C). Kim-1 expression in the proximal tubules is known to be markedly upregulated in the acute injured kidney.⁵³ As shown in Figure 2D, comparable Kim-1 upregulation was detected in mice with or without verteporfin 24 hours after IRI, but Kim-1 expression remained significantly higher at 7 days in the verteporfin-treated group. There was also comparable histologic injury at 24 hours, but more severe proximal tubule injury in the verteporfin-treated mice could be seen at day 7 post-IRI compared with the vehicle-treated mice (Figure 2E).

Figure 2.

Blocking Yes-associated protein (YAP)-TEA domain (TEAD) association delayed renal recovery from ischemia-reperfusion injury (IRI). Nine- to 10-weeks-old wild-type BALB/c mice (n=7–10 mice per group) were subjected to bilateral ischemia for 35 minutes followed by reperfusion for 24 hours or 7 days with or without administration of verteporfin (Vert; intraperitoneally, 100 mg/kg every other day starting after the mice recovered from anesthesia). (A) Immunoprecipitation studies indicated that Vert inhibited YAP interaction with TEAD and delayed functional recovery from IRI as indicated by the time course of (B) BUN and (C) serum creatinine determined at 7 days postsurgery. Vert also led to prolonged Kidney injury molecule 1 (Kim-1) expression in isolated mouse renal proximal tubule epithelial cells after AKI. (D) Representative histology of kidneys at days 2 and 7 after IRI (E) indicated more severe injury in the Vert-treated group, and tubule injuries were scored as described in Methods. Values are mean±SEM (n=5–10 for each group). Veh, vehicle. Original magnification: ×100 in E, upper panel; ×400 in E, lower panel. *P<0.05; ^#P<0.01.

Furthermore, the Yap^PTiKO or Yap^PTWT mice were subjected to bilateral IRI 2 weeks after tamoxifen or vehicle injection as shown in the schematic (Figure 3A). Immunoblotting analysis of isolated mouse renal proximal tubule cell lysates indicated that YAP expression was significantly decreased in the sham-operated Yap^PTiKO mice, and upregulation of YAP expression at 48 hours post-IRI was markedly inhibited in the isolated RPTC from Yap^PTiKO mice (Figure 3B). In addition, analysis of cytoplasmic and nuclear proteins prepared from isolated mouse renal proximal tubules showed inhibition in the Yap^PTiKO mice YAP nuclear distribution in response to IRI (Figure 3C), and immunofluorescence staining of kidneys confirmed decreased YAP nuclear translocation in the RPTC in the Yap^PTiKO mice in response to IRI (Figure 3D). Similar to verteporfin administration, selective proximal tubule YAP deletion delayed functional recovery (BUN: 29.2±2.1 versus 52.7±3.5 mg/dl; serum creatinine: 0.165±0.011 versus 0.21±0.010 mg/dl at 7 days post-IRI; n=6) (Figure 3, E and F). Kim-1 expression in isolated RPTC was also comparable 24 hours after IRI but remained significantly higher in the Yap^PTiKO mice compared with the Yap^PTWT mice at 7 days (Figure 3G). There was more severe kidney injury in the Yap^PTiKO mice at 7 days after IRI compared with that in the Yap^PTWT mice (Figure 3H).

Figure 3.

Inducible Yes-associated protein (YAP) deletion in renal proximal tubule epithelial cells (RPTCs) delayed renal recovery from ischemia-reperfusion injury (IRI). (A) Schematic of the generation of Yap^PTiKO and Yap^PTWT mice. (B) Decreased YAP expression from Yap^PTiKO mice subjected to sham or IRI that were studied after 48 hours. (C and D) IRI increased nuclear expression of YAP in Yap^PTWT mice 48 hours after sham or IRI surgery that was inhibited in Yap^PTiKO mice. After IRI, Yap^PTiKO mice had delayed functional recovery, indicated by time courses of (E) BUN and (F) serum creatinine 7 days postsurgery as well as (G) persistent Kidney injury molecule 1 (Kim-1) expression in isolated RPTCs. (H) There was also more severe histologic injury at days 2 and 7 after IRI in Yap^PTiKO mice (tubule injury was scored as described in Methods). Values are mean±SEM (n=5–10 for each group). IR, ischemia-reperfusion. Original magnification, ×600 in D; ×100 in H, upper panel; ×400 in H, lower panel. *P<0.05; ^#P<0.01.

YAP Activation in the RPTC Was Dependent on EGFR Activation in Response to AKI

YAP is inhibited by activation of the Hippo pathway kinase cascade through its phosphorylation at Ser127 directly by Lats.^54,55 However, we did not found any alteration of Lats phosphorylation in response to AKI (Figure 4, A and C). Our previous studies showed that EGFR activation is essential for renal recovery from AKI.²¹ We have also found that YAP was activated through an EGFR activation-dependent pathway in diabetic RPTC.³³ Therefore, we treated wild-type mice with the EGFR kinase inhibitor erlotinib, and we found that erlotinib not only inhibited EGFR and Akt phosphorylation but also, inhibited increased expression of both YAP and the EGFR ligand amphiregulin, a YAP target gene product^34,56 (Figure 4A). In addition, erlotinib treatment attenuated RPTC YAP nuclear translocation in response to IRI (Figure 4B). Expression of both YAP and amphiregulin and YAP nuclear translocation in the RPTC were also markedly inhibited in mice with conditional RPTC-specific EGFR deletion (EGFR^ptKO)^18,21 (Figure 4, C and D).

Figure 4.

Yes-associated protein (YAP) expression and nuclear translocation in renal proximal tubule epithelial cells (RPTCs) in response to ischemia-reperfusion injury (IRI) were EGF receptor (EGFR) activation dependent. (A and B) Administration of the EGFR tyrosine kinase inhibitor erlotinib by gavage (80 mg/kg per day starting 1 day before surgery) inhibited renal EGFR and AKT phosphorylation and amphiregulin expression. Lats phosphorylation was not altered in response to (A) IRI and (B) YAP nuclear expression. (C and D) Selective proximal tubule deletion of EGFR (EGFR^ptKO) also inhibited renal EGFR and Akt phosphorylation and amphiregulin expression, but (C) Lats phosphorylation was not altered in response to IRI. (D) YAP nuclear distribution in proximal tubule cells was deleted in the EGFR^ptKO mice (arrows indicate the cells in which EGFR was not completely deleted). Original magnification, ×600 in B and D. *P<0.05; ^#P<0.01.

To mimic IRI, cultured hRPTCs were exposed to hypoxia for 3 hours followed by reoxygenation for either 3 or 8 hours. YAP nuclear translocation increased in both nonconfluent (Figure 5A) and confluent cells (Supplemental Figure 4). Silencing EGFR expression by siRNA (Figure 5B) or inhibition of EGFR tyrosine kinase activity by treatment with erlotinib (Figure 5C, Supplemental Figure 4) decreased YAP nuclear translocation in response to hypoxia-reoxygenation. YAP primarily localized in cytosol, with minimal distribution in the nucleus before exposing the cells to hypoxia-reoxygenation. Distribution of YAP in the nucleus was increased, whereas cytoplasmic YAP protein was somewhat reduced in response to hypoxia-reoxygenation, and these changes were inhibited by pretreatment with erlotinib (Figure 5D).

Figure 5.

In cultured human renal proximal tubule epithelial cells (hRPTCs), Yes-associated protein (YAP) nuclear translocation in response to hypoxia-reoxygenation was EGF receptor (EGFR) activation dependent. (A) Subconfluent hRPTCs exposed to hypoxia for 3 hours followed by reoxygenation for 8 hours increased YAP nuclear translocation. (B) Silencing EGFR by siRNA-inhibited YAP nuclear translocation in response to hypoxia-reoxygenation in subconfluent hRPTC. (C) Inhibition of EGFR tyrosine kinase activity by treatment with erlotinib (Erl; 100 nM) inhibited YAP nuclear translocation in response to hypoxia-reoxygenation in the hRPTCs. (D) Erl treatment inhibited YAP nuclear distribution in response to hypoxia for 3 hours followed by reoxygenation for 3 or 8 hours. Ctrl, control; Veh, vehicle. Original magnification, ×600 in A–C. ^#P<0.01.

Phosphatidylinositol 3-Kinase–Akt1 Activation Mediates EGFR-Dependent YAP Activation in Response to AKI

Our previous study indicated that EGFR activated the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in response to IRI.²¹ A recent study in a cultured human mammary epithelial cell line suggested that activation of PI3K-PDK1 in response to EGF treatment activated YAP by suppressing the Hippo pathway.⁵⁷ We treated hRPTCs with two different PI3K inhibitors, LY294002 and wortmannin, and we found that both inhibitors not only inhibited Akt phosphorylation but also blocked YAP nuclear translocation in response to hypoxia-reoxygenation (Figure 6, A and B). The hRPTCs were transfected with PDK1 siRNA or AKT1 siRNA, and we found that silencing AKT1 but not PDK1 blocked YAP nuclear translocation (Figure 6, C and D). Moreover, either LY294002 treatment or AKT1 siRNA transfection reversed YAP nuclear translocation in response to hypoxia-reoxygenation (Figure 6E).

Figure 6.

In cultured human renal proximal tubule epithelial cells (hRPTCs), Yes-associated protein (YAP) nuclear translocation in response to hypoxia-reoxygenation was mediated by phosphatidylinositol 3-kinase (PI3K)-Akt but not by PI3K–phosphoinositide-dependent kinase 1 (PDK1) activation. (A) In hRPTCs, the PI3K inhibitors LY294002 (LY; 25 μM) and wortmannin (Wt; 100 nM) inhibited the increased Akt phosphorylation in hRPTC in response to hypoxia. (B) In confluent hRPTCs, LY or Wt inhibited YAP nuclear translocation induced by hypoxia-reoxygenation. (C) In hRPTC, transfection of PDK1 or AKT1 siRNA effectively silenced their respective protein expression. (D and E) YAP nuclear translocation induced by hypoxia-reoxygenation was inhibited by transfection of AKT1 but not PDK1 siRNA or by LY treatment. *P<0.05; **P<0.01. Ctrl, control; Veh, vehicle. Original magnification, ×600 in B and D.

Increased Cyclin D Expression and Retinoblastoma Protein Phosphorylation in AKI Were Dependent on YAP Activation

Dedifferentiation and proliferation of the surviving RPTCs play a critical role for kidney repair for mild to moderate AKI.^9,34,58–60 Sequential synthesis, activation, compartmentalization, and degradation of different cyclins and cyclin-dependent kinases are critical for controlling both entry into and exit from each cell cycle phase during cell proliferation.⁶⁰ Previous microarray-based expression profiling analysis suggested that large portions of YAP regulatory genes are conserved and that they are associated with the cell cycle.⁶¹ Activation of cyclin D–cyclin-dependent kinases 4 and 6 followed by phosphorylation of retinoblastoma protein (Rb) is important for the G1 to S phase transition.^56,62 We found increased cyclin D expression and increased Rb phosphorylation in response to IRI, both of which were inhibited in the verteporfin-treated mice and the Yap^PTiKO mice (Figure 7, A and B). A recent study showed that expression of amphiregulin was upregulated in kidneys of patients with AKI and acutely injured mouse kidneys.⁶³ We found that amphiregulin upregulation in response to IRI was also attenuated by administration of verteporfin or Yap gene deletion in RPTC (Figure 7, A and B). Cyclin D expression, Rb phosphorylation, and amphiregulin expression also increased in the isolated RPTC lysates from cisplatin-injected Yap^PTWT mice and were attenuated in the Yap^PTiKO mice (Supplemental Figure 2). The increased cyclin D expression and Rb phosphorylation in response to IRI were EGFR dependent, because they were inhibited in the EGFR^ptKO mice (Figure 7C).

Figure 7.

Amphiregulin and cyclin D expression and retinoblastoma protein (Rb) phosphorylation in kidney in response to ischemia-reperfusion injury (IRI) were mediated by EGF receptor (EGFR)–Yes-associated protein (YAP) signaling pathway activation. (A) Verteporfin or (B) selective proximal tubule deletion of YAP inhibited increased amphiregulin and cyclin D expression and Rb phosphorylation 48 hours post-IRI. (C) Proximal tubule deletion of EGFR also inhibited post-IRI increases in cyclin D and Rb phosphorylation. (D) Forty-eight hours post-IRI, Yap^PTiKO had decreased expression of Ki67 (a marker of cell proliferation). Green, LTA; purple, DAPI; red, Ki67. (E) siRNA knockdown of YAP in human renal proximal tubule epithelial cells (hRPTCs) decreased amphiregulin and cyclin D expression and Rb phosphorylation. (F) siRNA knockdown of YAP in hRPTC inhibited cell migration/proliferation in response to EGF (100 nM) in scratch wound assays. Cell images were captured at 24 hours. Original magnification, ×200 in D, columns 1 and 3; ×600 in D, columns 2 and 4; ×100 in F. Ctrl, control. ^#P<0.01 (compared between groups with or without inhibitory treatments).

RPTC proliferation indicated by Ki67 expression, a cell proliferation marker, decreased in the Yap^PTiKO mice 48 hours after IRI, although increased interstitial cell proliferation could still be detected in the Yap^PTiKO mice (Figure 7D). In cultured hRPTCs, we also observed upregulation of amphiregulin, cyclin D, and phospho-Rb 24 hours after hypoxia-reoxygenation, all of which were inhibited by silencing YAP expression (Figure 7E). Knocking down YAP expression also inhibited cultured hRPTC migration in response to exogenous EGF treatment after in vitro scratch injury (Figure 7F).

Activation of YAP but Not TAZ Promoted Renal Repair from IRI

The two key effectors of Hippo signaling pathway, YAP and TAZ, play redundant roles in some tissues.^47,64 To determine whether TAZ was also essential for renal recovery from IRI, we generated Taz^PTiKO or Yap/Taz^PTiDKO mice. As indicated in Figure 8A, the expression of TAZ or expressions of both TAZ and YAP were effectively deleted in the isolated renal proximal tubule lysates. Comparable injury developed in wild-type, Taz^PTiKO, or Yap/Taz^PTiDKO mice within 24 hour post-IRI (Figure 8, B–E), but BUN, serum creatinine, and Kim-1 expression were still much higher in Yap/Taz^PTiDKO mice than in the wild type or Taz^PTiKO (Figure 8, B–D). Histologic recovery from IRI was delayed only in the Yap/Taz double-gene deletion mice but not in the Taz single-gene deletion mice (Figure 8E), indicating that YAP but not TAZ is critical for renal recovery from IRI and suggesting that YAP and TAZ do not play redundant roles in response to IRI.

Figure 8.

YAP, but not TAZ activation in RPTC promoted kidney repair from AKI. Inducible Yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ) double-gene deletion but not Taz single-gene deletion in renal proximal tubule epithelial cells (RPTCs) delayed renal recovery from ischemia-reperfusion injury (IRI). (A) Effective deletion of either TAZ or both YAP and TAZ in RPTCs isolated from Taz^PTiKO or Yap/Taz^PTDiKO mice. (B and C) TAZ deletion alone did not affect functional recovery from IRI as determined by time course of (B) BUN or (C) serum creatinine 7 days post-IRI, and (D) there was persistent Kidney injury molecule 1 (Kim-1) expression in isolated RPTCs. (E) Selective deletion of TAZ alone did not alter structural recovery, but delayed recovery with combined YAP/TAZ deletion was similar to that seen with selective YAP deletion alone. Values are mean±SEM (n=5–10 for each group). (Original magnification, ×100 in E, rows 1 and 3; ×400 in E, rows 2 and 4. *P<0.05; ^#P<0.01.

Discussion

This study explored the potential role of YAP in renal recovery from AKI. We detected YAP nuclear translocation, a well known indicator of YAP activation, in RPTCs of kidneys of patients with AKI resulting from different insults. Similarly, YAP protein expression and nuclear YAP localization increased in ischemic mouse RPTCs within 24 hours after injury. Both selective renal proximal tubule EGFR deletion and administration of an EGFR tyrosine kinase inhibitor erlotinib inhibited YAP expression and nuclear translocation in response to IRI. Moreover, inducible deletion of YAP specifically in the RPTCs or administration of the YAP-TEAD association inhibitor verteporfin significantly delayed renal functional and structural recovery from IRI. The protective effects of YAP activation in the RPTCs were mediated by EGFR activation of PI3K/Akt. YAP activation promoted cell cycle progression through upregulation of cyclin D expression and Rb protein phosphorylation post-IRI as illustrated in Figure 9. In response to AKI, EGFR was mediated by YAP but not TAZ activation, because selective deletion of proximal tubule TAZ expression did not affect recovery from AKI.

Figure 9.

Suggested mechanism of Yes-associated protein (YAP) activation and its role in promoting kidney recovery from AKI. In response to IRI, EGF receptor (EGFR)-Akt activation mediates YAP activation and subsequent downstream target genes cyclin D and amphiregulin (AREG) expression, which activate cell proliferation and migration by phosphorylating retinoblastoma protein (Rb) and EGFR respectively, thereby promoting kidney recovery. In addition, YAP activation dependent expression of AREG serving as an EGFR ligand further activates EGFR-Akt dependent YAP activation. IRI, ischemia-reperfusion injury; Rb, retinoblastoma protein.

Activation of the canonical Hippo signaling pathway results in YAP phosphorylation and consequent sequestration of the phosphorylated YAP in the cytoplasm, thereby permitting degradation by the proteasome system.^54,65,66 YAP and TAZ play some redundant roles in the morula stage of mouse development and in controlling adult cardiac growth,^47,64 but they also have different or complementary functions in many situations. TAZ is expressed in all tissues, with the highest expression in the kidney, thymus, and peripheral blood leukocytes.²⁷ Under normal conditions, YAP expression is lower than TAZ in the kidney, with the exception of the podocyte.⁶⁷ However, studies by us and others^39,68 have shown that YAP expression and activation increase in response to chronic kidney injury and mediate progressive tubulointerstitial fibrosis. In contrast, our studies show an essential role for YAP activation to mediate renal epithelial cell regeneration after acute injury.

YAP plays distinct roles in kidneys that are not redundant with TAZ, because we only detected increased expression and nuclear translocation of YAP, but not TAZ, in the RPTCs in response to AKI. In data not shown, we did find upregulation of TAZ expression in isolated renal proximal tubules from the Yap^PTiKO mice and upregulation of YAP expression in isolated renal proximal tubules from the Taz^PTiKO mice, suggesting that there may be a reciprocal balance between the expression of YAP and TAZ in the RPTCs, but they are not necessarily able to compensate for each other. We speculate that TAZ expression may be important for maintaining structural integrity of normal renal tubules.^69–71

In these studies, all animals were subjected to moderate AKI. In wild-type mice, renal function almost completely recovered within 1 week, although histology still indicated some level of tubule dilation and epithelial simplification. With renal proximal tubule cell YAP deletion, renal function did not return back to normal until 2 weeks after injury, and there was more severe tubule dilation and epithelial simplification at both 2 and 7 days postsurgery. There is increasing evidence that severe AKI can transition to CKD; whether the delayed kidney recovery in Yap^PTiKO or Yap/Taz^PTiDKO mice ultimately will progress to chronic kidney injury will require further study.

Our previous study found activation of both EGFR-dependent PI3K-Akt and ERK1/2 signaling pathways in the RPTCs in response to ischemic insult.²¹ We believe that activation of both EGFR-PI3K-Akt and EGFR-MEK-ERK1/2 pathways is essential for renal recovery from IRI, because it is well known that PI3K-Akt activation is closely related to cell survival and migration and that ERK1/2 signaling pathway activation triggers cell proliferation. In our study, we found that EGFR-dependent PI3K-Akt signaling, but not MAPK (ERK1/2) activation (data not shown), is an upstream signal for YAP activation in response to AKI. How EGFR-MEK-ERK1/2 activation and EGFR-PI3K-Akt-YAP activation synchronize to promote cell proliferation and renal tubule cell regeneration will require further investigation. In addition, amphiregulin is an EGFR ligand, and amphiregulin is a downstream target gene for YAP activation. Further study will be required to determine if a positive feedback loop exists between EGFR activation and EGFR-dependent YAP activation–mediated amphiregulin production.

In summary, these studies show for the first time that activation of YAP signaling is an important component of the mechanisms by which renal epithelial cells recover from acute injury. Targeting the Hippo pathway or YAP activation may serve as potential targets for treatment of AKI.

Disclosures

None.