Research ArticleHepatologyNephrology Open Access | 10.1172/JCI175158

Manuel Chiusa,1 Youngmin A. Lee,2 Ming-Zhi Zhang,1 Raymond C. Harris,1,3 Taylor Sherrill,4 Volkhard Lindner,5 Craig R. Brooks,1 Gang Yu,6 Agnes B. Fogo,1,7 Charles R. Flynn,2 Jozef Zienkiewicz,3,4 Jacek Hawiger,3,4 Roy Zent,1,3 and Ambra Pozzi1,3

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Chiusa, M. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Lee, Y. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Zhang, M. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Harris, R. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Sherrill, T. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Lindner, V. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Brooks, C. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Yu, G. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Fogo, A. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Flynn, C. in: JCI | PubMed | Google Scholar |

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Zienkiewicz, J. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Hawiger, J. in: JCI | PubMed | Google Scholar

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Zent, R. in: JCI | PubMed | Google Scholar |

1Department of Medicine, Division of Nephrology and Hypertension, and

2Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

3Department of Veterans Affairs, Nashville, Tennessee, USA.

4Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

5Center for Molecular Medicine, Maine Health Institute for Research, Scarborough, Maine, USA.

6Department of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

7Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

Address correspondence to: Ambra Pozzi, Department of Medicine, Division of Nephrology and Hypertension, Medical Center North, B3115, Vanderbilt University, Nashville, Tennessee 37215, USA. Phone: 615.322.4637; Email: ambra.pozzi@vumc.og.

Find articles by Pozzi, A. in: JCI | PubMed | Google Scholar |

Published March 15, 2024 - More info

Published in Volume 134, Issue 6 on March 15, 2024
J Clin Invest. 2024;134(6):e175158. https://doi.org/10.1172/JCI175158.
© 2024 Chiusa et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published March 15, 2024 - Version history
Received: August 24, 2023; Accepted: January 17, 2024 View PDF Abstract

Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS–treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.

Graphical Abstract Introduction

Organ fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components (mainly collagen) within an injured organ that leads to the disruption of normal tissue architecture and loss of organ function. At present, therapeutic options for fibrosis are limited. A major obstacle to the development of antifibrotic therapies is that fibrosis is a multicellular event that requires physical as well as humoral crosstalk among different cell types within an injured organ. In addition, ECM homeostasis is regulated by several factors, including cellular receptors such as integrins and receptor tyrosine kinases (RTKs) (1, 2). We previously showed that the collagen-binding receptor integrin α1β1 is a negative regulator of fibrotic responses in kidney cells (3). A mechanism whereby this receptor downregulates collagen synthesis is by negatively modulating the phosphorylation/activation of the epidermal growth factor receptor (EGFR) via recruitment of the T cell protein tyrosine phosphatase PTPN2 (4).

EGFR activation has been implicated in the development of fibrosis in several organs, including kidney and liver. In the kidney, prolonged and/or aberrant EGFR signaling is a key determinant of progressive fibrotic injury (5), and glomerular activation of EGFR is a key step in the development of rapid progressive glomerulonephritis in both humans and mice (6). In the liver, EGF is upregulated in a rat model of liver fibrogenesis and in human cirrhotic liver tissues (7, 8), and a polymorphism in the human EGF gene that leads to increased EGF expression is associated with increased fibrosis and cirrhosis progression in patients with chronic hepatitis C (9).

Although EGFR plays a detrimental role in organ fibrosis, the use of EGFR inhibitors in humans leads to severe side effects, including skin toxicity (10). In addition, blocking EGFR might affect liver regeneration, as this process requires a functional EGF/EGFR axis in hepatocytes (7). Thus, a better understanding of selective fibrotic signaling activated downstream of the EGFR might lead to the development of safer and better-tolerated therapies for EGFR-mediated organ fibrosis.

We recently showed that a mechanism whereby EGFR regulates ECM production and in turn fibrotic responses is by promoting the nuclear translocation of the DNA/RNA-binding protein fused in sarcoma (FUS) in the mesangial cells of the kidney (11). Mechanistically, EGFR phosphorylates FUS on tyrosines 6 and 296, thus promoting FUS nuclear translocation. Nuclear FUS binds to the bidirectional promoter of collagen IV α1 and α2 chains, commencing its gene transcription (11). Consistent with this finding, mesangial cells lacking integrin α1β1 show not only increased baseline EGFR activation but also increased levels of nuclear FUS (11). Further corroborating a fibrotic role of FUS, we showed that mutating tyrosines 6 and 296 (FUS-Y6/296F) prevented EGF-induced FUS nuclear translocation and collagen IV production in mesangial cells in vitro (11). Consistent with this finding, downregulation of FUS in cardiac fibroblasts prevented PAX3 stability and, in turn, angiotensin II–induced collagen production (12). Further reinforcing the concept that prevention of FUS nuclear translocation has antifibrotic potential, patients affected by amyotrophic lateral sclerosis carrying mutations of FUS known to confer cytoplasmic gain of function have a decreased amount of collagen IV in skin, urine, and plasma (13–15). Finally, we showed that treatment of mesangial cells with the cell-penetrating peptide CP-FUS-NLS, which binds to transportin 1 (a nuclear import adaptor protein also known as karyopherin β2 or importin β2), inhibited FUS nuclear translocation and collagen IV transcription (11).

Although preventing FUS nuclear translocation inhibits collagen transcription, the fibrotic action of FUS has been investigated primarily in cell culture systems. Whether FUS has a role in governing fibrotic responses in vivo and whether preventing its nuclear translocation reduces fibrosis in vivo have not been investigated. We provide genetic evidence that mice expressing FUS carrying a mutation in the nuclear localization sequence (NLS) are protected from the development of fibrosis following kidney injury. Importantly, we provide pharmacologic evidence that mice treated with CP-FUS-NLS developed significantly less fibrosis following both kidney- and liver-induced injury. Our study shows that nuclear translocation of FUS in response to kidney and liver injury is linked to the promotion of fibrosis. As inhibition of FUS nuclear translocation ameliorates fibrotic responses in vivo, we propose that nuclear translocation of FUS can be viewed as a targetable step in organ fibrosis.

Results

Genetic or pharmacologic inhibition of EGFR kinase activity ameliorates adriamycin-induced kidney injury. We previously showed that EGFR promotes fibrotic responses in mesangial cells within the glomerulus of the kidney by phosphorylating FUS, which promotes its nuclear translocation and the commencement of collagen transcription (11). We also showed that fibrotic FUS and EGFR are activated in mesangial cells and in mice lacking integrin α1β1 (ItgA1KO) (4, 11), a negative regulator of EGFR phosphorylation/activation. To define the role of EGFR-mediated FUS nuclear translocation in glomerular injury, we induced glomerular injury in mice via administration of adriamycin (ADR), a well-established model of focal segmental glomerulosclerosis (16). We evaluated the degree of glomerular injury in ADR-treated BALB/c wild-type (WT) and ItgA1KO mice, as well as Wave2 mice and Wave2 mice crossed onto the ItgA1KO background (ItgA1KO/Wave2) (genetic approach). Wave2 mice possess a single-nucleotide mutation in the gene encoding EGFR, resulting in over 90% global reduction of EGFR tyrosine kinase activity (17). Renal injury was assessed by histologic examination 8 weeks after ADR treatment. As previously shown (3), both ADR-treated WT and ItgA1KO mice had severe mesangial expansion and well-developed glomerulosclerosis (Figure 1, A and B). However, the degree of injury was significantly higher in the ItgA1KO mice (Figure 1, A and B). Consistent with the glomerular injury, injured ItgA1KO mice had a significantly increased urinary albumin/creatinine ratio compared with WT mice (Figure 1C). Analysis of Wave2 and ItgA1KO/Wave2 mice revealed significantly reduced glomerular injury (Figure 1, A and B) and albuminuria (Figure 1C) in comparison with injured WT and ItgA1KO mice, respectively.

Figure 1

EGFR contributes to ADR-induced glomerular injury. (A) Representative images of periodic acid–Schiff–stained kidney sections from WT, ItgA1KO, Wave2, and ItgA1KO/Wave2 mice treated with ADR for 8 weeks. Scale bars: 20 μm. (B) Mesangial sclerosis index (MSI) of kidneys shown in A was evaluated and scored as described in Methods. Values are the mean ± SD, and symbols represent individual kidneys (n = 6 WT, n = 5 ItgA1KO, n = 5 Wave2, n = 6 ItgA1KO/Wave2, with ~20 glomeruli per kidney evaluated). (C) Albumin/creatinine ratio (ACR) was evaluated at baseline (n = 5 WT, n = 5 ItgA1KO, n = 5 Wave2, n = 5 ItgA1KO/Wave2) and 8 weeks (n = 22 WT, n = 29 ItgA1KO, n = 15 Wave2, n = 10 ItgA1KO/Wave2) after ADR injection. Symbols represent individual mice. (D) Representative images of periodic acid–Schiff–stained kidneys from WT and ItgA1KO mice treated with ADR for 8 weeks and treated with vehicle or erlotinib (ERL). Scale bars: 20 μm. (E) MSI of kidneys shown in D was evaluated and scored as described in Methods. Values are the mean ± SD, and symbols represent individual kidneys (n = 5 WT, n = 5 ItgA1KO, n = 5 WT+ERL, n = 5 ItgA1KO+ERL, with ~20 glomeruli per kidney). (F) ACR was evaluated at baseline (n = 3 WT, n = 3 ItgA1KO, n = 3 WT+ERL, n = 3 ItgA1KO+ERL) and 8 weeks (n = 5 WT, n = 5 ItgA1KO, n = 5 WT+ERL, n = 5 ItgA1KO+ERL) after ADR injection. Symbols represent individual mice. Statistical analysis: 1-way ANOVA followed by Dunnett’s multiple-comparison test (B, C, E, and F).

To further corroborate the role of EGFR in mediating glomerular injury, we investigated the degree of injury in ADR-treated WT and ItgA1KO mice treated with the selective EGFR inhibitor erlotinib for 8 weeks (pharmacologic approach). This inhibitor significantly reduced both glomerular injury and albuminuria in both WT and ItgA1KO (Figure 1, D–F), suggesting that activation of EGFR plays a deleterious role in ADR-mediated kidney injury.

Genetic or pharmacologic inhibition of EGFR kinase activity decreases ADR-induced kidney fibrosis. One of the key features of glomerulosclerosis is increased collagen deposition. Loss of integrin α1β1 leads to increased collagen production via EGFR activation (3). Thus, we investigated the levels of nonfibrillar collagen IV and fibrillar collagens by immunohistochemistry and Masson’s trichrome staining, respectively. Both techniques clearly revealed more collagen staining in the glomeruli of injured ItgA1KO mice compared with WT mice (Figure 2, A–H). Collagen accumulation was significantly decreased in Wave2 and ItgA1KO/Wave2 mice (Figure 2, A–D). Similarly, treatment of ItgA1KO mice with erlotinib resulted in greater reductions of both nonfibrillar and fibrillar collagen deposition compared with erlotinib-treated WT mice (Figure 2, E–H).

Figure 2

EGFR contributes to ADR-induced glomerulosclerosis. (A, C, E, and G) Representative light microscopy of collagen IV–stained (A and E) or Masson’s trichrome–stained (C and G) kidney sections from the mice indicated 8 weeks after ADR injection. Asterisks indicate single glomeruli. Scale bars: 25 μm. (B, D, F, and H) The amount of collagen IV per glomerulus (B, n = 5 WT, n = 4 ItgA1KO, n = 5 Wave2, n = 4 ItgA1KO/Wave2; F, n = 5 WT, n = 4 ItgA1KO, n = 6 WT+ERL, n = 7 ItgA1KO+ERL) or fibrillar collagen per microscopic field (D, n = 4 WT, n = 6 ItgA1KO, n = 5 Wave2, n = 6 ItgA1KO/Wave2; H, n = 5 WT, n = 5 ItgA1KO, n = 5 WT+ERL, n = 5 ItgA1KO+ERL) was evaluated using ImageJ as described in Methods. Values are the mean ± SD, and symbols represent individual kidneys (with an average of at least 12 glomeruli per kidney or three ×20 microscopic fields per kidney). Statistical analysis: 1-way ANOVA followed by Dunnett’s multiple-comparison test (B, D, F, and H).

Genetic or pharmacologic inhibition of EGFR kinase activity decreases glomerular nuclear level of FUS. To determine whether EGFR kinase activity positively regulates nuclear translocation of FUS in injured kidneys, we evaluated the levels of phosphorylated EGFR in injured glomeruli by immunofluorescence. Significantly higher levels of phosphorylated EGFR were detected in the glomeruli of ADR-injured ItgA1KO mice compared with injured WT mice (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI175158DS1). ItgA1KO mice crossed onto the Wave2 background or treated with erlotinib showed significant reduction of activated EGFR upon injury (Supplemental Figure 1, A and B). Consistent with the levels of activated EGFR, analysis of kidneys 8 weeks after ADR injury revealed significantly higher levels of nuclear FUS in glomeruli of ItgA1KO mice, which were significantly decreased in ItgA1KO/Wave2 mice or following erlotinib treatment (Figure 3, A–D). Thus, injury-mediated EGFR activation leads to increased FUS nuclear localization.

Figure 3

EGFR contributes to nuclear FUS translocation in ADR-induced injury. (A and C) Representative confocal images of kidneys from the mice indicated 8 weeks after ADR injections stained with anti-FUS antibody (green) or DAPI (blue). Scale bars: 15 μm. (B and D) The number of FUS-positive glomerular cells was counted and expressed as percentage of FUS-positive cells per glomerulus. Values are the mean ± SD, and symbols represent individual kidneys (B, n = 4 WT, n = 6 ItgA1KO, n = 5 Wave2, n = 6 ItgA1KO/Wave2; D, n = 4 WT, n = 5 ItgA1KO, n = 5 WT+ERL, n = 5 ItgA1KO+ERL, with an average of at least 4 glomeruli per kidney). Statistical analysis: 1-way ANOVA followed by Dunnett’s multiple-comparison test (B and D).

FUSR521G mice show reduced ADR-induced glomerular injury. To better define the role of nuclear FUS in regulating fibrotic responses, we induced glomerular injury in mice that express human FUS mutated in the NLS (R521G), which prevents its nuclear translocation (18). This mouse carries a CAG promoter, a floxed LacZ gene, the human FUS cDNA with the R521G mutation, and an IRES-EGFP (enhanced green fluorescent protein) coding sequence (18). We crossed this mouse with the Pdgfrb-cre mouse (19) to generate cagFUSR521G; Pdgfrb-cre mice (hereafter referred to as FUSR521G) (Supplemental Figure 2A), which express PGDFR-β in glomeruli (including mesangial cells [ref. 20]). Staining of kidneys from control (Cre) and FUSR521G mice (Supplemental Figure 2B) or Western blot analysis of kidney lysates (Supplemental Figure 2C) with anti-GFP antibody showed positive GFP staining or bands in glomeruli or kidney cortices of FUSR521G mice only. To ensure that expression of mutated FUS did not affect the basal levels of activated EGFR, we stained kidney sections with anti–phosphorylated EGFR. No overall differences in the basal levels of phosphorylated EGFR were observed in glomeruli of Cre or FUSR521G mice (Supplemental Figure 2, D and E).

Next, we induced ADR-mediated glomerular injury in control and FUSR521G mice. As these mice are on the C57BL/6J background, we followed the protocol described by Heikkilä et al. (21) and sacrificed the mice 2 weeks after injection. Compared with control (Cre) mice, FUSR521G mice showed reduced glomerular injury and accumulation of proteinaceous casts and significantly decreased albumin/creatinine ratio following injury (Figure 4, A and B). This protective effect was also accompanied by overall decreased Picrosirius red staining (Figure 4, C and D).

Figure 4

Reduced ADR-induced glomerulosclerosis in FUSR521G mice. (A and C) Representative images of periodic acid–Schiff–stained (A) or Picrosirius red–stained (C) kidney sections from uninjured (Cre or FUSR521G) mice and control (Cre) or FUSR521G mice treated with ADR for 2 weeks. Scale bars: 25 μm. (B) ACR was evaluated at 0, 1, and 2 weeks after ADR injection. Values are the mean ± SD, and symbols represent individual mice (n = 8 Cre, n = 7 FUSR521G). (D) The amount of glomerular fibrillar collagen was evaluated using ImageJ as described in Methods. Values are the mean ± SD, and symbols represent individual kidneys (n = 5 Cre 0-wk, n = 8 Cre 2-wk, n = 7 FUSR521G, with an average of at least 12 glomeruli per kidney). Statistical analysis: 1-way ANOVA followed by Dunnett’s multiple-comparison test (B and D).

FUSR521G mice show decreased nuclear FUS levels and glomerulosclerosis following kidney injury. To determine whether the protective effect observed in ADR-injured FUSR521G mice was due to reduced nuclear levels of FUS, we analyzed kidney nuclear fractions of uninjured as well as injured Cre control and FUSR521G mice by Western blot. Significantly higher levels of nuclear FUS were detected in ADR-treated Cre but not ADR-treated FUSR521G mice, compared with injured mice (Figure 5, A and B). Immunofluorescence analysis confirmed a significant increase in the number of FUS-positive nuclei in glomeruli of injured Cre mice compared with that detected in injured FUSR521G mice (Figure 5, C and D). Consistent with reduced FUS nuclear levels, FUSR521G mice also showed significant reduction in glomerular collagen IV deposition (Figure 5, E and F) and mRNA levels of Col1A2 and Col4A2 (Figure 5, G and H) compared with injured Cre mice.

Figure 5

Reduced ADR-induced FUS nuclear translocation in FUSR521G mice. (A) Nuclear fractions (50 μg/lane) of kidney cortices from uninjured mice and mice treated with ADR for 2 weeks were analyzed by Western blot for levels of FUS. Histone H3 and GAPDH were used to verify the purity of nuclear and nonnuclear fractions, respectively. (B) FUS bands were quantified by densitometry analysis, and values were expressed as FUS/histone H3 ratio. Values are the mean ± SD, and symbols represent individual kidneys (n = 3 uninjured, n = 4 Cre, n = 4 FUSR521G). (C) Representative confocal images of kidneys from uninjured mice or mice treated with ADR for 2 weeks stained with anti-FUS antibody (red) or DAPI (blue). Scale bars: 20 μm. (D) The number of FUS-positive glomerular cells was counted and expressed as percentage of FUS-positive cells per glomerulus. Values are the mean ± SD, and symbols represent individual kidneys (n = 4 uninjured, n = 10 Cre, n = 7 FUSR521G, with an average of at least 10 glomeruli per kidney). (E) Representative images of kidney sections from uninjured mice or mice treated with ADR for 2 weeks stained with anti–collagen IV antibody. Scale bar: 20 μm. (F) The intensity of glomerular collagen IV was evaluated using ImageJ as described in Methods. Values are the mean ± SD, and symbols represent individual kidneys (n = 4 uninjured, n = 8 Cre, n = 7 FUSR521G, with an average of at least 10 glomeruli per kidney). (G and H) mRNA expression of Col1A2 (n = 6 uninjured, n = 16 Cre, n = 8 FUSR521G) and Col4A2 (n = 7 uninjured, n = 8 Cre, n = 7 FUSR521G) chains in kidney cortices of the mice indicated was analyzed by reverse transcription real-time quantitative PCR. Values are the mean ± SD, and symbols represent individual kidneys. Statistical analysis: 1-way ANOVA followed by Dunnett’s multiple-comparison test (B, D, and F–H).

Pharmacologic inhibition of FUS nuclear translocation ameliorates ADR-mediated glomerular injury. To translate the genetic findings to a more clinically relevant setting, we used a cell-penetrating peptide inhibitor of FUS nuclear translocation that we recently generated (11). This penetrating chimeric peptide (named CP-FUS-NLS), but not the mutated inactive peptide (CP-mutFUS-NLS), inhibits FUS nuclear translocation in cells by preventing its interaction with transportin 1/karyopherin β2. As we showed that CP-FUS-NLS prevents EGF-induced FUS nuclear translocation and collagen production by mesangial cells in vitro (11), we tested whether this peptide exerts an antifibrotic action in vivo.

First, we determined whether this peptide reaches the kidneys by performing an acute injection of CP-FUS-NSL conjugated to fluorescein amidite. To do this, the fluorescent peptide was injected every 2 hours i.p. (1 mg/kg BW) for a total of 6 hours. Two hours after the last injection, the kidneys were collected, and frozen kidney sections were analyzed under an epifluorescence microscope. Compared with vehicle-treated mice, kidneys from mice treated with fluorescein amidite–conjugated CP-FUS-NSL showed green fluorescence in both tubules and glomeruli (Supplemental Figure 3, A and B), indicating that this peptide reaches the kidneys.

Next, we induced ADR-mediated glomerular injury in WT male BALB/c mice. Mice were then divided into 3 groups: one group received vehicle (PBS), one group received CP-FUS-NSL (1 mg/kg — which corresponds to 0.3 nM — via i.p. injection twice a day, 3 times per week), and one group received CP-mutFUS-NSL (0.95 mg/kg — which corresponds to 0.3 nM — via i.p. injection twice a day, 3 times per week). After 2 weeks the mice were sacrificed and organs collected for analysis.

Mice with ADR injury treated with CP-FUS-NLS showed reduced glomerular injury (Figure 6A) compared with injured mice treated with vehicle or CP-mutFUS-NLS, indicating that CP-FUS-NLS decreased glomerular injury.

Figure 6

Pharmacologic inhibition of FUS nuclear translocation ameliorates ADR-induced glomerulosclerosis. (A) Representative images of periodic acid–Schiff–stained or Picrosirius red–stained kidney sections from uninjured mice and mice treated for 2 weeks with ADR alone or in combination with CP-mut-FUS-NLS or CP-FUS-NLS. Scale bars: 25 μm. (B) mRNA expression of Col1A2 chain in kidney cortices of the mice indicated was analyzed by reverse transcription quantitative PCR. Values are the mean ± SD, and symbols represent individual kidneys (n = 8 uninjured, n = 9 ADR, n = 10 ADR+CP-mutFUS-NLS, n = 9 ADR+CP-FUS-NLS). (C) Representative images of kidney sections from the mice described in A stained with anti–collagen IV antibody. Scale bar: 20 μm. (D) The intensity of glomerular collagen IV was evaluated using ImageJ as described in Methods. Values are the mean ± SD, and symbols represent individual kidneys (n = 8 uninjured, n = 7 ADR, n = 10 ADR+CP-mutFUS-NLS, n = 8 ADR+CP-FUS-NLS, with an average of at least 10 glomeruli per kidney). (E) Representative images of kidneys from the mice described in A stained with anti-FUS antibody (green) or DAPI (blue). Scale bars: 20 μm. (F) The number of FUS-positive glomerular cells was counted and expressed as percentage of FUS-positive cells per glomerulus. Values are the mean ± SD, and symbols represent individual kidneys (n = 8 uninjured, n = 7 ADR, n = 10 ADR+CP-mutFUS-NLS, n = 8 ADR+C