A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a Mouse Model of Alport Syndrome
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A Neutralizing IL-11 Antibody Improves Renal Function and
Increases Lifespan in a Mouse Model of Alport Syndrome
Journal: Journal of the American Society of Nephrology
Manuscript ID JASN-2021-04-0577.R1
Manuscript Type: Original Article - Basic Research
Date Submitted by the
08-Dec-2021
Author:
Complete List of Authors: Widjaja, Anissa; Duke-NUS Medical School, Cardiovascular and Metabolic
Disorders Program
Shekeran, Shamini; Duke-NUS Medical School, Cardiovascular and
Metabolic Disorders Program
Adami, Eleonora ; Duke-NUS Medical School, Cardiovascular and
Metabolic Disorders Program; Max Delbruck Centre for Molecular
Medicine in the Helmholtz Association, Cardiovascular and Metabolic
Sciences
Goh, Joyce; Duke-NUS Medical School, Cardiovascular and Metabolic
Disorders Program
Tan, Jessie; National Heart Centre Singapore, National Heart Research
Institute Singapore
Viswanathan, Sivakumar; Duke-NUS Medical School, Cardiovascular and
Metabolic Disorders Program
Lim, Sze Yun; Duke-NUS Medical School, Cardiovascular and Metabolic
Disorders Program
Tan, Puay Hoon; Duke-NUS Medical School, Cardiovascular and
Metabolic Disorders Program; Singapore General Hospital, Department of
Anatomical Pathology; National University Singapore Yong Loo Lin School
of Medicine, Department of Anatomy
Hubner, Norbert; Max Delbruck Centre for Molecular Medicine in the
Helmholtz Association, Cardiovascular and Metabolic Sciences; DZHK;
Charité Universitätsmedizin Berlin
Coffman, Thomas; Duke-NUS Medical School, Cardiovascular and
Metabolic Disorders Program
Cook, Stuart; Duke-NUS Medical School, Cardiovascular and Metabolic
Disorders Program; National Heart Centre Singapore, National Heart
Research Institute Singapore; MRC London Institute of Medical Sciences
Alport syndrome, fibrosis, Interleukin 11, podocyte, Therapy, glomerular
Keywords:
disease, glomerulosclerosis, chronic kidney disease
Journal of the American Society of Nephrology
Page 1 of 23
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3 Authors: Widjaja, Anissa; Shekeran, Shamini; Adami, Eleonora ; Goh, Joyce; Tan, Jessie; Viswanathan,
4
Sivakumar; Lim, Sze Yun; Tan, Puay Hoon; Hubner, Norbert; Coffman, Thomas; Cook, Stuart
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6
7 Title: A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a Mouse Model
8 of Alport Syndrome
9
10 Running title: IL11 antibody therapy in mouse model of Alport syndrome
11
12 Manuscript Type: Original Article - Basic Research
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14
15 Manuscript Category: Chronic kidney disease
16
17
Funders: Fondation Leducq, (Grant / Award Number: '16CVD03')
18 National Medical Research Council, (Grant / Award Number: 'MOHâ€CIRG18novâ€0002','NMRC
19 Centre Grant ','NMRC/OFYIRG/0053/2017','NMRC/STaR/0029/2017')
20 Medical Research Council, (Grant / Award Number: )
21 European Union Horizon 2020 Research And Innovation, (Grant / Award Number: 'AdG788970')
22 Tanoto Foundation, (Grant / Award Number: )
23
24
Financial Disclosure: CUST_FINANCIAL_DISCLOSURE :No data available. S.A.C. is a co-inventor of the
25
26
patent applications: WO/2017/103108 (TREATMENT OF FIBROSIS), WO/2018/109174 (IL11
27 ANTIBODIES), WO/2018/109170 (IL11RA ANTIBODIES). S.A.C. and A.A.W are co-inventors of the patent
28 application: US US2020/0270340A1 (Treatment of Kidney Injury) and GB2009292.0. (Treatment and
29 prevention of disease caused by Type IV collagen dysfunction). S.A.C. is a cofounder and shareholder of
30 Enleofen Bio PTE LTD, a company that made anti IL11 therapeutics, which were acquired for further
31 development by Boehringer Ingelheim in 2019. A. Widjaja reports Patents and Inventions: Boehringer
32 Ingelheim. S. Cook reports Research Funding: Boehringer Ingelheim. T. Coffman reports Advisory or
33 Leadership Role: Editorial Boards: Cell Metabolism and JCI, Singapore Health Services Board of Directors,
34
Singapore Eye Research Institute, and Kidney Research Institute University of Washington. P. Tan
35
36 reports Honoraria for delivering talk on Prostate cancer - AstraZeneca. All other authors declare no
37 competing interest.
38
39 Study Group/Organization Name: CUST_STUDY_GROUP/ORGANIZATION_NAME :No data available.
40
41 Study Group Members’ Names: CUST_STUDY_GROUP_MEMBERS :No data available.
42
43
44
Total number of words: 3385
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46 Abstract: Background: Alport syndrome is a genetic disorder characterized by a defective glomerular
47 basement membrane, tubulointerstitial fibrosis, inflammation, and progressive renal failure. IL-11 was
48 recently implicated in fibrotic kidney disease but its role in Alport syndrome is unknown
49 Methods: We determined IL-11 expression by molecular analyses and in an Alport syndrome mouse
50 model. We assessed the effects of a neutralizing IL-11 antibody (X203) versus an IgG control in Col4a3-/-
51 mice (lacking the gene encoding a type IV collagen component) on renal tubule damage, function,
52
fibrosis, and inflammation. Effects on lifespan of X203, the IgG control, an angiotensin-converting
53
54 enzyme inhibitor (ramipril), or ramipril+X203 were also studied.
55 Results: In Col4a3 mice, as kidney failure advanced, renal IL-11 levels increased and IL-11 expression
56 localized to tubular epithelial cells. The IL-11 receptor IL11RA is expressed in tubular epithelial cells and
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60 Journal of the American Society of Nephrology
Page 2 of 23
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3 podocytes and is upregulated in tubular epithelial cells of Col4a3 mice. Administration of X203 reduced
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albuminuria, improved renal function, and preserved podocyte numbers and levels of key podocyte
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6
proteins that are reduced in Col4a3 mice; these effects were accompanied by reduced fibrosis and
7 inflammation, attenuation of epithelial-tomesenchymal transition, and increased expression of
8 regenerative markers. X203 attenuated pathogenic ERK and STAT3 pathways, which were activated in
9 Col4a3 mice. Median lifespan of Col4a3 mice was prolonged 22% by ramapril, 44% with X203, and 99%
10 with amipril+X203.
11 Conclusions: In an Alport syndrome mouse model, renal IL-11 is upregulated, and neutralization of IL-11
12 reduces epithelial-to-mesenchymal transition, fibrosis, and inflammation, while improving renal
13 function. Anti-IL-11 combined with ACE inhibition synergistically extends lifespan. This suggests that a
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therapeutic approach targeting IL-11 holds promise for progressive kidney disease in Alport syndrome.
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60 Journal of the American Society of Nephrology
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Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 Significance Statement
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6 Alport syndrome, a genetic disorder of the glomerular basement membrane, frequently leads
7 to end-stage renal failure. In an animal model of Alport syndrome—mice lacking the gene
8 Col4a3—angiotensin-converting enzyme (ACE) inhibition is protective. The authors show
9 that IL-11 is upregulated in the renal tubular epithelia of Col4a3-/- mice; IL-11’s receptor,
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expressed on podocytes and tubule cells, is upregulated in the diseased kidneys of Col4a3-/-
12 mice. Giving 6-week-old Col4a3-/- mice a neutralizing IL-11 antibody (X203) reduced
13 pathological ERK and STAT3 activation and limited epithelial-to-mesenchymal transition;
14 reduced kidney fibrosis, inflammation, and tubule damage; and improved kidney function.
15
Median lifespan of Col4a3-/- mice was prolonged 22% by ramapril alone, 44% with X203
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17 alone, and 99% with ramipril+X203. These data suggest that anti-IL-11 therapies hold
18 promise for treating kidney disease in Alport syndrome.
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Journal of the American Society of Nephrology
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Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a
4 Mouse Model of Alport Syndrome
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Anissa A. Widjaja1†*, Shamini Guna Shekeran1†, Eleonora Adami1,2, Joyce Goh Wei Ting1,
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Jessie Tan3, Sivakumar Viswanathan1, Lim Sze Yun1, Tan Puay Hoon1,4,5, Norbert
10 Hϋbner2,6,7, Thomas M. Coffman1, Stuart A. Cook1,3,8*
11
12 Affiliations:
1Cardiovascular and Metabolic Disorders Program, Duke-National University of Singapore
13
14 Medical School, Singapore.
15 2Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the
16 Helmholtz Association (MDC), 13125 Berlin, Germany.
17 3National Heart Research Institute Singapore, National Heart Centre Singapore, Singapore.
18 4Department of Anatomical Pathology, Singapore General Hospital, Singapore, Singapore.
19 5Department of Anatomy, Yong Loo Lin School of Medicine, National University of
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21 Singapore, Singapore, Singapore.
6DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, 13347 Berlin,
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23 Germany.
7Charité-Universitätsmedizin, 10117 Berlin, Germany.
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25 8MRC-London Institute of Medical Sciences, Hammersmith Hospital Campus, London, UK.
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28 †These authors contributed equally to this work
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30 *Correspondence to:
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Anissa A. Widjaja and Stuart A. Cook
33 Email: anissa.widjaja@duke-nus.edu.sg and stuart.cook@duke-nus.edu.sg
34 8 College Road 169857
35 Duke-NUS Medical School, Singapore
36 Phone: (65) 660102584
37
38 Fax: (65) 6221 2534
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Running title: IL-11 Antibody and Alport Syndrome
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53 Abstract
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56 Background Alport syndrome is a genetic disorder characterized by a defective glomerular
57 basement membrane, tubulointerstitial fibrosis, inflammation, and progressive renal failure.
58 IL-11 was recently implicated in fibrotic kidney disease but its role in Alport syndrome is
59
unknown.
60
Journal of the American Society of Nephrology
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Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 Methods We determined IL-11 expression by molecular analyses and in an Alport syndrome
4
mouse model. We assessed the effects of a neutralizing IL-11 antibody (X203) versus an
5
6 IgG control in Col4a3-/- mice (lacking the gene encoding a type IV collagen component) on
7 renal tubule damage, function, fibrosis, and inflammation. Effects on lifespan of X203, the
8 IgG control, an angiotensin-converting enzyme inhibitor (ramipril), or ramipril+X203 were
9 also studied.
10
11
12
Results In Col4a3-/- mice, as kidney failure advanced, renal IL-11 levels increased and IL-11
13 expression localized to tubular epithelial cells. The IL-11 receptor IL11RA is expressed in
14 tubular epithelial cells and podocytes and is upregulated in tubular epithelial cells of Col4a3-/-
15 mice. Administration of X203 reduced albuminuria, improved renal function, and preserved
16
podocyte numbers and levels of key podocyte proteins that are reduced in Col4a3-/- mice;
17
18 these effects were accompanied by reduced fibrosis and inflammation, attenuation of
19 epithelial-to-mesenchymal transition, and increased expression of regenerative markers.
20 X203 attenuated pathogenic ERK and STAT3 pathways, which were activated in Col4a3-/-
21 mice. Median lifespan of Col4a3-/- mice was prolonged 22% by ramapril, 44% with X203, and
22
99% with amipril+X203.
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25 Conclusions In an Alport syndrome mouse model, renal IL-11 is upregulated, and
26 neutralization of IL-11 reduces epithelial-to-mesenchymal transition, fibrosis, and
27 inflammation, while improving renal function. Anti-IL-11 combined with ACE inhibition
28 synergistically extends lifespan. This suggests that a therapeutic approach targeting IL-11
29
holds promise for progressive kidney disease in Alport syndrome.
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Journal of the American Society of Nephrology
Page 6 of 23
Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 Introduction
4
Alport syndrome (AS) is caused by mutation in the A3/4/5 genes that encode chains of type
5
6 IV collagen1,2. These mutations lead to abnormalities in glomerular basement membrane
7 (GBM) collagen composition, integrin-mediated podocyte dysfunction, glomerular
8 hypertension, and ultrafiltration3,4. AS affects up to 60,000 people in the United States and is
9 associated with hearing loss, ocular abnormalities, and chronic renal disease (CKD).
10
11
In the commonest form of disease due to X-linked mutation of COL4A5, 90% of
12 affected males develop end-stage kidney failure by the age of 405. Early disease can
13 manifest as hematuria, microalbuminuria or proteinuria and while there are no specific
14 therapies, affected children are commonly treated with an angiotensin converting enzyme
15
inhibitor (ACEi), based in part on extrapolation of studies conducted in Col4a3-/- mice6 and
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17 supported by more recent data from clinical trials7,8.
18 The Col4a3-/- mouse strain is widely viewed as one of the best animal models of
19 progressive AS. In seminal studies, treatment of four-week-old Col4a3-/- mice with an ACEi
20 (ramipril), prior to onset of proteinuria and tubulointerstitial fibrosis, attenuated kidney
21
dysfunction and prolonged lifespan6. However, if ramipril treatment of Col4a3-/- mice was
22
23 delayed until seven weeks of age, after proteinuria was established, there was limited
24 beneficial effect6,9. There are no specific or second-line medical therapies for AS and renal
25 transplantation is the prefered treatment for progressive CKD in AS10.
26 Kidney dysfunction in AS is initiated in the glomerulus, related to altered GBM
27
mechanics and podocyte dysfunction. However, as in other primary glomerular diseases, a
28
29 major determinant of progressive kidney failure is in the associated tubulointerstitial
30 disease4. Indeed, similar to other forms of CKD, kidney function in AS patients correlates
31 most strongly with the degree of tubulointerstitial fibrosis, rather than glomerular pathology11.
32 Disease pathogenesis in AS is complex, involving renin angiotensin system and TGFβ
33
34 activation, inflammation, partial epithelial-mesenchymal transition (pEMT) of tubular
35 epithelial cells (TECs)/podocytes and fibrosis, among other factors9,12,13. Increasingly the role
36 of pEMT, a failed-repair proximal tubule cell (FR-PTC) state14, is viewed as an initiating
37 factor for renal fibrosis, inflammation and failure, particularly as it prevents TEC proliferation
38
and renal repair15–20.
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40 Here we investigated whether (1) IL11, recently implicated as important for
41 tubulointerstitial fibrosis and renal dysfunction21, is involved in the kidney pathology of AS
42 and (2) a neutralizing IL11 antibody given to Col4a3-/- mice with established renal disease
43 and proteinuria could improve molecular pathology, renal structure and function, and delay
44
onset of death due to kidney failure.
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46
47 Methods
48 Antibodies
49 Cyclin D1 (55506, CST), E-Cadherin (3195, CST), p-ERK1/2 (4370, CST), ERK1/2 (4695,
50
51
CST), GAPDH (2118, CST), GFP (ab6673, Abcam), IgG (11E10, Aldevron), neutralizing
52 anti-IL11 (X203, Aldevron), anti-IL11RA (X209, Aldevron), NHPS2/Podocin (ab181143,
53 Abcam), PCNA (13110, CST), αSMA (19245, CST; WB), SNAI1 (3879, CST, WB), p-STAT3
54 (4113, CST), STAT3 (4904, CST), TGFβ (3711, CST), Wilms’ Tumor 1 (ab89901, Abcam, IF
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and IHC), Wilms’ Tumor 1 (ab267377, Abcam, WB), anti-goat Alexa Fluor 488 (ab150129,
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57 Abcam), anti-rabbit Alexa Fluor 647 (ab150067, Abcam), anti-rabbit HRP (7074, CST), anti-
58 mouse HRP (7076, CST).
59
60 Ethics statements
Journal of the American Society of Nephrology
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3 Animal studies were carried out in compliance with the recommendations in the Guidelines
4
on the Care and Use of Animals for Scientific Purposes of the National Advisory Committee
5
6 for Laboratory Animal Research (NACLAR). All experimental procedures were approved
7 (SHS/2019/1482) and conducted in accordance with the SingHealth Institutional Animal
8 Care and Use Committee.
9
10
11
Mouse model of Alport
12 Col4a3-/- (Col4a3tm1Dec) mice were purchased from The Jackson Laboratory
13 (https://www.jax.org/strain/002908). Mice were housed in temperatures of 21-24℃ with 40-
14 70% humidity on a 12 h light/12 h dark cycle and provided with food and water ad libitum.
15
For treatment study, Col4a3-/- were administered 20mg/kg of anti-IL11 (X203) or IgG isotype
16
17 control (11E10) by intraperitoneal (IP) injection starting from 6 weeks of age twice a week for
18 2.5 weeks; wild-type littermates were used as controls. Mice were sacrificed for blood and
19 kidney collection when they were 8.5-week-old. For lifespan study mice were
20 intraperitoneally administered either X203 (twice a week, 20 mg/kg) or 11E10 (twice a week,
21
20 mg/kg) alone or in combination with ramipril (10 mg/kg; 6 days/week) starting from 6
22
23 weeks of age, until death ensued.
24 Col4a3-/--Il11:EGFP
25 Col4a3-/- mice were crossed to transgenic mice with EGFP constitutively knocked-in to the
26 Il11 gene22 to generate hybrid cross of Col4a3-/- -Il11:EGFP+/-. Age-matched Col4a3+/+ -
27
28
Il11:EGFP+/- littermates were used as controls. Mice were sacrificed at 7.5 weeks of age;
29 kidneys were excised and OCT-embedded for immunofluorescence staining.
30
31 Western Blot
32 Western blot was carried out on total protein extracts from mouse kidney tissues. Kidneys
33
34 were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and
35 phosphatase inhibitors (Thermo Scientifics), followed by centrifugation to clear the lysate.
36 Protein concentrations were determined by Bradford assay (Bio-Rad). Protein lysates were
37 separated by SDS-PAGE, transferred to PVDF membrane, and subjected to immunoblot
38
analysis for various antibodies (1:1000 in TBST) as outlined in the main text, figures, or
39
40 and/or figure legends. Proteins were visualized using the ECL detection system (Pierce) with
41 the appropriate secondary antibodies: anti-rabbit HRP or anti-mouse HRP (1:2000 in TBST).
42
43 Quantitative polymerase chain reaction (qPCR)
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Total RNA was extracted from snap-frozen kidney tissues using Trizol (Invitrogen) followed
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46 by RNeasy column (Qiagen) purification. cDNAs were synthesized with iScriptTM cDNA
47 synthesis kit (Bio-Rad) according to manufacturer’s instructions. Gene expression analysis
48 was performed on duplicate samples with either TaqMan (Applied Biosystems) or fast SYBR
49 green (Qiagen) technology using StepOnePlusTM (Applied Biosystem) over 40 cycles.
50
51 Expression data were normalized to GAPDH mRNA expression and fold change was
52 calculated using 2-∆∆Ct method. The sequences of specific TaqMan probes and SYBR green
53 primers are available upon request.
54
55
Colorimetric assays
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57 The levels of blood urea nitrogen (BUN) and creatinine in mouse serum were measured
58 using Urea Assay Kit (ab83362, Abcam) and Creatinine Assay Kit (ab65340, Abcam),
59 respectively. Urine albumin and creatinine levels were measured using Mouse Albumin
60
Journal of the American Society of NephrologyPage 8 of 23
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3 ELISA kit (ab108792, Abcam) and Creatinine Assay Kit (ab204537, Abcam), respectively. All
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ELISA and colorimetric assays were performed according to the manufacturer’s protocol.
5
6
7 Histology
8 Kidney tissues were fixed for 48 hours at RT in 10% neutral-buffered formalin (NBF),
9 dehydrated, embedded in paraffin, and sectioned at 7μm. Transverse kidney sections were
10
11
then stained with periodic acid Schiff (PAS) and Masson’s Trichrome according to standard
12 protocol. Images of the sections were captured by light microscopy and blue-stained fibrotic
13 areas were semi-quantitatively determined with Image-J software (color deconvolution-
14 Masson’s Trichrome) from the whole kidney area (100X field, n=4 kidneys/group). Kidney
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sections (n=4-7/group) were independently scored for tubulointerstitial fibrosis (from
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17 Masson’s Trichrome- stained kidney sections) and for glomerulosclerosis and tubular
18 atrophy (from PAS-stained kidney sections) by a renal pathologist in a blinded fashion with
19 the following criteria:
20 ● Interstitium (0, no fibrosis; 1, less than 25% fibrosis; 2, 25% to 50% fibrosis; 3, more
21
than 50% fibrosis).
22
23 ● Glomeruli (0, no sclerosis; 1, less than 25% sclerosis; 2, 25% to 50% sclerosis; 3,
24 more than 50% sclerosis).
25 ● Tubules (0, no atrophy; 1, less than 25% atrophy; 2, 25% to 50% atrophy; 3, more
26 than 50% atrophy).
27
● Total score is the sum of interstitial fibrosis score, glomeruli score, and tubule atrophy
28
29 score.
30 Treatment and genotypes were not disclosed to investigators performing the histology and
31 generating semi-quantitative readouts.
32
33
34 Immunohistochemistry
35 Kidneys were fixed in 10% neutral-buffered formalin (NBF), paraffinized, cut into 7μm
36 sections, incubated with primary antibodies overnight and visualized using the appropriate
37 ImmPRESS HRP IgG polymer detection kit: anti-rabbit (MP-7401, Vector Laboratories), anti-
38
mouse (MP-7402, Vector Laboratories) with ImmPACT DAB Peroxidase Substrate (SK-
39
40 4105, Vector Laboratories). Quantification of WT+ve cells were performed in a blinded fashion
41 from 4 images (200X field)/kidney (n=3-4 kidneys/group).
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43 Immunofluorescence
44
Kidneys were rinsed in cold PBS and patted dry with a lint free paper and cryo-molded in
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46 OCT compound (4583, Tissue-TekⓇ). After the OCT compound is frozen, kidney specimens
47 were wrapped in aluminium foil and stored in -80oC. Cryo-embedded kidneys were
48 cryosectioned (-20°C) at 7 µm thickness and allowed to dry on the slides for 1 hour (RT).
49
Kidney sections were fixed in cold acetone for 15 min prior to brief PBS washes,
50
51 permeabilized with 0.1% TritonX-100 (T8787, Sigma), and blocked with 2.5% normal horse
52 serum (S-2012, Vector Labs) for 1 hour (RT). Kidney sections were incubated with GFP and
53 WT1 (1:500 in PBS containing 0.1% Tween20) primary antibodies overnight (4°C), followed
54 by incubation with the appropriate Alexa Fluor 488/647 secondary antibodies (1:250) for 1
55
56
hour (RT). DAPI was used to stain the nuclei prior to imaging by fluorescence microscope
57 (Leica).
58
59 Statistical analyses
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3 Statistical analyses were performed using GraphPad Prism software (version 8). Statistical
4
significance between control and experimental groups were analysed by two-sided Student’s
5
6 t tests or by one-way ANOVA as indicated in the figure legends. P values were corrected for
7 multiple testing according to Tukey when several conditions were compared to each other
8 within one experiment. Comparison analysis for two parameters from two different groups
9 were performed by two-way ANOVA. The criterion for statistical significance was P < 0.05.
10
11
12 Results
13 IL11 is upregulated in the kidneys of Col4a3-/- mice.
14 IL11 is not expressed in normal healthy tissues but its induction is commonly seen in
15
fibroinflammatory diseases23. We profiled the Il11 mRNA expression in kidneys of Col4a3-/-
16
17 mice and found it to be upregulated (17.8-fold, PPage 10 of 23
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3 mass (Figure 2C) and had significantly less kidney fibrosis by both biochemical and
4
histological assessments (Figure 2D-F).
5
6
7
8 Gene expression analyses showed renal levels of extracellular matrix genes (Col1a1,
9 Col1a2, Col3a1 and Fn), the myofibroblast marker Acta2 and pro-fibrotic factors (Il11 and
10
11
Tgfb1) were all reduced by X203 as compared to IgG (Figure 3A). The effect seen on
12 transcript expression was confirmed at the protein level for alpha-smooth muscle actin
13 (⍺SMA) and fibronectin (Figure 3B).
14 At the signaling level, IL11 is known to activate ERK across cell types and this
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pathway has been mechanistically linked with IL11-driven fibrosis26,28,29. IL11 inhibition in
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17 vivo can also be associated with reduced STAT3 activation, which is thought to be largely a
18 secondary phenomenon reflecting lesser stromal-driven inflammation26,30. As compared to
19 wild-type mice, kidneys from Col4a3-/- mice treated with IgG exhibited elevated ERK and
20 STAT3 activation, in contrast ERK and STAT3 phosphorylation was largely diminished in
21
kidneys of X203-treated Col4a3-/- mice (Figure 3,B and C). These data are consistent with
22
23 X203 target engagement in the kidney, reduced ERK activation and diminished
24 inflammation.
25 In many kidney diseases, it is thought that damaged TECs transition to a pEMT/FR-
26 PCT state, which is central to the subsequent development of tubulointerstitial fibrosis and
27
28
CKD15–18. TEC pEMT/FR-PCT14 is characterised by increased SNAI1 expression and
29 reciprocal downregulation of E-Cadherin that is regulated, in part, by TGFβ15,16. As
30 compared to wild-type controls, Col4a3-/- mice receiving IgG exhibited a strong molecular
31 signature of EMT with increased SNAI1 and decreased E-Cadherin expression (Figure 3,B
32 and C). In contrast, SNAI1 and E-Cadherin levels in Col4a3-/- mice receiving X203 were
33
34 similar to those seen in wild-type mice. Thus, anti-IL11 reduces TEC pEMT in the kidneys of
35 Col4a3-/- mice.
36 A specific feature of injured TECs that enter a pEMT/FR-PCT state is their inability to
37 replicate, a process that relates to SNAI1 repression of Cyclins D1/2 that blocks G1/S
38
transitions19,20,31. We profiled levels of Cyclin D1 and those of PCNA, a marker of G1/S, in
39
40 kidneys of Col4a3-/- mice treated with either IgG or X203 and also in WT controls. Col4a3-/-
41 mice receiving X203 showed marked upregulation of Cyclin D1 and PCNA, as compared to
42 WT mice and Col4a3-/- mice receiving IgG (Figure 3,B and C). This suggests anti-IL11
43 inhibits pEMT/FR-PCT transitions in the kidneys of Col4a3-/- mice and releases TECs to
44
reenter G1/S, to replicate and to repair damaged tubules.
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46
47 Podocyte preservation and lesser renal inflammation is associated with inhibition of
48 IL11 signaling in Col4a3-/- mice.
49 AS affects GBM composition leading to podocyte dysfunction/loss that relates to TGFβ
50
51 activity and pEMT processes in both podocytes and TECs9,18. Immunohistochemistry
52 analysis of the podocyte marker WT1 revealed a greater staining in wild-type mice and
53 X203-treated Col4a3-/- mice, as compared to IgG-treated Col4a3-/- mice (Figure 4A).
54 Quantification of the number of WT1-positive cells (podocytes) was carried out in a blinded
55
fashion and confirmed significant (P=0.0002) preservation of podocyte integrity in Col4a3-/-
56
57 mice receiving X203 as compared to Col4a3-/- mice receiving IgG (Figure 4B). Preservation
58 of podocytes in X203-treated Col4a3-/- mice was further ascertained by immunoblotting and
59 findings were extended to Podocin, a second podocyte marker (Figure 4, C and D).
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3 TGFβ upregulation in podocytes and tubular cells, which coincides with the onset of
4
proteinuria in the Col4a3-/- mouse9,32, is thought of importance for disease pathogenesis in
5
6 AS. We thus examined TGFβ levels and observed that X203, but not IgG, significantly
7 reduced the degree of TGFβ upregulation in the kidneys of Col4a3-/- mice (Figure 4, C and
8 D). Apoptosis of podocytes and tubule cells is implicated in AS and caspase activity is
9 reduced in Col4a3-/- mice given Olmesartan32. We observed caspase 3 activation in the IgG-
10
11
treated Col4a3-/- mice that was reduced by X203 administration (Figure 4, C and D).
12 Tnf⍺ expression in podocytes is of particular importance in AS and leads to podocyte
13 apoptosis and glomerulosclerosis13. It was therefore notable that X203 reduced Tnf⍺
14 expression in Col4a3-/- mice, as compared to IgG treated controls (Figure 4E). Markers of
15
tubule damage and inflammation were also assessed. As compared to wild-type mice,
16
17 control Col4a3-/- mice had elevated indicators of tubule damage (Kim1 and Ngal), which were
18 restored by X203 administration towards the levels seen in WT mice (Figure 4E).
19 Proinflammatory interleukins (Il6 and Il1b) and CC chemokines (Ccl2 and Ccl5) were also
20 elevated in Col4a3-/- mice receiving IgG and were equally diminished by administration of
21
X203 (Figure 4E).
22
23
24
25 Inhibition of IL11 signaling improves kidney histopathology and function
26 Next, we tested whether inhibition of IL11 signaling, which mitigated intermediate
27
28 phenotypes of kidney pathology in Col4a3-/- mice, also improved renal structure and function.
29 To determine the effect of anti-IL11 therapy on kidney pathology, kidney sections stained
30 with periodic acid Schiff (PAS) and Masson’s Trichrome were evaluated by a renal
31 pathologist who was blinded to treatments, and the severity of pathological features
32
including fibrosis, glomerulosclerosis and tubular atrophy were assigned a composite score
33
34 (see methods for scoring details). Compared to IgG-treated mice, X203 treatment was
35 associated with diminished levels of tubular atrophy and interstitial fibrosis, consistent with
36 our internal analysis (Figure 2F), while glomerulosclerosis was completely abrogated, and
37 the overall damage score was significantly reduced (Figure 5A-D).
38
To assess renal function, we measured blood urea nitrogen (BUN), serum creatinine
39
40 (Cr) and urinary albumin:creatinine ratios at the end of the anti-IL11 monotherapy study (at
41 8.5 weeks of age). As compared to wild-type mice, IgG-treated Col4a3-/- mice had elevated
42 BUN, Cr and urinary ACR levels (fold elevation compared to wild-type: 12.4, 7.3, 13.6,
43 respectively), whereas administration of X203 from week 6 lowered BUN, Cr and urinary
44
45
ACR to levels seen in WT mice, consistent with a significant overall improvement in renal
46 function (Figure 5E-G).
47
48 Anti-IL11 extends lifespan in Col4a3-/- mice
49 Death from progressive renal failure typically occurs in Col4a3-/- mice starting from 8.5 weeks
50
51 of age and mean survival times are reproducibly reported at around 10 weeks (e.g. 71 days6;
52 69 days33). Untreated Col4a3-/- mice used for the studies described here had a mean survival
53 of 62.7±1.9 days. Previous studies have shown that administration of ramipril from four
54 weeks of age, prior to the onset of proteinuria and before IL11 is expressed (Figure 1B),
55
extends lifespan in Col4a3-/- mice whereas initiation of ramipril from seven weeks of age,
56
57 when disease is established, does not6. We sought to determine if anti-IL11 could extend
58 lifespan when initiated at six weeks of age, after proteinuria is present and IL11 is
59 upregulated in the kidneys, and compared its effects to ramipril alone or ramipril combined
60 with anti-IL11, also administered from six weeks of age (Figure 6A).
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3 As compared to untreated controls, administration of IgG had no effect on survival
4
(Figure 6, B and C). In contrast, ramipril significantly extended median lifespan by 22% (14
5
6 days), whereas anti-IL11 alone increased median lifespan by 44% (29 days) (Figure 6, B
7 and C). Notably, anti-IL11 combined with ramipril acted synergistically to further extend
8 median lifespan of Col4a3-/- mice by 99% (62 days) (Figure 6, B and C). This suggests that
9 anti-IL11 and ramipril inhibit different pathological processes in the diseased kidneys of
10
11
Col4a3-/- mice.
12
13 Discussion
14 Blockade of the renin-angiotensin system (RAS) is a mainstay of therapy for patients with AS
15
and other forms of CKD, but unfortunately progression to end-stage renal failure is typical in
16
17 individuals with aggressive AS5,10. This shortcoming likely reflects the complex renal
18 pathology of progressive AS, involving GBM-specific initiating factors and generic
19 tubulointerstitial disease mechanisms that cannot be completely ameliorated by RAS
20 blockade alone. Here we identify IL11 as a novel cause of kidney injury in AS and show that
21
inhibition of IL11 has independent and additive therapeutic benefits relative to ACE inhibition
22
23 in Col4a3-/- mice.
24 IL11 is a misunderstood cytokine22,23 that is secreted from a variety of stromal and
25 epithelial cells in response to cellular injury to act in an autocrine and paracrine manner
26 causing epithelial cell dysfunction, stromal cell activation and inflammation23. In the kidney
27
parenchyma, IL11RA is expressed on TECs throughout the nephron and in podocytes, two
28
29 key cell lineages that can be affected by pEMT, an initiating factor for kidney fibrosis,
30 inflammation and failure in a range of different kidney diseases15–18. IL11RA is also
31 expressed on stromal fibroblasts and vascular smooth muscle cells34 and is important for
32 myofibroblast transformation23.
33
34 One mechanism for kidney protection by inhibition of IL11 signaling in Col4a3-/- mice
35 could be through inhibition of pEMT. In support of this, X203 reduced SNA1 expression,
36 central to EMT/pEMT15,16,35, and restored E-cadherin levels31, while preserving podocyte
37 numbers and expression of podocyte-specific proteins17 (Figure 7). Furthermore, X203
38
dosing of Col4a3-/- mice induced substantial upregulation of renal Cyclin D1 and PCNA
39
40 expression associated with SNAI1 inhibition, suggesting escape of TECs from pEMT/FR-
41 PCT, restoration of TEC proliferation, and kidney repair19,20,31.This effect could be related to
42 suppression of TGFβ, a determinant of pEMT in the kidney15,16,18, as anti-IL11 lowered TGFβ
43 expression. However, while inhibition of TGFβ signaling is proinflammatory36 we show here,
44
as we have in other tissues, that inhibition of IL11 reduces inflammation26,34. Thus pEMT/FR-
45
46 PCT in the kidney may be driven by IL11 itself, similar to reports of its effects in fibrotic lung
47 disease and cancer37,38.
48 While pEMT of damaged podocytes and TECs may initiate renal pathology, the
49 consequent activation of stromal and inflammatory cells is needed for disease progression.
50
51
Indeed, deletion of Il11ra1 in fibroblasts diminishes pathogenic ERK signaling and protects
52 against kidney dysfunction in folic acid nephropathy21. It is therefore likely that some of the
53 effects of X203 in Col4a3-/- mice, are mediated through inhibition of IL11-dependent
54 myofibroblast activation (Figure 7), which in addition to reducing fibrosis also STAT3
55
phosphorylation and IL6 levels. IL6 is linked with a range of kidney diseases and, while not a
56
57 therapeutic target itself, IL6 may serve as a biomarker for latent IL11 activity39. Interestingly,
58 urinary IL11 levels correlate with proteinuria in IgA nephropathy and lupus nephritis and
59 perhaps might be useful for patient stratification40. This also suggests IL11 may be important
60 in other diseases of the renal glomerulus, which remains to be explored.
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4
The Col4a3-/- mouse line has been a useful model of progressive AS and death from
5
6 kidney failure typically occurs between 63 (this study) and 71 days6,33. A previous study
7 showed that ACEi extended lifespan of Col4a3-/- mice by 79 days (110%) when initiated at
8 four weeks of age, before renal IL11 is upregulated. However, there was no survival benefit
9 if ACEi was initiated at seven weeks of age, after kidney injury was established. Here we find
10
11
that ACEi therapy started in six-week-old Col4a3-/- mice modestly extended lifespan by 14
12 days (22%). In contrast, anti-IL11 monotherapy begun at six weeks of age was more
13 effective than ramipril alone, increasing lifespan by 29 days (44%). Most notably, the
14 combination of anti-IL11 and ramipril prolonged survival of Col4a3-/- mice by 62 days (99%),
15
which is substantially longer than ACEi alone, the current standard of care in AS patients.
16
17 Except for limited and incompletely penetrant developmental defects of teeth and
18 skull sutures, humans with loss-of-function of IL11RA appear well with normal immune
19 function. A similar phenotype is seen in Il11ra1 null mice. Interestingly, two recently and
20 separately developed Il11 null mice appear normal with no obvious bony deficits41,42
21
suggesting that inhibiting IL11 might have advantages in side effect profile over targeting
22
23 IL11RA. Taken together, the mild phenotypes of humans and mice lacking IL11RA or IL11,
24 along with absence of untoward effects with lengthy anti-IL11RA and anti-IL11 treatment in
25 mice26, provide an encouraging safety signal for long-term inhibition of IL11 signaling in
26 chronic diseases, like AS23,25.
27
We end by suggesting that inhibition of IL11 signaling may be considered as a novel
28
29 therapeutic approach for patients with AS, and perhaps other progressive forms of CKDs.
30 Anti-IL11 therapy combined with RAS blockade may be of particular interest given the near
31 ubiquitous use of ACEi/ARB in CKD and the synergistic interaction between anti-IL11 and
32 ACEi therapy shown here. With anti-IL11/anti-IL11RA drugs nearing the clinic it will be
33
34 interesting to see if a therapeutic approach for AS discovered in the Col4a3-/- mouse
35 translates to patients, for a second time6,7.
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3 Funding
4
This research was supported by the National Medical Research Council (NMRC), Singapore
5
6 STaR awards (NMRC/STaR/0029/2017), NMRC Centre Grant to the NHCS,
7 MOH‐CIRG18nov‐0002, MRC-LMS (UK), Tanoto Foundation to S.A.C. A.A.W. is supported
8 by NMRC/OFYIRG/0053/2017. N.H. and S.A.C. are supported by a grant from the Leducq
9 Foundation (16CVD03). N.H. is recipient of an ERC advanced grant under the European
10
11 Union Horizon 2020 Research and Innovation Program (AdG788970).
12
13 Author Contributions
14 CRediT Taxonomy
15
Anissa Widjaja: Conceptualization, Formal analysis, Investigation, Methodology,
16
17 Supervision, Writing – original draft, Writing – review & editing
18 Shamini Shekeran: Investigation, Methodology, Validation
19 Eleonora Adami: Visualization
20 Joyce Goh: Investigation
21
Jessie Tan: Investigation
22
23 Sivakumar Viswanathan: Investigation
24 Sze Yun Lim: Investigation
25 Puay Hoon Tan: Formal analysis, Investigation
26 Norbert Hubner: Funding acquisition
27
28
Thomas Coffman: Methodology, Writing – review & editing
29 Stuart Cook: Conceptualization, Formal analysis, Funding acquisition, Methodology,
30 Supervision, Writing – original draft, Writing – review & editing
31
32 Acknowledgements
33
34 None
35
36 Disclosures
37 S.A.C. is a co-inventor of the patent applications: WO/2017/103108 (TREATMENT OF
38
FIBROSIS), WO/2018/109174 (IL11 ANTIBODIES), WO/2018/109170 (IL11RA
39
40 ANTIBODIES). S.A.C. and A.A.W are co-inventors of the patent application: US
41 US2020/0270340A1 (Treatment of Kidney Injury) and GB2009292.0. (Treatment and
42 prevention of disease caused by Type IV collagen dysfunction). S.A.C. is a co-founder and
43 shareholder of Enleofen Bio PTE LTD, a company that made anti-IL11 therapeutics, which
44
were acquired for further development by Boehringer Ingelheim in 2019. A. Widjaja reports
45
46 Patents and Inventions: Boehringer Ingelheim. S. Cook reports Research Funding:
47 Boehringer Ingelheim. T. Coffman reports Advisory or Leadership Role: Editorial Boards:
48 Cell Metabolism and JCI, Singapore Health Services Board of Directors, Singapore Eye
49 Research Institute, and Kidney Research Institute University of Washington. P. Tan reports
50
51 Honoraria for delivering talk on Prostate cancer - AstraZeneca. All other authors declare no
52 competing interest.
53
54
55
56
57
58
59
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3 Figure Legends
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5
6 Figure 1. IL11 is upregulated in kidneys of Col4a3-/- mice and IL11RA is expressed in
7 podocytes and renal tubular epithelial cells.
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47 A-C. Renal (A) Il11 RNA (n=8-11/group) and (B-C) IL11 protein expression (n=3/group) in
48 wild-type and Col4a3-/- mice. D. Immunohistochemistry staining of IL11RA with anti-IL11RA
49 (X209) or IgG (11E10) as control on the kidneys of wild-type and Il11ra1-/- mice (scale bars,
50
20 µm; representative of n=3 datasets/group). E. Comparison of Il11ra1 and gp130
51
52 expression in mouse kidney cells based on single cell transcriptomic analysis by Park et.
53 al.24. F. Immunohistochemistry staining of IL11RA with X209 on the kidneys of wild-type and
54 Col4a3-/- mice (scale bars, 20 µm; representative of n=3 datasets/group). G.
55 Immunofluorescence images (scale bars, 75 µm; representative of n=3 datasets/group) of
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57 EGFP and Wilm’s Tumor 1 expression in the kidneys of Col4a3+/+-Il11:EGFP+/- and Col4a3-/--
58 Il11:EGFP+/-mice. (A) Data are shown as box-and-whisker with median (middle line), 25th–
59 75th percentiles (box) and min-max values (whiskers); 2-tailed Student’s t-test, (C) data are
60 shown as mean±SD; one-way ANOVA with Dunnett’s correction. FC: fold change.
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3 Figure 2. In Col4a3-/- mice, a neutralizing IL11 antibody (X203) preserves kidney mass
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and reduces renal fibrosis.
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44 A. Schematic showing therapeutic dosing of Col4a3-/- mice for experimental data shown in
45 B-I. Six-week-old Col4a3-/- mice were administered IgG/X203 (20 mg/kg, 2x/week) for 2.5
46 weeks; wild-type littermates were used as controls (n=8-11/group). B. Body weight (shown
47
as a percentage (%) of initial body weight). C. Kidney weight. D. Total renal collagen content
48
49 per mg of kidney weight as measured by quantitative colorimetric determination of
50 hydroxyproline residues obtained by acid hydrolysis of collagen (see methods for more
51 details). E-F. (E) Representative and (F) quantification (from 100X field images) of Masson
52 Trichrome’s staining (representative datasets from n=4/group). (B, F) Data are shown as
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mean±SD, (C, D) data are shown as box-and-whisker with median (middle line), 25th–75th
55 percentiles (box) and min-max values (whiskers); (B) 2-way ANOVA with Tukey’s correction,
56 (C, D, F,) one-way ANOVA with Tukey’s correction.
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Journal of the American Society of NephrologyPage 20 of 23
Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 Figure 3. X203 reduces renal ERK and STAT3 activation, fibrosis, and a signature of
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epithelial-to-mesenchymal transition in mice with Alport syndrome.
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29 A. Relative renal mRNA expression of pro-fibrotic markers (Col1a1, Col3a1, Il11, Col1a2,
30 Fn, Acta2, and Tgfβ) (n=8-11/group). B-C. (B) Western blots and (C) densitometry analysis
31 of p-ERK, ERK, p-STAT3, STAT3, ⍺SMA, Fibronectin, E-cadherin, SNAI1, PCNA, Cyclin D1,
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33 and GAPDH (n=4/group). (A) Data are shown as box-and-whisker with median (middle line),
34 25th–75th percentiles (box) and min-max values (whiskers), (C) data are shown as
35 mean±SD; (A, C) one-way ANOVA with Tukey’s correction. FC: fold change
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Journal of the American Society of NephrologyPage 21 of 23
Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 Figure 4. Inhibition of IL11 signaling with a neutralizing IL11 antibody preserves
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podocytes and reduces renal inflammation and tubule damage in Col4a3-/- mice. A-E.
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36 Data for experiments shown in schematic Figure 2A. A-B. (A) Representative images
37 (representative datasets from n=3/group) and (B) quantification (from 200X field images) of
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Wilms’ Tumor 1 (WT1) staining. C-D. (C) Western blots and (D) densitometry analysis of
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40 TGFβ, Cleaved Caspase 3, Caspase 3, Podocin, WT1, and GAPDH (n=4/group). E. Relative
41 renal mRNA expression of kidney injury markers (Kim1 and Ngal), podocyte marker
42 (Podocin), and pro-inflammation markers (Il6, Ccl2, Ccl5, Tnf⍺, and Il1β) (n=8-11/group). (B,
43 E) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles
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45 (box) and min-max values (whiskers), (D) data are shown as mean±SD; one-way ANOVA
46 with Tukey’s correction. FC: fold change
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Journal of the American Society of NephrologyPage 22 of 23
Copyright 2022 by ASN, Published Ahead of Print on 2/9/22, Accepted/Unedited Version
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3 Figure 5. Inhibition of IL11 signaling in Col4a3-/- mice improves renal histology and
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function.
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27 A-G. Data for experiments shown in schematic Figure 2A. (A) Interstitial fibrosis, (B)
28 glomerulosclerosis, (C) tubular atrophy, and (D) total histology composite scores (n=4-
29 7/group). (E) Blood urea nitrogen (BUN), (F) serum Creatinine, (G) urinary
30 albumin:creatinine ratios (n=8-11/group). (A-D) Data are shown as mean±SD, (E-G) data are
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shown as box-and-whisker with median (middle line), 25th–75th percentiles (box) and min-
33 max values (whiskers); one-way ANOVA with Tukey’s correction.
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