Journal: Nature Communications

Article Title: Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

doi: 10.1038/s41467-022-35306-1

Figure Lengend Snippet: a Schematic of X203 dose-finding experiments for experiments shown in ( b , c ). IgG (11E10, 10 mg/kg) or X203 (1–10 mg/kg) was administered to mice 1 day before FA (200 mg/kg) injection. Mice were sacrificed at day 21 post FA (IgG ( n = 6), X203 1 mg/kg ( n = 5), X203 5 mg/kg ( n = 8), X203 10 mg/kg ( n = 7)). b Kidney collagen content by hydroxyproline assay and c BUN levels. d Schematic and e representative kidney gross anatomy for mice that were subjected to X203 and MAB218 treatment regimen following FA-mediated AKI. Mice were randomly divided into 7 groups with the number of mice/group indicated in the table. 10 or 50 mg/kg of IgG, 10 mg/kg of X203 or MAB218 (a commercial anti-IL11 from R&D Systems) were administered twice a week intraperitoneally starting from 1 day before FA injection until the mice were sacrificed (day 21). f Schematic of therapeutic comparison of X203 and anti-TGFβ: 20 mg/kg IgG (11E10), X203, or anti-TGFβ (1D11) was administered to mice 1 day before FA (200 mg/kg) injection for experiments shown in ( g – l ). Mice were sacrificed on day 28 post FA. g Representative Masson’s Trichrome images of whole kidney cross section (scale bars: 500 µm, representative dataset from n = 5/group), h quantification of collagen area from Masson’s Trichrome-stained kidney sections ( n = 5/group), i kidney collagen content by hydroxyproline assay (NaHCO 3 , FA + IgG, FA + 1D11 ( n = 6/group), FA + X203 ( n = 7)), j BUN, k urine ACR. j , k NaHCO 3 ( n = 4), FA + IgG, FA + 1D11 ( n = 6/group), FA + X203 ( n = 8). l Western blots of ERK, STAT3, and SMAD2 activation (representative dataset from n = 5/group). b , c Data are shown as mean ± SD, h – k data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers). b , c One-way ANOVA with Dunnett’s correction, h , i one-way ANOVA with Tukey’s correction, k Kruskal–Wallis with Dunn’s correction. Source data are provided as a Source data file.

Article Snippet: 12-week-old male C57BL/6J mice (InVivos, Singapore (SG)) were administered 20 mg/kg (2x/week) of anti-IL11 (X203) or IgG for 6 weeks, starting from day 21 post FA (200 mg/kg) injection.

Techniques: Injection, Hydroxyproline Assay, Comparison, Staining, Western Blot, Activation Assay, Whisker Assay

Journal: Nature Communications

Article Title: Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

doi: 10.1038/s41467-022-35306-1

Figure Lengend Snippet: a Schematic of X203 therapeutic dosing of FA-injured mice for experiments shown in ( b – k ). IgG/X203 (10 mg/kg) was administered biweekly starting from day 3 following FA injury. Blood and kidney phenotypes were analyzed on day 28. b Representative kidney gross anatomy, c kidney weights, d Western blots of pERK, ERK, pp90RSK, p90RSK, pGSK3β, GSK3β, SNAI1, ZEB, E-Cadherin, Cyclin D1, αSMA, FN1, and GAPDH (representative dataset from n = 5/group), e representative Masson’s Trichrome images of whole kidney cross section (scale bars: 500 µm, representative dataset from n = 5/group), f quantification of collagen area from Masson’s Trichrome-stained kidney sections ( n = 5/group), g kidney collagen contents by hydroxyproline assay, the levels of h BUN, i serum creatinine, and j urine ACRs, k relative mRNA expression of kidney injury ( Ngal , Kim1 ) and pEMT ( Snai1 , Zeb ) markers. c , f – k Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers). c , g – k NaHCO 3 ( n = 5), FA + IgG ( n = 9), FA + X203 ( n = 8). c Kruskal–Wallis with Dunn’s correction, f – k one-way ANOVA with Tukey’s correction. FC: Fold change. Source data are provided as a Source data file.

Article Snippet: 12-week-old male C57BL/6J mice (InVivos, Singapore (SG)) were administered 20 mg/kg (2x/week) of anti-IL11 (X203) or IgG for 6 weeks, starting from day 21 post FA (200 mg/kg) injection.

Techniques: Western Blot, Staining, Hydroxyproline Assay, Expressing, Whisker Assay

Journal: Nature Communications

Article Title: Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

doi: 10.1038/s41467-022-35306-1

Figure Lengend Snippet: a Representative immunofluorescence images (scale bars: 100 µm) of EGFP and E-Cadherin expression in UUO and control kidneys of Il11 EGFP/+ mice (representative dataset from n = 3/group) and b Western blots of IL11 protein expression in control and UUO kidneys (representative dataset from n = 5/group). Kidneys were collected at day 10 and contralateral kidneys were used as controls. c Schematic of X203 therapeutic dosing of mice subjected to left unilateral ureteral obstruction for experiments shown in ( d – h ). IgG/X203 (10 mg/kg) was administered on day 4 and day 8 post UUO and kidneys were collected on day 10; contralateral (right) kidneys from the IgG group were used as controls. d Western blots of pERK, ERK, pp90RSK, p90RSK, pGSK3β, GSK3β, SNAI1, ZEB, E-Cadherin, Cyclin D1, αSMA, FN1, and GAPDH (representative dataset from n = 5/group), e kidney collagen content by hydroxyproline assay (control, UUO + X203 ( n = 7/group), UUO + IgG ( n = 6)), f representative Masson’s Trichrome images of whole kidney cross section (scale bars: 500 µm, representative dataset from n = 5/group), g quantification of collagen area from Masson’s Trichrome-stained kidney sections ( n = 5/group), and h relative renal mRNA expression of pro-inflammatory markers ( Tnfα , Il6, Ccl2 , Ccl5 , Il1β ), fibrosis markers ( Col1a1 , Col1a2 , Col3a1 , Fn1 , Acta2) , and kidney injury markers ( Ngal , Kim1 ) ( n = 7/group). e , g , h Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers); one-way ANOVA with Tukey’s correction except for ( h , Ccl2 and Ccl5 ) which were analyzed by Kruskal–Wallis with Dunn’s correction. FC: Fold change. Source data are provided as a Source data file.

Article Snippet: 12-week-old male C57BL/6J mice (InVivos, Singapore (SG)) were administered 20 mg/kg (2x/week) of anti-IL11 (X203) or IgG for 6 weeks, starting from day 21 post FA (200 mg/kg) injection.

Techniques: Immunofluorescence, Expressing, Control, Western Blot, Hydroxyproline Assay, Staining, Whisker Assay

Journal: Nature Communications

Article Title: Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

doi: 10.1038/s41467-022-35306-1

Figure Lengend Snippet: a Representative IF images of αSMA +ve and SNAI1 +ve cells and Collagen 1 immunostaining (scale bars: 100 µm, representative dataset from n = 3/group) and b Western blots of pERK, ERK, pp90RSK, p90RSK, pGSK3β, GSK3β, SNAI1, ZEB, E-Cadherin, PCNA, Cyclin D1, αSMA, and GAPDH (representative dataset from n = 4/group) from TECs stimulated with either IL11 or TGFβ in the presence of IgG/X203. c Heatmaps showing quantification of αSMA +ve and SNAI1 +ve cells and Collagen 1 intensity/area ( n = 14/group; values are shown in Supplementary Fig. ) and d Western blots of pERK, ERK, pp90RSK, p90RSK, pGSK3β, GSK3β, SNAI1, ZEB, E-Cadherin, PCNA, Cyclin D1, αSMA, and GAPDH for TECs stimulated with various pro-fibrotic factors in the presence of IgG/X203 (representative dataset from n = 4/group). e Quantification of αSMA +ve and SNAI1 +ve cells and Collagen 1 intensity/area from TECs stimulated with TGFβ or IL11 subjected to siRNA knockdown for SNAI1 (NT: non-targeting siRNA control) (24 h; n = 14/group; Bsl, baseline). f Schematic showing mechanism and signaling pathway by which IL11 induces repression of SNAI1 transcription. g Schematic and h quantification of αSMA +ve cells and Collagen 1 immunostaining of HKFs ( n = 14/group) for media transfer experiments in which conditioned media from control or TGFβ-stimulated TECs (24 h) were used to treat primary human kidney fibroblasts (HKFs) in the presence of anti-TGFβ (1D11) alone or with either IgG or X203. a – h AngII (100 nM), bFGF (10 ng/ml), Endothelin 1 (ET-1, 250 ng/ml), H 2 O 2 (0.2 mM), IL11 (5 ng/ml), PDGF (20 ng/ml), TGFβ1 (5 ng/ml), IgG (2 μg/ml), X203 (2 μg/ml), anti-TGFβ (clone 1D11, 2 μg/ml), siNT/siSNAI1 (25 nM); 24-h stimulation. e , h Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers); one-way ANOVA with Tukey’s correction (except for H, αSMA +ve which was analyzed by Kruskal–Wallis with Dunn’s correction). Bsl: Baseline; I/A: Intensity per area. Source data are provided as a Source data file.

Article Snippet: 12-week-old male C57BL/6J mice (InVivos, Singapore (SG)) were administered 20 mg/kg (2x/week) of anti-IL11 (X203) or IgG for 6 weeks, starting from day 21 post FA (200 mg/kg) injection.

Techniques: Immunostaining, Western Blot, Knockdown, Control, Whisker Assay

Journal: Nature Communications

Article Title: Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

doi: 10.1038/s41467-022-35306-1

Figure Lengend Snippet: a Schematic and Western blots (representative dataset from n = 4/group) of pERK, ERK, pp90RSK, p90RSK, pGSK3β, GSK3β, SNAI1, ZEB, E-Cadherin, PCNA, Cyclin D1, αSMA, and GAPDH for TEC reversal experiment: TECs were stimulated with TGFβ for 72 h followed by addition of IgG/X203 for another 24 h. b Representative IF images of αSMA +ve and SNAI1 +ve cells and Collagen 1 immunostaining (scale bars: 100 µm, representative dataset from n = 3/group), and c quantification ( n = 14/group) and d representative IF images (scale bars: 100 µm, representative dataset from n = 3/group) of EdU +ve cells for TEC reversal experiments as shown in ( a ). c Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers); Kruskal–Wallis with Dunn’s correction. Bsl: Baseline. Source data are provided as a Source data file.

Article Snippet: 12-week-old male C57BL/6J mice (InVivos, Singapore (SG)) were administered 20 mg/kg (2x/week) of anti-IL11 (X203) or IgG for 6 weeks, starting from day 21 post FA (200 mg/kg) injection.

Techniques: Western Blot, Immunostaining, Whisker Assay

Journal: Nature Communications

Article Title: Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease

doi: 10.1038/s41467-022-35306-1

Figure Lengend Snippet: a Schematic of X203 therapeutic dosing regimen for CKD reversal experiments shown in ( b – l ): X203 or IgG (IP, 10 mg/kg) was administered 2X/week for 12 weeks from day 21 (week 3) after FA injury, when CKD is established and sustained. Blood and kidneys were collected every 3 weeks; full course serum and kidney phenotyping study were performed at the study end-point (week 15: 12 weeks after the start of X203 therapy); blood and kidneys from control mice i.e., those receiving IP injection of vehicle control (0.3 M NaHCO 3 ) were collected at week 3 and 15. b Representative kidney gross anatomy, c kidney weights (NaHCO 3 (W3), FA (W3) ( n = 11/group), FA + IgG (W6, W9), FA + X203 (W6, W12) ( n = 4/group), FA + X203 (W9), FA + IgG (W12), NaHCO 3 (W15) ( n = 5/group), FA + IgG/X203 (W15) n = 13/group)), d renal collagen content by hydroxyproline assay, e representative Masson’s Trichrome images of whole kidney cross section (scale bars: 500 µm, representative dataset from n = 4/group for kidneys collected at week 6, 9, 12 or from n = 5/group for week 3 and 15), f Western blots showing renal ERK and p90RSK activation, GSK3β inactivation, and SNAI1, ZEB, E-Cadherin, Cyclin D1, αSMA, FN1, GAPDH expression (representative dataset from n = 5/group), g BUN, h serum creatinine, i urine ACR (NaHCO 3 (W3, W15) ( n = 5/group), FA (W3) ( n = 6), FA + IgG/X203 (W15) ( n = 9/group). d , g , h NaHCO 3 (W3), FA (W3) ( n = 11/group), FA + IgG (W6, W9), FA + X203 (W6) ( n = 4/group), FA + X203 (W9, W12), FA + IgG (W12), NaHCO 3 (W15) ( n = 5/group), FA + IgG/X203 (W15) ( n = 13/group). j Heatmaps of renal mRNA expression of fibrosis, inflammation, kidney injury, and EMT markers (values are shown in Supplementary Fig. ) from control, IgG- and X203-injected mice (week 15). k Representative immunofluorescence images of EdU (green) and E-Cadherin (red) expression and l quantification of EdU +ve E-Cadherin +ve cells in the kidneys of mice receiving either IgG or X203 reversal dosing regimen for 6 weeks starting from day 21 post FA administration (representative dataset from n = 3/group for NaHCO 3 and n = 4/group for FA + IgG and FA + X203 group; scale bars: 100 µm). c , d , g – i Data are shown as mean ± SD; k data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers). c , d , g , h two-way ANOVA with Sidak’s correction; i two-tailed Student’s t-test; k one-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.

Article Snippet: 12-week-old male C57BL/6J mice (InVivos, Singapore (SG)) were administered 20 mg/kg (2x/week) of anti-IL11 (X203) or IgG for 6 weeks, starting from day 21 post FA (200 mg/kg) injection.

Techniques: Control, Injection, Hydroxyproline Assay, Western Blot, Activation Assay, Expressing, Immunofluorescence, Whisker Assay, Two Tailed Test