A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a Mouse Model of Alport Syndrome

Abstract

Background: Alport syndrome is a genetic disorder characterized by a defective glomerular basement membrane, tubulointerstitial fibrosis, inflammation, and progressive renal failure. IL-11 was recently implicated in fibrotic kidney disease, but its role in Alport syndrome is unknown.

Methods: We determined IL-11 expression by molecular analyses and in an Alport syndrome mouse model. We assessed the effects of a neutralizing IL-11 antibody (×203) versus an IgG control in Col4a3^-/- mice (lacking the gene encoding a type IV collagen component) on renal tubule damage, function, fibrosis, and inflammation. Effects of ×203, the IgG control, an angiotensin-converting enzyme (ACE) inhibitor (ramipril), or ramipril+X203 on lifespan were also studied.

Results: In Col4a3^-/- mice, as kidney failure advanced, renal IL-11 levels increased, and IL-11 expression localized to tubular epithelial cells. The IL-11 receptor (IL-11RA1) is expressed in tubular epithelial cells and podocytes and is upregulated in tubular epithelial cells of Col4a3^-/- mice. Administration of ×203 reduced albuminuria, improved renal function, and preserved podocyte numbers and levels of key podocyte proteins that are reduced in Col4a3^-/- mice; these effects were accompanied by reduced fibrosis and inflammation, attenuation of epithelial-to-mesenchymal transition, and increased expression of regenerative markers. X203 attenuated pathogenic ERK and STAT3 pathways, which were activated in Col4a3^-/- mice. The median lifespan of Col4a3^-/- mice was prolonged 22% by ramipril, 44% with ×203, and 99% with ramipril+X203.

Conclusions: In an Alport syndrome mouse model, renal IL-11 is upregulated, and neutralization of IL-11 reduces epithelial-to-mesenchymal transition, fibrosis, and inflammation while improving renal function. Anti-IL-11 combined with ACE inhibition synergistically extends lifespan. This suggests that a therapeutic approach targeting IL-11 holds promise for progressive kidney disease in Alport syndrome.

Keywords: Alport syndrome; chronic kidney disease; fibrosis; glomerular disease; glomerulosclerosis; interleukin 11; podocyte; therapy.

Graphical abstract

Figure 1.

IL-11 is upregulated in the kidneys of Col4a3^−/− mice, and IL-11RA is expressed in podocytes and renal tubular epithelial cells. (A–C) Renal (A) Il-11 RNA (n=8–11/group) and (B–C) IL-11 protein expression (n=3/group) in WT and Col4a3^−/− mice. (D) IHC staining of IL-11RA with anti-IL-11RA (×209) or IgG (11E10) as control on the kidneys of WT and Il-11ra1^−/− mice (scale bars, 20 µm; representative of n=3 datasets/group). (E) Comparison of Il-11ra1 and gp130 expression in mouse kidney cells on the basis of single-cell transcriptomic analysis by Park et al. (F) IHC staining of IL-11RA with ×209 on the kidneys of WT and Col4a3^−/− mice (scale bars, 20 µm; representative of n=3 datasets/group). (G) IF images (scale bars, 75 µm; representative of n=3 datasets/group) of EGFP and WT1 expression in the kidneys of Col4a3^+/+-Il-11:EGFP^+/− and Col4a3^−/−-Il-11:EGFP^+/−mice. (A) Data are shown as box and whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers); two-tailed Student’s t test. (C) Data are shown as mean±SD; one-way ANOVA with Dunnett’s correction. FC, fold change.

Figure 2.

In Col4a3^−/− mice, a neutralizing IL-11 antibody (×203) preserves kidney mass and reduces renal fibrosis. (A) Schematic showing therapeutic dosing of Col4a3^−/− mice for experimental data shown in B-I. Six-week-old Col4a3^−/− mice were administered IgG/X203 (20 mg/kg, twice a week) for 2.5 weeks; WT littermates were used as controls (n=8–11/group). (B) Body weight (shown as a percentage of initial body weight). (C) Kidney weight. (D) Total renal collagen content per milligram of kidney weight as measured by quantitative colorimetric determination of hydroxyproline residues obtained by acid hydrolysis of collagen (see Methods for more details). (E) Representative and (F) quantification (from 100× field images) of Masson’s trichrome staining (representative datasets from n=4/group). (B and F) Data are shown as mean±SD. (C and D) Data are shown as box and whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers). (B) Two-way ANOVA with Tukey’s correction. (C, D, and F) One-way ANOVA with Tukey’s correction.

Figure 3.

X203 reduces renal ERK and STAT3 activation, fibrosis, and a signature of epithelial-to-mesenchymal transition in mice with AS. (A) Relative renal mRNA expression of profibrotic markers (Col1a1, Col3a1, Il-11, Col1a2, Fn, Acta2, and Tgf-β; (n=8–11/group). (B) WB and (C) densitometry analysis of p-ERK, ERK, p-STAT3, STAT3, α-smooth muscle actin, fibronectin, E-cadherin, SNAI1, PCNA, cyclin D1, and GAPDH (n=4/group). (A) Data are shown as box and whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers). (C) Data are shown as mean±SD. (A and C) One-way ANOVA with Tukey’s correction.

Figure 4.

Inhibition of IL-11 signaling with a neutralizing IL-11 antibody preserves podocytes and reduces renal inflammation and tubule damage in Col4a3^−/− mice. (A–E) Data for experiments shown in schematic Figure 2A. (A) Representative images (representative datasets from n=3/group) and (B) quantification (from 200× field images) of WT1 staining. (C) WB and (D) densitometry analysis of TGF-β, cleaved caspase 3, caspase 3, podocin, WT1, and GAPDH (n=4/group). (E) Relative renal mRNA expression of kidney injury markers (Kim1 and Ngal), podocyte marker (podocin), and proinflammation markers (Il-6, Ccl2, Ccl5, Tnf-α, and Il1-β; n=8–11/group). (B and E) Data are shown as box and whisker with median (middle line), 25th–75th percentiles (box), and minimum–maximum values (whiskers). (D) Data are shown as mean±SD; one-way ANOVA with Tukey’s correction.

Figure 5.

Inhibition of IL-11 signaling in Col4a3^−/− mice improves renal histology and function. (A–G) Data for experiments shown in schematic Figure 2A. (A) Interstitial fibrosis, (B) glomerulosclerosis, (C) tubular atrophy, and (D) total histology composite scores (n=4–7/group). (E) BUN, (F) serum Cr, and (G) urinary albumin-creatinine ratios (n=8–11/group). (A–D) Data are shown as mean±SD. (E–G) 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.

Figure 6.

Therapeutic targeting of IL-11 extends lifespan when given alone or in combination with ramipril. (A) Schematic showing therapeutic dosing of Col4a3^−/− mice in lifespan study for data shown in (B) and (C) (n=8–12/group). Col4a3^−/− mice were administered IgG/X203 (20 mg/kg, twice a week, intraperitoneally) alone or in combination with ramipril (10 mg/kg per day, 6 days/week, intraperitoneally) starting from 6 weeks of age until death ensued. (B) Survival curves and (C) percentage of lifespan extension (over untreated Col4a3^−/− mice) of mice treated with IgG, X203, ramipiril+IgG, or ramipril+X203. (C) 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.

Figure 7.

Proposed mechanism for IL-11-induced renal failure in AS. Glomerular basement membrane disruption due to Col4A3 mutation causes podocyte dysfunction and consequent tubular stress. Injured TECs, and podocytes, upregulate IL-11, leading to autocrine pEMT (SNAI1 upregulation and E-cadherin downregulation) of TECs and podocytes and paracrine activation of stromal myofibroblasts, which themselves secrete IL-11 that amplifies the fibrotic response and stimulates the recruitment/activation of immune cells. Neutralizing antibodies against IL-11 reduce renal dysfunction and extend life in Col4a3-deleted mice.