Dual inhibition of the endothelin and angiotensin receptor ameliorates renal and inner ear pathologies in Alport mice

Abstract

Alport syndrome (AS), a type IV collagen disorder, leads to glomerular disease and, in some patients, hearing loss. AS is treated with inhibitors of the renin-angiotensin system; however, a need exists for novel therapies, especially those addressing both major pathologies. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist (DEARA) under clinical development for focal segmental glomerulosclerosis and IgA nephropathy. We report the ability of sparsentan to ameliorate both renal and inner ear pathologies in an autosomal-recessive Alport mouse model. Sparsentan significantly delayed onset of glomerulosclerosis, interstitial fibrosis, proteinuria, and glomerular filtration rate decline. Sparsentan attenuated glomerular basement membrane defects, blunted mesangial filopodial invasion into the glomerular capillaries, increased lifespan more than losartan, and lessened changes in profibrotic/pro-inflammatory gene pathways in both the glomerular and the renal cortical compartments. Notably, treatment with sparsentan, but not losartan, prevented accumulation of extracellular matrix in the strial capillary basement membranes in the inner ear and reduced susceptibility to hearing loss. Improvements in lifespan and in renal and strial pathology were observed even when sparsentan was initiated after development of renal pathologies. These findings suggest that sparsentan may address both renal and hearing pathologies in Alport syndrome patients. © 2023 Travere Therapeutics, Inc and The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: Alport syndrome; basement membrane; chronic kidney disease; extracellular matrix; genetic disorders; sparsentan; stria vascularis.

Figure 1.. Schematic overview of studies in AS mice.

Studies examining the ability of sparsentan (SP) or losartan (LS) to delay the progression of the renal and inner ear pathology and dysfunction in AS mice were run in several different paradigms. (1) EI and LI: Treatment in AS mice was started at 3 wk of age (EI) or 5 wk of age (LI). Studies were terminated at 7 wk of age for collection of serum and removal and processing of kidneys for assessment of pathology. RNA from isolated glomeruli and cortex harvested from mice in EI studies were used to analyze gene expression. (2) GFR: In AS mice treated from 4 wk of age, assessment of GFR was conducted at 9 wk of age, and GS and FS were determined at 10 wk of age. (3) Lifespan: Treatment of AS mice was initiated at 4, 5, 6, or 7 wk of age, and UP/C was determined on a weekly basis. (4) Hearing loss: AS mice were treated from 3 wk of age (EI) or 5 wk of age (LI). Pre-noise ABR was assessed at 8.5 wk of age, and a second ABR test was performed 5 days following noise exposure. In EI studies, cochleae were excised following the second ABR test. Striae were isolated and processed for TEM to histology and measure SCBM width.

Figure 2.. Sparsentan delays development of GS, fibrosis, and GBM morphology more than losartan in AS mice.

(A, B) Trichrome staining of representative kidney sections from EI and LI studies (n=8, except for AS-LS 5–7 wk of age, n=7). Compared to the kidney section from a WT-V mouse treated from 3–7 wk of age, the representative kidney section from a 3–7 wk of age AS-V mouse contained an increased number of sclerotic glomeruli, as indicated by the enlarged glomerulus denoted by black arrows in the inset. In kidney sections from AS-SP or AS-LS EI mice, sclerotic glomeruli were largely absent such that the pathology was comparable to that of the WT-V mice. The representative section from an AS-UT 5-wk-old mouse depicts the increase in GS at baseline before treatment in LI, as indicated by the enlarged glomerulus (black arrows in the inset). The GS in AS-SP 5–7-wk-old mice tended to be less than that in AS-LS 5–7-wk-old mice, as indicated by the difference in the number of black arrows between the images. (C) Representative cortical sections from AS mice in LI studies showing that fluorescence was attenuated in AS-SP 5–7-wk-old mice compared to those from AS-V 3–7-wk-old mice following staining with anti-COLI antibodies (red) or anti-CD45 antibodies (green), illustrating the antifibrotic and anti-inflammatory effects of sparsentan. (D) Quantification of GS following IF with anti-fibronectin antibodies showed that in AS-SP 5–7-wk-old mice, the percentage of sclerotic glomeruli in kidney sections was significantly ameliorated (**P<0.01) compared to that in AS-V 3–7-wk-old mice. GS in AS-UT 5-wk-old mice was also significantly lower than that in AS-V 3–7-wk-old mice (**P<0.01). (E) Quantification of TIF in LI studies via anti-COLI IF depicting significant prevention of TIF in AS-SP 5–7-wk-old mice (*P<0.05) compared to AS-V 3–7-wk-old mice. TIF in AS-UT 5-wk-old mice was also significantly lower than that in AS-V 3–7-wk-old mice (**P<0.01). (F) Glomerular ultrastructural images from WT-V 3–7-wk-old mice, AS-V 3–7-wk-old mice, AS-SP 5–7-wk-old mice, and AS-LS 5–7-wk-old mice illustrate the GBM thickening and dysmorphology (asterisks) and podocyte effacement (black arrows) and preservation of GBM morphology in AS-SP 5–7-wk-old mice compared to AS-V 3–7-wk-old mice. AS-LS 5–7-wk-old mice had little alterations in GBM compared to AS-V 3–7-wk-old mice. Panel D data were statistically analyzed using Brown-Forsythe and Welch 1-way ANOVA tests with Dunnett’s T3 multiple comparison test. Panel E data were statistically analyzed using the Kruskal–Wallis test with Dunn’s multiple comparison test.

Figure 3.. Sparsentan delayed the decline in GFR and renal pathology in aged AS mice.

GFR and renal pathology in WT and AS mice treated with vehicle or sparsentan from 4 wk of age. (A) Comparison of GFR at 9 wk of age. ***P<0.001, ****P<0.0001, WT-V (n=6) or AS-SP (n=15) compared to AS-V mice (n=10). Preservation of GFR in AS-SP mice compared to AS-V mice was associated with amelioration at 10 wk of age of (B) GS and (C) TIF. **P<0.01 AS-SP (n=7) compared to AS-V (n=4). (D) Representative sections from three WT-V, AS-V, and AS-SP mice following staining with an anti-COLI antibody, indicating the attenuation of fibrosis in 10-wk-old AS-SP mice. Data in panel A are shown as their median; data in B and C are shown as their mean±SD. Analysis of panel A data used 1-way ANOVA followed by Tukey’s multiple comparison test. Analysis of panels B and C data was conducted using a 2-tailed Student’s t test. All analyses were performed using GraphPad Prism.

Figure 4.. Sparsentan treatment attenuated expression of proinflammatory/profibrotic genes in both the cortical and glomerular compartments of the kidney.

Heat map of DE genes from the mouse fibrosis microarray in the (A) cortex and (B) glomeruli of AS mice treated in EI studies from 3–7 wk old with vehicle or sparsentan (120 mg/kg) and compared to tissue from WT-V mice. Of the 84 genes in the panel investigated, 32 and 17 genes were found differentially expressed (P<0.05) in the renal cortex and glomeruli of AS mice, respectively, relative to WT mice. Changes in expression levels of several of the DE genes were ameliorated or trended toward normalization when AS mice were treated with sparsentan. Statistical analysis is described in the Methods section under Gene expression analysis.

Figure 5.. Sparsentan extended lifespan and delayed proteinuria even when treatment initiated in mice with developed renal structural changes.

Lifespan and UP/C of AS-V, AS-LS, or AS-SP mice treated from 4, 5, 6, or 7 wk of age, where renal structural changes and proteinuria were evident from 5 wk of age. (A) Kaplan–Meier plot showing the percent survival of AS-V, AS-LS, or AS-SP mice with treatment initiated at different ages. (B) Mean UP/C over the course of the lifespan study in mice shown in panel A. Dashed line indicates the mean UP/C of AS-V mice at 10 wk of age. (C) Sclerotic glomeruli fraction in untreated AS mice at 5, 6, and 7 wk of age (n=6, n=6, and n=7, respectively). (D) TIF in untreated AS mice at 5, 6, and 7 wk of age (n=6, n=6, and n=7, respectively). Dosing with study treatment AS-LS, 10 mg/kg; AS-SP, 120 mg/kg. The number of mice surviving at each week during the life-span studies is presented in supplementary material, Table S7.

Figure 6.. Sparsentan but not losartan protected against hearing loss in AS mice.

Hearing loss from a metabolic noise stress in AS or WT mice following treatment with vehicle, losartan, or sparsentan from 3–9 wk of age (EI) or 5–9 wk of age (LI; sparsentan only) was assessed by ABR measurements at frequencies of 8–40 kHz. AS-V (A) and AS-VW (B) were susceptible to the metabolically stressful noise as evidenced by hearing loss 5 days following exposure versus the recovery by WT-V or WT-VW mice, respectively (*P<0.05 for WT-V versus AS-V), (C) Recovery of hearing loss in AS-SP mice was equivalent to that of WT-SP mice, (D) Hearing loss in AS-LS mice was worse compared to AS-VW mice (*P<0.05 WT-LS versus AS-LS), (E) SCBM width in WT and AS mice treated with vehicle, SP, or LS from 3–9.5 wk of age in EI studies (n=5), determined from ultrastructural images obtained following excision of the cochlea after the post-noise ABR measurement. Both SP and LS significantly prevented the increase in SCBM width observed in AS-V mice. *P<0.05 for AS-V versus WT-SP; **P<0.01 for AS-V versus WT-V; ***P<0.001 for AS-V versus AS-SP or AS-LS. Notably, the average SCBM in the AS-LS mice had thinned to less than the normal basement membrane width of 50–60 nm, while the remaining three mice showed average SCBM widths consistent with those from WT-V mice. (F) Late treatment at 5–9 wk of age with sparsentan significantly attenuated susceptibility to hearing loss in AS mice compared to AS-V mice at all frequencies tested: WT-V, n=16; AS-V, n=10; AS-SP, n=11. ###P<0.001, #P<0.05 for AS-V versus WT-V; ***P<0.001, **P<0.01, *P<0.05 for AS-V versus AS-SP. Data in (A–E) are shown as mean dB SPL±SEM. Data in (E) are shown for each stria as well as the group mean±SD (nm). Analysis (A–D and F) was performed using 2-way ANOVA followed by the Holm-Šídák multiple comparison test (GraphPad Prism). Analysis in (E) was performed using 1-way ANOVA and Tukey’s post-hoc multiple comparison test (GraphPad Prism).