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

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.


Introduction
Alport syndrome (AS) is a congenital basement membrane collagen disorder associated with progressive failure of the glomerular filtration barrier, hearing loss, and anterior lenticonus. AS culminates in end-stage renal disease (ESRD) in some patients as early as the third decade of life [1]. The vast majority of AS cases are due to COL4A3/4 haploinsufficiency, while autosomal-recessive inheritance is rare ($1%) [2]. Approximately 5% of the cases are X-linked caused by pathogenic variants in COL4A5 and are at much higher risk for chronic kidney disease (CKD) [3]. In AS, the glomerular basement membrane (GBM) of the kidney and strial capillary basement membrane (SCBM) in the inner ear lack the physiological association of three type IV collagen chains (α3, α4, and α5). Instead, only the type IV collagen (α1)2α2 GBM network remains, which is thinner and contains fewer interchain crosslinks [4]. In the kidney, this abnormal GBM composition results in a compromised ability to withstand biomechanical stresses on the glomerular filtration barrier, leading to glomerular disease [5]. For similar reasons, AS is also frequently associated with progressive hearing loss.
Current standard-of-care (SOC) renin-angiotensinaldosterone system (RAAS) inhibitors, including angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs), have beneficial renal effects in AS patients that could be mediated by pleiotropic nephroprotective effects, including a reduction in intraglomerular pressure resulting in alleviation of biomechanical stresses on the GBM [6][7][8][9][10]. However, Endothelin 1, a vasoactive peptide with pleiotropic effects in renal pathophysiology, has emerged as an important factor in the progression of AS. Endothelin-1 (ET-1), a vasoactive peptide, is an agonist for two G-protein-coupled receptors, endothelin type A receptor (ET A R) and endothelin type B receptor (ET B R). Activation of ET A R results in vasoconstriction, proliferation, inflammation, extracellular matrix production, and fibrosis.
We showed that ET-1 is induced in the endothelium of Alport glomeruli, where it binds to ET A Rs on mesangial cells. The mechanism downstream of ET A R remains only partially elucidated. Activation of CDC42/RAC1 leads to mesangial filopodial invasion of glomerular capillaries, resulting in the deposition of mesangial proteins, including laminin α2, in the GBM [13]. Laminin α2 directly injures podocytes by activating focal adhesion kinase and NF-κB [14]. ET A R blockade has been shown to prevent mesangial filopodial invasion and to ameliorate glomerular disease in Alport mice [13].
Inner ear pathology in AS is associated with progressive thickening of the SCBM [15]. The stria vascularis (see supplementary material, Figure S1) plays an important role in maintaining the endocochlear potential and, thus, hearing [16]. In mouse models of AS, SCBM thickens due to accumulation of basement membrane proteins, leading to hypoxia, metabolic stress, and elevated susceptibility to noise-induced hearing loss [17]. In parallel with the beneficial effects of ET A R antagonism in the AS kidney, amelioration of pathologies in the stria vascularis was also observed [17], indicating a role of ET-1 in the pathophysiology of AS-associated hearing loss. While the molecular mechanisms downstream of ETAR activation in the stria are not clearly understood, it is likely that pathways similar to those in the mesangial cells may play a role. However, whether ET A R antagonism lessens susceptibility to functional hearing loss was not determined.
Sparsentan is a single molecule that antagonizes both ET A R and angiotensin II subtype 1 receptor (AT 1 R). It is currently undergoing phase 3 clinical trials for the treatment of focal segmental glomerulosclerosis (FSGS) and IgA nephropathy (IgAN) [18,19]. Studies in experimental models support a wide spectrum of beneficial effects of dual inhibition of the RAAS and ET system in the treatment of kidney diseases [20]. We hypothesized that dual inhibition of ET and AngII receptors with sparsentan could serve as a potentially efficacious treatment for glomerular and renal tubulointerstitial disease in the autosomal-recessive Col4a3 À/À mouse model of AS, while also delaying the onset of inner ear pathology and susceptibility to functional hearing loss.

Mice
Male and female Col4a3 knockout (KO) mice, on the 129/Sv background, were used [21]. All experiments were performed in accordance with approved IACUC protocols at Boystown National Research Hospital and Washington University, with full attention to and compliance with USDA recommendations for the care and use of animals in research (https://www.fs.usda. gov/research/about/animalcare, last accessed 31 March 2023). Animals were housed in rooms at 70 F temperature, 55% relative humidity, and with a 14/10-h light/ dark cycle. Animals had free access to rodent chow (Envigo, Indianapolis, IN, USA) and water. The genetic status of Col4a3 KO mice was confirmed by a genomic PCR test on DNA isolated from tail clippings. Mice were caged together based on sex. The experimental design was focused to minimize the number of animals employed and to minimize pain and distress.

Animal studies
Male and female Col4a3 KO mice were randomized and stratified by weight into treatment groups. Mice were given vehicle (0.5% methylcellulose 4,000 cps, #64625, Sigma, St. Louis, MO, USA; 0.25% Tween-80 in distilled water, #P1754, Sigma) or sparsentan by oral gavage once daily. Losartan was given in the drinking water (DW) to deliver 10 mg/kg daily. In studies where losartan was initiated at 3 weeks of age, losartan (10 mg/kg) was administered by oral gavage between 3 and 4 weeks of age. In lifespan studies, AS mice were dosed until they lost more than 15% of peak body weight. For the hearing loss studies, additional control groups were included using AS mice that were given water by oral gavage between 3 and 4 weeks of age (VW, vehicle water). A schematic of the studies is depicted in Figure 1.
Blood pressure (BP) measurements BP was measured using a tail cuff and non-invasive blood pressure system (CODA2 High Throughput System; Kent Scientific, Torrington, CT, USA).
Measurements of urine protein to creatinine ratio (UP/C) Urine was collected weekly. UP was measured using a standard Bradford assay (BioRad Inc., Hercules, CA, USA). UP was normalized to creatinine (UP/C) (BioAssay Systems Creatinine Assay kit, #DICT-500; BioAssay Systems, Hayward, CA, USA).

Immunofluorescence (IF) microscopy
Fresh frozen OCT-embedded kidneys were sectioned at 8 μm and fixed in acetone for 10 min at À20 C. Slides were incubated overnight at 4 C in the appropriate primary antibody and blocking solution. Dual staining was performed by incubating slides using antibodies for CD45 at 1 Tubulointerstitial fibrosis (TIF) and glomerulosclerosis (GS) scoring TIF and GS scoring was performed blinded by two individual scorers. For GS, both fibronectin and Masson's trichrome were employed. Masson's trichrome staining was performed by the University of Nebraska Medical Center's Tissue Science Facility. The total numbers of sclerotic (>50% glomerular area) and healthy glomeruli were recorded and scored for each cryosection and the sclerotic proportion was expressed as a percentage.

Transmission electron microscopy
The GBM and SCBM were prepared and examined as described previously [13,14,22].

Gene expression analysis
Kidney cortical glomeruli were isolated following transcardiac perfusion with 4-μm Dynabeads (Life Technologies, Carlsbad, CA, USA) using a magnet, as described previously [23]. Gene expression levels were determined using the RT 2 Profiler PCR Array (Array: PAMM-120ZA; Qiagen, Hilden, Germany), which assesses 84 genes involved in dysregulated tissue remodeling during the repair and healing of wounds. Raw counts were normalized using the average value across biological replicates of five reference genes (Actb, B2m, Gapdh, Hprt1, and Rplp0). Fold-changes (FCs) were calculated as the ratio of the normalized gene expression in the test sample to the normalized gene expression in the control sample (2 ÀΔCt  Transdermal glomerular filtration rate (GFR) method GFR was determined using a transdermal device (MediBeacon, Creve Coeur, MO, USA), as previously described [24]. For device placement and injection of (1) EI and LI: Treatment in AS mice was started at 3 weeks of age (EI) or 5 weeks of age (LI). Studies were terminated at 7 weeks 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 was used to analyze gene expression. (2) GFR: In AS mice treated from 4 weeks of age, assessment of GFR was conducted at 9 weeks of age, and GS and FS were determined at 10 weeks of age. (3) Lifespan: Treatment of AS mice was initiated at 4, 5, 6, or 7 weeks of age, and UP/C was determined on a weekly basis. (4) Hearing loss: AS mice were treated from 3 weeks of age (EI) or 5 weeks of age (LI). Pre-noise ABR was assessed at 8.5 weeks 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 for histology and measurement of SCBM width.
Dual inhibition of endothelin type-A and angiotensin II AT1 receptors in Alport mice 355 FITC-labeled sinistrin, mice were anesthetized with isoflurane. An imager device was mounted onto each animal's back. Background signal was recorded for 2 min prior to the retro-orbital injection of 150 mg/kg FITC-sinistrin (in PBS) (# NC1570801; Fisher Scientific, part of Thermo Fisher Scientific). Data were analyzed using Mannheim Pharma and Diagnostics Lab software (MediBeacon) [25]. GFR (ml/min) was calculated from the decrease in fluorescence intensity over time. T 1/2 was calculated in minutes from the injection start to the 50% overall luminance point [26].

Inner ear studies
All procedures for hearing studies were conducted at Washington University (St. Louis, MO, USA). The colony was established using mice from the Cosgrove lab to ensure consistency with the renal studies. Dosing was performed as described for the renal studies.
Hearing evaluation with the auditory brainstem response (ABR) Threshold evaluations were conducted in mice at 8.5 and 9.5 weeks of age (following exposure to noise stress) that had been treated with vehicle, vehicle-water, sparsentan, or losartan from 3 or 5 weeks of age (vehicle or sparsentan only). Mice were sedated using avertin (2,2,2-tribromoethanol, 250 mg/kg; Sigma-Aldrich) and underwent ABR testing as described previously [17]. The magnitude of the hearing loss post-noise was calculated by subtracting the ABR hearing threshold for pre-noise from that of post-noise hearing testing.

Metabolic stress via exposure to noise
Following an initial hearing assessment, mice were exposed to a metabolically stressful noise (106 dB SPL OB centered at 10 kHz for 10 h); this noise has been previously shown not to cause damage to the organ of Corti and hair cells [17,27]. Following the noise exposure, the mouse was removed and returned to vivarium housing. Five days after the noise exposure, the mouse underwent a post-noise hearing assessment followed by euthanasia.

Basement membrane width
Cochleae were removed for analysis following the post-noise ABR measurement. Preparation of the cochlea for transmission electron microscopy (TEM) was conducted as described previously [28]. SCBM thickness measurements were calculated as described previously [17].

Statistical analyses
Data are shown as mean ± SD, mean, or median (CI) as indicated. The number of replicates for each experiment is shown in the figure legends. Significance was set at a probability level of less than 0.05. The statistical method used for comparison of groups is indicated in the figure or table legend and was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) software or SAS version 9.4 (SAS Institute, Cary, NC, USA) as indicated.

Results
Studies comparing the effects of sparsentan with those of losartan on renal and auditory function were conducted in several experiments with different designs as illustrated in Figure 1. The dose of sparsentan was selected following a pilot study (supplementary material, Table S1). Early intervention (EI) studies compared the effects of sparsentan (AS-SP; 120 mg/kg) in AS mice with those in losartan-treated AS mice (AS-LS; 10 mg/kg) in mice treated from ages 3 to 7 weeks. Sparsentan-and losartan-treated AS mice showed significant attenuation of the development of proteinuria (p < 0.05), TIF, and GS (p < 0.001 and p < 0.01, respectively; Figure 2A and supplementary material, Figure S2). In late intervention (LI) studies, sparsentan attenuated UP/C at 7 weeks (p < 0.05), whereas the anti-proteinuric effect in losartan-treated mice was not significant (supplementary material, Table S2). Sparsentan also significantly ameliorated GS and TIF in 7-week-old mice to levels comparable to those in untreated 5-week-old AS mice (AS-UT) (p < 0.01 and p < 0.05, respectively; Figure 2), whereas the effect of losartan on GS ( Figure 2D) or the TIF score ( Figure 2E) was not significant compared with 7-weekold vehicle-treated AS mice (AS-V).
Ultrastructural analysis of glomeruli in mice from EI studies demonstrated that sparsentan largely prevented GBM dysmorphology and ameliorated podocyte effacement (supplementary material, Figure S2B) to a greater extent than observed in AS-LS mice. In LI studies, AS-SP mice exhibited an intermediate GBM dysmorphology and foot process effacement, but these abnormalities were qualitatively less than those observed in AS-V 7-week-old mice ( Figure 2F). Losartan had no appreciable effect on GBM dysmorphology when administered in LI studies ( Figure 2F).

Sparsentan delayed the decline in kidney function
The beneficial effects of sparsentan on renal function were also assessed by measuring GFR. Figure 3A shows that while the GFR was markedly reduced in the 9-week-old AS-V mice (p < 0.001), the GFR in 9-week-old AS-SP mice was, after 5 weeks of treatment, significantly higher than that in AS-V mice (p < 0.0001) and was maintained at a level comparable to that of vehicle-treated wild-type (WT-V) mice. Moreover, examination of kidney sections from 10-week-old mice (after 6 weeks' treatment) revealed that preservation of kidney function in AS-SP mice coincided with a significant attenuation of the development of GS (p < 0.01; Figure 3B) and TIF (p < 0.01) compared with AS-V mice ( Figure 3C,D). . Compared with the kidney section from a WT-V mouse treated from 3 to 7 weeks of age, the representative kidney section froma 3-to7-week-old AS-V mouse contained anincreased number of sclerotic glomeruli, as indicated by the enlarged glomerulus denoted by black arrowheads 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-week-old mouse depicts the increase in GS at baseline before treatment in LI, as indicated by the enlarged glomerulus (black arrowheads in the inset). The GS in AS-SP 5-to 7-week-old mice tended to be less than that in AS-LS 5-to 7-week-old mice, as indicated by the difference in the number of black arrowheads between the images. (C) Representative cortical sections from AS mice in LI studies showing that fluorescence was attenuated in AS-SP 5-to 7-week-old mice compared with that from AS-V 3-to7-week-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 5to 7-week-old mice, the percentage of sclerotic glomeruli in kidney sections was significantly ameliorated (**p < 0.01) compared with that in AS-V 3-to 7-week-old mice. GS in AS-UT 5-week-old mice was also significantly lower than that in AS-V 3-to 7-week-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-to 7-week-old mice (*p < 0.05) compared with AS-V 3-to7-week-old mice. TIF in AS-UT 5-week-old mice was also significantly lower than that in AS-V 3-to 7-week-old mice (**p < 0.01). (F) Glomerular ultrastructural images from WT-V 3-to7-week-old mice, AS-V 3-to7-week-old mice, AS-SP 5-to 7-week-old mice, and AS-LS 5-to 7-week-old mice illustrate the GBM thickening and dysmorphology (asterisks) and podocyte effacement (black arrowheads) and preservation of GBM morphology in AS-SP 5to 7-week-old mice compared with AS-V 3-to 7-week-old mice. AS-LS 5-to 7-week-old mice had little alterations in GBM compared with AS-V 3to 7-week-old mice. Panel D data were statistically analyzed using Brown-Forsythe and Welch one-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.

Dual inhibition of endothelin type-A and angiotensin II AT1 receptors in Alport mice 357
Sparsentan treatment attenuated profibrotic/ pro-inflammatory gene expression in both the glomerular and the cortical compartments of the kidney Of the 84 genes in the mouse fibrosis array panel, 32 and 17 genes were found to be differentially expressed (DE; significance level p < 0.05) in the renal cortex and isolated glomeruli, respectively, of AS mice relative to WT mice in EI studies where mice were treated from 3 to 7 weeks of age ( Figure 4). By contrast, the expression levels of multiple DE genes in the cortex and glomeruli, such as the pro-inflammatory and profibrotic mediators Ccl2 and Ccr2, Il1b, and other signal transduction genes, such as serpin family E member 1 (Serpine1), tissue inhibitor of metalloproteinase-1 (Timp1), thrombospondin 2 (Thbs2), and Myc, in sparsentan-treated AS mice tended to be closer to those observed in the WT mice ( Figure 4A,B).

Sparsentan treatment extended lifespan even when initiated in mice with renal pathology
Sparsentan treatment started at 4 weeks significantly extended lifespan compared with vehicle ( Figure 5A, median lifespan, AS-V mice, 67.5 days; AS-SP mice, 118.0 days; p < 0.01; supplementary material, Table S3). Losartan treatment started at 4 weeks was less effective than sparsentan in extending lifespan (median lifespan, AS-LS mice, 84.0 days; p < 0.05; supplementary material, Table S3). These beneficial effects were not associated with differences in BP between the groups (supplementary material, Table S4). However, these effects paralleled the delay in the development of proteinuria in AS-SP mice. For AS mice started on treatment at 4 weeks, the increase in UP/C was delayed in animals receiving sparsentan compared with those treated with vehicle ( Figure 5B). The median UP/C in AS-SP mice was significantly lower than that in AS-V mice at 7 (p < 0.05), 8 (p < 0.01), 9 (p < 0.01), and 10 (p < 0.05) weeks of age (supplementary material, Table S5). The median UP/C of AS-LS mice, however, was significantly lower than that of AS-V mice only at 8 weeks of age (p < 0.05) and was significantly higher than that of AS-SP mice from 7 to 11 weeks of age. Moreover, the median lifespan of AS-SP mice was significantly increased compared with that of AS-V mice even when sparsentan treatment was started at 5, 6, or 7 weeks of age (p < 0.01; supplementary material, Table S3), despite progressive increases in GS and TIF in untreated mice at these ages ( Figure 5C,D).

Sparsentan lessened susceptibility to hearing loss in AS mice
To determine whether sparsentan impacted susceptibility to hearing loss, we exposed mice to a metabolically Analysis of panels B and C data was conducted using a two-tailed Student's t test. All analyses were performed using GraphPad Prism.

D Cosgrove et al
stressful noise to which the WT mice are resistant. This noise enhances the hypoxic stress on the inner ear of the AS mice [16,17]. AS mice and their WT littermates were treated from 3 to 9.5 weeks of age with sparsentan, losartan, or their vehicles before examining hearing function using the ABR technique. Hearing was assessed before (at 8.5 weeks of age) and 5 days after exposure to the noise. AS-V and AS-VW (AS mice vehicle water) mice were more susceptible to hearing loss than their WT counterparts ( Figure 6A,B, respectively), particularly in the low-to-mid test frequencies (supplementary material, Table S6). AS-SP and WT-SP mice showed equal ability to recover from the noise stress ( Figure 6C). AS-LS mice exhibited no recovery of hearing loss; in fact, hearing loss was significantly greater at 16 kHz ( p < 0.05) in AS-LS mice than in AS-VW ( Figure 6D). Preliminary studies showed equivalent SCBM thickness in AS-V and AS-VW mice; therefore, SCBM comparisons used only AS-V mice (supplementary material, Figure S3). SCBM thickness in Figure 6E revealed, as previously described [17], that AS-V mice had a significantly wider SCBM than did WT-V mice (p < 0.01). By contrast, the SCBM thickness in AS mice following treatment with sparsentan or losartan was equivalent to that in WT-V mice and significantly thinner than that in AS-V mice (p < 0.001). Interestingly, several AS-LS mice had very thin SCBMs. Examination of the strial ultrastructure (supplementary material, Figure S4) showed markedly fewer occurrences of lucent vacuoles and intracellular edema in AS-SP compared with AS-V mice, whereas AS-LS mice had pathology reminiscent of that observed in the AS-V mice. Importantly, although losartan treatment in the AS mice prevented SCBM thickening, this did not translate into a functional hearing improvement. The SCBM differences between sparsentan and losartan were further underscored in pilot EI studies during which AS mice were treated with vehicle, sparsentan (200 mg/kg), or losartan (10 mg/kg). SCBMs were examined by IF for expression of laminin α2, laminin α5, and collagen (IV) α1 chain [17]. The results (supplementary material, Figure S5) show that in EI studies from 3 to 7 weeks old with vehicle or sparsentan (120 mg/kg) and compared with tissue from WT-V mice. Of the 84 genes in the panel investigated, 32 and 17 genes were found to be differentially expressed (p < 0.05) in the renal cortex and glomeruli of AS mice, respectively, relative to WT mice. Changes in the 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 Materials and methods section under 'Gene expression analysis'.
Dual inhibition of endothelin type-A and angiotensin II AT1 receptors in Alport mice 359 these ECM molecules were equally present in WT-V and AS-SP mouse SCBMs but were more abundant in the SCBMs of AS-V mice. Losartan did not prevent the ECM protein accumulation seen in AS mouse SCBMs. Importantly, sparsentan intervention begun in 5-week-old mice with renal pathology still protected against the noise susceptibility of the AS-V mouse ( Figure 6F). Compared with WT-V mice, AS-V mice had a significantly elevated hearing threshold across test frequencies (8-32 kHz, p < 0.001; 40 kHz, p < 0.05). Moreover, in comparison to the AS-V mice, AS-SP mice exhibited a significant reduction of hearing loss at all test frequencies (8 kHz, p < 0.01; 16-32 kHz, p < 0.001; 40 kHz, p < 0.05) ( Figure 6F).

Discussion
In this study, dual inhibition of ET A R and AT 1 R by sparsentan in AS mice prevented the development of proteinuria, renal molecular and structural changes; ameliorated the development of the hearing loss; and extended lifespan when administered either early in disease development or late post-development of glomerular structural changes. Lifespan improvement in AS mice has been previously linked to preservation of kidney function [29][30][31][32][33]. This study also demonstrated preservation of GFR with sparsentan treatment, consistent with the treatmentassociated extension of lifespan in the AS mice. Previous studies exploring the effects of ET and RAAS inhibition have suggested the involvement of both systems in the pathophysiology of renal complications of AS. In fact, AT 1 R antagonists or ACE inhibitors (ACEi) are considered to be SOC for AS patients. In this study, comparisons were made with losartan as an AT 1 R antagonist, a relevant comparator to the ARB moiety of sparsentan. Sparsentan offered greater renal protection and lifespan extension than losartan, particularly when treatment was initiated in mice later in their disease, suggesting additive nephroprotective effects of ET A R to SOC treatment approaches. While ACEi show good renoprotection in both mice and man when administered early (preemptive therapy), renoprotection is lost when administered after renal disease is established [9,30].  Table S7.

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Previous studies [13] provided strong evidence for the involvement of ET-1 acting via ET A R in the pathophysiology of AS renal dysfunction. The ET A R antagonist sitaxentan improved GS, TIF, proteinuria, and glomerular filtration rate (GFR), and normalized GBM ultrastructure and profibrotic gene expression [13]. The effects of RAAS inhibition on the development of kidney injury and extension of lifespan in AS models have been more extensively studied. Gross et al [30] found that ramipril ameliorated the development of proteinuria and structural changes in AS mice (129/Sv Col4a3 À/À mice as used in this study). Ramipril significantly prolonged lifespan when administered from 4 weeks of age to a greater extent than was observed with sparsentan in the present studies. 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 to 9 weeks of age (EI) or from 5 to 9 weeks of age (LI; sparsentan only) was assessed by ABR measurements at frequencies of 8-40 kHz. AS-V (A) and AS-VW (B) mice 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 with 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 to 9.5 weeks 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 two of 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 weeks of age with sparsentan significantly attenuated susceptibility to hearing loss in AS mice compared with AS-V mice at all the 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 panels A-D, F are shown as mean dB SPL ± SEM. Data in panel E are shown for each stria as well as the group mean ± SD (nm). Analysis of panels A-D and F was performed using two-way ANOVA followed by the Holm-Šídák multiple comparison test (GraphPad Prism). Analysis in panel E was performed using one-way ANOVA and Tukey's post hoc multiple comparison test (GraphPad Prism).
Dual inhibition of endothelin type-A and angiotensin II AT1 receptors in Alport mice 361 However, in AS mice, when ramipril treatment was started at 7 weeks of age, there was no significant effect of lifespan [30]. This markedly contrasts with the ability of sparsentan in this study with treatment initiated at 7 weeks of age to extend median lifespan by 15 days, suggesting the potential benefit of dual ET A R/AT 1 R inhibition over current SOC therapies.
In the microarray study, DE genes between AS and WT mice align well with a previous study where wholetranscriptome analysis was performed on AS mouse kidney tissue [34]. In the renal cortical and glomerular compartment, sparsentan treatment of AS mice attenuated changes in the expression of a number of pro-inflammatory, profibrotic, proliferative, and adhesion pathway genes previously implicated in the AS pathogenesis. Some DE genes were attenuated by ET A R antagonism based on previous studies, such as several Mmps, Timp1, Ccl2 and Ccr2, Il1b, and Thbs2 [13,29,35,36]. Notably upregulation of Serpine1, which plays several roles, including regulation of MMPs, and impacting podocyte loss in a mouse FSGS model, was ameliorated by sparsentan in both the cortex and the glomeruli [37]. Also of interest is Myc [38], important in the developing mouse kidney [39] and a key player in renal fibrosis [40]. Thus, at the molecular level, sparsentan treatment impacts a wide range of pathways associated with development of renal disease in AS mice. In further support for sparsentan attenuating changes in matrix deposition in the GBM, dual immunostaining in glomeruli demonstrated an absence of laminin α2 in the GBM and of integrin α8 in the glomerulus of sparsentantreated mice compared with AS-V mice (supplementary material, Figure S6).
Hearing loss frequently accompanies AS, particularly in patients with COL4A5 mutations [41]. Thus far, no therapeutic intervention ameliorated the inner ear pathology and/or function in AS mice. The connection between ET A R-mediated dysregulation of SCBM proteins and SCBM thickening is supported by the fact that the strial and glomerular pathologies are functionally similar [13,17,42]. Here, we showed that sparsentan indeed does preserve stria vascularis morphology and SCBM width. Moreover, sparsentan, most likely through its ET A R antagonism, allowed mice to recover from a metabolically stressful noiseevenwhen administered after proteinuriaand renaldisease were established. Notably, losartan was ineffective in preventing stress-related hearing loss in AS mice.
Sparsentan has recently been shown to ameliorate a variety of functional, structural, and molecular characteristics of renal injury in other models of glomerular diseases [43][44][45] and, even more importantly, to have a profound anti-proteinuric effect in patients with FSGS [46]. Whether the renoprotection offered by dual antagonism at the ET A R and AT 1 R in the AS mouse studies will translate to the clinic will depend on the results from a recently initiated trial with sparsentan in pediatric patients with selected glomerular diseases (EPPIK; https://clinicaltrials.gov/ct2/ show/NCT05003986), also open to AS patients, and from an ongoing phase 2 clinical trial with the ET A R antagonist atrasentan in AS patients (https://clinicaltrials.gov/ct2/ show/NCT04573920).
In summary, our study demonstrates that dual ET A R/AT 1 R antagonism with sparsentan in the AS mouse model shows two significant advantages over treatment with an ARB alone. First, it significantly improved renal function and lifespan even when treatment was started in animals with established proteinuria, TIF, and GS. Second, sparsentan treatment prevented sensitivity to hearing loss, again, even when administered after significant renal damage was established. If translated to the clinic, sparsentan might have the potential to preserve both hearing and renal function in patients with Alport syndrome.

SUPPLEMENTARY MATERIAL ONLINE
Supplementary materials and methods Figure S1. Structural pathology of the stria vascularis Figure S2. In early intervention (EI) studies, sparsentan delays development of GS, fibrosis, and GBM morphology more than losartan in AS mice Figure S3. Sparsentan prevented thickening of the SCBM in AS mice Figure S4. Sparsentan attenuated strial pathology in Alport mice Figure S5. Sparsentan, but not losartan, prevents ECM accumulation in the SCBM of AS mice Figure S6. Sparsentan inhibits mesangial filopodial invasion of glomerular capillary loops in AS mice Table S1. Dose-dependent effects of sparsentan in AS mice treated from 3-7 wk of age in pilot EI studies Table S2. UP/C in 7-wk-old mice in EI and LI studies and TIF score in EI Table S3. Median life span of AS-V, AS-SP, or AS-LS mice starting treatment at different ages Table S4. BP during life span studies with treatment started at 4 wk of age Table S5. Median UP/C of AS-V, AS-SP, or AS-LS at different ages of treatment initiation Table S6. Hearing loss following metabolic noise stress in early intervention studies Table S7. The number of mice surviving at each week during the life-span studies (relates to Figure 5)