Sparsentan is superior to losartan in the gddY mouse model of IgA nephropathy

Abstract

Background: The mechanism leading to the development of immunoglobulin A nephropathy (IgAN) remains to be completely understood. Endothelin-1 (ET-1) as well as angiotensin II (AngII) promote glomerular injury, tubulointerstitial inflammation and fibrosis leading to chronic kidney disease. Sparsentan, a dual endothelin angiotensin receptor antagonist, recently received accelerated approval in the USA for the reduction of proteinuria in adults with IgAN at high risk of disease progression. To elucidate the mechanisms by which sparsentan is efficacious in IgAN, we examined the effect of treatment in gddY mice, a spontaneous IgAN mouse model, versus the monoselective angiotensin II type 1 receptor (AT1R) antagonist, losartan, on the development of renal injury at doses resulting in similar blood pressure lowering.

Methods: Four-week-old gddY mice were given control chow, chow containing sparsentan or drinking water containing losartan until 12 or 20 weeks old.

Results: Remarkably, the albumin:creatine ratio (ACR) was attenuated more rapidly and to a greater extent in mice treated with sparsentan than those treated with losartan. The decrease in ACR from baseline after 4 weeks of treatment correlated with beneficial effects of sparsentan on glomerulosclerosis and protection of podocytes and glycocalyx after 16 weeks of treatment across treatment groups; thus, sparsentan treatment delayed development of renal injury to a greater extent than losartan. Expression of mRNA for ET-1, endothelin type A receptor and AT1R and proinflammatory genes was upregulated in 12-week-old gddY mice and was prevented by sparsentan and losartan to a comparable extent.

Conclusions: The results of this study, and in light of the results of the phase 3 PROTECT trial, provide a novel perspective and understanding of the mechanisms by which sparsentan has a beneficial renoprotective effect against IgAN compared with AT1R antagonism alone.

Keywords: IgA nephropathy; albuminuria; angiotensin II type 1 receptor antagonist; endothelin receptor antagonist; sparsentan.

Graphical Abstract

Figure 1:

Schematic of study to compare sparsentan and losartan in gddY mice. The 4-week-old gddY mice received sparsentan (900 or 1800 ppm) in their diet or losartan (10 or 30 mg/kg) in drinking water for 8 or 16 weeks, during which urine was collected every 2 weeks for albumin and creatinine determination. Blood pressure was recorded when mice were 8, 12 and 18 weeks old. After 8 weeks of treatment, a subgroup of mice were sacrificed, and the remainder were treated for a total of 16 weeks. Blood was taken for generation of plasma, and kidney samples were removed and processed for assessment of glomerulosclerosis, WT-1 positivity, glycocalyx (20 weeks age only) and gene expression. Created with Biorender.com by W.C. RT-PCR, real-time polymerase chain reaction.

Figure 2:

Effects of sparsentan (SP) and losartan (LS) on blood pressure (BP), serum creatinine (CRE) and ACR in gddY mice. (A) sBP levels were increased in control gddY mice but attenuated in gddY mice treated with SP or LS. SP (900 ppm) and LS (30 mg/kg) groups had similar antihypertensive effects in 8-, 12- and 18-week-old gddY mice. ***P < .001, **P < .01, *P < .05 for SP900 or SP1800 compared with control gddY, ^@@@P < .001, ^@@P < .01, ^@P < .05 for LS30, and P < .001 and P < .01 for LS10 compared with control gddY mice following one-way ANOVA and Tukey's multiple comparison (GraphPad PRISM). Data are shown as mean ± SD. (B) Serum creatinine remained stable at 12 and 20 weeks of age and was not significantly affected by any treatment during the study. Data are shown as mean ± SD. (C) Log change in ACR from baseline over the course of the study. Data are shown as mean ± SD. (D) Percentage change in ACR from baseline at 6, 12 and 18 weeks of age (2, 8 and 14 weeks of treatment). Data are shown as mean ± SD for gddY mice C, SP1800 (n = 10, 412 weeks old; n = 6, 1420 weeks old); SP900, LS10 or LS30 (n = 12, 412 weeks old; n = 7, 1420 weeks old).

Figure 3:

Sparsentan protects from development of glomerulosclerosis, podocyte injury and glomerular glycocalyx to a greater extent than losartan. (A) PAS staining, (B) staining with anti-WT-1 antibodies, (C) immunofluorescence following incubation with FITC-labeled anti-lectin antibodies of representative kidney sections in BALB/c mice or control gddY mice or gddY mice treated with sparsentan 900 ppm in diet or losartan 30 mg/kg/day in drinking water for 16 weeks. Scale bars: 50 μm. (A, D) A statistically significant increase in the glomerulosclerosis score was observed in gddY control mice compared with BALB/c mice (P < .01). A significant attenuation in glomerulosclerosis was observed in gddY SP900 mice compared with gddY control mice or losartan-treated mice (P < .001). (B, E) Sparsentan significantly prevented podocyte loss compared with gddY control mice and losartan-treated mice (P < .001). Losartan also attenuated podocyte loss in gddY mice but not to the extent of sparsentan, which resulted in gddY mice having a comparable number of podocytes per glomerulus to healthy BALB/c mice. (C, F) Sparsentan significantly prevented glycocalyx damage to a greater extent when compared with gddY control mice and losartan-treated mice (P < .001). Data are shown as mean ± SD. Statistical analysis in (E) and (F) was performed by one-way ANOVA with post hoc analysis using Tukey's multiple comparison test, with the analysis in (E) using ln transformed data. Analysis in (D) for gddY groups was also performed by one-way ANOVA of ln transformed data with post hoc analysis using Tukey's multiple comparison test. Comparison of BALB/c and gddY groups in (D) was performed using unpaired t-test. ***P < .0001; gddY C vs SP900; ^###P < .0001, ^##P < .01 vs BALB/c; P < .0001; P < .001, P < .05 vs LS30 following one-way ANOVA.

Figure 4:

Renal structural changes at weeks 8 and 16 of treatment are correlated with change in ln (ACR) at week 4 of treatment. Relationship between change (delta) in ln (ACR) from week 0 to week 4 of treatment and (A) glomerulosclerosis after week 8 of treatment, (B) podocyte number (WT-1 immunostaining) after week 8 of treatment, (C) glomerulosclerosis after week 16 of treatment. (D) podocyte number (WT-1 immunostaining) after 16 weeks of treatment. (E) Glycocalyx % lectin positive area after 16 weeks of treatment. r = correlation coefficient. Change in ln (ACR) at week 4 is significantly correlated with glomerulosclerosis [P < .0001 (A), P < .01 (C)], podocyte number [P < .01 (B), P < .0001 (D)] and glycocalyx damage [P < .001 (E)].

Figure 5:

Upregulation of ET-1, ET_AR and AT₁R mRNA in 12-week-old gddY mice was prevented by treatment with sparsentan and losartan treatment. Real-time PCR was performed in mRNA isolated from kidney tissue taken from mice at 12 weeks of age. Statistical analysis was performed using Mann–Whitney U test. *P < .05 compared with gddY C, ^#P < .05, ^##P < .01 compared with BALB/c mice. Data are shown as mean fold-change ± SEM relative to expression in BALB/c mice. BALB/c, SP900 and LS30, n = 5; gddY C, n = 4.

Figure 6:

Upregulation of inflammation genes in 12-week-old gddY mice were prevented by sparsentan or losartan. Real-time PCR was performed in mRNA isolated from kidney tissue taken from BALB/c or gddY mice at 12 weeks of age. Statistical analysis was performed using the Mann-Whitney U test. *P < .05 compared with gddY C, ^#P < .05, ^##P < .01 compared with BALB/c mice. Data are shown as mean fold-change ± SEM relative to expression in BALB/c mice. BALB/c, n = 5 (except for IL-6, n = 2); SP900 and LS30, n = 5; gddY C, n = 4.

Figure 7:

Circulating IgA levels at 12 and 20 weeks of age in gddY mice. Serum levels of IgA were assessed in gddY C, or gddY mice treated with sparsentan or losartan at 12 and 20 weeks of age. Individual animal data are shown with mean and SD.