Sterol-O-acyltransferase-1 has a role in kidney disease associated with diabetes and Alport syndrome

Abstract

Defective cholesterol metabolism primarily linked to reduced ATP-binding cassette transporter A1 (ABCA1) expression is closely associated with the pathogenesis and progression of kidney diseases, including diabetic kidney disease and Alport Syndrome. However, whether the accumulation of free or esterified cholesterol contributes to progression in kidney disease remains unclear. Here, we demonstrate that inhibition of sterol-O-acyltransferase-1 (SOAT1), the enzyme at the endoplasmic reticulum that converts free cholesterol to cholesterol esters, which are then stored in lipid droplets, effectively reduced cholesterol ester and lipid droplet formation in human podocytes. Furthermore, we found that inhibition of SOAT1 in podocytes reduced lipotoxicity-mediated podocyte injury in diabetic kidney disease and Alport Syndrome in association with increased ABCA1 expression and ABCA1-mediated cholesterol efflux. In vivo, Soat1 deficient mice did not develop albuminuria or mesangial expansion at 10-12 months of age. However, Soat1 deficiency/inhibition in experimental models of diabetic kidney disease and Alport Syndrome reduced cholesterol ester content in kidney cortices and protected from disease progression. Thus, targeting SOAT1-mediated cholesterol metabolism may represent a new therapeutic strategy to treat kidney disease in patients with diabetic kidney disease and Alport Syndrome, like that suggested for Alzheimer's disease and cancer treatments.

Keywords: Alport syndrome; SOAT1 inhibitor; cholesterol; diabetic kidney disease; podocyte injury; renal function.

Figure 1.. SOAT1 inhibitor (SI) treatment of human podocytes reduces cholesterol ester (CE), triglyceride (TG) and lipid droplet (LD) content by increasing ABCA1 expression and ABCA1-mediated cholesterol efflux.

Human podocytes were treated with SOAT1 inhibitor (SI, Sandoz 58–035, 4μM) or DMSO (Ctrl) for 48hrs. (a) Bar graph analysis showing the quantification of the images shown in (e). Lipid content was measured via Nile red fluorescent intensity per cell (n=15–20), (b) Cholesterol content represented as CE/TC (%) (n=5), (c) TG content (n=5). (d) Bar graph analysis showing cytotoxicity normalized to viability (n=4–5). (e) Representative images (original magnification x20) showing Nile red (green) and DAPI (blue) staining. (f) Quantitative real-time PCR analysis of ABCA1 expression in DMSO-treated (Ctrl) or SI-treated (SI) human podocytes (n=5). (g) Quantification of the percentage of cholesterol effluxed via the ABCA1 transporter in DMSO-treated (Ctrl) or SI-treated (SI) human podocytes (n=5). Kruskal-Wallis test (g), or Mann-Whitney test (all other panels).

Figure 2.. SOAT1 inhibitor (SI) treatment increases ABCA1 expression and protects podocytes from injury.

(a) Quantitative real-time PCR analysis of SOAT1 expression in podocytes that were exposed to NP and P patient sera from T2D patients (n=4). (b) Quantitative real-time PCR analysis of ABCA1 expression in DMSO-treated (Ctrl) or SI-treated normal human podocytes that were exposed to NP sera and P sera (n=5). (c) Bar graph analysis showing cytotoxicity normalized to viability in DMSO-treated (Ctrl) or SI-treated normal human podocytes that were exposed to NP and P patient sera (n=6–7). (d) Bar graph analysis showing cytotoxicity normalized viability in DMSO-treated or SI-treated siCO and siABCA1 podocytes that were exposed to P sera (n=5). Mann-Whitney test (a) or Kruskal-Wallis test (all other panels).

Figure 3.. Soat1 knockout mice do not develop albuminuria or mesangial expansion at 10–12 months of age.

(a) Quantitative real-time PCR analysis of SOAT1 expression in kidney cortices of WT and Soat1^−/− mice (n=6–7). (b) Bar graph analysis showing the quantification of CE/TC (%) from kidney cortices of WT and Soat1^−/− (n=6–7) mice at 10–12 months of age. (c) Quantitative real-time PCR analysis of ABCA1 expression in kidney cortices of WT and Soat1^−/− mice (n=8–9). (d, e) Representative images (d) and bar graph quantification (e) of Oil red O staining of kidney cortices (scale bar: 20 μm, n=7). (f) Summary of body weight, fast plasma glucose, albumin/creatinine, BUN in WT and Soat1^−/− mice (n=8–9). (g, h) Representative images (g) and bar graph quantification of the mesangial expansion score (h) determined by PAS staining (scale bar: 20 μm, n=5–6). Mann-Whitney test.

Figure 4.. Soat1 deficiency improves renal function in diabetic mice (db/db).

Mice were sacrificed and analyzed at 16 weeks of age. (a) db/+;Soat1^+/+, db/db;Soat1^+/+, and db/db;Soat1^−/− littermates were analyzed for albumin-to-creatinine ratios (ACR) determined at 16 weeks of age (n=5–9). (b) Bar graph analysis showing the kidney cortices cholesterol content represented as CE/TC(%) in db/+;Soat1^+/+, db/db;Soat1^+/+, and db/db;Soat1^−/− mice (n=5–10). (c) Quantitative real-time PCR analysis of ABCA1 expression in glomeruli of db/+;Soat1^+/+, db/db;Soat1^+/+, and db/db;Soat1^−/− mice (n=5–8). (d,e) Representative images of PAS staining (d) and bar graph analysis of the mesangial expansion score (e) in db/+;Soat1^+/+, db/db;Soat1^+/+, and db/db;Soat1^−/− (scale bar: 20 μm) (n=5–6). (f, g) Representative images of WT1 staining (f) and bar graph analysis of the number of WT1-positive cells per glomerular cross section (g) in db/+;Soat1^+/+, db/db;Soat1^+/+, and db/db;Soat1^−/− mice (scale bar: 25 μm) (n=5–6). (h, i) Representative images of TEM images (h) and bar graph quantification of the number of foot processes effacement (blue arrowheads) per μm of GBM (i) in db/+;Soat1^+/+, db/db;Soat1^+/+, and db/db;Soat1^−/− mice (scale bars: 500 nm) (n=5–6). Kruskal-Wallis test.

Figure 5.. SOAT1 Inhibitor (SI) improves renal function in a mouse model of AS.

(a) Bar graph analysis showing SOAT1 activity in immortalized primary podocytes isolated from WT and AS mice (n=9–10). (b) Immortalized primary podocytes of WT and AS mice treated with SI or DMSO for 48hr. Bar graph analysis showing apoptosis normalized to viability (n=6). (c-f) 4-week-old AS mice received daily i.p injected of SI (15mg/kg) for 4 weeks. Three groups of mice were analyzed: WT (n=7), AS (n=7) and AS+SI (n=5–6) mice. (c) Bar graph analysis showing the quantification of albumin-to-creatinine ratios (ACR), (d) blood urea nitrogen (BUN), (e) serum creatinine levels, (f) body weight (BW). Mann-Whitney test (a) or Kruskal-Wallis test (all other panels).

Figure 6.. SOAT1 Inhibitor (SI) improves renal histology in a mouse model of AS.

4-week-old AS mice received daily i.p injected of SI (15mg/kg) for 4 weeks. Three groups of mice were analyzed: WT (n=5–8), AS (n=5–8) and AS+SI (n=5–6) mice. (a, b) Representative images of PAS staining (a) and bar graph analysis of the mesangial expansion score (b) in WT, AS and AS+SI mice (scale bar: 20 μm). (c, d) Representative images (c) quantification (d) of Oil red staining of kidney cortices (scale bar: 20 μm). (e) Kidney cortices cholesterol content represented as CE/TC (%). (f, g) Representative images (f) and bar graph quantification of glomerular and interstitial fibrosis by Picrosirius Red staining (g) (scale bar: 20 μm). (h-j) Representative images of transmission electron micrograph (TEM) (h) and bar graph quantification of foot process effacement (blue arrowheads) (i) and glomerular basement membrane thickness (orange arrowheads) (j) (scale bar: 500 nm). Kruskal-Wallis test.