The Novel Compound Sul-121 Preserves Endothelial Function and Inhibits Progression of Kidney Damage in Type 2 Diabetes Mellitus in Mice

Figures

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  Figure 1. Effects of Sul-121 treatment on metabolic parameters and albuminuria in db/db mice and in lean mice. (A) Body weight and (B) non-fasting plasma glucose levels throughout the duration of the experiment. (C) The progression in urinary albumin excretion rate was attenuated by Sul-121 treatment in db/db Sul-121 vs db/db vehicle, from week 6 onwards. *p < 0.05 db/db Sul-121 vs db/db vehicle, #p < 0.05 lean vs db/db.

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  Figure 2. Effects of Sul 121 treatment on glomerular histology. Representative pictures of PAS stainings are shown for lean vehicle (A), lean Sul-121 (B), db/db vehicle (C) and db/db Sul-121 (D). Inserts show details of glomerular staining. (E) Glomerular size was increased in db/db vehicle and normalized in db/db Sul-121. (F) Glomerulosclerosis scores were increased in db/db vehicle and significantly reduced in db/db Sul-121. *p < 0.05 db/db vehicle vs lean vehicle, #p < 0.05 db/db Sul-121 vs db/db vehicle.

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  Figure 3. Effect of Sul-121 treatment on renal fibronectin, WT1 and Collagen I expression. (A–D) Stainings of fibronectin (green), actin (red), and dapi (blue) are shown for lean vehicle (A), lean Sul-121 (B), db/db vehicle (C) and db/db Sul-121 (D). Increased fibronection staining is observed only in db/db vehicle (C). (E,F) Stainings of podocyte marker WT1 (red), fibronectin (green) and dapi (blue) shown for lean vehicle only. (E) Nuclear staining of WT1. (F) Overlay of fibronectin and dapi. (G) Overlay of WT1, fibronectin and dapi. (H–K) Representative pictures of polarization contrast microscopy of collagen I (indicated by white arrows) in lean vehicle (H), lean Sul-121 (I), db/db vehicle(J) and db/db Sul-121(K). (L) Renal fibronectin expression was additionally examined using Western blotting. Insert showing representative cropped blots for fibronectin and beta-actin as loading control. Renal fibronectin was significantly upregulated in db/db vehicle. Sul-121 reduced fibronectin expression in db/db Sul-121. (M) Quantification of the average number of WT1 positive nuclei per glomerulus. Decreased numbers of WT1 positive cells were present in db/db vehicle and db/db Sul-121. (N)…

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  Figure 4. Effect of Sul-121 treatment on expression of markers of interstitial damage and inflammation. Expression of Collagen 1 (A), Smooth muscle actin (SMA) (B), Macrosialin (CD68) (C), TNF-alpha (D) and IL-1B (E) was assessed by real-time PCR. SMA expression was increased in db/db vehicle and db/db Sul-121(B). IL-1B was increased in db/db vehicle(E). *p < 0.05 db/db vehicle or db/db Sul-121 vs lean vehicle.

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  Figure 5. Effect of Sul-121 treatment on ROS status. (A) Plasma H2O2 levels are increased in db/db vehicle and normalized in db/db Sul-121) (B) Urinary H2O2 excretion is increased similarly in both db/db vehicle and db/db Sul-121. (C) SOD1 expression is decreased in db/db vehicle and normalized in db/db Sul-121. (D) SOD2 expression is increased in db/db vehicle and further increased in db/db Sul-121. (E) SOD3 expression was not significantly different between groups. (F) COX2 expression was increased in db/db vehicle. (G) Glomerular protein carbonylation was increased in lean-Sul-121 and further increased in db/db vehicle. Protein carbonylation in db/db Sul-121 was similarly increased as in db/db vehicle. (H) Effect of Sul-121 on in-vitro ROS production. Stimulation of cultured mesangial cells with high glucose/insulin increased ROS production which could be inhibited by Sul-121 pretreatment. (I) Acute effects of Sul-121 on renal mitochondrial ROS production ex-vivo. Increased mitochondrial ROS production in db/db was decreased by Sul-121. *p < 0.05 db/db vehicle vs lean vehicle, #p < 0.05 db/db Sul-121 vs db/db, **p < 0.05 as indicated by horizontal line, †p < 0.05 glucose vs control, ‡P…

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  Figure 6. Sul-121 treatment restores endothelium mediated relaxation. (A) Relaxations to acetylcholine were reduced in aortic rings of db/db vehicle indicating endothelial dysfunction. Sul-121 restored relaxation to lean vehicle values. (B) Dissection of the components involved in relaxation demonstrated a significant increase of the combined NO + PG component in db/db Sul-121 vs db/db vehicle. *p < 0.05 db/db Sul-121 vs db/db vehicle. (C) Sensitivity of the aortic rings to the NO donor SNP demonstrated decreased NO sensitivity in db/db vehicle. *p < 0.05 db/db vehicle vs lean vehicle, #p < 0.05 db/db Sul-121 vs db/db vehicle.

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  Figure 7. Albuminuria correlates with ROS status and endothelial function. (A) AER correlates significantly with plasma H2O2 concentrations (p < 0.05). (B) AER correlates significantly with maximal response to acetylcholine (p < 0.05).