Diallyl Trisulfide Attenuates Ischemia-Reperfusion-Induced ER Stress and Kidney Dysfunction in Aged Female Mice

Figures

• 

  Figure 1. DATS attenuated ER stress in IRI of the aged kidney. (A) H2S content in the kidney. Values are expressed as mean ± SD, n = 6/group, * p < 0.01 vs. other groups, † p < 0.01 vs. ND+IRI. (B) Protein expression of phospho-inositol requiring enzyme 1 alpha (p-IRE1α), phosphor PRKR-like endoplasmic reticulum kinase (p-PERK), and activating transcription factor 6 (ATF6). (C) Densitometry analysis of protein bands normalized to β-actin, values are mean ± SD, n = 6/group, * p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI. (D) Fold change of mRNA levels of ER stress markers, glucose-regulated protein 78 (GRP78), glucose-regulated protein (GRP94), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Values are expressed as mean ± SD, n = 6/group, * p < 0.05 vs. ND, † p < 0.01 vs. ND+IRI.

• 

  Figure 2. DATS treatment attenuated ER stress in aged kidney IRI. (A) Expression of unfolded protein response sensors, ATF6, and p-IRE1α. ATF6 was upregulated in the glomeruli and tubules (white and yellow arrows, respectively) and p-IRE1α was upregulated predominantly in the glomeruli compared to the tubules (green and red arrows, respectively). Scale bar, 20 μm. (B) Data are mean ± SD, n = 6/group, * p < 0.01 vs. ND, † p < 0.01 vs. ND and DATS, ‡ p < 0.01 vs. ND+IRI.

• 

  Figure 3. DATS supplementation in IRI enhanced the expression of angiogenesis markers in the kidney. (A) Representative bands of HIF-1α, VEGF-A, erythropoietin (EPO), and eNOS. (B) Densitometric analysis of protein expression as mean ± SD, n = 6/group, * p < 0.01 vs. ND or DATS alone, † p < 0.01 vs. ND+IRI.

• 

  Figure 4. DATS treatment increased angiogenesis after IRI in the aged kidney. (A) Expression of angiogenesis markers, VEGF-A, and CD31 was decreased in the ND+IRI kidney (green and red arrows, respectively) and increased with DATS (white and yellow arrows, respectively). Scale bar, 20 μm (B) Data are mean ± SD, n = 6/group, * p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI.

• 

  Figure 5. DATS activated p-PI3K, p-AKT, p-mTOR, and p-MAPK pathways in renal IRI. (A) Representative image of protein expression of signaling molecules of endothelial proliferation and survival. (B) Densitometry analysis of protein expression as mean ± SD, n = 6/group, *, ϕ p < 0.01 vs. ND, † p < 0.05 vs. ND+IRI.

• 

  Figure 6. DATS treatment suppressed IRI-induced renal inflammation and fibrosis markers in the aged kidney. (A) Representative bands showing expression of renal injury marker, LCN2 (lipocalin 2), and inflammatory markers TNF-α, IL17, and IL-1β. (B) Data are mean ± SD, n = 6/group, * p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI. (C) Representative bands for TGF-β, Col1A1, and NF-kB. (D) Data are mean ± SD, n = 6/group, * p < 0.01 vs. ND, †, ϕ p < 0.01 vs. ND+IRI.

• 

  Figure 7. DATS improved renal cortical blood flow in the IRI kidney. (A) Representative laser Doppler flowmetry images showing renal cortical perfusion. The kidney is outlined by black dashed lines. Reduction of blood flow in ND+IRI kidney is shown as a low-intensity signal (green areas, black arrows), and improvement in blood flow is shown as pink areas (white arrows) in DATS+IRI kidney. DATS alone increased blood flow in the DATS kidney (yellow arrows). (B) Line traces from the kidney. Black trace is from the aorta, red trace is from the renal artery, blue trace is from the renal vein, and pink trace is from the renal cortex (blue arrow). (C) Data are expressed as mean ± SD, n = 6/group, *, ϕ p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI.

• 

  Figure 8. DATS treatment reduced the increased resistive index (RI) of outer medullary arteries caused by IRI in the aged kidney. (A) Representative ultrasound images of the kidney showing color Doppler of vessels in the outer cortico-medullary area. Pulsed-wave Doppler was placed on the outer medullary arteries to acquire data. The resistive index (RI) was measured using the formula RI = (PSV − EDV)/PSV. RI—resistive index, PSV—peak systolic velocity, EDV—end-diastolic velocity. (B) Values are mean ± SD, n = 6/group, * p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI.

• 

  Figure 9. Renal IRI caused functional vascular rarefaction in the aged kidney and DATS treatment improved renal vascular density. (A) Representative barium sulfate angiography images of renal vasculature characterized by narrowing of lobar arteries (white arrows), loss of arcuate and interlobular branches (red and blue arrows, respectively), DATS rescues renal vasculature in WT+DATS+IRI kidney (yellow, green, and black arrows). (B) Vessel segmentation analysis images of total vasculature against the background. Vessel segmentation was conducted using VesSeg software (VesSeg_V0.1.4, University of Lubeck, Lubeck, Germany), (C) Data are mean ± SD, n = 6/group, *, ϕ p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI.

• 

  Figure 10. DATS rescued the aged kidney from severe dysfunction caused by IRI. (A) Representative images of glomerular filtration rate (GFR) estimated by non-invasive FITC-sinistrin method. (B) GFR was calculated using the formula, GFR [μL/min/100 g b.w.] = 14,616.8 [μL/100 g b.w.]/t1/2 (FITC-sinistrin) [min]. (C) Plasma creatinine was measured using QuantiChromTM creatinine assay kit by colorimetric method. Plasma creatinine concentration was calculated using the following formula: [(OD sample 5 − ODsample 0)/(OD standard 5 − OD standard 0)] × [STD] (mg/dL). Data are mean ± SD, n = 6/group, * p < 0.01 vs. ND, † p < 0.01 vs. ND+IRI.