Salvianolic Acid B Protects Against Fatty Acid-Induced Renal Tubular Injury via Inhibition of Endoplasmic Reticulum Stress

Abstract

Background/Aims: Obesity-related kidney disease is associated with elevated levels of saturated free fatty acids (SFA). SFA lipotoxicity in tubular cells contributes to significant cellular apoptosis and injury. Salvianolic acid B (SalB) is the most abundant bioactive molecule from Radix Salviae Miltiorrhizae. In this study, we investigated the effect of SalB on SFA-induced renal tubular injury and endoplasmic reticulum (ER) stress, in vivo and in vitro. Methods: C57BL/6 mice were assigned to five groups: a control group with normal diet (Nor), high-fat diet group (HFD), and HFD with three different SalB treatment doses, low (SalBL; 3 mg/kg), medium (SalBM; 6.25 mg/kg), and high (SalBH; 12.5 mg/kg) doses. SalB was intraperitoneally injected daily for 4 weeks after 8 weeks of HFD. After 12 weeks, mice were sacrificed and kidneys and sera were collected. Apoptosis and ER stress were induced in human proximal tubule epitelial (HK2) cells by palmitic acid (PA, 0.6 mM), tunicamycin (TM, 1 μg/ml), or thapsigargin (TG, 200 nM) in vitro. Results: C57BL/6 mice fed a high-fat diet (HFD) for 12 weeks exhibited increased apoptosis (Bax and cleaved caspase-3) and ER stress (BIP, P-eIF2α, ATF4, CHOP, ATF6, IRE1α, and XBP1s) markers expression in the kidney, compared with control mice, which were remarkably suppressed by SalB treatment. In vitro studies showed that PA (0.6 mM) induced apoptosis and ER stress in cultured HK2 cells. SalB treatment attenuated all the adverse effects of PA. However, SalB failed to inhibit TM or TG-induced ER stress in HK2 cells. Conclusion: The study indicated that SalB may play an important role in obesity-related kidney injury via mediating SFA-induced ER stress.

Keywords: ER stress; apoptosis; renal tubular injury; salvianolic acid B; saturated fatty acid.

FIGURE 1

SalB inhibited tubular injury in the kidneys of HFD mice (A) H&E staining illustrated vacuolated proximal convoluted tubular cells (arrowheads) in HFD mice were attenuated by SalB treatment (magnification ×400) (B) The kidney lipid content in the mice was observed using oil red O staining (arrowheads) (magnification ×200). HFD was associated with increased lipid deposition in the kidney of the mice, which was ameliorated with SalB treatment (C) Quantitative analysis of Oil red O staining (D,E) Western blotting showed increased ICAM-1 and VCAM-1 expression in the kidney cortex of the HFD mice was ameliorated with SalB treatment. The corresponding quantifications were shown as well (F,G,H) Analysis of renal mRNA expression levels by quantitative real-time PCR for IL-1β, IL-6 and TNF-α in HFD-fed mice with or without SalB treatment. Nor, mice fed with normal diet; HFD, mice fed with high-fat diet; SalBL, SalBM, or SalBH, mice fed with high-fat diet and treated with low dosage of SalB (3 mg/kg), medium dosage of SalB (6.25 mg/kg), or high dosage of SalB (12.5 mg/kg), respectively. Data are represented as means ± SEM (n = 6). *p < 0.05 and **p < 0.01, compared with Nor; ^# p < 0.05 and ^## p < 0.01, compared with HFD.

FIGURE 2

SalB suppressed HFD-induced apoptosis of renal tubular cells in mice (A,B) Apoptosis (arrowheads) induced by HFD in kidney cells was detected by TUNEL assays (green) (magnification ×200). The corresponding quantitative analysis of TUNEL assays was shown as well. Nuclei were counterstained with Hoechst 33258 (blue) (C,D) Western blotting and the corresponding quantitative analysis showed HFD-induced decrease in Bcl-2 and increase in BAX and cleaved caspase-3 protein levels in the kidney cortex, which were ameliorated with SalB treatment. Nor, mice fed with normal diet; HFD, mice fed with high-fat diet; SalBL, SalBM, or SalBH, mice fed with high-fat diet and treated with low dosage of SalB (3 mg/kg), medium dosage of SalB (6.25 mg/kg), or high dosage of SalB (12.5 mg/kg), respectively. Data are represented as means ± SEM (n = 6). *p < 0.05 and **p < 0.01, compared with Nor; ^# p < 0.05 and ^## p < 0.01, compared with HFD.

FIGURE 3

SalB attenuated HFD-induced ER stress in the kidney cortex of mice (A,C) Representative photomicrographs of BIP-stained kidney sections and the corresponding quantitative analysis (B,C) Protein levels of P-eIF2α, eIF2α, ATF4, CHOP, ATF6, IRE1α, and XBP1s in the kidney cortex were detected by western blotting. The corresponding quantitative analysis was shown as well. Nor, mice fed with normal diet; HFD, mice fed with high-fat diet; SalBL, SalBM, or SalBH, mice fed with high-fat diet and treated with low dosage of SalB (3 mg/kg), medium dosage of SalB (6.25 mg/kg), or high dosage of SalB (12.5 mg/kg), respectively. Data are represented as means ± SEM (n = 6–8). *p < 0.05 and **p < 0.01, compared with Nor; ^# p < 0.05 and ^## p < 0.01, compared with HFD.

FIGURE 4

SalB inhibited PA-induced HK2 cells apoptosis (A,B) CCK-8 assay detection of HK2 cell viability after treatment with PA (A) or SalB (B) for 24 h (C) HK2 cells were preincubated with SalB, 1 μM, 10 μM, or 100 μM for 1 h, followed by treatment with 0.6 mM PA for another 24 h. CCK-8 assay showed SalB treatment in HK2 cells attenuated PA-induced cell death (D,E) Apoptosis ratios of HK2 cells were determined by flow cytometry, with Annexin-V/PI double staining (F) Western blotting and the corresponding quantitative analysis showed PA-induced decrease in BCL-2, and increase in BAX and cleaved caspase-3 protein levels in HK2 cells, which were ameliorated with SalB treatment. Data are represented as means ± SEM (n = 6). *p < 0.05 and **p < 0.01, compared with Con or DMSO; ^# p < 0.05 and ^## P < 0.01, compared with PA. Con, The control group; DMSO, DMSO treatment group; PA, palmitic acid treatment group; PA + SalB 1 μM, palmitic acid plus 1 μM SalB treatment; PA + SalB 10 μM, palmitic acid plus 10 μM SalB treatment; PA + SalB 100 μM, palmitic acid plus 100 μM SalB treatment.

FIGURE 5

SalB attenuated PA-induced ER stress in HK2 cells (A,B) HK2 cells were preincubated with SalB, 1 μM, 10 μM, or 100 μM for 1 h, followed by treatment with 0.6 mM PA for another 24 h. Protein levels and the corresponding quantitative analysis of BIP, P-eIF2α, eIF2α, ATF4, CHOP, ATF6, IRE1α, and XBP1s in HK2 cells. Data are represented as means ± SEM (n = 4–9). *p < 0.05 and **p < 0.01, compared with DMSO; ^# p < 0.05 and ^## p < 0.01, compared with PA. DMSO, DMSO treatment group; PA, palmitic acid treatment group; PA + SalB 1 μM, palmitic acid plus 1 μM SalB treatment; PA + SalB 10 μM, palmitic acid plus 10 μM SalB treatment; PA + SalB 100 μM, palmitic acid plus 100 μM SalB treatment.

FIGURE 6

SalB did not inhibit tunicamycin or thapsigargin-induced ER stress in HK2 cells (A) Western blotting and the corresponding quantitative analysis showed that treatment with TM (1 μg/ml, 2 μg/ml, 5 μg/ml, respectively) for 24 h stimulated BIP and CHOP protein expression in HK2 cells (B) HK2 cells were preincubated with SalB, 1 μM, 10 μM, or 100 μM for 1 h, followed by treatment with 1 μg/ml TM for another 24 h. TM-induced upregulated expression of the ER stress markers BIP, P-eIF2α, eIF2α, ATF4, CHOP, and ATF6 in HK2 cells. Pretreatment with SalB did not attenuate the TM-induced ER stress. IRE1α expression was not increased after TM treatment in HK2 cells (C) HK2 cells were preincubated with SalB, 1 μM, 10 μM, or 100 μM for 1 h, followed by treatment with 200 nM TG for another 24 h. SalB did not decrease the expression of BIP, P-eIF2α/eIF2α, ATF4, CHOP, ATF6, and IRE1α induced by TG in HK2 cells. DMSO, DMSO treatment group; TM, tunicamycin treatment group; TG, thapsigargin treatment group; TM + SalB 1 μM, tunicamycin plus 1 μM SalB treatment; TM + SalB 10 μM, tunicamycin plus 10 μM SalB treatment; TM + SalB 100 μM, tunicamycin plus 100 μM SalB treatment; TG + SalB 1 μM, thapsigargin plus 1 μM SalB treatment; TG + SalB 10 μM, thapsigargin plus 10 μM SalB treatment; TG + SalB 100 μM,thapsigargin plus 100 μM SalB treatment. Data are represented as means ± SEM (n = 3–7). *p < 0.05 and **p < 0.01, compared with DMSO; ^# p < 0.05 and ^## p < 0.01, compared with TM or TG.