Intrarenal renin-angiotensin system mediates fatty acid-induced ER stress in the kidney

Abstract

Obesity-related kidney disease is related to caloric excess promoting deleterious cellular responses. Accumulation of saturated free fatty acids in tubular cells produces lipotoxicity involving significant cellular dysfunction and injury. The objectives of this study were to elucidate the role of renin-angiotensin system (RAS) activation in saturated fatty acid-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) and in mice fed with a high-fat diet. Treatment with saturated fatty acid palmitic acid (PA; 0.8 mM) for 24 h induced ER stress in HK2, leading to an unfolded protein response as reflected by increased expressions of the ER chaperone binding immunoglobulin protein (BiP) and proapoptotic transcription factor C/EBP homologous protein (CHOP) protein as evaluated by immunoblotting. PA treatment also induced increased protein expression of inositol requiring protein 1α (IRE1α), phosphorylated eukaryotic initiation factor-α (eIF2α), and activating transcription factor 4 (ATF4) as well as activation of caspase-3. PA treatment was associated with increased angiotensin II levels in cultured medium. The angiotensin II type 1 receptor (AT1R) blocker valsartan or renin inhibitor aliskiren dramatically suppressed PA-induced upregulation of BiP, CHOP, IRE1α, p-eIF2α, and ATF4 in HK2 cells. In contrast, valsartan or aliskiren did not prevent ER stress induced by tunicamycin. C57BL/6 mice fed with a high-fat diet for 14 wk exhibited increased protein expressions of BiP and CHOP compared with control mice, which were significantly attenuated by the valsartan treatment. Increased angiotensin II levels in serum and urine were observed in mice fed with a high-fat diet when compared with controls. It is suggested that the intrarenal RAS activation may play an important role in diabetic kidney injury via mediating ER stress induced by saturated fatty acid.

Keywords: angiotensin II; kidney; saturated fatty acid; valsartan.

Fig. 1.

Valsartan or aliskiren prevented palmitic acid (PA; 0.8 mM)-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) cells after a 24-h treatment. A and B: protein abundance of binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) was unchanged in HK2 cells treated with BSA (2 and 10 mg/ml) and with or without valsartan (10⁻⁶ M) or aliskiren (10⁻⁷ M). C: MTT assays of HK2 cells. HK2 cells were incubated with PA at different concentrations for 24 h. After the cells were incubated with tetrazolium salt solution for 2 h, the quantity of formazan product was determined from the absorbance at 560 nm. *P < 0.05, compared with PA concentration 0; #P < 0.05, compared with PA concentration 0.8 mM. D: PA induced upregulation of the ER stress markers BiP, phosphorylated eukaryotic initiation factor (eIF)2α/eIF2α, activating transcription factor 4 (ATF4), and CHOP expression in HK2 cells, whereas pretreatment with valsartan (10⁻⁶ M) or aliskiren (10⁻⁷ M) attenuated ER stress induced by PA. E: quantitative analysis of ER stress marker levels normalized to β-actin. For the control condition (CTL), the BSA concentration was equivalent to cells exposed to 0.8 mM PA complexed to BSA. Representative results of 3 independent experiments are shown. PA, palmitic acid treatment group; PA + Val, palmitic acid plus valsartan treatment; PA + Ali, palmitic acid plus aliskiren treatment. *P < 0.05, compared with CTL; #P < 0.05, compared with PA.

Fig. 2.

Valsartan or aliskiren treatment decreased BiP (A) and CHOP (B) expression in HK2 cells treated with PA. A: immunofluorescence staining of BiP in cultured HK2 cells. In CTL (a, e, and i), BiP scarcely localizes intracellular cytoplasm of HK2 cells, whereas PA (0.8 mM) induced significantly increased labeling of BiP (b, f, and j), which was clearly suppressed by either valsartan (10⁻⁶ M; c, g, and k) or aliskiren (10⁻⁷ M; d, h, and l) treatment. B: immunofluorescence staining of CHOP in cultured HK2 cells. CTL exhibited weak staining of CHOP in nuclei and cytoplasm of HK2 cells (a, e, and i), in contrast, PA treatment was associated with abundant nuclear expression of CHOP (b, f, and j), which was inhibited by either valsartan (c, g, and k) or aliskiren treatment (d, h, and l). C: quantitative analysis of BiP- and CHOP-positive staining. *P < 0.05, compared with CTL; #P < 0.05, compared with PA.

Fig. 3.

Valsartan or aliskiren attenuated PA-induced HK2 cell apoptosis by inhibiting renin-angiotensin system (RAS). A: Western blot analysis of PA-induced active, cleaved caspase-3 protein steady-state levels in HK2 cells, which was inhibited by valsartan or aliskiren treatment. B: corresponding densitometric analyses of levels of cleaved caspase-3 protein normalized to β-actin. C and D: representative terminal deoxynucleotidyl transferase-mediated deoxyuridintriphosphate nick end labeling (TUNEL) staining of PA-treated HK2 cells and corresponding quantitative analysis. Apoptotic nuclei (arrowheads) were observed in PA-treated HK2 cells. The percentage of apoptotic cells (TUNEL-positive cells) was obtained in at least 10 fields from the PA treatment group (b), PA + Val group (c), and PA + Ali group (d). *P < 0.05, compared with CTL; #P < 0.05, compared with PA. E: PA treatment was associated with increased release of angiotensin II (ANG II) in medium of cultured HK2 cells. *P < 0.05, compared with CTL. F: mRNA levels of RAS components in PA-stimulated HK2 cells with pretreatment of valsartan or aliskiren. *P < 0.05, compared with CTL; #P < 0.05, compared with PA. G and H: Western blots and quantitative analysis of upregulated BiP and CHOP induced by ANG II. Renin treatment did not induce ER stress in HK2 cells. *P < 0.05, compared with CTL.

Fig. 4.

Valsartan or aliskiren treatment did not prevent tunicamycin (TM; 2 μg/ml) induced ER stress in HK2 cells. A: tunicamycin induced upregulation of the ER markers BiP, IRE1α, p-eIF2α/eIF2α, ATF4, and CHOP expression in HK2 cells; neither pretreatment with valsartan (10⁻⁶ M) nor aliskiren (10⁻⁷ M) attenuated ER stress induced by TM. B: quantitative analysis of ER stress marker levels normalized to β-actin. Representative results of 3 independent experiments are shown. TM, tunicamycin treatment group; TM + Val, tunicamycin plus valsartan treatment; TM + Ali, tunicamycin plus aliskiren treatment. *P < 0.05, compared with CTL.

Fig. 5.

Valsartan prevented increased kidney injury molecule 1 (KIM-1) in mice fed with high-fat diet (HFD). A: mRNA levels of neutrophil gelatinase-associated lipocalin (NGAL) and KIM-1 in the kidney cortex of HFD-fed mice with or without valsartan treatment. B: urinary levels of KIM-1 measured by ELISA. HFDV, mice fed with high-fat diet and valsartan. *P < 0.05, compared with CTL; #P < 0.05, compared with mice fed with HFD diet; n = 6 in each group.

Fig. 6.

High-fat diet was associated with RAS activation in mouse kidneys. A and B: serum and urine ANG II concentration was significantly increased in mice fed with HFD. *P < 0.05, compared with CTL; #P < 0.05 compared with mice without valsartan treatment; n = 10–12 in each group. C: mRNA levels of RAS components in the kidneys of HFD-fed mice with or without valsartan treatment. ATG, angiotensinogen; ReninR, renin receptor; ACE, angiotensin-converting enzyme; AT₁R, ANG II type 1 receptor. *P < 0.05, compared with CTL; #P < 0.05, compared with mice fed with HFD; n = 6 in each group.

Fig. 7.

Valsartan inhibited increased BiP and CHOP protein expression in the kidney cortex of mice fed with HFD. A: Western blots analysis of BiP and CHOP expression. HFD treatment was associated with increased levels of BiP and CHOP protein expression in the kidney cortex, which was prevented by valsartan. B: corresponding densitometric analyses of BiP and CHOP levels normalized to β-actin. C: analysis of mRNA expression levels by quantitative real-time PCR for BiP and CHOP in HFD-fed mice with or without valsartan. *P < 0.05, compared with CTL; #P < 0.05, compared with HFD; n = 6 in each group.

Fig. 8.

Valsartan treatment improved tubular histology and BiP expression from mice on high-fat diet. Representative photomicrographs illustrated vacuolated proximal convoluted tubular cells (arrowheads) and impaired brush border in HFD mice (B, E, and H), which were attenuated by valsartan treatment (C, F, and I). Immunostaining of BiP (arrows) was observed in cytoplasm of proximal tubular cells, with typical perinuclear labeling in CTL mice (D and G). In HFD mice (E and H), immunolabeling of BiP was seen more extensive than CTL and surrounding vacuoles in proximal tubule cells. There was clearly reduced BiP immunostaining in HFD mice treated with valsartan (F and I). Magnification: ×400 for A–F; ×1,000 for G, H, and I.