AdipoRon Protects against Tubular Injury in Diabetic Nephropathy by Inhibiting Endoplasmic Reticulum Stress

Abstract

Endoplasmic reticulum (ER) stress has been reported to play a pivotal role in diabetic nephropathy (DN). AdipoRon is a newly developed adiponectin receptor agonist that provides beneficial effects for diabetic mice; however, its underlying mechanism remains to be delineated. Here, we demonstrated increased expression levels of ER stress markers, accompanied by upregulated levels of proinflammatory cytokines and increased expression of collagen I, fibronectin, Bax, and cleaved caspase 3 in the kidneys of db/db mice compared with control mice. Decreased expression of adiponectin receptor 1 (AdipoR1) and phosphorylated 5'AMP-activated kinase (p-AMPK) was also observed in the kidneys of db/db mice. However, these alterations were partially reversed by intragastric gavage with AdipoRon. In vitro, AdipoRon alleviated high-glucose-induced ER stress, oxidative stress, and apoptosis in HK-2 cells, a human tubular cell line. Moreover, AdipoRon restored the expression of AdipoR1 and p-AMPK in HK-2 cells exposed to high-glucose conditions. Additionally, these effects were partially abrogated by pretreatment with AdipoR1 siRNA, but this abrogation was ameliorated by cotreatment with AICAR, an AMPK activator. Furthermore, the effects of AdipoRon were also partially abolished by cotreatment with compound C. Together, these results suggest that AdipoRon exerts favorable effects on diabetes-induced tubular injury in DN by inhibiting ER stress mediated by the AdipoR1/p-AMPK pathway.

Figure 1

Effects of AdipoRon on renal functional and morphological changes in db/db mice: (a) body weight changes in three mouse groups from 16 weeks to 20 weeks; (b) blood glucose levels; (c) serum creatinine concentrations; (d) proteinuria/24 h in each group; (e) urinary 8-OHdG levels; (f) serum adiponectin levels; (g) HE (upper panels), PAS (middle panels), and Masson staining (bottom panels) of renal tissue sections from db/m, db/db, db/db mice treated with AdipoRon, and 16-week-old db/db mice before the treatment. n = 8 mice per group. Bars = 100 μm. (h, i) Quantitative analysis of the tubular interstitial damage score and mesangial expansion score in each group. The data are expressed as the means ± SDs. ^∗P < 0.05 versus db/m control mice; ^#P < 0.05 versus db/db mice. HE: hematoxylin and eosin; PAS: periodic acid-Schiff.

Figure 2

Effects of AdipoRon on inflammation, apoptosis, and fibrosis in the kidneys of db/db mice. (a) Representative immunofluorescence images of collagen I (A–C) and FN (D–F) expression in the renal tissues of db/m, db/db, and db/db mice treated with AdipoRon. Oxidative stress and apoptosis in renal tissues were detected using DHE reagents (G–I) and TUNEL staining (J–L), respectively. n = 8 mice per group. Bars = 100 μm. (b) Semiquantitative analysis of the protein levels of collagen I and FN detected by IF. (c) Semiquantitative assessment of oxidative stress and apoptosis in each group. (d) Western blot analysis of cleaved caspase 3, Bcl-2, Bax, FN, and collagen I. β-Actin was used as a loading control. n = 8 mice per group. (e) Relative band density of cleaved caspase 3, Bcl-2/Bax, FN, and collagen I expression. (f–h) mRNA levels of MCP-1, IL-6, and TNFα in the renal tissues of each group determined by qRT-PCR. The data are shown as the mean ± SD. ^∗P < 0.05 versus db/m control; ^#P < 0.05 versus db/db mice. FN: fibronectin; DHE: dihydroethidium; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MCP-1: monocyte chemotactic protein-1; IL-6: interleukin-6; TNFα: tumor necrosis factor α; qRT-PCR: quantitative reverse-transcriptase PCR.

Figure 3

Role of AdipoRon in the expression of AdipoR1 and p-AMPK and ER stress in the kidneys of db/db mice. (a) Representative immunofluorescence images of AdipoR1 (A–C), GRP78 (D–F), and CHOP (G–I) in the renal tissues of three mouse groups. n = 8 per group. Bars = 100 μm. (b) Semiquantitative analysis of AdipoR1 and p-AMPK expression. (c) Semiquantitative analysis of nuclear CHOP positive cells (%). (d) Representative immunoblots of AdipoR1, p-AMPK, and T-AMPK. β-Actin was used as a loading control. (e) Relative band intensity. (f) Representative immunoblots of GRP78, p-PERK, PERK, and CHOP. (g) Relative band intensity. n = 8 mice per group. The data are shown as the means ± SDs. ^∗P < 0.05 versus db/m control mice; ^#P < 0.05 versus db/db mice. AdipoR1: adiponectin receptor 1; GRP78: glucose-regulated protein of 78 kDa; CHOP: C/EBP homologous protein; p-AMPK: phosphorylated 5′AMP-activated kinase; PERK: protein kinase-like endoplasmic reticulum kinase; p-PERK: phospho-PERK.

Figure 4

Effects of AdipoRon treatment on ER stress, inflammation, apoptosis, and fibrosis in HK-2 cells under HG conditions. (a) Representative confocal images of AdipoR1 in HK-2 cells treated or not treated with different concentrations of AdipoRon (5 μM, 10 μM, and 50 μM) under HG conditions. n = 3 per group. Bars = 10 μm. (b) Quantification of the average immunofluorescence intensity of AdipoR1 in HK-2 cells. (c) The levels of intracellular ROS in treated HK-2 cells as assessed by DCFH-DA staining. n = 3 per group. Bars = 10 μm. (d) Bars graphs depict intracellular ROS content in treated HK-2 cells. (e) Representative western blot bands of AdipoR1, p-AMPK, T-AMPK, GRP78, p-PERK, PERK, and CHOP in HK-2 cells treated with different concentrations of AdipoRon under HG conditions. β-Actin was used as a loading control. n = 3 per group. (f) Relative band density of WB. (g) Immunoblot assays of Bcl-2, Bax, cleaved caspase 3, collagen I, and FN in treated HK-2 cells. n = 3 per group. (h) mRNA levels of MCP-1, IL-6, and TNFα in HK-2 cells treated or not treated with different concentrations of AdipoRon (5 μM, 10 μM, and 50 μM) under HG conditions. n = 3 per group. The data are shown as the means ± SDs. ^∗P < 0.05 versus LG; ^#P < 0.05 versus HG. LG: low glucose; HG: high glucose; ROS: reactive oxygen species; μM: μmol/L.

Figure 5

Effect of AdipoR1 siRNA, AICAR, and compound C on the levels of p-AMPK and ER stress in HK-2 cells treated with AdipoRon under HG conditions. (a) Immunoblot assays of the protein expression of p-AMPK, T-AMPK, GRP78, p-PERK, PERK, and CHOP in HK-2 cells treated with AdipoRon, AdipoR1 siRNA, AICAR, or compound C under HG ambience. n = 3 per group. (b) Relative band density. (c) Representative western blot bands of Bcl-2, Bax, cleaved caspase 3, collagen I, and FN in HK-2 cells treated with AdipoRon, AdipoR1 siRNA, AICAR, or compound C under HG conditions. n = 3 per group. (d) Relative band density. The data are shown as the means ± SDs. ^∗P < 0.05 compared to the LG group; ^#P < 0.05 compared to the HG group. ^$P compared to the HG+AdipoRon group. ^@P < 0.05 compared to the HG+AdipoRon+AdipoR1 siRNA group. ^&P compared to the HG+AdipoRon group. AICAR: 5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide.