TRB3 is stimulated in diabetic kidneys, regulated by the ER stress marker CHOP, and is a suppressor of podocyte MCP-1

Abstract

The prevalence of diabetic nephropathy continues to rise, highlighting the importance of investigating and discovering novel treatment strategies. TRB3 is a kinase-like molecule that modifies cellular survival and metabolism and interferes with signal transduction pathways. Herein, we report that TRB3 expression is increased in the kidneys of type 1 and type 2 diabetic mice. TRB3 is expressed in conditionally immortalized podocytes; however, it is not stimulated by elevated glucose. The diabetic milieu is associated with increased oxidative stress and circulating free fatty acids (FFA). We show that reactive oxygen species (ROS) such as H(2)O(2) and superoxide anion (via the xanthine/xanthine oxidase reaction) as well as the FFA palmitate augment TRB3 expression in podocytes. C/EBP homologous protein (CHOP) is a transcription factor that is associated with the endoplasmic reticulum stress response. CHOP expression increases in diabetic mouse kidneys and in podocytes treated with ROS and FFA. In podocytes, transfection of CHOP increases TRB3 expression, and ROS augment recruitment of CHOP to the proximal TRB3 promoter. MCP-1/CCL2 is a chemokine that contributes to the inflammatory injury associated with diabetic nephropathy. In these studies, we demonstrate that TRB3 can inhibit basal and stimulated podocyte production of MCP-1. In summary, enhanced ROS and/or FFA associated with the diabetic milieu induce podocyte CHOP and TRB3 expression. Because TRB3 inhibits MCP-1, manipulation of TRB3 expression could provide a novel therapeutic approach in diabetic kidney disease.

Fig. 1.

TRB3 expression increases in diabetic mouse kidneys. A: C57BL/6 mice were treated with low-dose streptozotocin (STZ) and kidneys were harvested 8 wk later. Real-time PCR studies revealed a 5-fold increase in TRB3 expression in the STZ-treated mice (n = 5 mice per group). B: TRB3 expression was also increased in the kidneys of 24-wk-old db/db mice (n = 5 mice per group). *P < 0.05 vs. control mice, **P < 0.05 vs. db/m mice, Student's t-test and bars represent SE. Frozen sections of control and STZ-treated (×600) mice (C) and db/m and db/db (×1,000) mice (D) were stained with anti-TRB3 and anti-podocin. There is an upregulation of TRB3 staining in the glomeruli of the diabetic mice (STZ, db/db) when compared with the controls (control, db/m).

Fig. 2.

High-glucose conditions do not regulate TRB3 expression. Conditionally immortalized podocytes were differentiated and incubated for 14 days in high-glucose conditions. Mannitol was used as an osmotic control. The results are the average of 3 studies performed.

Fig. 3.

Reactive oxygen species (ROS) augment TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 4 h with H₂O₂ (A) and 3 h with xanthine and xanthine oxidase (B). Real-time PCR demonstrated that ROS augment TRB3 mRNA expression. Each study was performed at least 3 times and the averages of the results are shown. *P < 0.05 vs. control mice; 1-way ANOVA (A) and Student's t-test (B).

Fig. 4.

Free fatty acids (FFA) increase TRB3 expression. Fully differentiated, conditionally immortalized podocytes were treated for 24 h with graded concentrations of palmitate:BSA and BSA controls. A: real-time PCR studies demonstrated that FFA induce TRB3 expression. *P < 0.05 vs. control, **P < 0.05 vs. 100 μM palmitate. B: fully differentiated podocytes were treated with palmitate, BSA, and H₂O₂ overnight. Cellular lysates were harvested and Western blotting was performed and blotted for TRB3 and actin. The following blot is representative of studies performed at least 3 times. C: palmitate and H₂O₂ were added simultaneously and separately and do not appear to have an additive effect on TRB3 expression. *P < 0.05 vs. control (BSA, 1-way ANOVA).

Fig. 5.

ROS and FFA induce C/EBP homologous protein (CHOP) expression and augment recruitment of CHOP and C/EBPβ to the proximal TRB3 promoter. Fully differentiated podocytes were treated for 4 h with H₂O₂ (A) and 24 h with palmitate (Palm) or BSA control (B). CHOP expression was assessed by real-time PCR. *P < 0.05 vs. control (1-way ANOVA). C: as above, podocytes were treated for 24 h with Palm, BSA, H₂O₂, and tunicamycin. Western blotting was performed and CHOP protein expression was assessed. This blot is representative of studies performed at least 3 times. D: Western blotting for GRP78/BiP expression was also performed. E: differentiated podocytes were treated for 4 h with 50 μM H₂O₂ or control and chromatin immunoprecipitation (ChIP) studies were performed. Immunoprecipitation (IP) was performed with antibodies directed to CHOP, C/EBPβ, and the control IgG. E: represents real-time PCR amplification. *P < 0.05 vs. control, 1-way ANOVA. F: represents the 5′ region of the murine TRB3 promoter with the potential CHOP/C/EBPβ binding site at −61. Arrows represent the site of primers used for the ChIP studies. G: represents semiquantitiative PCR ChIP results (34 cycles). H: fully differentiated podocytes were transfected with pcDNA3-CHOP or control vector and 24 h later, TRB3 expression was assessed by Western blotting.

Fig. 6.

CHOP increases in diabetic mouse kidneys. Kidneys from STZ-treated (A) and db/db mice (B) and controls were harvested and mRNA expression of CHOP was evaluated by real-time PCR. There was a significant increase in CHOP expression in the db/db mice compared with the db/m controls and a trend for an increase in the STZ-treated mice (n = 5 mice per group, Student's t-test).

Fig. 7.

A: TRB3 inhibits MCP-1 expression in podocytes. Fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3 (750 ng/well, 96-well plate), stimulated with phorbol 12-myristate 13-acetate (PMA), and supernatatant MCP-1 expression was assessed 24 h after transfection. Results are the average of studies performed at least 3 times. *P < 0.05 vs. pcDNA3-transfected, -unstimulated cells. **P < 0.05 vs. pcDNA3-transfected, PMA-stimulated cells. B: fully differentiated podocytes were transfected with pcDNA3 or pcDNA3-HA-TRB3, and 24 h later HA and TRB3 expression were assessed by Western blotting. TRB3 is efficiently transfected into differentiated podocytes.

Fig. 8.

Proposed therapeutic potential of TRB3 in diabetic kidney disease. Hyperglycemia and elevated FFA associated with diabetes augment ROS, which induce endoplasmic reticulum (ER) stress and activation of MAPK pathways. We propose that enhanced TRB3 expression could inhibit MAPK signaling thereby reducing NF-κB and AP-1 activation of inflammatory genes such as MCP-1, transforming growth factor (TGF)-β, and plasminogen activator-1 (PAI)-1.