Crosstalk between endoplasmic reticulum stress and oxidative stress in the progression of diabetic nephropathy

Abstract

Increasing evidence in substantiating the roles of endoplasmic reticulum stress, oxidative stress, and inflammatory responses and their interplay is evident in various diseases. However, an in-depth mechanistic understanding of the crosstalk between the intracellular stress signaling pathways and inflammatory responses and their participation in disease progression has not yet been explored. Progress has been made in our understanding of the cross talk and integrated stress signaling network between endoplasmic reticulum stress and oxidative stress towards the pathogenesis of diabetic nephropathy. In this present study, we studied the crosstalk between the endoplasmic reticulum stress and oxidative stress by understanding the role of protein disulfide isomerase and endoplasmic reticulum oxidase 1α, a key player in redox protein folding in the endoplasmic reticulum. We had recruited a total of 90 subjects and divided into three groups (control (n = 30), type 2 diabetes mellitus (n = 30), and diabetic nephropathy (n = 30)). We found that endoplasmic reticulum stress markers, activating transcription factor 6, inositol-requiring enzyme 1α, protein kinase RNA-like endoplasmic reticulum kinase, C/EBP homologous protein, and glucose-regulated protein-78; oxidative stress markers, thioredoxin-interacting protein and cytochrome b-245 light chain; and the crosstalk markers, protein disulfide isomerase and endoplasmic reticulum oxidase-1α, were progressively elevated in type 2 diabetes mellitus and diabetic nephropathy subjects. The association between the crosstalk markers showed a positive correlation with endoplasmic reticulum stress and oxidative stress markers. Further, the interplay between endoplasmic reticulum stress and oxidative stress was investigated in vitro using a human leukemic monocytic cell line under a hyperglycemic environment and examined the expression of protein disulfide isomerase and endoplasmic reticulum oxidase-1α. DCFH-DA assay and flow cytometry were performed to detect the production of free radicals. Further, phosphorylation of eIF2α in high glucose-exposed cells was studied using western blot. In conclusion, our results shed light on the crosstalk between endoplasmic reticulum stress and oxidative stress and significantly contribute to the onset and progression of diabetic nephropathy and therefore represent the major therapeutic targets for alleviating micro- and macrovascular complications associated with this metabolic disturbance. Graphical abstract.

Keywords: Crosstalk; Diabetic nephropathy; ER oxidase 1α; Endoplasmic reticulum stress; Oxidative stress; Protein disulfide isomerase.

Graphical abstract

Fig. 1

The expression levels of ER stress markers such as PERK, IRE1, ATF6, ER chaperone protein GRP78, and pro-apoptotic protein CHOP in PBMCs of study subjects were measured using qRT-PCR. A total of twenty-seven samples were analyzed for gene expression studies. Data are expressed as fold change over control and presented as mean ± SD of three independent experiments. Statistical analysis was performed by the Kruskal-Wallis test. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 2

The expression levels of selected oxidative stress markers TXNIP and p22^pHox in PBMCs of study subjects were measured using the qRT-PCR analysis. A total of twenty-seven samples were analyzed for gene expression studies. Data are expressed as fold change over control and presented as mean ± SD of three independent experiments. Statistical analysis was performed by the Kruskal-Wallis test. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

The levels of crosstalk genes such as PDIA2 and ERO1A in PBMCs of study subjects were measured using qRT-PCR analysis. A total of twenty-seven samples were analyzed for gene expression studies. Data are expressed as fold change over control and presented as mean ± SD of three independent experiments. Statistical analysis was performed by the Kruskal-Wallis test. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

a Effect of ER and oxidative stress markers in high glucose (33.3 mM) treated in THP-1 cells under different time points. b Effect of crosstalk markers in high glucose stimulated THP-1 cells under different time intervals and assessed the gene expression using qRT-PCR. Data are expressed as fold change over control and presented as mean ± SD of three separate experiments. Statistical analysis was performed using unpaired “t” test with the Welch corrections. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 5

Effect of high glucose (33.3 mM) on eIF2α phosphorylation by western blotting in THP-1 cells. Data are expressed as p-eIF2α(S51)/eIF2α ratio and presented as mean ± SD of three separate experiments. Statistical analysis was performed using by the Kruskal-Wallis test. *p < 0.05

Fig. 6

Determination of reactive oxygen species generation via detection of DCFDA by flow cytometry. THP-1 cells were incubated with high glucose for different time points and stained with DCFDA. The fluorescence intensity was measured by flow cytometry. A shift to the right indicates increased reactive oxygen species levels (a). The mean fluorescence intensity (MIF) values are presented as means ± SD of three experiments (b). Statistical analysis was performed using by the Kruskal-Wallis test. *p < 0.05