Unlike type 1 diabetes, which is induced by an inherited disorder of insulin production in pancreatic islet β-cells [1], type 2 diabetes is caused by a variety of factors, including a lack of exercise, alcohol intake, genetic factors, and diet [2]. Diabetic nephropathy occurring in proximal renal epithelial cells is one of the main complications of diabetes [3]. Expression of receptors for advanced glycation end products (RAGE) is upregulated by abundant advanced glycation end products (AGEs) during diabetes-associated complications. The AGE–RAGE axis is involved in the onset of diseases such as Alzheimer’s disease, cancer, and osteoporosis [4,5,6]. According to recent studies, AGEs affect the glomerular filtration rate, resulting in chronic renal failure [7]. Alikhani et al. (2005) reported that N-ε-(carboxymethyl) lysine (CML)–collagen induced the apoptosis of fibroblasts, as well as increased caspase-3, -8, and -9 activity in an in vivo test [8]. Glyceraldehyde-derived AGEs induce apoptosis in human dermal fibroblasts by increasing reactive oxygen species (ROS) and activating the NLRP3 inflammasome [9]. Methylglyoxal (MGO), a major electrophilic dicarbonyl compound, is generated as a nonenzymatic breakdown product of a triosephosphate intermediate in the glycolytic process and has been linked to dicarbonyl stress, which leads to the development of AGEs and related cellular dysfunction [10,11]. We previously showed that the treatment of NRK-52E kidney cells with MGO-derived AGEs (AGE4) leads to an increase in the protein levels of matrix metalloproteinase-2 (MMP-2) and MMP-9 via AGE4–RAGE interactions [12]. Endoplasmic reticulum (ER) stress is an important mechanism that induces diabetes [13]. Moreover, mitochondrial functions include calcium homeostasis in cells, respiration, and biogenesis regulation through c-Jun NH2-terminal kinase (JNK) pathways [14,15,16], which are also closely related to cell apoptosis response pathways [17]. Although ER stress affects mitochondria directly or indirectly [18], the exact effects of AGE4 on the signaling pathways associated with ER stress and cell apoptosis are unknown. Therefore, we used in vitro and in vivo models to investigate the involvement of the AGE4–RAGE axis and specific signaling pathways that induce ER stress and mitochondrial dysfunction, which contribute to apoptosis.

3. Discussion

Takeuchi et al. employed various sugar sources, including glucose, α-hydroxyaldehydes, and dicarbonyl compounds, to glycate proteins and classified AGEs into glucose-derived AGEs (AGE1); glyceraldehyde-derived AGEs (AGE2), glycolaldehyde-derived AGEs (AGE3), methylglyoxal-derived AGEs (AGE4), glyoxal-derived AGEs (AGE5), and 3-deoxyglucosone-derived AGEs (AGE6) [29,30]. Methylglyoxal, a reactive dicarbonyl produced during glucose metabolism, accumulates in diabetic patients [30], and its derived AGE4 is known to cause several diseases, including diabetes and cancer [11].

Our results clearly indicated that a significant decrease in cyclin D1 protein level was caused by AGE4. This decrease may result in an accumulation of cells in the sub-G1 and G1 phases. Cyclin D1 has been shown to be necessary for progression through the G1 process of the cell cycle in order to cause cell migration [31]. In addition, the activation of NF-κB by AGE4 treatment can be regulated through the JNK signaling pathway, according to our findings. RAGE–AGE4 axis-induced apoptosis is correlated with the induction of ER stress markers, such as CHOP, ATF4, and GRP78, via JNK pathway. In the previous study, AGE4 treatment induced RAGE-dependent cell inflammation in rat kidney epithelial cells (NRK-52E) through the ERK, JNK, and NF-κB pathways [12]. JNK and NF-κB are the main oncogenic signaling pathways linked to ER stress, which leads to nonalcoholic steatohepatitis and hepatocellular carcinoma [32].

The major source of ROS generation is the mitochondrial respiratory complexes, and the JNK pathway is involved in mitochondrial apoptosis [33,34]. AGEs play an important role in developing various diseases such as myocardial dysfunction and atherosclerosis [35,36], induce ROS through interaction with receptors, and regulate the JNK signal pathway [37,38]. In this study, AGE4 treatment of HK-2 human kidney cells induced ROS production through interacting with RAGE and activating the JNK signaling pathway, both of which play important roles in cellular apoptosis. Recent studies have shown that chronic inflammation induced by intracellular ROS is related to cell signaling in mitochondria and the development of metabolic diseases such as diabetes [39,40,41]. Cellular apoptosis is caused by oxidative stress-induced signal cascades related to cellular apoptosis involving Bcl-2 family proteins [42,43]. When Bcl-2 family proteins interact with the outer mitochondrial membrane, apoptosis proteins are activated [44,45]. On the other hand, due to physiological or pathological conditions, ER stress triggers inflammation, apoptosis, and cell death [46,47,48]. ER stress activates GRP78 and ATF4 through the phosphorylation of eIF2α and promotes the CHOP gene belonging to the C/EBP family, leading to cellular apoptosis [49].

5. Conclusions

In summary, the following points are suggested in this study: (1) RAGE–AGE4 can cause ER stress that leads to apoptosis in kidney cells. (2) The functions of the ER and mitochondria are interconnected, and the cellular mechanism of disorders is induced by the JNK signal pathway through AGE4–RAGE axis. Understanding the mechanism by which AGE4 induces ER stress and mitochondrial dysfunction during diabetic nephropathy will serve as a basis for a new therapeutic approach. Since AGE4, a type of AGE, causes cellular renal apoptosis, future research addressing the active component in AGE4 that causes diabetic nephropathy will aid in the delivery of treatments for diabetic complications.