Patients with chronic kidney disease often develop cardiovascular disease and die before reaching end-stage renal disease (Jankowski et al., 2021). More severe renal impairment is associated with a higher incidence of cardiovascular events, and patients with heart failure have a higher degree of renal impairment than healthy individuals. Furthermore, it has been reported that anemia, which frequently complicates both, leads to reciprocal and progressive deterioration of the heart and kidney and is a poor prognostic factor for heart failure and chronic kidney disease (McCullough, 2021). Thus, cardiovascular disease, chronic kidney disease, and anemia adversely affect each other (Silverberg et al., 2006). This mutually detrimental condition, called cardiorenal anemia syndrome (CRAS), is a complex disease associated with adverse clinical outcomes, increased risk of hospitalization and death, and decreased quality of life (McCullough, 2021).

Currently, there are few satisfactory clinical treatments for CRAS. Clinical management of patients with CRAS consists of excreting toxins or artificially supplementing with organ-protective substances. Erythropoietin (EPO), which was administered as a therapeutic agent for renal anemia under the policy of supplementation of protective substances, was not effective in humans, although it was effective in restoring cardiac function in animal experiments (Swedberg et al., 2013). On the contrary, it has been reported that exogenous EPO enhances catecholamine, angiotensin II, and endothelin; increases blood pressure; and worsens prognosis (Parfrey et al., 2005; Agarwal, 2018). Recently, however, hypoxia-inducible factor and prolyl-hydroxylase inhibitors have been reported to improve renal anemia by promoting EPO production (Maxwell and Eckardt, 2016). However, the effects of hypoxia-inducible factor and prolyl-hydroxylase inhibitors on CRAS has not been reported. We focused on inflammation as a common feature of the three components of CRAS (Schiattarella et al., 2021).

Dimethyl fumarate (DMF) is a Food and Drug Administration–approved effective treatment of relapsing-remitting multiple sclerosis and is an antioxidant and anti-inflammatory agent (Grzegorzewska et al., 2017). Multiple pathways have been reported as the mechanisms of action of DMF. The most prominent is the Nrf2 pathway activator, which induces its potent anti-inflammatory response in part by directly targeting the nuclear factor κ B (NF-κB) signaling pathway (Grzegorzewska et al., 2017). Its mechanism of action includes immunomodulatory and cytoprotective effects. We hypothesized that anti-inflammatory DMF could ameliorate CRAS, an inflammatory condition common to cardiovascular disease (CVD), chronic kidney disease (CKD), and anemia. Therefore, we investigated whether DMF, an anti-inflammatory agent, is effective for CRAS.

Focusing on the inflammation that occurs commonly in CRAS, we hypothesized that DMF, an anti-inflammatory drug, could suppress CRAS. The results showed that DMF significantly ameliorated anemia, provided cardiac and renal protection, and prolonged overall survival. Therefore, DMF was suggested to be effective in CRAS.

Since the DS rats used this time are a model of CRAS due to hypertension, the trend of blood pressure was investigated. A previous study reported that DMF has a blood pressure–lowering effect (Ahmed et al., 2018). In this experiment, a significant decrease was observed in the HS+DMF group compared with the HS group at 10 and 12 weeks of age. At 14 weeks of age, there was a decreasing trend, but no significant difference was observed. The reason why there was a significant difference in the previous study but not in the current study may be because the models used were different. In this model, the increase in blood pressure was greater than in the previous model, so it might not be possible to lower it completely. In this study, sampling was performed at week 14, when no significant difference in antihypertensive effect was observed, suggesting that DMF might affect other effects than just antihypertensive effects.

A previous study reported that DMF increased fetal hemoglobin, provided heme detoxification, and corrected anemia in sickle cell disease (Pierini et al., 2017). In this study, red blood cell count, serum hemoglobin level, and hematocrit were significantly decreased in the HS group compared with the control group but significantly improved by DMF. Since the kidney is the site of erythropoietin production, the effect of DMF on renal injury in this model might have affected erythropoietin production function and contributed to the improvement of red blood cell numbers. On the other hand, reticulocytes were significantly increased in the HS group compared with the control group but were significantly suppressed by DMF. We previously reported that anemia in DS rats fed an HS diet is caused by shortened red blood cell lifespan (Manabe et al., 2020). These findings suggest that DMF might improve anemia.

Although few reports have examined the effects of DMF on heart failure, several studies have reported cardioprotective effects. DMF has been shown to prevent cardiovascular disease by inhibiting dendritic cell maturation and may exert protective effects on myocardial ischemia/reperfusion models (Kuang et al., 2020; Sun et al., 2022). Myocardial remodeling, which is the cause of heart failure, is thought to be formed by the degeneration and necrosis of myocardial cells, the infiltration of inflammatory cells, and tissue fibrosis (Szekely and Arbel, 2018). In this study, we investigated cardiomyocyte degeneration and fibrosis. H&E staining showed disorganized cellular structure and edematous cardiomyocytes in the HS group. Furthermore, when fibrosis was evaluated by MTC staining, significant fibrosis occurred in the HS group. These abnormalities were significantly inhibited by DMF. In addition, TGF-β1 mRNA expression in heart tissue was also examined, and TGF-β1 was increased in the HS group, but DMF suppressed it. These findings suggest that suppression of fibrosis in cardiac tissue was associated with suppression of TGF-β1.

We examined cytokines in cardiac tissue. Regardless of the underlying etiology, the development and progression of heart failure are closely associated with inflammation, and the induction and activation of chemokines are one of the hallmarks of the inflammatory response in heart failure. IL-1β is known to induce fibrosis and cardiac remodeling after acute myocarditis in mice. Inflammation is involved in the pathogenesis of many heart diseases, and IL-1β is one of the major mediators in this inflammatory process (Szekely and Arbel, 2018). Ccl2 functions in the development and progression of cardiovascular diseases such as heart failure. ICAM1 is an immunoglobulin-like adhesion molecule that mediates leukocyte arrest and transendothelial migration from the bloodstream to the sites of inflammation. ICAM1 regulates cardiac inflammation and pathologic cardiac remodeling by mediating T-cell recruitment to the left ventricle, contributing to cardiac dysfunction and heart failure (Salvador et al., 2016). In this study, DMF suppressed the increase in IL-1β, Ccl2, and ICAM1 mRNA expression, suggesting that DMF may have suppressed inflammation in heart tissue. NOX4 is known to be the major source of reactive oxygen species (ROS) in myocardial remodeling (Matsushima et al., 2016). In this study, NOX4, which increased in the HS group, was suppressed in the HS+DMF group, suggesting the possibility that ROS production was suppressed and cardiomyocyte abnormalities were suppressed. From the above, DMF has been shown to exert anti-inflammatory and antifibrotic effects and to have cardioprotective effects associated with the suppression of various cytokines.

Recent studies strongly support a protective effect of DMF against renal damage (Yang et al., 2021). In the present study, serum creatinine was significantly higher in the HS group compared with the control group, but this increase was significantly decreased in the HS+DMF group compared with the HS group. This suggested that DMF has a renal protective effect.

Furthermore, in renal tissues, PAS staining revealed an increase in tubular atrophy in the HS group, but it was suppressed in the HS+DMF group. In addition, according to the results of MTC staining for evaluating fibrosis, fibrosis significantly increased in the HS group but was significantly suppressed by DMF. A previous study reported that DMF, which plays an important role in the development of renal fibrosis, prevents renal fibrosis through suppression of TGF-β1 signaling (Oh et al., 2012). In addition, PDGFb is an important factor involved in renal interstitial fibrosis (Floege et al., 2008). In the present results, the TGF-β1 and PDGFb mRNA expression that increased in the HS group was suppressed by DMF. We examined the mRNA expression of KIM1. KIM1 is highly expressed in renal tubular cells after injury and is commonly considered an early biomarker of acute kidney injury (Vaidya et al., 2006). As a result, a significant increase was observed in the HS group and a significant suppression in the HS+DMF group. Furthermore, DMF significantly decreased urinary total protein and albumin, which were significantly increased in the HS group. These findings suggested that DMF inhibits renal tissue damage such as fibrosis and tubular damage.

We examined mRNA expression of cytokines associated with inflammation in renal tissue. Inflammation plays an important role in the pathogenesis of kidney disease (Kashyap et al., 2018). Stimulation of IL-1β signaling within the kidney itself is also already known to exacerbate the progression of renal injury via activation of the NLRP3 inflammasome (McKnight et al., 2020). ICAM1 has also been shown to be expressed in renal tubular cells during inflammation (Wu et al., 2007). Ccl2 is an important mediator of interstitial inflammation, fibrosis, and tubular atrophy in chronic kidney disease (Kashyap et al., 2018; Xu et al., 2019). In this study, IL-1β, ICAM1, and Ccl2, which were increased in the HS group, were suppressed in the HS+DMF group. These findings suggest that DMF exerts a protective effect on the kidneys by suppressing inflammation at the genetic level. In addition, we examined the expression of NF-κB in renal tissue by western blot and found that DMF suppressed the elevated expression in the HS group. This suggests that inflammation is suppressed even at the protein level. In the present study, NOX4 mRNA expression, which increased in the HS group, was suppressed in the HS+DMF group. NOX4 is involved in ROS generation. Increased ROS causes oxidative stress and contributes to anemia. These findings suggest that DMF suppresses ROS generation and may also be involved in suppressing anemia symptoms. These support the results of previous studies that DMF can act as a nephroprotectant in part through regulation of oxidative stress, mitochondrial function, and inflammation (Ashari et al., 2023).

In the present study, damage occurred to vascular endothelial cells, vascular smooth muscle cells, and renal secretory cells in the HS group, which may impair some of the inflammatory cytokines and growth factors that we measured. This leads to immune cell chemoattraction and transmigration, enabled by ICAM. A high-salt diet can also stimulate immune cells such as macrophages, dendritic cells, and T cells, which ultimately amplify inflammation, oxidative stress, and injury in chronic kidney disease (CKD) and cardiovascular disease (CVD). DMF is thought to have cardiorenal protective effects by inhibiting these damaging mechanisms.

There are several limitations to this study. The lack of a DMF monotherapy control group is a potential weakness in the study design. We did not establish a DMF alone group in this study because we believed that DMF monotherapy had little effect in the absence of disease. Therefore, we believe that this absence does not unduly compromise the interpretation of the results. Although cytokines were examined in this research, it was not possible to examine them one by one using knockout mice. Therefore, it was not possible to investigate what kind of changes would be caused by the deficiency of each cytokine. Regarding heart failure, this study was limited to histologic analysis and cytokine mRNA analysis, and an echo examination was not performed, so further examination is necessary.t