Renal fibrosis associated with inflammation is considered as the final common pathway by which chronic kidney disease (CKD) leads to end stage renal disease (ESRD) (1, 2). Occurrence of CKD and ESRD are very common in patients with inflammatory diseases such as cardiometabolic syndrome, diabetes, and hypertension (2–4). These are the most common chronic diseases of the modern world that cause CKD and high mortality (3, 4). The high mortality and morbidity associated with CKD often linked to the lack of an effective anti-fibrotic and anti-inflammatory agents that could target renal fibrosis (4).

There is an unmet need of novel combined anti-inflammatory and anti-fibrotic agents, particularly as an effective therapeutic approach for CKD and its progression to ESRD (5–7). Angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers are the current therapeutic choices for the clinical management and treatment of CKD (8). However, these mainstay treatment options have limited efficacy to prevent or treat renal fibrosis, the major pathological event that leads to the progression of CKD to ESRD (5, 6). More recently, finerenone, a nonsteroidal selective mineralocorticoid receptor antagonist, has been demonstrated to decrease albuminuria and risk of CKD progression in chronic heart failure and diabetes (9, 10). Other novel approaches that have been attempted include the nuclear factor E2-related factor 2 inducer bardoxolone and the endothelin receptor blocker avosentan which proved largely ineffective in treating inflammation and associated renal fibrosis (11, 12). Findings from these studies clearly underscore a critical need for agent that can effectively treat renal fibrosis, the final common pathophysiological event in the progression of CKD to ESRD.

Potential targets for combating inflammation and renal fibrosis in CKD are the nuclear farnesoid X receptor (FXR) and the soluble epoxide hydrolase (sEH) enzyme (13, 14). FXR expression is decreased in the human and rodent models of kidney disease and this decrease correlated with the level of inflammation and fibrosis in the kidney (13, 15). Activation of FXR demonstrated kidney protective actions in animal models by decreasing kidney inflammation, oxidative stress, and fibrosis (16, 17). FXR is expressed on immune cells and FXR activation has anti-inflammatory actions (17, 18). These findings indicated an important role for FXR in kidney disease. Similar to FXR activation, inhibition of sEH that metabolizes and inactivates kidney protective epoxyeicosatrienoic acids (EETs) can combat inflammation and kidney disease (14, 19, 20). Experimental studies demonstrated beneficial kidney actions of EETs and sEH genetic deletion or inhibition in animal models of renal fibrosis and CKD (19, 20). Interestingly, it has also been demonstrated that FXR activation can induce the expression of EET producing cytochrome P450 enzymes and several polyunsaturated fatty acids including arachidonic acid act as FXR ligands (21, 22). Intriguingly, our research group has developed a dual acting FXR agonist and sEH inhibitor, DM509 which could decrease renal inflammation and fibrosis that occurs in CKD (23, 24).

Previous studies have demonstrated in mice that DM509 protected against liver and kidney fibrosis (23, 25, 26). Our previous kidney study demonstrated that DM509 administered one day prior to unilateral ureteral obstruction (UUO) prevented development of renal fibrosis (26). Unfortunately, the clinical treatment of CKD occurs after significant renal inflammation and fibrosis are evident. Thus, in the present study DM509 was tested in a clinically relevant manner.

In the current study we investigated interventional treatment with DM509 to reduce renal inflammation and fibrosis to halt the progression of CKD. Our findings demonstrate the therapeutic potential for the dual acting agent DM509 to combat renal inflammation and fibrosis associated with CKD progression.

Fibrosis, a characteristic of all chronic kidney diseases (CKD), is recognized to be an independent predictor of disease progression (1, 3, 27). CKD and ESRD are major public health problems with increasing prevalence worldwide and in the US almost 15% (37 million) people are suffering from CKD (28, 29). Unfortunately, at present, there are no effective therapies to prevent or slow the progression of renal inflammation and fibrosis, and the treatment options for patients with ESRD are limited to dialysis and renal transplant (4–6). The lack of novel drugs targeting the pathological events of renal inflammation and fibrosis often give mixed results with unwanted side effects (5, 6). The shortcomings of these experimental therapies are likely due to the fact that the pathogenesis of fibrosis is complex. Renal fibrosis is linked to expansion of the interstitium with activated myofibroblasts, elevated inflammation, tubular atrophy, and microvascular injury (2, 30). In previous study our laboratory demonstrated that DM509 administered in a preventive manner reduced renal fibrosis in UUO mice (26). A major shortcoming of the previous study is that the clinical treatment CKD takes place after renal damage is evident. The current study administered DM509 to UUO mice at three days where renal inflammation and fibrosis is established (19, 20). DM509 is a dual acting FXR agonist and sEH inhibitor that possesses low nanomolar dual potency (31). Previous studies have demonstrated that following a single 10 mg/kg dose of DM509 to mice results in a robust increase in the EET/DHET (sEH substrate product) ratio and modulation of FXR regulated gene expression in the liver (31). In the present study we have demonstrate the unique ability of DM509 given in an interventional mode to reduce renal inflammation and slow progression of renal fibrosis in the mouse UUO model. Our findings demonstrate promising anti-inflammatory and anti-fibrotic effects of interventional DM509 treatment in regard to its ability to affect all renal fibrotic pathological events.

During renal fibrosis there is marked accumulation of myofibroblasts in the tubular interstitial space and these are the primary cells to synthesize and deposit pathological components of fibrillar matrix namely collagen and fibronectin (30, 32). Accumulation of fibrotic matrix progressively destroys the normal kidney tissue architecture by contraction and increased stiffness, resulting in disrupted blood flow supply and nephron function, as well as by increasing the space between vascular and tubular structures (30, 32). In the present study, we have used an established UUO mouse model that demonstrates marked renal fibrosis with accumulation of activated myofibroblast and collagen formation. These critical fibrotic events are evident from elevated renal collagen level and high expression of activated myofibroblast markers α-SMA and FSP-1. Interventional treatment with the dual FXR agonist and sEH inhibitor, DM509 attenuated collagen and myofibroblast formation and demonstrated a robust anti-fibrotic effect in the UUO kidney. The observed anti-fibrotic ability of DM509 can be attributed to the biological actions of both FXR agonist and sEH inhibitor pharmacophores.

Several earlier studies reported marked anti-fibrotic actions of sEHi in animal models of CKD and renal fibrosis, including the mouse UUO model (19, 20, 33). Indeed, genetic or pharmacological sEH inhibition markedly reduced renal fibrosis and extracellular matrix protein formation in UUO mice by acting on multiple fibrotic pathophysiological events (19, 20). The other pharmacophore of DM509, FXR is highly expressed within the kidney, and studies have demonstrated that FXR activation can mitigate renal injury (13, 16). FXR levels were inversely correlated with CKD progression in mice and the degree of interstitial fibrosis in humans (34). FXR activation using obeticholic acid also decreased kidney inflammation and fibrosis in an ischemic reperfusion model of renal injury (34). It has been demonstrated that the renal expression of FXR is decreased in UUO mice (35). In the same study, it was also demonstrated that a novel FXR agonist EDP-305 reduced renal fibrosis (35). Consistent with these findings, we suggest that the anti-fibrotic actions of interventional DM509 treatment is due to both FXR agonism and sEH inhibition.

Renal fibrosis is complex process consisting of several mechanisms that ultimately leads to myofibroblast formation and extracellular matrix synthesis (36–38). Initial cell injury in response to UUO results in cell stress and necrosis which leads to release of damage-associated molecular patterns (DAMPs) leading to inflammation (36, 37). Several mechanisms including cellular activation, cellular proliferation, immune cell infiltration, EMT, mesothelial-to-mesenchymal transition (MMT), and endothelial-to-mesenchymal transition (EndoMT) are responsible for increasing the net pool of myofibroblast (37, 38). The current study evaluated the ability for interventional DM509 treatment to reduce EMT, inflammation and EndoMT in UUO mice.

EMT is one of several contributors to renal fibrosis in UUO mice (39, 40). Consistent with these findings, in the present study the renal fibrosis in UUO kidney was accompanied by EMT with a markedly reduced expression of epithelial marker E-cadherin and higher expression of mesenchymal markers α-SMA and FSP-1. UUO mice also demonstrated marked renal fibrosis with collagen formation and myofibroblast formation. It has been reported that the anti-fibrotic effect of the FXR agonist, EDP-305 could be associated with its ability to reduce mesenchymal expression and restoration of epithelial expression within the renal tubules (35). The ability of DM509 to also act as an sEH inhibitor to maintain and elevate epoxyeicosatrienoic acids (EETs) levels could contribute to the reduction in EMT and anti-fibrotic actions. EETs have strong cardiovascular and renal actions and are kidney protective with potent anti-fibrotic actions (14, 33). Interestingly, FXR agonist have the ability to affect the production of EETs from arachidonic acid by inducing CYP P450 (CYP) epoxygenases (21, 22). Indeed, it has been reported that in a liver fibrosis model FXR activation reprogrammed arachidonic acid metabolism by inducing CYP epoxygenase expression and EET production (21). The current study demonstrated that the anti-fibrotic actions of DM509 in UUO model is associated with a reduction in EMT. The anti-fibrotic action of DM509 was associated with its ability to reduce renal tubular EMT. Our findings suggest that the reduction in EMT induced by DM509 is due to FXR agonism, sEH inhibition and the important interactive relationship of these two DM509 pharmacophore activities.

During renal fibrosis, inflammation plays a key role in the activation of fibroblast and extracellular matrix formation in the kidney (2, 30, 41). Upon activation by profibrotic cytokines, resident interstitial fibroblasts progressively gain the myofibroblast phenotype, which was consistently seen in various CKD models including the UUO model (39, 42). Indeed, it was found that the monocytes/macrophages are the most abundant immune cells in most CKD models, and the presence of macrophages in human CKD biopsies is associated with tubular interstitial fibrosis and poor renal survival (43). In the present study, we demonstrated marked renal inflammation with elevated kidney MCP-1 levels and renal infiltration of immune cells in the UUO kidney. Elevated kidney chemokine levels and presence of immune cells in the UUO kidney were accompanied by elevated renal expression of key profibrotic cytokines. These findings are consistent with our earlier findings in the UUO renal fibrosis model (19, 20, 39). The dual acting molecule DM509 administered as an interventional treatment markedly reduced renal inflammation by reducing MCP-1 levels, infiltration of immune cells, and cytokine expression in UUO mice. The anti-inflammatory action of DM509 can be attributed to the biological actions of both FXR agonism and sEH inhibition activities. Anti-inflammatory actions of sEH inhibition in reducing kidney injury are well established in CKD animal models (19, 20, 44). Inhibition of sEH is known to reduce chemotaxis by reducing chemokines, renal infiltration of immune cells, and cytokine production in CKD (14, 20, 33). The sEH inhibitory anti-inflammatory actions in reducing renal fibrosis are also well established (14, 20). It has been reported that sEH inhibition either by genetic manipulation or by a pharmacological inhibition provides robust anti-inflammatory actions and attenuates renal fibrosis progression in UUO mice (19, 20). In UUO mice, sEH inhibition resulted in a marked decrease in renal infiltration of different types of immune cells and reduced profibrotic cytokine production which are linked to renal fibrotic pathophysiology (19, 20). The increase in EET levels in response to sEH inhibition could have direct actions on immune cells. A recent study provided evidence that renal EETs may act locally to regulate the ENaC activity in circulating monocytes entering the kidney to reduce inflammation in salt-sensitive hypertension (45). FXR activation has also been shown to suppress the inflammatory response by affecting several critical aspects of inflammatory processes (13, 17). FXR activation inhibited inflammation in various organ systems including liver, lung, and kidney (17). In several studies using rodent models of type I diabetes, high-fat diet-induced cardiometabolic syndrome, and type 2 diabetes mellitus, FXR expression and its target genes were down regulated in the kidney (46–48). Interestingly, such changes in the renal expression of FXR and its target genes in these models with renal pathology correlated with elevated inflammation and fibrosis in the kidney (46–48). A role of FXR was also reported in the UUO renal fibrosis model where it was demonstrated that FXR activation reduced renal inflammation and fibrosis by decreasing renal immune cell infiltration and expression of profibrotic cytokines (35). Like sEH inhibition and increased EET levels, FXR could act directly on immune cells. Indeed, previous studies have demonstrated that FXR agonists inhibit gastrointestinal inflammation through direct actions on immune cells to preserve the intestinal barrier during inflammatory bowel disease (49). A link to sEH inhibition could also exists as the FXR mediated anti-inflammatory actions require active CYP epoxygenases (21). Overall, the well-known anti-inflammatory actions of sEH inhibition and FXR agonism indicate that the robust anti-inflammatory actions of the novel dual acting DM509 are associated with the biological consequences of both molecular activities.

Renal inflammation caused by elevated chemokines, infiltrating immune cells and cytokine productions cause tubular injury and this tubular injury is considered an important event in renal fibrosis and CKD progression (3, 30, 42). Indeed, tubular degeneration evokes a vivid peritubular environment, and the injured tubular cells initiate a local inflammation, that finally leads to the removal of all tubular remnants and the formation of fibrosis (30, 42). An interesting finding from a recent reversal UUO study found an important contribution for F4/80 positive macrophages but not CD3+ T-cells in salt-sensitive hypertension (44). Our research group has previously demonstrated that DM509 treatment to non-alcoholic steatohepatitis mice decreased liver CXCR3, CXCL9, CXCL10, TNFα, IL-1β, and TGF-β expression (23). In the current study we demonstrate a similar DM509 anti-inflammatory action in UUO. Kidney MCP- levels, CD45 positive immune cells, and TNFα, IL-1β, and IL-6 IL gene expression were decreased by DM509 treatment. Previous studies with sEH inhibitors or genetic deletion have demonstrated decreased transforming growth factor beta (TGF-β) in UUO mice (19, 20, 50). Likewise, FXR has been demonstrated to have anti-fibrotic actions in UUO mice via regulating TGFβ-Smad3 pathway (16, 34). FXR expression can be found on macrophages, tissue resident macrophages, and dendritic cells (18, 51). Future studies are necessary to evaluate the actions of interventional DM509 treatment on TGF-β and specific immune cells. Nevertheless, TGF-β is a critical regulator that is likely inhibited by DM509 to decrease myofibroblast activation, renal fibrosis, and tubular injury.

In the present study, we demonstrated tubular injury with elevated renal KIM-1 and NGAL expression. These findings are consistent with findings of a recent study with the UUO renal fibrosis model (26). The current study also demonstrated reduced expression of claudins in the UUO mice. Claudins exhibit a specific expression pattern in the kidney and are considered to be essential in tight junction formation to establish close connections between epithelial cells, thereby maintaining cell polarity and tubular integrity (52, 53). Loss of tubular integrity and polarity of the tubular epithelial cells are associated with tubular epithelial injury and renal fibrosis (53, 54). Interventional treatment with dual acting FXR agonist and sEH inhibitor, DM509 reduced renal tubular injury and restored tubular epithelial integrity in UUO mice. We suggest that this DM509 action is a consequence of both FXR agonism as well as sEH inhibition. Several studies have demonstrated that sEH inhibition can reduce renal tubular injury (14, 19, 20). A similar renal tubular protective effect of sEH inhibition has also been reported in ob/ob mice with diabetic nephropathy and renal fibrosis (55). These findings indicate that sEH inhibitory activity byDM509 protects renal tubules. FXR agonism has also demonstrated to be renal tubule protective in several studies (34, 56). Obeticholic acid, a FXR agonist provided renal protection with reduced renal inflammation, tubular injury, and fibrosis in a lipopolysaccharide-induced acute kidney injury model (56). The FXR agonist obeticholic acid demonstrated a similar renal tubular protective and anti-fibrotic effect in a rodent model of renal ischemia injury (34). The findings of the current study demonstrate that interventional treatment with the dual FXR agonists and sEH inhibitor, DM509 acts through combined reduction in inflammation, attenuation of EMT, and decreased tubulointerstitial fibrosis significantly decreased renal injury in UUO mice.

Similar to renal tubular injury, another important pathophysiological event in CKD progression is vascular injury followed by vascular loss (56, 57). Intact microvasculature is a prerequisite for normal tubular structure and functions and peritubular vascular injury is associated with CKD progression (56–58). Indeed, a decrease in CD34+ tubulointerstitial capillaries has been observed together with tubular interstitial fibrosis in the UUO model (59). In a recent study, we demonstrated marked renal vascular loss in the UUO kidney with a decrease in CD31+ renal vasculature (50). Consistent with these previous findings, the current study demonstrated marked renal vascular loss in UUO mice with reduced renal CD31+ vascular levels. UUO mice also demonstrated kidney vascular inflammation with elevated expression of adhesion molecules ICAM and VCAM and a leaky endothelial barrier with elevation in claudin-5 and VE-cadherin. Claudin-5 and VE-Cadherin are specifically expressed in endothelial cells to maintain vascular barrier functions and regulate endothelial permeability (60, 61). In the UUO model reduction of renal Claudin-5 and VE-Cadherin was demonstrated to be associated with renal fibrosis progression (62). Interestingly, in the present study the dual acting molecule DM509 reduced CD31+ renal vascular loss in UUO mice. DM509 interventional treatment also reduced renal vascular inflammation and endothelial permeability in the UUO kidney by reducing ICAM and VCAM expression and also by attenuating the loss of claudin-5 and VE-Cadherin.

It is important to note that in the UUO kidney, we demonstrated markedly higher expressions of mesenchymal markers α-SMA and FSP-1 and reduced expression of endothelial markers VE-cadherin and CD31. This difference in the expressions of endothelial and mesenchymal markers indicate a possible endothelial-to-mesenchymal transition (EndoMT) in the UUO kidney. The EndoMT is a complex process by which certain endothelial cell subsets lose endothelial characteristics and transform into mesenchymal or smooth muscle cells (63, 64). EndoMT is often linked to the development of fibrosis in animal models, including the UUO model (65). We suggest that the effects of DM509 on the renal vasculature in UUO mice is caused by both its FXR agonistic property and ability to inhibit sEH. FXR activation by obeticholic acid, a synthetic FXR agonist, attenuated vascular remodeling of pulmonary vasculature in a rat model of pulmonary hypertension (66). This study suggested that FXR activation mediated anti-inflammatory actions to reduce EndoMT (66). Likewise, sEH inhibition has been demonstrated to stabilize EET levels, inhibit vascular inflammation, and promote vascular repair through neovascularization (14, 67, 68). An increase in EET levels induced by treatment with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) promoted vascular repair in human coronary arterial endothelial cells (68). Pharmacological sEH inhibition also reduced coronary artery inflammation in a mouse model of heart disease associated with coronary artery aneurysms and myocardial infarction (68). Similar to AUDA, another sEH inhibitor t-AUCB dose dependently increased the expression of the angiogenic factor vascular endothelial growth factor (VEGF) (67, 69). Overall, we demonstrated that the novel dual acting molecule DM509 given as an interventional treatment reduced vascular loss during renal fibrosis by reducing vascular inflammation, restoring renal vasculature, and restoring vascular endothelial integrity. Our data also indicated a possible role of DM509 on EndoMT to reduce renal vascular injury in UUO mice.

We demonstrated renal anti-fibrotic actions of a novel first-in-class dual acting molecule DM509 that simultaneously act as a FXR agonist and sEH inhibitor. Interventional treatment with DM509 reduced renal fibrosis in UUO mice by acting on several critical inflammatory mediated pathophysiological events. Our data demonstrated that DM509 uniquely reduced extracellular matrix formation and renal tubular and vascular injury in UUO mice. Our data also suggest that DM509 anti-inflammatory and anti-fibrotic actions are associated with its ability to reduce renal EMT and EndoMT. Overall, these data reveal that DM509 given in a clinically relevant manner is a promising dual acting anti-fibrotic and anti-inflammatory therapeutic that attenuates renal epithelial and vascular pathological events associated with progression of CKD to ESRD.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Medical College of Wisconsin Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.

MK: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. BN: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. AS: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. DM: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Resources, Writing – review & editing. JI: Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing.