4 DISCUSSION
Diabetic nephropathy is a major cause of ESRD worldwide. According to the World Health Organization (WHO), 1 in every 11 adults worldwide are diabetic (Saran et al., 2020). This high incidence of diabetes is linked to the high incidence of diabetic nephropathy and consequent ESRD. Approximately 20-40% of all diabetic patients develop diabetic kidney disease in their lifetime, which often progresses to ESRD (Isomaa et al., 2001; Grundy, 2006). Unfortunately, despite the recent progress in our understanding, the complicated pathophysiology of diabetic nephropathy has limited our success to treat and manage diabetic nephropathy. Hence, there is growing interest in developing novel therapies that will target multiple pathophysiological factors of type 2 diabetes and its renal complications (Roche et al., 2015).
Over the years, several studies demonstrated critical etio-pathological roles of eicosanoids in the type 2 diabetes and associated kidney disease (Lorthioir et al. 2012; Molinar-Toribio et al., 2015). Indeed, a large number of studies demonstrated that eicosanoid metabolites are associated with type 2 diabetes, blood pressure, lipid levels, and insulin signaling (Imig, 2018; Bellucci et al., 2017; Nasrallah et al., 2016; Harris, 2008). In the present interventional study in overt type 2 diabetic obese ZSF1 rats, we investigated the effect of a novel molecule that concurrently acts on two pathways of arachidonic acid metabolism in type 2 diabetes. Our findings demonstrate that interventional treatment with the dual acting sEH/COX-2 inhibitor, PTUPB, reduces diabetic kidney injury in type 2 diabetic, hypertensive, and hyperlipidemic obese ZSF1 rats.
In type 2 diabetic obese ZSF1 rats, we compared the efficacy of interventional PTUPB treatment with an angiotensin converting enzyme (ACE) inhibitor, enalapril. It is important to note that ACE inhibitors are widely used for blood pressure control and are particularly beneficial in hypertensive type 2 diabetic subjects to treat diabetic nephropathy (Batlle et al., 2012). Interestingly, we found that PTUPB did not reduce hyperglycemia and hyperinsulinemia in obese ZSF1 diabetic rats. This is consistent with the findings of an earlier study where we reported that PTUPB prevents development of type 2 diabetes in Zucker diabetic fatty (ZDF) rats (Hye Khan et al., 2016). PTUPB treatment prevented development of insulin resistance and elevation of blood glucose in ZDF rats. PTUPB treatment was given prior to an elevation in blood glucose and insulin resistance to the ZDF rats. Indeed, the objective of that earlier study was to investigate if PTUPB could prevent development of type 2 diabetes. Findings of this previous study prompted us to carry out the present study to investigate the ability of PTUPB interventional treatment to reduce diabetes and its complication in a rat model of severe type 2 diabetes and diabetic nephropathy. Our findings demonstrate that interventional PTUPB treatment failed to reduce blood glucose or improve glucose homeostasis in obese ZSF1 rats. Similar to interventional PTUPB treatment, interventional ACE inhibitor treatment with enalapril did not affect hyperglycemia and insulin resistance in obese ZSF1 rats. These findings are in accord to our earlier findings on the effects of enalapril in obese ZSF1 diabetic rats (Hye Khan et al., 2018). In contrast to our findings, there are studies that demonstrate beneficial enalapril effects on blood glucose and insulin sensitivity. These studies found that enalapril improved insulin sensitivity in fructose-fed spontaneously hypertensive and Cohen Diabetic rats (Vuorinen-Markkola and Yki-Järvinen, 1995). Enalapril is also reported to improve glucose storage and insulin sensitivity in hypertensive type 1 diabetic patients (Rosenthal et al., 1995). The discrepancy between our findings in obese ZSF1 rats and these earlier studies could be due to the different interventional experimental design and the use of different diabetic rat models. Overall, we demonstrate that dual sEH/COX-2 inhibitor PTUPB or enalapril interventional treatment did not reduce type 2 diabetes in obese ZSF1 rats.
An important finding of the present study is the reduction in kidney injury by interventional PTUPB treatment in type 2 diabetic obese ZSF1 rats. The obese ZSF1 rats develop diabetic nephropathy with marked kidney functional and structural injuries (Hye Khan et al., 2018; Bilan et al., 2011; Su et al., 2018). We demonstrate potent renal actions of PTUPB in reducing renal functional and structural injuries in type 2 diabetic obese ZSF1 rats.
The pathophysiology of diabetic nephropathy is complex due to the presence of several comorbid conditions in type 2 diabetic patients. Most often these comorbid conditions are hypertension and hyperlipidemia (Ritz et al., 2001). In clinical studies, it is demonstrated that better blood pressure control in type 2 diabetes decreased the onset or degree of kidney injury and vascular complications (Xie et al., 2016). In the present study, interventional PTUPB treatment demonstrated marked anti-hypertensive actions in obese ZSF1 rats. A similar anti-hypertensive action of PTUPB has been reported in an earlier study (Hye Khan et al., 2016). This anti-hypertensive action of PTUPB could be related to the sEH inhibitor activity. The sEH inhibitors are widely reported to be anti-hypertensive, and this effect has been attributed to its ability to increase the ratio of epoxyeicosatrienoic acids (EETs) to their less biologically active diols (Imig et al., 2002; Neckář et al., 2012). Unlike sEH inhibition, COX-2 inhibition is not anti-hypertensive (Zhao et al., 2005; Cheng and Harris, 2004) and COX-2 inhibitors do not affect blood pressure in humans and animals (Bombardier et al., 2000). However, it reported that COX-2 inhibitor celecoxib increased systolic, diastolic and mean blood pressure in rat with normal blood pressure (Safaeian et al., 2018). Additionally, some clinical studies have shown that chronic COX-2 inhibition can induce hypertension in patients. It is reported that the COX-2 inhibitor Vioxx increase blood pressure in in human and it is considered as a significant adverse effect (Cho et al., 2003). Hence, it is likely that the anti-hypertensive effect of PTUPB in this study is due to sEH inhibitory activity. Moreover, it led us to suggest that use of PTUPB will be beneficial in patients who need COX-2 inhibition without developing COX-2 related adverse effect.
In chronic kidney disease, including diabetic nephropathy, disease progression is associated hyperlipidemia which is a common co-morbid condition of type 2 diabetes (Ferro et al., 2018). Important and beneficial renal outcomes of current lipid lowering therapies are known on complications in type 2 diabetes patients (Cases and Coll, 2005; Ferro et al., 2018). In the present study, interventional PTUPB treatment demonstrated an interesting lipid lowering action in type 2 diabetic obese ZSF1 rats. This lipid lowering action of PTUPB can be attributed to its sEH inhibitory activity as the lipid lowering effect of sEH inhibition has been reported in several studies (EnayetAllah et al., 2008). A polymorphism in the sEH gene (EPHX2 ) has been reported in humans with marked lipid abnormalities. It is reported that the R287Q variant of sEH is associated with elevated plasma cholesterol and triglycerides in familial hypercholesterolemia (EnayetAllah et al., 2008). Additionally, animal studies in sEH null (EPHX2 -/-) mice demonstrated lower plasma total cholesterol levels and lower HMG-CoA reductase activity (EnayetAllah et al., 2008). A similar lipid lowering action has been reported for COX-2 inhibition (Imig et al., 2005). These earlier findings are in accord with our current findings in obese ZSF1 rats and led us to suggest that the marked lipid lowering actions of PTUPB is caused by actions on COX-2 and sEH pathways.
Unlike PTUPB, interventional enalapril treatment did not cause any beneficial effect on lipid profile of obese ZSF1 rats. As reported in an earlier study, it is possible that 12-24 weeks long enalapril treatment could affect lipid profile in obese ZSF1 rats (Bilan et al., 2011). However, it should be noted that in this previous study enalapril treatment was given prior the development of hyperlipidemia. Moreover, it is not yet known if enalapril or any ACE inhibitor can alleviate established hyperlipidemia in a pre-clinical type 2 diabetic nephropathy model like the obese ZSF1 rat.
Elevated renal inflammation is an important pathophysiological factor of diabetic nephropathy. Indeed, chronic inflammation is a hallmark of metabolic dysfunctions including type 2 diabetes, hypertension, and hyperlipidemia. It is reported that the severity of renal inflammation and its renal consequence depends on the presence of different metabolic pathologies (Hotamisligil, 2006; Zhang and Lerman, 2016). During metabolic dysfunctions the normal physiological regulatory system is disrupted and initiates a cascade of deleterious inflammatory responses in multiple organs including the kidney (Hotamisligil, 2006; Furman et al., 2019). During metabolic diseases like type 2 diabetes, infiltration of immune cells and cytokine production occur in the abdominal and peri-renal fat and acts as vital source of inflammation in the kidney (Ma et al., 2006). In the present study, we demonstrate marked renal inflammation with renal macrophage infiltration and elevated chemokine production in obese ZSF1 rats. Similar to this finding, we earlier demonstrated renal injury associated with increased renal chemokine and elevated renal infiltration of immune cells in other metabolic disease models (Hye Khan et al., 2018; Imig et al., 2012). Interestingly, interventional PTUPB treatment reduced renal inflammation by reducing renal infiltration of immune cells and chemokine MCP-1 production in type 2 diabetic obese ZSF1 rats. We also demonstrate that renal inflammation in obese ZSF1 rats was ameliorated by enalapril and this finding is in agreement with the findings of a recent study in same rat model (Hye Khan et al., 2018).
Several recent studies demonstrated marked anti-inflammatory actions of PTUPB in multiple pathological conditions and in multiple organs including the kidney. In a mice sepsis model, PTUPB reduced systemic inflammation and reduced liver and kidney injury (Zhang et al., 2020). Certain chemotherapy drugs cause treatment limiting macrophage driven cytokine surge and PTUPB treatment prevented such cytokine surge during chemotherapy (Gartung et al., 2019). In an earlier study, we demonstrated that PTUPB prevented development of renal inflammation in ZDF rats by preventing macrophage infiltration in the kidney (Hye Khan et al., 2016). In these earlier studies we and others have provided evidence that PTUPB has anti-inflammatory activities and PTUPB multiple organ protective actions are associated with strong anti-inflammatory actions. However, it should be noted that in these earlier studies PTUPB treatment was used in preventive manner. In the present study PTUPB was used in interventional manner and it reduced renal inflammation in an event when disease was already well established and where inflammation is an important pathophysiological factor.
Anti-inflammatory actions of interventional PTUPB treatment are most likely caused by the inhibition of both COX-2 and sEH pathways, as inhibitors of each of these pathways have ability to reduce renal inflammation (Gassler et al., 2001; Bombardier et al., 2000). sEH inhibition demonstrated renal anti-inflammatory action in hypertension and diabetes animal models. Global sEH knockout (EPHX2 -/-) mice treated with deoxycorticosterone acetate in combination with high salt (DOCA-salt) had lower inflammatory gene expression and lesser degree of renal macrophage infiltration compared to wild type mice (Manhiani et al., 2009). Moreover, in a renal fibrosis model, sEH inhibition either by gene knockout or by pharmacological inhibition provided antifibrotic action in the kidney by reducing renal inflammation (Kim et al., 2015). Not only sEH inhibition but also COX-2 inhibition demonstrated anti-inflammatory action in the kidney. In ZDF rats, the diabetic kidney injury associated with elevated renal inflammation is reduced by COX-2 inhibition (Dey et al., 2004). Indeed, several studies demonstrated marked anti-inflammatory actions of COX 2 inhibition in multiple renal pathologies including type 2 diabetes (Honma et al., 2013; Fujihara et L., 2003). In an earlier study, we demonstrated that COX-2 inhibitor rofecoxib reduced renal tubular glomerular injury in type 2 diabetic obese ZDF rats, and the renal action of rofecoxib was associated with its anti-inflammatory effect (Dey et al., 2004). These earlier findings led us to suggest that the anti-inflammatory actions of COX-2 and sEH inhibition contributed to the marked renal anti-inflammatory actions of PTUPB. Overall, we demonstrate a unique biological action of the dual acting sEH/COX-2 inhibitor PTUPB in treating renal inflammation and injury in a rat model with established renal dysfunction.
In diabetic nephropathy, along with tubular injury, glomerular injury is a pathophysiological hallmark of the kidney injury and dysfunction. We demonstrated that the type 2 diabetic obese ZSF1 rats had marked glomerular injury and damage in the glomerular filtration barrier as assessed from reduced expression of slit diaphragm component nephrin. Interventional PTUPB treatment markedly reduced renal injury in type 2 diabetic obese ZSF1 rats. In the preceding sections we have discussed the diabetic kidney injury treating ability of PTUPB in terms of its beneficial actions on metabolic dysfunctions such as hypertension, hyperlipidemia, and also marked renal inflammation in obese ZSF1 rats. Apart from these approaches, we further investigated renal action of PTUPB in an in vitro study using isolated glomeruli. We determined the ability of PTUPB in maintaining glomerular permeability, an important functional feature of glomeruli for their efficient filtration capacity. We demonstrated that PTUPB directly maintains normal glomerular permeability. The findings of this in vitrostudy suggest that the renal injury treating ability of PTUPB in diabetic nephropathy is not only caused by its ability to reduce renal inflammation but also due to its direct effects on the glomerular filtration barrier. Indeed, an important role of endogenous CYP450 metabolites of arachidonic acid in maintaining the glomerular protein permeability barrier has been reported. It has been shown that EETs play an important role in maintaining normal glomerular permeability (Williams et al., 2007). Increased COX-2 expression in podocytes also leads to increased glomerular permeability (Cheng et al., 2007). Thus, PTUPB likely decreases glomerular barrier injury through inhibitory actions on both sEH and COX-2.
Apart of diabetic nephropathy, in type 2 diabetes co-morbid conditions like hyperlipidemia and obesity often contribute to multiple organ injury. Type 2 diabetic patients with co-morbid hyperlipidemia are at high risk to develop chronic liver disease, particularly non-alcoholic fatty liver disease (NAFLD) (Younossi et al., 2019). In the present study, as well as in an earlier study, we demonstrated that diabetic nephropathy is accompanied by liver dysfunction and steatosis in type 2 diabetic obese ZSF1 rats (Hye Khan et al., 2018). Interestingly, interventional PTUPB but not enalapril treatment markedly reduced liver injury and steatosis in obese ZSF1 rats. Our findings are in accord with an earlier study which reported that the ACE inhibitor enalapril did not treat liver dysfunction and steatosis in obese ZSF1 rats (Bilan et al., 2011).
In regard to the action of PTUPB, a recent study demonstrated liver protective effect of PTUPB. It is shown that PTUPB reduced liver weight, liver lipid content, steatosis, and the liver expression of lipolytic/lipogenic and lipid uptake related genes in a high fat diet induced NAFLD mouse model. PTUPB treatment also arrested liver fibrosis with a decreased collagen deposition and expression of several fibrotic markers including α-smooth muscle action (Sun et al., 2020). It is suggested that the effects of PTUPB on liver dysfunction particularly on hepatostetosis is associated with its lipid lowering and anti-inflammatory actions (Sun et al., 2020). The liver protective actions of PTUPB can also be related to the effect of sEH inhibition on the liver. sEH inhibitors reduced liver steatosis in a high-fat diet induced metabolic disease model (Liu et al., 2012). A strong anti-fibrotic action of sEH inhibitor is also reported in carbon tetrachloride induced cirrhotic hepatitis model (Harris et al., 2015). Activation of COX-2 is reported to be involve in liver injury, hence, COX-2 inhibitor will have a beneficial liver protective effect (Horrillo et al., 2007). The liver effects of PTUPB are also likely linked to the anti-inflammatory and lipid lowering actions. Overall, we demonstrated that the dual acting sEH/COX-2 inhibitor, PTUPB, has promising effects on liver dysfunction that is associated with type 2 diabetes and other similar metabolic disorders.
In summary, we tested a unique dual acting sEH/COX-2 inhibitor, PTUPB that given in an interventional manner is not anti-diabetic but can effectively treat diabetic renal injury and several co-morbid conditions in type 2 diabetes. Interventional PTUPB treatment has multiple actions including decreasing diabetic kidney injury, lowering blood pressure, and reducing hyperlipidemia. We further found that PTUPB has promising effect on type diabetes related liver disease. We believe the small molecule dual acting sEH/COX-2 inhibitor PTUPB has promising drug development potential for diabetic nephropathy and other diabetic complications.