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.