Coagulation, Protease-Activated Receptors, And Diabetic Kidney Disease: Lessons From ENOS-Deficient Mice

Endothelial nitric oxide synthase (eNOS) dysfunction is known to exacerbate the progression and prognosis of diabetic kidney disease (DKD). One of the mechanisms through which this is achieved is that low eNOS levels are associated with hypercoagulability, which promotes kidney injury. In the extrinsic coagulation cascade, the tissue factor (factor III) and downstream coagulation factors, such as active factor X (FXa), exacerbate inflammation through activation of the protease-activated receptors (PARs). Recently, it has been shown that the lack of or reduced eNOS expression in diabetic mice, as a model of advanced DKD, increases renal tissue factor levels and PAR1 and 2 expression in their kidneys. Furthermore, pharmaceutical inhibition or genetic deletion of coagulation factors or PARs ameliorated inflammation in DKD in mice lacking eNOS. In this review, we summarize the relationship between eNOS, coagulation, and PARs and propose a novel therapeutic option for the management of patients with DKD. 

Keywords: diabetic glomerulosclerosis; factor Xa; inflammation; tissue factor

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Introduction 

The number of patients with diabetic kidney disease (DKD) is increasing worldwide (de Boer et al. 2011; Kainz et al. 2015; Ogurtsova et al. 2017). DKD is one of the major causes of mortality in patients with diabetic complications (Afkarian et al. 2013). Furthermore, DKD is progressive and is the main cause of end-stage kidney disease requiring renal replacement therapy (Gregg et al. 2014; Liyanage et al. 2015). In Japan, diabetic nephropathy accounts for more than 39% of new dialysis patients (Nitta et al. 2020). Therefore, the development of novel therapeutic options is required to manage patients with DKD.

There is increasing interest in the role of the coagulation system in DKD pathogenesis. Hypercoagulability is associated with DKD (Goldberg 2009; Domingueti et al. 2016). An elevated fibrinogen level in blood was shown to be associated with low estimated glomerular filtration rate, high proteinuria, and severe histological injury, and it was a predictor of the progression to end-stage kidney disease (Dalla Vestra et al. 2005; Pan et al. 2018; Zhang et al. 2018). Some studies have shown a correlation between the level of D-dimer, a fibrin degradation product, and renal dysfunction in DKD (Domingueti et al. 2018; Pan et al. 2018). Hypercoagulability in DKD is likely determined by multiple factors. For example, the renin-angiotensin-aldosterone system activates tissue factor (TF, factor III) and increases thrombotic events (Dielis et al. 2005). Otherwise, hyperglycemia, dyslipidemia, inflammation, or endothelial dysfunction are involved in the pro-thrombotic state under DKD pathogenesis (Goldberg 2009). 

Endothelial dysfunction is present in DKD that progresses to microvascular complications (Goldberg 2009; Nakagawa et al. 2011). Impaired endothelial nitric oxide synthase (eNOS) production or reduced eNOS activity (e.g., impaired eNOS phosphorylation) is a hallmark of endothelial dysfunction in DKD (Nakagawa et al. 2011; Cheng et al. 2012). We have shown that a loss of eNOS expression was linked to the elevation in the TF level and the extrinsic coagulation system activity (Li et al. 2010; Wang et al. 2011a), which is closely associated with thrombotic events in patients with chronic kidney diseases (Kolachalama et al. 2018).

In addition to the elevated risk of thrombotic events, coagulation proteases, such as active factor FVII (FVIIa), active factor (FXa), and thrombin, which are present in the extrinsic coagulation cascade, mediate tissue injury through a protease-activated receptor (PAR)-dependent mechanism (Madhusudhan et al. 2016; Posma et al. 2019). In this review, we summarize the findings obtained from diabetic mice lacking eNOS and discuss the relationship between the coagulation-PAR pathway, eNOS levels, and DKD pathogenesis.

Tissue Factor/Protease-Activated Receptors Pathway

Tissue factor (TF), known as factor III, is a 47 kDa transmembrane protein that interacts with factor VII (FVII) (Grover and Mackman 2018). The TF/FVIIa complex is an activator of the extrinsic coagulation cascade and catalyzes the activation of FX and FIX. FXa and activated co-factor V (FVa) form a prothrombotic complex that generates thrombin. Finally, thrombin converts fibrinogen to fibrin, resulting in the formation of thrombi (Grover and Mackman 2018). TF is expressed in both vascular smooth muscle cells and adventitial fibroblasts. Under inflammatory conditions, its expression is induced in endothelial cells or circulating cells such as monocytes (Østerud and Bjørklid 2006).

Protease-activated receptors (PARs) are members of the G-protein-coupled receptor superfamily, comprising four PAR proteins (PAR1-4). PARs undergo cleavage by proteases at the N-terminal end and are activated upon binding to a new N-terminus containing a tethered ligand. The four members of PARs (PAR1-4) are activated by specific coagulation proteases: the TF and FVIIa complex activates PAR2, factor Xa activates both PAR1 and PAR2, and thrombin activates PAR1, PAR3, and PAR4 (Camerer et al. 2000; Coughlin 2005; Rothmeier and Ruf 2012; Zhao et al. 2014). The relationship between TF, coagulation proteases, and PARs is shown in Fig. 1.

PARs are known to be widely expressed in renal cells and are involved in the pathophysiology of kidney injury. Both PAR1 and PAR2 are expressed in glomerular endothelial cells, mesangial cells, and kidney tubular cells derived from human or mouse; PAR2, PAR3, and PAR4 are expressed in human podocytes; PAR1, PAR3, and PAR4 are expressed in murine podocytes (Tanaka et al. 2005; Vesey et al. 2005; Madhusudhan et al. 2012; Dong et al. 2015; Madhusudhan et al. 2016).

Although the harmful or protective effects of PARs in kidney injury have been demonstrated, accumulating data suggest that PARs exacerbate inflammation by stimulating the production of cytokines and chemokines (Rothmeier and Ruf 2012; Isermann 2017; Posma et al. 2019). Consistent with this finding, PAR1 and PAR2 agonists promote the expression of inflammatory mediators, such as monocyte chemotactic protein 1 (MCP1) and plasminogen activator inhibitor-1 (PAI-1), and pro-fibrotic molecules in endothelial, mesangial, and kidney tubular cells (Vesey et al. 2005; Vesey et al. 2013; Ellinghaus et al. 2016; Waasdorp et al. 2016; Oe et al. 2019). In in vivo studies, the lack of PAR1 or the presence of PAR1 inhibitors reduced inflammation in models of crescentic glomerulonephritis or obstructive kidney injury (Cunningham et al. 2000; Waasdorp et al. 2019; Lok et al. 2020). Similarly, the therapeutic effects of PAR2 inhibition in kidney injuries were accompanied by a reduction in inflammation in the kidneys (Hayashi et al. 2016; Du et al. 2017; Han et al. 2019; Watanabe et al. 2019).

Endothelial Nitric Oxide Synthase Polymorphisms in DKD

Endothelial nitric oxide synthase (eNOS) is one of the three NOS isoforms, and it contributes to the production of NO in the vascular endothelium (Walford and Loscalzo 2003). NO produced by eNOS in the vascular endothelium plays a crucial role in regulating vascular relaxation, anti-inflammation, and prevention of thrombus formation (Walford and Loscalzo 2003). Impaired eNOS expression is associated with the development of DKD. Recently conducted meta-analyses on humans have revealed that G894T (rs1799983), C-786T (rs2070744), and intron 4b/4a (rs869109213) in eNOS (NOS3) genes are close associated with the development of DKD (Dellamea et al. 2014; Zhang et al. 2015; Dong et al. 2018). Of these variants, the role of the G894T (Glu298Asp) polymorphism in eNOS function has been well characterized. The reduced production of NO or nitrite accumulation was indicated in transfected CHO cells with 298Asp compared to those with 298Glu (Noiri et al. 2002). Collectively, these results indicate that the reduced production of NO by eNOS dysfunction is important for the progression of DKD in humans.

Fig. 1. Relationship between coagulation factors and protease-activated receptors (PARs). In the extrinsic coagulation cascade, the tissue factor (TF) and active FVII (FVIIa) complex activates protease-activated receptor 2 (PAR2), active FX (FXa) activates both PAR1 and PAR2, and thrombin targets PAR1, PAR3, and PAR4. Cleavage of the N-terminal sequence of PARs by coagulation proteases reveals a new N-terminal sequence that acts as a tethered ligand and promotes inflammation.

Diabetic Mice Lacking eNOS as a Model of Human Diabetic Nephropathy 

The establishment of reliable preclinical models resembling human DKD is essential for the study of novel therapeutic options. Based on the evidence showing an association between eNOS polymorphism and DKD, several studies have demonstrated that eNOS knock-out models of type I and type II DM are some of the successful models mimicking human DKD (Brosius et al. 2009; Azushima et al. 2018). It was demonstrated that streptozotocin-induced diabetic mice lacking eNOS develop severe albuminuria, mesangial expansion, thickening of the glomerular basement membrane, and arteriolar hyalinosis, resembling human DKD (Nakagawa et al. 2007). Similarly, type II diabetic mice (db/db) lacking eNOS showed severe glomerulosclerosis and albuminuria (Zhao et al. 2006).

Elevated TF in Diabetic Mice Lacking eNOS

NO inhibits thrombus formation and platelet aggregation (Walford and Loscalzo 2003). Because glomerular thrombus formation was observed in diabetic mice lacking eNOS (Nakagawa et al. 2007), impaired eNOS expression is likely associated with the increase in TF-dependent coagulation. The association between eNOS and TF in DKD has been addressed in our previous reports (Li et al. 2010; Wang et al. 2011a; Oe et al. 2016) (Fig. 2).

Reduced or a lack of eNOS expression increases renal TF activity in diabetic Akita mice 

We characterized TF expression in diabetic Akita (Ins2Akita/+) mice, a model of type I DM, with various expression levels of eNOS (eNOS+/+, eNOS+/-, and eNOS-/-) (Wang et al. 2011a). We found that the severity of DKD was associated with reduced eNOS expression. Urinary albumin excretion, glomerulosclerosis, and reduction in glomerular filtration rate were exacerbated in the following order: eNOS+/+; Ins2Akita/+ < eNOS+/-; Ins2Akita/+ < eNOS-/-; Ins2Akita/+. Interestingly, kidney TF expression and activity were increased in eNOS+/-; Ins2Akita/+ and eNOS-/-; Ins2Akita/+ mice compared with those in eNOS+/+; Ins2Akita/+ mice. Glomerular fibrin deposition was also remarkable in eNOS+/-; Ins2 Akita/+ and eNOS-/-; Ins2 Akita/+ mice. Furthermore, renal Tf mRNA expression was correlated with disease severity, urinary albumin excretion, and renal inflammatory cytokine expression.