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

High-fat diet and lack of eNOS synergistically increase TF in diabetic mice 

Many studies have demonstrated a connection between obesity and thrombosis (Samad and Ruf 2013). For example, upregulation of the TLR4-NF-kB pathway under metabolic disease conditions increases TF levels (Lv et al. 2009; Owens et al. 2012; Rogero and Calder 2018). Furthermore, saturated fatty acid palmitate induces histone H3 release via ROS and JNK pathway-dependent mechanisms and extracellular histones, which increase TF expression in monocytes (Shrestha et al. 2013). Focusing on the relationship between coagulation, eNOS, and dyslipidemia, our previous study has demonstrated the combined effects of eNOS deficiency and a high-fat diet on TF expression (Li et al. 2010). In the study, eNOS-null mice were treated with low-dose STZ to develop DM and were fed a high-fat diet. The lack of eNOS expression increased urinary albumin excretion and histological injuries, which were further exacerbated by a high-fat diet. The expression and activity of TF were increased in mouse kidneys because of a lack of eNOS expression and a high-fat diet in a synergistic manner. Interestingly, colocalization experiments with a glomerular monocyte/macrophage marker demonstrated an increase in the level of immunoreactive TF.

Neutralizing Antibodies against TF Ameliorate Inflammation in Diabetic Kidneys in eNOS-Knockout Mice

An increase in TF-dependent coagulation is associated with increased inflammation (Witkowski et al. 2016). Inhibition of the TF-dependent coagulation system ameliorated inflammatory diseases, such as LPS-induced sepsis (Pawlinski et al. 2010). To elucidate the causal link between inflammation and TF in DKD, we examined the short-term effect of neutralizing antibodies against TF on inflammation of diabetic kidneys in eNOS-deficient mice (Li et al. 2010). The results showed that administration of anti-TF antibodies markedly reduced the renal expression levels of inflammation- and fibrosis-related genes such as Tnfa, Ccl2, Tgfb, and Col4 mRNA in mouse kidneys four days after TF neutralization.

CLICK HERE TO GET NATURAL ORGANIC CISTANCHE EXTRACT WITH 25% ECHINACOSIDE AND 9% ACTEOSIDE FOR KIDNEY FUNCTION

Supportive Service Of Wecistanche-The largest cistanche exporter in the China:

Email:wallence.suen@wecistanche.com 

Whatsapp/Tel:+86 15292862950

Shop For More Specifications Details:

https://www.xjcistanche.com/cistanche-shop

Coagulation FXa Inhibitor Ameliorates Kidney Injury in Diabetic Mice Lacking eNOS 

Coagulation FXa, located downstream of TF/VIIa, also contributes to inflammation through the PAR-dependent pathway. Its role in DKD has been demonstrated in several animal models. Sumi et al. (2011) demonstrated that fondaparinux, an FXa inhibitor, reduced urinary protein levels, glomerular hypertrophy, and fibrin deposition in db/ db mice. The therapeutic effect of FXa inhibition in DKD mice lacking eNOS was demonstrated by our group (Oe et al. 2016). Edoxaban (50 mg/kg/day), an oral FXa inhibitor, was administered to eNOS-/-; Ins2Akita/+ mice for three months, and the results showed amelioration of histological injuries, such as mesangial matrix proliferation. The expression levels of inflammatory genes in the kidneys were reduced by edoxaban (Table 1). FXa activates both PAR1 and PAR2. To elucidate the mechanism of FXa-mediated kidney injury, we demonstrated that the anti-inflammatory effects of FXa inhibitors are similar to those found in PAR2-/- mice and PAR2-/- mice with FXa inhibitors. These findings suggested that FXa likely caused inflammation through a PAR2-dependent mechanism in DKD. In contrast, the redox ban did not improve glomerular injury in eNOS+/+; Ins2Akita/+ mice, suggesting that the therapeutic effect of FXa inhibition was associated with eNOS-dependent hypercoagulability in type I diabetic mice.

Other Coagulation Factors in DKD 

Because thrombin targets PAR1, which progresses vascular inflammation (Chen and Dorling 2009), thrombin likely exacerbates DKD. However, the protective or harmful roles of thrombin in DKD pathogenesis have been demonstrated; low-dose thrombin (50 pM) prevented, whereas high-dose thrombin (20 nM) aggravated, glucose-induced apoptosis in podocytes (Wang et al. 2011b). The effects of thrombin inhibitors such as dabigatran on diabetic mice lacking eNOS should be elucidated in the future.

Fibrinogen is involved in various inflammatory statuses. Interestingly, partial reduction or the absence of fibrinogen was beneficial to renal ischemia-reperfusion models or obstructive kidney fibrosis (Sörensen et al. 2011; Craciun et al. 2014). Because glomerular fibrin deposition is increased in diabetic mice lacking eNOS (Nakagawa et al. 2007; Li et al. 2010; Wang et al. 2011a), elucidating its role in DKD pathogenesis deserves further research.

Expression of PAR1 and PAR2 in Diabetic Mice Lacking eNOS 

Increased expression of PARs is associated with kidney injury. We and others have demonstrated that the expression levels of Par1 and/or Par2 mRNA were increased in animal models with diabetic nephropathy, adenine-induced kidney injury, obstructive renal fibrosis, and cisplatin-induced kidney injury (Chung et al. 2013; Hayashi et al. 2016; Oe et al. 2016; Watanabe et al. 2019). Furthermore, glomerular PAR2 protein levels were increased in db/db mice, a model of type II DM (Sumi et al. 2011). We demonstrated the association between PAR expression and eNOS deficiency in diabetic mice (Oe et al. 2016). The expression level of Par1 mRNA was significantly higher in eNOS−/−; Ins2Akita/+ mice than in eNOS−/−; Ins2Akita/+ and non-DM mice. Similarly, the expression of Par2 was significantly higher in eNOS−/−; Ins2Akita/+ mice than in non-DM mice. In contrast, Par4 mRNA levels did not differ between the genotypes. A lack of eNOS exacerbates inflammation in DKD (Wang et al. 2011a). Because pro-inflammatory cytokines reportedly increase PARs (Nystedt et al. 1996), elevated inflammation caused by a lack of eNOS likely induces renal Pars expression in DKD. Collectively, the results of our studies suggested that the expression levels of PAR1 and PAR2, similar to the case of TF, were increased in diabetic kidneys when eNOS was lacking (Fig. 2).

PAR2 Deletion Ameliorated Diabetic Kidney 

Injury in Mice with Reduced eNOS

We and others have addressed the role of PAR2 in DKD in previous studies. Par2 deletion did not affect glomerular injury in diabetic wild-type Akita mice (Ins2Akita/+) with eNOS (Oe et al. 2016). Furthermore, STZ-induced diabetic mice lacking PAR2 showed reduced albuminuria compared to diabetic wild-type mice but increased meangoal expansion (Waasdorp et al. 2017). Collectively, these results demonstrate that there are no or mild therapeutic effects of PAR2 inhibition on kidney injury in the early and mild models of DKD. In contrast, we have demonstrated the effects of PAR2 deficiency on DKD in diabetic Akita mice with reduced expression of eNOS (eNOS+/-; Ins2Akita/+) (Oe et al. 2016). The lack of PAR2 significantly reduced the levels of urinary albumin excretion, mesangial expansion, and thickness of the GBM. The expression levels of pro-inflammatory and fibrosis-related genes, including Tnfa, Tgfb, and Col4, were also reduced in mouse kidneys. These findings suggest that PAR2 is pathogenic not in early, but in the relatively advanced diabetic glomerular injury caused by eNOS deficiency (Table 1).

Dual Blockade of PAR1 and PAR2 in Diabetic Mice with Reduced eNOS

We have demonstrated that PAR1 and PAR2 cooperatively contribute to DKD pathogenesis (Mitsui et al. 2020). In this study, male type I diabetic Akita mice heterozygous for eNOS (Ins2Akita/+; eNOS+/-) were used as a model of DKD. These mice were treated with vehicle, PAR1 antagonist (E5555, 60 mg/kg/day), PAR2 antagonist (FSLLRY, 3 mg/kg/day), or E5555 + FSLLRY for 4 weeks. Administration of the PAR1 or PAR2 antagonist alone attenuated glomerular injury, such as mesangial expansion and collagen IV deposition, compared to administration of a vehicle. Synergistic therapeutic effects of both PAR1 and PAR2 inhibition were observed, and the urinary albumin-to-creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle administration. Furthermore, dual blockade of PAR1 and PAR2 by E5555 + FSLLRY synergisticcally ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and deposition of type IV collagen. The expression levels of inflammation- and fibrosis-related genes in the kidneys were also reduced (Table 1). 

We focused on the pro-inflammatory effects of PAR1 and PAR2 agonists on human endothelial cells (Mitsui et al. 2020). The results showed that stimulation with both PAR1 and PAR2 agonists synergistically increased the expression levels of MCP1 and PAI1 mRNA. The effect of the PAR1 agonist was blocked by an NF-κB inhibitor, whereas that of the PAR2 agonist was blocked by NF-kB and MAPK inhibitors. Collectively, PAR1 and PAR2 cooperatively contribute to vascular inflammation and DKD through different signaling pathways (Fig. 3).

Conclusion

In this review, we focused on the relationship between the coagulation protease-PAR pathway and eNOS deficiency in diabetic mice. Low production of eNOS is linked to hypercoagulability and increased PAR signaling, which are harmful to DKD. These findings may indicate their pathological roles in the advanced or later phase of DKD (e.g., with renal failure and/or massive proteinuria). Oral FXa inhibitors are widely used to prevent thrombosis (Patel et al. 2011; Robertson et al. 2015). Their use in the treatment of DKD is a promising option. Furthermore, some PAR1 antagonists, including atopaxar and vorapaxar, can be used in antiplatelet therapy to prevent acute coronary syndrome (Goto et al. 2010; Tricoci et al. 2012). In addition, there is remarkable progress in the development of PAR2 antagonists (Lim et al. 2013; Cheng et al. 2017; Jiang et al. 2018), and they can be novel therapeutic options to treat patients with DKD. 

Acknowledgments 

This study was supported by Gonryo Medical Foundation. We would like to thank Editage (https://www. editage.com) for English language editing.

Conflict of Interest The authors declare no conflict of interest. 

References 

Afkarian, M., Sachs, M.C., Kestenbaum, B., Hirsch, I.B., Tuttle, K.R., Himmelfarb, J. & de Boer, I.H. (2013) Kidney disease and increased mortality risk in type 2 diabetes. J. Am. Soc. Nephrol., 24, 302-308. 

Azushima, K., Gurley, S.B. & Coffman, T.M. (2018) Modelling diabetic nephropathy in mice. Nat. Rev. Nephrol., 14, 48-56. Brosius, F.C., Alpers, C.E., Bottinger, E.P., Breyer, M.D., Coffman, T.M., Gurley, S.B., Harris, R.C., Kakoki, 

M., Kretzler, M., Leiter, E.H., Levi, M., McIndoe, R.A., Sharma, K., Smithies, O., Susztak, K., et al. (2009) Mouse models of diabetic nephropathy. J. Am. Soc. Nephrol., 20, 2503-2512. Camerer, E., Huang, W. & Coughlin, S.R. (2000) Tissue factorand factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc. Natl. Acad. Sci. U. S. A., 97, 5255-5260. 

Chen, D. & Dorling, A. (2009) Critical roles for thrombin in acute and chronic inflammation. J. Thromb. Haemost., 7 Suppl 1, 122-126. Cheng, H., Wang, H., Fan, X., Paueksakon, P. & Harris, R.C. (2012) Improvement of endothelial nitric oxide synthase activity retards the progression of diabetic nephropathy in db/ db mice. Kidney Int., 82, 1176-1183. Cheng, R.K.Y., Fiez-Vandal, C., Schlenker, O., Edman, K., Aggeler, B., Brown, D.G., Brown, G.A., Cooke, R.M., Dumelin, C.E., Doré, A.S., Geschwindner, S., Grebner, C., Hermansson, N.O., Jazayeri, A., Johansson, P., et al. (2017) Structural insight into allosteric modulation of protease-activated receptor 2. 

Nature, 545, 112-115. Chung, H., Ramachandran, R., Hollenberg, M.D. & Muruve, D.A. (2013) Proteinase-activated receptor-2 transactivation of epidermal growth factor receptor and transforming growth factor-β receptor signaling pathways contributes to renal fibrosis. J. Biol. Chem., 288, 37319-37331. Coughlin, S.R. (2005) Protease-activated receptors in hemostasis, thrombosis and vascular biology. J. Thromb. Haemost., 3, 1800-1814. Craciun, F.L., Ajay, A.K., Hoffmann, D., Saikumar, J., Fabian, S.L., 

Bijol, V., Humphreys, B.D. & Vaidya, V.S. (2014) Pharmacological and genetic depletion of fibrinogen protects from kidney fibrosis. Am. J. Physiol. Renal Physiol., 307, 

F471- 484. Cunningham, M.A., Rondeau, E., Chen, X., Coughlin, S.R., Holdsworth, S.R. & Tipping, P.G. (2000) Protease-activated receptor 1 mediates thrombin-dependent, cell-mediated renal inflammation in crescentic glomerulonephritis. J. Exp. Med., 191, 455-462.