Introduction
Diabetic retinopathy is one of the most common microvascular complications of diabetes and an important cause of non-traumatic blindness in adults [
1,
2]. Diabetic retinopathy develops in two stages: non-proliferative and proliferative. The early stages mainly involve vascular cell loss, vascular leakage and the destruction of the blood–retinal barrier. As the disease progresses, neovascularisation gradually occurs and the proliferative diabetic retinopathy stage begins. Among the types of diabetic retinopathy, diabetic macular oedema is the most common and is the main cause of visual impairment and blindness in people with diabetes. The main pathophysiology involves increased vascular leakage and accumulation of fluid in the macula, leading to macular oedema and increased retinal thickness [
3,
4]. Many studies have shown that oxidative stress, inflammation and apoptosis play important roles in the early stages of diabetic retinopathy [
5‐
8]. Therefore, inhibiting oxidative stress, inflammation and apoptosis may prevent diabetic retinopathy.
Kallikrein is a serine protease that exists as serum kallikrein and tissue kallikrein, which are key enzymes of the kallikrein–kinin system (KKS) [
9]. Many studies have shown that the occurrence of diabetic microvascular complications is associated with the KKS, especially kallikrein. Montanari et al found that kallikrein promotes glucose utilisation and lipid metabolism and improves diabetic cardiomyopathy [
10]. Some studies have reported that exogenous kallikrein reduces proteinuria and improves the pathological structure of the kidney, fibrosis, inflammation and oxidative stress to protect against diabetic nephropathy [
11,
12]. However, the effects of plasma and tissue kallikrein on diabetic retinopathy are controversial. Clermont et al demonstrated that plasma kallikrein contributes to retinal vascular permeability [
13], while other studies have shown that tissue kallikrein improves diabetic retinopathy by inhibiting retinal vascular permeability and vascular endothelial growth factor (VEGF) increases in rat models of diabetes [
14].
In this study, we evaluated the retinal protective role of pancreatic kallikrein in KKAy mice (a mouse model of spontaneous type 2 diabetes) and a mouse model of high-fat diet (HFD)/streptozotocin (STZ)-induced type 2 diabetes. We investigated whether pancreatic kallikrein could alleviate diabetic retinopathy by changing the KKS and improving pathological structural features of the retina, and explored the potential mechanisms of action.
Discussion
The KKS is a complex and multifunctional endogenous peptidergic system consisting mainly of kininogen, serum or tissue kallikreins, bradykinin and kinin. Among these constituents, kallikrein is a key enzyme of the KKS; it was originally found in human urine and used as a vasodilatory substance in antihypertensive therapy [
23,
24]. There are two major classes of kallikrein, plasma kallikrein and tissue kallikrein, which differ greatly in their molecular mass, substrate, immunological properties, gene structure and type of kinins released. The effects of both classes on diabetic retinopathy are also very different. Some studies have suggested that plasma kallikrein contributes to retinal vascular dysfunctions in diabetic rats [
13,
25] and that its inhibitors can improve diabetic macular oedema [
26]. Other studies have shown that tissue kallikrein improves diabetic retinopathy by inhibiting retinal vascular permeability and VEGF increases in diabetic rats [
14]. Therefore, our study mainly explored whether pancreatic kallikrein could protect against retinopathy and the possible mechanisms by which this might occur.
In this study, we selected two mouse models of type 2 diabetes for further validation. One was the KKAy mouse model, which was developed by Japanese scholars who transferred the yellow obese
Ay gene into KK mice. These mice exhibit hyperglycaemia, severe obesity, hyperlipidaemia and insulin resistance, and are considered to be spontaneous type 2 diabetic mice [
15,
27]. Many studies have shown that KKAy mice develop proteinuria, mesangial matrix accumulation and glomerular basement membrane thickening, making them good models for studying diabetic nephropathy [
28,
29]. Because diabetic nephropathy and retinopathy involve microvascular lesions, the pathological changes are similar. Therefore, KKAy mice have also been used to study diabetic retinopathy [
30]. Our second model in this study used an HFD combined with STZ injection to induce type 2 diabetes. This model is the most widely accepted animal model for evaluating retinal complications in type 2 diabetes [
31‐
33]. Our results showed that KKAy mice developed hyperglycaemia (blood glucose >16.7 mmol/l) after 12 weeks, and most mice in the HFD group developed hyperglycaemia (blood glucose >16.7 mmol/l) after i.p. injection of STZ. Therefore, both models were successfully established and were in the early stages of diabetic retinopathy due to the short study duration.
Our results demonstrated that pancreatic kallikrein was unable to improve metabolic abnormalities such as body weight, blood glucose, liver function, renal function or lipids in either diabetic mouse model. However, pancreatic kallikrein significantly improved retinal pathological structural features, increasing retinal thickness and ameliorating retinal acellular vessel formation and pericyte loss in both models. The results from the two models were similar, but there were some differences such as in body weight and ALT, which may have resulted from differences in genetic background, individual variations among different mice, sensitivity to STZ or severity of hyperglycaemia. After confirming the retinal protective effect of pancreatic kallikrein, we explored the underlying mechanism. Hyperglycaemia leads to increased production of ROS in the body. The retina is particularly sensitive to oxidative stress because of its high polyunsaturated fatty acid content, high consumption of oxygen and glucose oxidation [
34]. Many studies have shown that oxidative stress is elevated in people with diabetic retinopathy and animal models of the disease, and that it plays a crucial role in the pathogenesis of diabetic retinopathy [
5]. Liu et al demonstrated that kallikrein could inhibit nitrotyrosine and increase glomerular-stimulating hormone, inhibiting oxidative stress and thus improving diabetic nephropathy [
11]. In agreement with the results of the above-mentioned studies, our results demonstrated that pancreatic kallikrein treatment could reduce the production of ROS, downregulating NOX2 and upregulating the antioxidant SOD2 in both type 2 diabetic mouse models.
In addition, many studies have shown that inflammation is a major pathogenic factor of diabetic retinopathy that can lead to retinal blood vessel loss and vascular leakage in the early stages of the disease [
6]. Furthermore, hyperglycaemia-induced oxidative stress can also lead to the production of inflammatory factors such as TNF-α and IL-1β [
35]. In this study, we found that vascular leakage occurred in both diabetic mouse models and that pancreatic kallikrein treatment attenuated the vascular permeability. Previous studies [
36,
37] have shown that VEGF is an important proinflammatory factor that plays an important role in causing vascular leakage. Kato et al found that kallidinogenase (kallikrein) could inhibit VEGF expression and improve retinal vascular permeability [
14]. Nakamura et al also found that tissue kallikrein inhibited retinal neovascularisation via the cleavage of VEGF
164 in an oxygen-induced retinopathy model [
38]. In agreement with the results of the above-mentioned studies, our results demonstrated that VEGF expression was elevated in the diabetic groups but reduced by pancreatic kallikrein. We also examined the expression of the inflammatory cytokines TNF-α and IL-1β, and found that pancreatic kallikrein decreased the expression of these cytokines in the retina under diabetic conditions.
In addition to directly leading to diabetic retinopathy, oxidative stress and inflammation can also induce apoptosis, which further aggravates diabetic retinopathy [
21]. Further, TNF-α also plays an important role in the loss of diabetic microvascular cells [
39]. Therefore, we next explored the effect of pancreatic kallikrein on retinal apoptosis. Many studies have shown that loss of pericytes is an early and important change in diabetic retinopathy [
40,
41]. In this study, we observed loss of pericytes and an increase in acellular capillaries in the two diabetic mouse models, and found that pancreatic kallikrein treatment improved these phenomena. In addition, analysis of TUNEL and apoptosis-related indicators (cleaved caspase 3 and BAX/Bcl-2 ratios) further confirmed that pancreatic kallikrein could improve apoptosis in diabetic retinopathy.
What effect does pancreatic kallikrein have on the KKS? Since pancreatic kallikrein acts primarily on kininogen to produce bradykinin, which acts on its receptors (B1R and B2R), we examined the expression of B1R and B2R. B1R is known to be minimally expressed under physiological conditions but strongly induced in pathological states; in contrast, B2R is constitutively expressed. Previous studies have shown that B1R and B2R expression is upregulated in high glucose-stimulated endothelial cells [
42], vascular smooth muscle cells [
43] and STZ-induced diabetic rats [
44]. However, in our two diabetic models, the protein levels of B1R and B2R were not significantly different in the diabetic groups compared with the normal groups. This discrepancy may be due to differences in the animal models, the severity of hyperglycaemia or the duration of diabetes. Moreover, this study showed that the expression of B1R and B2R was significantly increased after pancreatic kallikrein treatment, indicating that the upregulation might be mediated by ligand-induced effects. Interestingly, some studies have shown that B1R is involved in the inflammatory cascade and in vascular permeability in diabetic retinopathy, and that inhibiting B1R can improve diabetic retinopathy [
45‐
47]. The discrepancy between the results of those studies and ours may be due to differences in the animal models, genetic backgrounds or severity of hyperglycaemia. More importantly, the KKS and renin–angiotensin system are key proteolytic systems that control a wide spectrum of systemic and local physiological activities. Multiple interactions between these two systems indicate that they are co-dependent; changes in one system are unavoidable in the other. Kallikrein generates kinins and also converts prorenin to renin. Thus, it participates in both kinin-dependent and angiotensin-dependent pathways [
48]. In addition, after systemic pancreatic kallikrein treatment, pancreatic kallikrein may act on kininogen to produce bradykinin, which is not only related to B1R but also closely related to the action of B2R. Therefore, this cascade effect is far different from the effect of direct application of B1R inhibitors or agonists. Moreover, Kakoki et al demonstrated that a lack of both bradykinin B1R and B2R can enhance nephropathy, neuropathy and bone mineral loss in Akita diabetic mice [
49]. Further studies are warranted to examine the mechanism of the retinal protective role of pancreatic kallikrein in diabetic retinopathy.
In conclusion, our results demonstrate that pancreatic kallikrein has retinal protective effects in KKAy and HFD/STZ-induced type 2 diabetic mice. These effects include improving pathological structural changes in the retina, ameliorating diabetes-induced retinal oxidative stress and inflammation, and attenuating apoptosis. These effects may be attributed, at least in part, to upregulation of bradykinin receptors, but other mechanisms cannot be ruled out. Therefore, exogenous pancreatic kallikrein may represent a novel therapeutic agent for the early stages of diabetic retinopathy.
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