Pancreatic kallikrein protects against diabetic retinopathy in KK Cg-Ay/J and high-fat diet/streptozotocin-induced mouse models of type 2 diabetes
Many studies have shown that tissue kallikrein has effects on diabetic vascular complications such as nephropathy, cardiomyopathy and neuropathy, but its effects on diabetic retinopathy are not fully understood. Here, we investigated the retinoprotective role of exogenous pancreatic kallikrein and studied potential mechanisms of action.
We used KK Cg-Ay/J (KKAy) mice (a mouse model of spontaneous type 2 diabetes) and mice with high-fat diet/streptozotocin (STZ)-induced type 2 diabetes as our models. After the onset of diabetes, both types of mice were injected intraperitoneally with either pancreatic kallikrein (KKAy + pancreatic kallikrein and STZ + pancreatic kallikrein groups) or saline (KKAy + saline and STZ + saline groups) for 12 weeks. C57BL/6J mice were used as non-diabetic controls for both models. We analysed pathological changes in the retina; evaluated the effects of pancreatic kallikrein on retinal oxidative stress, inflammation and apoptosis; and measured the levels of bradykinin and B1 and B2 receptors in both models.
In both models, pancreatic kallikrein improved pathological structural features of the retina, increasing the thickness of retinal layers, and attenuated retinal acellular capillary formation and vascular leakage (p < 0.05). Furthermore, pancreatic kallikrein ameliorated retinal oxidative stress, inflammation and apoptosis in both models (p < 0.05). We also found that the levels of bradykinin and B1 and B2 receptors were increased after pancreatic kallikrein in both models (p < 0.05).
Pancreatic kallikrein can protect against diabetic retinopathy by activating B1 and B2 receptors and inhibiting oxidative stress, inflammation and apoptosis. Thus, pancreatic kallikrein may represent a new therapeutic agent for diabetic retinopathy.
KeywordsApoptosis Diabetic retinopathy Inflammation Oxidative stress Pancreatic kallikrein
NADPH oxidase 2
Optical coherence tomography
Quantitative real-time PCR
Reactive oxygen species
Superoxide dismutase 2
Vascular endothelial growth factor
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, 6, 7, 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) . 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 . 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 , 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 .
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.
KK Cg-A y/J mice
Male KK Cg-Ay/J (KKAy) mice were originally developed by Japanese scholars, who transferred the Ay gene into KK mice . After they had acclimated for 2 weeks, the mice were randomly divided into two groups. One group underwent daily i.p. injections of saline (154 mmol/l NaCl; n = 16, 10 ml/kg, KKAy + saline group), while the other group underwent daily i.p. injections of pancreatic kallikrein (Qianhong Biochemical Pharmaceutical Company, Changzhou, China) (n = 16, 40 U/kg, KKAy + pancreatic kallikrein group) for 12 weeks. Both groups of mice were fed KK feed. In addition, age-matched C57BL/6J mice (n = 16, C57 group) were used as controls. See electronic supplementary materials (ESM) Methods for details.
HFD/STZ-induced type 2 diabetic mice
For the second mouse model of type 2 diabetes, male C57BL/6J mice were chosen and diabetes was induced as previously described . First, 16 mice were randomly chosen to receive a standard diet (normal control group), while the others were fed an HFD (n = 35) for 12 weeks. Subsequently, mice in the HFD group were injected with STZ (30 mg/kg; Sigma-Aldrich, St Louis, MO, USA) i.p. for 7 consecutive days, while the mice in the normal control group were injected i.p. with citrate–phosphate buffer. One week after the final injection, blood glucose levels were tested and mice with random blood glucose levels above 16.7 mmol/l were considered to be type 2 diabetic mice. The diabetic mice were then randomly separated into two groups, one of which was treated with pancreatic kallikrein (n = 16, STZ + pancreatic kallikrein group) and the other with saline (n = 16, STZ + saline group). The injection method, dose and time of pancreatic kallikrein administration were the same as for the KKAy mice. Three mice were excluded because their blood glucose did not reach the threshold. Body weight and blood glucose were measured weekly until the end of the study. After 12 weeks of pancreatic kallikrein treatment, all mice were killed. See ESM Methods for details.
All mice were housed two per cage in polycarbonate cages with corncob bedding and maintained on a 12 h light/12 h dark cycle and 10% humidity at 20 ± 4°C with ad libitum access to diet. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the Animal Welfare Act guidelines. The protocols were approved by the ethical committee of Tianjin Medical University.
In the HFD/STZ-induced diabetic mice, an IPGTT was performed after 12 weeks of an HFD to assess insulin resistance. See ESM Methods for details.
Optical coherence tomography
After 12 weeks of treatment, all mice were subjected to optical coherence tomography (OCT). Mice were weighed and anaesthetised using an i.p. injection of 1% (vol./vol.) sodium pentobarbital (30 mg/kg), and 0.5% tropicamide solution was used for eye dilation. The mice were placed on an adjustable box for spectral domain OCT (Heidelberg Engineering, Heidelberg, Germany). OCT scanning was initially directed from the posterior pole to the central portion of the mouse eye, which was facilitated by aiming the laser beam of the scanner. Retinal thickness was evaluated using Image-Pro Plus 6.0 analysis software (Media Cybernetics, Rockville, MD, USA).
Eyeballs were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 μm sections. As it is thought that retinal layer thickness varies throughout the retina, all sections were cut in the same orientation and always through the optic nerve head, and the regions were quantified at a constant distance from the optic nerve head. After deparaffinisation, H&E staining was performed and observed under a light microscope. Retinal thickness was evaluated using Image-Pro Plus 6.0 analysis software.
Paraffin-embedded retinas were cut into 4 μm sections for immunohistochemistry analysis (see ESM Methods for details). The stained sections were observed and imaged under a light microscope, and staining was quantified using Image-Pro Plus 6.0 analysis software.
Retinal trypsin digestion
Retinal trypsin digestion was performed as previously described . Acellular vessels and pericytes were quantified from six random fields per retina under ordinary light microscopy according to a documented protocol . See ESM Methods for details.
Retinal vascular immunofluorescence staining
Next, we performed retinal immunofluorescence staining and observed the results under a laser scanning confocal microscope (LSM 710, Carl Zeiss, Oberkochen, Germany). Retinal capillary density was analysed using Image J software (National Institutes of Health, Bethesda, MD, USA). See ESM Methods for details.
Reactive oxygen species (ROS) detection
Cryosections (4 μm) of eyecups were stained with dihydroethidium (Vigorous Biotechnology, Beijing, China) for 45 min at 37°C as previously described . Fluorescence images were observed under a fluorescence microscope and positive staining was quantified using Image-Pro Plus 6.0 analysis software.
Apoptosis detection was performed using a TUNEL apoptosis detection kit (biotin-labelled POD method; Universal, Nanjing, China) according to the manufacturer’s instructions. See ESM Methods for details.
Serum bradykinin levels were determined by using an ELISA kit (JL12234, Jianglaibio, Shanghai, China). All ELISA steps were conducted in accordance with the manufacturer’s protocols.
Western blot analysis
Western blotting was performed to determine the retinal expression of NADPH oxidase 2 (NOX2), superoxide dismutase 2 (SOD2), IL-1β, TNF-α, VEGF, cleaved caspase 3, BAX, Bcl-2, B1 receptor (B1R) and B2 receptor (B2R). Densitometry analyses of the bands were performed using Image J software. See ESM Methods for details.
Quantitative real-time PCR (qPCR)
qPCR was used to determine the relative expression levels of mRNA (see ESM Methods for details). Primer sequences for B1r (also known as Bdkrb1), B2r (also known as Bdkrb2) and Klk1 (which encodes kallikrein 1) are shown in ESM Table 1.
Statistical analysis was performed using Prism 5.0 software (GraphPad, La Jolla, CA, USA). Values are shown as the means ± SEM. The powers of each experiment were calculated using PASS software (PASS 11, Kaysville, UT, USA) and are shown in ESM Table 2. The significance of differences among different groups was evaluated using ANOVA; p < 0.05 was considered statistically significant.
Data on body weight and blood glucose, creatinine, alanine aminotransferase (ALT) and triacylglycerol levels after 12 weeks of pancreatic kallikrein treatment for the KKAy mice are summarised in ESM Table 3. Compared with those of the C57 group, the body weight and blood glucose, creatinine, ALT and triacylglycerol levels of the KKAy + saline group were significantly elevated (p < 0.05). However, these variables did not change significantly with pancreatic kallikrein treatment. The characteristics of the HFD/STZ-induced type 2 diabetic mice are presented in ESM Table 4. Unlike for the KKAy mice, the ALT level in the STZ + pancreatic kallikrein group was decreased compared with the STZ + saline group, although there was no statistical difference (p = 0.18); there were also no significant differences in creatinine levels among the STZ + pancreatic kallikrein, STZ + saline and normal control groups (p = 0.0839).
Pancreatic kallikrein protects against retinal structure changes
Pancreatic kallikrein attenuates vascular damage in the retina
A retinal trypsin digestion assay was performed to investigate acellular capillary formation and the number of pericytes. Compared with the C57 group, the KKAy + saline group had significant increases in retinal acellular capillaries and fewer pericytes. However, pancreatic kallikrein reduced these pathological changes (Fig. 1e). This finding was confirmed by semiquantitative analysis (Fig. 1f, g). Similarly, in the STZ + saline group, we observed acellular capillaries and a loss of pericytes, while pancreatic kallikrein reversed these pathological changes (Fig. 2d, f, g).
Pancreatic kallikrein ameliorates retinal oxidative stress
Pancreatic kallikrein suppresses retinal inflammation
Pancreatic kallikrein improves retinal apoptosis
Pancreatic kallikrein activates expression of the KKS
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 . Other studies have shown that tissue kallikrein improves diabetic retinopathy by inhibiting retinal vascular permeability and VEGF increases in diabetic rats . 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 . 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, 32, 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 . 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 . Liu et al demonstrated that kallikrein could inhibit nitrotyrosine and increase glomerular-stimulating hormone, inhibiting oxidative stress and thus improving diabetic nephropathy . 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 . Furthermore, hyperglycaemia-induced oxidative stress can also lead to the production of inflammatory factors such as TNF-α and IL-1β . 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 . Nakamura et al also found that tissue kallikrein inhibited retinal neovascularisation via the cleavage of VEGF164 in an oxygen-induced retinopathy model . 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 . Further, TNF-α also plays an important role in the loss of diabetic microvascular cells . 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 , vascular smooth muscle cells  and STZ-induced diabetic rats . 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, 46, 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 . 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 . 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.
We would like to thank C. Li (NHC Key Laboratory of Hormones and Development [Tianjin Medical University], Tianjin Key Laboratory of Metabolic Diseases, Tianjin Medical University Metabolic Diseases Hospital & Tianjin Institute of Endocrinology) for her helpful discussion.
YiC, XY, JZ, YuC, MX, XL, LC and BS contributed to the acquisition and analysis of the data. YiC, XY, YL, TL, ZM, LS, LC and BS designed experiments, analysed results and interpreted the data. YiC, JZ, LS, LC and BS drafted the manuscript. YuC, MX, XL, YL, TL and ZM revised the manuscript. All authors reviewed and approved the final version of the manuscript. LC and BS take full responsibility for the work and the conduct of the study, had access to the data and controlled the decision to publish.
This work was supported by the National Natural Science Foundation of China (no. 81470187 and 31800722), the Natural Science Foundation of Tianjin (no. 15ZXHLSY00460, 18JCYBJC26100 and 18JCZDJC35500), a China Postdoctoral Science Foundation Grant (2015M581309), and the Postgraduate Innovation Fund of ‘13th Five-Year Comprehensive Investment’, Tianjin Medical University (YJSCX201815).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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