Pancreatic kallikrein protects against diabetic retinopathy in KK Cg-Ay/J and high-fat diet/streptozotocin-induced mouse models of type 2 diabetes

Aims/hypothesis 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. Methods 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. Results 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). Conclusions/interpretation 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. Electronic supplementary material The online version of this article (10.1007/s00125-019-4838-9) contains peer reviewed but unedited supplementary material, which is available to authorised users.


METHODS
KK Cg-A y /J mice Male KK Cg-A y /J (KKAy) mice (weight 25-29 g, 8 weeks old) were obtained from HFK Bio-Technology Co., Ltd. (Beijing, China). This strain was obtained by Japanese scholars who transferred the Ay gene into KK mice [15]. After they had acclimated for 2 weeks, the mice were randomly divided into two groups. One group underwent daily i.p.
HFD/STZ-induced type 2 diabetic mice For another type 2 diabetic mouse model, male C57BL/6J mice (weight 18-25 g, 8 weeks old) were obtained from HFK Bio-Technology Co., Ltd., and diabetes was induced as described previously [16]. First, sixteen mice were randomly chosen to receive a standard diet (n=16, NC group), while the others were fed a high-fat diet (n=35, HFD group) for 12 weeks. Standard diet and high-fat diet purchased from HFK Bio-Technology Co., Ltd. (Beijing, China). The HFD consisted of 78.7% standard diet, 10% glucose, 10% animal fat, 1% total cholesterol and 0.3% sodium cholate. Subsequently, mice in the HFD group were injected with STZ (30 mg/kg, Sigma-Aldrich, St.Louis, MO, USA) intraperitoneally for 7 consecutive days, while the mice in the NC group were injected intraperitoneally with citrate-phosphate buffer and served as the normal control group (n= 12, NC group). One week after injection, blood glucose levels were tested, and mice with random blood glucose levels above 16.7 mmol/l were considered type 2 diabetic mice. The diabetic mice were then randomly separated into two groups, a PK-treated group (n=16, STZ+PK group) and a diabetic group treated with saline (n=16, STZ+NS group). The injection method, dose and time of PK administration were the same as those for the KKAy mice. Three mice were excluded because their blood glucose did not reach the standard. After 12 weeks of PK treatment, all mice were sacrificed. Body weight and blood glucose were measured weekly until the end of this study.
The mice were housed two per cage in polycarbonate cages with corncob bedding at 20 ± 4°C with a 12 h light/12 h dark cycle and 10% humidity. All experiments in this study were randomized. Researchers and animal caretakers were blinded for each group. 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.

Intraperitoneal glucose tolerance test
In the HFD/STZ-induced diabetic model mice, an intraperitoneal glucose tolerance test (IPGTT) was performed after 12 weeks of a high-fat diet to assess insulin resistance. Mice in both groups were fasted for 12h and then injected intraperitoneally with 10% (wt/vol.) glucose solution at 2 g/kg body weight. Blood samples were taken from the tail and measured with a glucometer (Roche, Basel, Germany) at 0, 15, 30, 60, 90 and 120 min after glucose injection. Then, a curve was drawn, and the area under the curve (AUC) was calculated.

Immunohistochemistry
After deparaffinization, antigen retrieval was performed with heated Tris-EDTA, and endogenous peroxidase was blocked with 3% (vol./vol.) hydrogen peroxide. The sections were incubated with primary antibodies against cleaved caspase3 (1:200; no.A2156; Abclonal， MA, USA) at 4°C overnight. The sections were stained with a diaminobenzidine (DAB) kit after incubation with HRP-conjugated secondary antibody. The stained sections were observed and imaged under a light microscope, and the staining was quantified with Image-Pro Plus 6.0 analysis software.

Retinal trypsin digestion
Retinal trypsin digestion was performed as previously described [17]. Briefly, eyeballs were fixed in 4% paraformaldehyde for at least 48 h. The retinas were isolated and washed overnight with gentle shaking followed by 3% (wt/vol.) trypsin (no.0458; Amresco 1:250; PA, USA) digestion for 45 minutes at 37°C. Then, vasculature was gently separated and stained with periodic acid Schiff (PAS). Acellular vessels and pericytes were quantified from six random fields per retina under ordinary light microscopy according to a documented protocol [18].

Retinal vascular immunofluorescence staining
Next, we performed retinal immunofluorescence staining. Briefly, eyeballs were fixed in 4% paraformaldehyde for 40 min at room temperature. Then, the retinas were dissected under a dissection microscope and transferred to 4% paraformaldehyde for incubation at 4°C overnight. Next, the retinas were permeabilized with permeabilization buffer (PBS pH 6.8, 1% (wt/vol.) BSA, 0.5% (vol./vol.) Triton X-100) and stained with isolectin B4 (1:500, no.121413; Thermo Fisher scientific, MA, USA) at 4℃ overnight. Finally, the retinas were fixed and sealed with DAPI. The slides were observed under a laser scanning confocal microscope (LSM 710, Carl Zeiss AG, Oberkochen, Germany). In each group, eight fields were randomly selected and capillary density was analysed with Image J (NIH, MD, USA) software.

TUNEL Assay
Apoptosis detection was performed using a TUNEL apoptosis detection kit (Biotin-labelled POD method, universal, Nanjing, China) according to the manufacturer's instructions. Briefly, paraffin sections were deparaffinized, permeabilized with proteinase K, blocked with 3% (vol./vol.) hydrogen peroxide, linked with TDT enzyme, and labelled with streptavidin-HRP.
Then, the sections were stained with a DAB kit. Images were obtained under a light microscope, and staining was quantified with Image-Pro Plus 6.0 analysis software.

Western Blot Analysis
We used highly efficient RIPA lysis buffer plus PMSF to lyse retinal tissue. Proteins from the different experimental groups were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% (wt/vol.) non-fat milk, incubated with primary antibodies at 4°C overnight, and subsequently incubated with HRP-conjugated

Quantitative real-time PCR (qPCR)
Total RNA was extracted from retinal samples using TRIzol reagent (9108, TaKaRa, Biotech, Japan) according to the manufacturer's instructions. Next, RNA was converted to cDNA by synthesis (AT301-03, TransGen Biotech, Beijing, China), and quantitative PCR was conducted on a CFX96 Real-Time PCR System (Bio-Rad, USA) with aSYBR Green PCR Reagent kit (AQ131-04, TransGen Biotech, Beijing, China). GAPDH was used as a housekeeping gene. All primers are shown in ESM Table 1.