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

Abstract

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-A^y/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.

Keywords: Apoptosis; Diabetic retinopathy; Inflammation; Oxidative stress; Pancreatic kallikrein.

Fig. 1

Pancreatic kallikrein protects the retinal structure in KKAy mice. (a) Representative photomicrographs from retinal OCT in C57 and KKAy mice. The green line indicates the position near the optic nerve head. Scale bar, 200 μm; n=6 experimental samples per group. (b) Quantification of the total retinal thickness in the circular area close to the optic nerve head. (c) Representative images of retinal H&E staining in C57 and KKAy mice and (d) quantification of the total retinal thickness in the circular area around the optic nerve head. Scale bar, 50 μm. (e) Representative images of retinal trypsin digestion assay in C57 and KKAy mice. White arrows indicate acellular vessels. Scale bar, 50 μm. (f, g) Acellular vessels and pericytes were quantified in flat-mounted retinas of C57 and KKAy mice. Data are expressed as means ± SEM; n=5 experimental samples per group. *p<0.05 vs the C57 group; ^‡p<0.05 vs the KKAy + saline (S) group. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PK, pancreatic kallikrein; S, saline

Fig. 2

Pancreatic kallikrein protects the retinal structure in HFD/STZ-induced type 2 diabetic mice. (a) Experimental procedure for HFD/STZ-induced type 2 diabetes. (b, c) Curve and AUC for the results of the IPGTT; n=6 experimental samples per group. (d) Representative images of retinal H&E staining and a trypsin digestion assay in HFD/STZ-induced type 2 diabetic mice. The white arrows indicate acellular vessels. Scale bars, 50 μm. (e) Quantification of the total retinal thickness in the circular area around the optic nerve head. (f, g) Acellular vessels and pericytes were quantified in flat-mounted retinas. Data are expressed as means ± SEM; n=5 experimental samples per group. ^§p<0.05 vs the normal control (NC) group; ^†p<0.05 vs the STZ + saline (S) group. GCL, ganglion cell layer; INL, inner nuclear layer; NC, normal control; ONL, outer nuclear layer; PK, pancreatic kallikrein; S, saline

Fig. 3

Effect of pancreatic kallikrein on retinal vessels in KKAy and HFD/STZ-induced type 2 diabetic mice. (a) Representative immunofluorescence images of peripheral area vessels of the retina and (b) quantification of peripheral area vessel densities; (c) representative immunofluorescence images of central area vessels of the retina and (d) quantification of central area vessel densities in the C57, KKAy + saline (S) and KKAy + pancreatic kallikrein (PK) groups. (e) Representative immunofluorescence images of peripheral area vessels of the retina and (f) quantification of peripheral area vessel densities; (g) representative immunofluorescence images of central area vessels of the retina and (h) quantification of central area vessel densities in the normal control, STZ + S and STZ + PK groups. White arrows indicate vascular leakage. Scale bars, 200 μm. Data are expressed as means ± SEM; n=5 experimental samples per group. *p<0.05 vs the C57 group; ^‡p<0.05 vs the KKAy + S group; NC, normal control; PK, pancreatic kallikrein; S, saline

Fig. 4

Pancreatic kallikrein suppresses retinal oxidative stress in KKAy and HFD/STZ-induced type 2 diabetic mice. (a, f) The production of ROS, measured by dihydroethidium staining and (b, g) quantification of the fluorescence intensity in each experimental group shown. Scale bars, 50 μm. n=5 experimental samples per group. (c, h) Western blot analysis of NOX2 and SOD2 protein and (d, e, i, j) densitometric analysis for band intensities normalised to GAPDH in each experimental group shown. Data are expressed as fold of the control for that experiment (C57 or normal control [NC]), or as a ratio, and as means ± SEM; n=6 experimental samples per group.*p<0.05 vs the C57 group; ^‡p<0.05 vs the KKAy + saline (S) group; ^§p<0.05 vs the NC group; ^†p<0.05 vs the STZ + S group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; INL, inner nuclear layer; NC, normal control; ONL, outer nuclear layer; PK, pancreatic kallikrein; S, saline

Fig. 5

Pancreatic kallikrein ameliorates retinal inflammation in KKAy and HFD/STZ-induced type 2 diabetic mice. (a, e) Western blot analysis of TNF-α, IL-1β and VEGF protein, with GAPDH run for each gel, and (b–d, f–h) densitometric analysis for band intensities normalised to GAPDH in each experimental group. Data are expressed as means ± SEM; n=6 experimental samples per group. *p<0.05 vs the C57 group; ^‡p<0.05 vs the KKAy + saline (S) group; ^§p<0.05 vs the normal control (NC) group; ^†p<0.05 vs the STZ + S group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, normal control; PK, pancreatic kallikrein; S, saline

Fig. 6

Pancreatic kallikrein improves retinal apoptosis in KKAy and HFD/STZ-induced type 2 diabetic mice. (a) Representative images of TUNEL staining in the C57, KKAy + saline (S) and KKAy + pancreatic kallikrein (PK) groups. (b) Quantification of the number of TUNEL-positive cells in six randomly selected fields. (c) Representative immunohistochemical micrographs of retinas stained for cleaved caspase 3 in the three groups and (d) relative expression of cleaved caspase 3. n=5 experimental samples per group. (e) Western blot analysis of cleaved caspase 3, BAX and Bcl-2 protein, with GAPDH run for each gel. (f, g) Densitometric analysis for band intensities normalised to GAPDH in each experimental group shown. n=6 experimental samples per group. (h, i) Representative photomicrographs of TUNEL staining and the percentage of TUNEL-positive cells in the normal control, STZ + S and STZ + PK groups. (j, k) Immunohistochemical micrographs of retinas stained for cleaved caspase 3 and the relative expression in the three groups. n=5 experimental samples per group. (l) Western blot analysis of cleaved caspase 3, BAX and Bcl-2, with GAPDH run for each gel, and (m, n) densitometric analysis for band intensities normalised to GAPDH in each experimental group shown. n=6 experimental samples per group. Scale bars, 50 μm in (a, c, h, j); inset boxes in (a, h) indicate enlarged images, scale bars, 20 μm. Data are expressed as means ± SEM. Fold of control data are calculated using the control for that experiment (C57 or NC). *p<0.05 vs the C57 group; ^‡p<0.05 vs the KKAy + S group; ^§p<0.05 vs the normal control (NC) group; ^†p<0.05 vs the STZ + S group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCL, ganglion cell layer; INL, inner nuclear layer; NC, normal control; ONL, outer nuclear layer; PK, pancreatic kallikrein; S, saline

Fig. 7

Pancreatic kallikrein increases the expression of the KKS. (a, e) Retinal bradykinin levels were measured by ELISA in each experimental group. qPCR analysis for the expression of B1r, B2r and Klk1 in (b–d) KKAy and (f–h) HFD/STZ-induced type 2 diabetic mice, and their controls. (i, l) Western blot analysis of B1R and B2R and (j, k, m, n) densitometric analysis of band intensities normalised to GAPDH in each experimental group. Fold of control data are calculated using the control for that experiment (C57 or normal control [NC]). Data are expressed as means ± SEM; n=5−6 experimental samples per group. *p<0.05 vs the C57 group; ^‡p<0.05 vs the KKAy + saline (S) group; ^§p<0.05 vs the NC group; ^†p<0.05 vs the STZ + S group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, normal control; PK, pancreatic kallikrein; S, saline