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Abstract
PURPOSE: To determine the involvement of rat kallikrein-binding protein (RKBP) in the development of diabetic retinopathy. METHODS: Diabetes was induced by streptozotocin (STZ) (55 mg/kg body weight in 0.05 M citrate buffer, pH 4.5) in male Sprague-Dawley rats (150 to 175 g, 6 weeks old) as confirmed by hyperglycemia and reduced body weight. Retinas were dissected from animals at 1, 2, and 4 months of diabetes. The functional activity of RKBP in retinal homogenates was determined by its complex formation with tissue kallikrein. Immunoreactive RKBP levels were measured by enzyme-linked immunosorbent assay. The RKBP messenger RNA (mRNA) levels in the retina were measured by Northern blot analysis using the RKBP complementary DNA probe. The activity of total Na+,K(+)-ATPase was determined by a radioassay. Total protein concentration was determined by a protein assay. RESULTS: The kallikrein-binding activity was reduced in the retinas of STZ-diabetic rats at 1 (59%), 2 (50%), and 4 (38%) months of diabetes compared to those of age-matched control subjects. Levels of immunoreactive RKBP were significantly lower in the diabetic animals at each time point examined compared to those of control subjects. At 1 and 2 months of diabetes, RKBP levels (nanogram/milligram protein) were decreased significantly to 6.9 +/- 0.7 (n = 8) and 10.6 +/- 1.0 (n = 8), respectively, compared to those of age-matched control subjects (14.1 +/- 0.7, n = 8, P < 0.001, and 14.1 +/- 1.2, n = 8, P < 0.01). At 4 months of diabetes, retinal RKBP levels were lower in both control and diabetic groups, but RKBP levels in diabetic groups were significantly lower (5.8 +/- 0.6, n = 8) than those of the age-matched control subjects (8.4 +/- 0.9, n = 8, P < 0.01). Similarly, Northern blot analysis showed that RKBP mRNA levels were reduced in the retina of each group of STZ-diabetic rats, suggesting that the decrease in RKBP occurred at the level of transcription. CONCLUSIONS: The results show that STZ-induced diabetic rats have decreased retinal RKBP; moreover, this suggests that RKBP may contribute to diabetic retinopathy.