Kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions

Abstract

[한글]

[영문]Background: Recent studies have shown that podocyte injury plays an important role in the pathogenesis of various proteinuric glomerular diseases, including diabetic nephropathy. The number of podocytes is decreased in diabetic glomeruli and angiotensin II (AII)-mediated apoptosis is known to be involved in the process of podocyte loss under diabetic conditions. The kallikrein-kinin system (KKS) is known to closely interact with the renin-angiotensin system (RAS) and to serve as the physiologic counterbalance to the RAS. Since the RAS is activated in diabetic glomeruli and is considered to play an important role in glomerular injury, the KKS is supposed to have a protective effect on the pathogenesis of diabetic nephropathy. However, the results of recent studies, which investigated the role of the KKS on diabetic nephropathy, were absolutely contrary. Moreover, the presence of a local KKS in podocytes and the changes of its components under diabetic conditions have never yet been explored. In this study, I examined whether a local KKS existed in podocytes and whether the expression of the components of the KKS and bradykinin (BK) production were changed in diabetic glomeruli and in cultured podocytes exposed to high glucose medium. I also elucidated the functional role of BK in podocyte apoptosis, which is implicated as a potential mechanism of podocyte loss characterized in diabetic nephropathy.Methods: In vivo, 32 Sprague-Dawley rats were injected either with diluent (n=16, C) or with streptozotocin intraperitoneally (IP) (n=16, DM), and 8 rats from each group were treated with BK (0.5 μg/hour) via subcutaneously implanted osmotic minipumps for 6 weeks. In vitro, immortalized mouse podocytes were cultured in media containing normal glucose (5.6 mM, NG), NG+24.4 mM mannitol (NG+M), NG+10-7 M AII (NG+AII), high glucose (30 mM, HG) with or without 6-hour pretreatment of 10-8 M BK. BK levels in sieved glomeruli and cell lysates were measured by ELISA. Real-time PCR and Western blot for kallikrein, kininogen, BK B1-receptor (B1R), and B2-receptor (B2R) mRNA and protein expression, respectively, were performed with sieved glomeruli and cell lysates. For the assessment of apoptosis, Western blot for Bax, Bcl-2, and active fragments of caspase-3 were performed. TUNEL assay and Hoechst 33342 staining were also performed with renal tissue and cultured podocytes.Results: 24-hour urinary albumin excretion was significantly higher in DM compared to C rats, and this increment was ameliorated by BK treatment in DM rats. Not only kininogen, kallikrein, B1R, and B2R mRNA and protein expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG medium. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli and HG- and AII-stimulated podocytes were significantly abrogated by BK treatment. The antiapoptotic effect of BK in experimental diabetic glomeruli and in cultured podocytes under diabetic conditions seemed to be mediated through the proapoptotic p38 mitogen-activated protein kinase pathway.Conclusion: I demonstrate for the first time that the expression of all components of the KSS is decreased in diabetic glomeruli and in cultured podocytes exposed to high glucose, and this suppressed KKS is associated with podocyte apoptosis. In addition, BK treatment ameliorated podocyte apoptosis under diabetic conditions. These findings suggest that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.