Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention (6, 19, 35, 64). However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of this study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS+Transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat (autotransplantation, ATx). Complement proteins (C3 and C5b-9) were evaluated with western blotting (reducing and non-reducing conditions) and immunostaining. Western blot of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS+Transplant compared to ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS+Transplant but was absent in Sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS+Transplant. Real-time PCR (SYBR Green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS activates the complement system in renal transplants.
Apoptotic gene expression in potentially fertile and subfertile men
