Aberrant activation of complement system in renal grafts is mediated by cold storage

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.

Progression of Interstitial Fibrosis and Tubular Atrophy in Low Immunological Risk Renal Transplants Monitored by Sequential Surveillance Biopsies: The Influence of TAC Exposure and Metabolism

The combination of tacrolimus (TAC) and mycophenolate is the most widely employed maintenance immunosuppression in renal transplants. Different surrogates of tacrolimus exposure or metabolism such as tacrolimus trough levels (TAC-C0), coefficient of variation of tacrolimus (CV-TAC-C0), time in therapeutic range (TTR), and tacrolimus concentration dose ratio (C/D) have been associated with graft outcomes. We explore in a cohort of low immunological risk renal transplants (n = 85) treated with TAC, mycophenolate mofetil (MMF), and steroids and then monitored by paired surveillance biopsies the association between histological lesions and TAC-C0 at the time of biopsy as well as CV-TAC-C0, TTR, and C/D during follow up. Interstitial inflammation (i-Banff score ≥ 1) in the first surveillance biopsy was associated with TAC-C0 (odds ratio (OR): 0.69, 95% confidence interval (CI): 0.50–0.96; p = 0.027). In the second surveillance biopsy, inflammation was associated with time below the therapeutic range (OR: 1.05 and 95% CI: 1.01–1.10; p = 0.023). Interstitial inflammation in scarred areas (i-IFTA score ≥ 1) was not associated with surrogates of TAC exposure/metabolism. Progression of interstitial fibrosis/tubular atrophy (IF/TA) was observed in 35 cases (41.2%). Multivariate regression logistic analysis showed that mean C/D (OR: 0.48; 95% CI: 0.25–0.92; p = 0.026) and IF/TA in the first biopsy (OR: 0.43, 95% CI: 0.24–0.77, p = 0.005) were associated with IF/TA progression between biopsies. A low C/D ratio is associated with IF/TA progression, suggesting that TAC nephrotoxicity may contribute to fibrosis progression in well immunosuppressed patients. Our data support that TAC exposure is associated with inflammation in healthy kidney areas but not in scarred tissue.