The introduction of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus greatly reduced the rate of allograft rejection, although their chronic use is marred by a range of side effects, among them vascular toxicity. In transplant patients, it is proved that innate immunity promotes vascular injury triggered by ischemia-reperfusion damage, atherosclerosis and hypertension. We hypothesized that activation of the innate immunity and inflammation may contribute to CNI toxicity, therefore we investigated whether TLR4 mediates toxic responses of CNIs in the vasculature. Cyclosporine and tacrolimus increased the production of proinflammatory cytokines and endothelial activation markers in cultured murine endothelial and vascular smooth muscle cells as well as in ex vivo cultures of murine aortas. CNI-induced proinflammatory events were prevented by pharmacological inhibition of TLR4. Moreover, CNIs were unable to induce inflammation and endothelial activation in aortas from TLR4−/− mice. CNI-induced cytokine and adhesion molecules synthesis in endothelial cells occurred even in the absence of calcineurin, although its expression was required for maximal effect through upregulation of TLR4 signaling. CNI-induced TLR4 activity increased O2−/ROS production and NF-κB-regulated synthesis of proinflammatory factors in cultured as well as aortic endothelial and VSMCs. These data provide new insight into the mechanisms associated with CNI vascular inflammation.

Cyclosporine A (CsA) successfully prevents allograft rejection, but nephrotoxicity is still a dose-limiting adverse effect. TLR4 activation promotes kidney damage but whether this innate immunity receptor mediates CsA nephrotoxicity is unknown. The in vivo role of TLR4 during CsA nephrotoxicity was studied in mice co-treated with CsA and the TLR4 inhibitor TAK242 and also in TLR4 mice. CsA-induced renal TLR4 expression in wild-type mice. Pharmacological or genetic targeting of TLR4 reduced the activation of proinflammatory signaling, including JNK/c-jun, JAK2/STAT3, IRE1α and NF-κB and the expression of Fn14. Expression of proinflammatory factors and cytokines was also decreased, and kidney monocyte and lymphocyte influx was prevented. TLR4 inhibition also reduced tubular damage and drastically prevented the development of kidney fibrosis. In vivo and in vitro CsA promoted secretion of the TLR ligand HMGB1 by tubular cells upstream of TLR4 activation, and prevention of HMGB1 secretion significantly reduced CsA-induced synthesis of MCP-1, suggesting that HMGB1 may be one of the mediators of CsA-induced TLR4 activation. These results suggest that TLR4 is a potential pharmacological target in CsA nephrotoxicity.

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The tubular epithelium may be intrinsically involved in promoting kidney injury by junctional instability, epithelial-mesenchymal transition (EMT) and extracellular matrix remodelling. In this work, we investigated whether the pleiotropic and proinflammatory cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), could be able to disturb junctional protein expression and to induce EMT of tubular cells. In cultured murine proximal tubular cells TWEAK induced phenotypic changes that were accompanied by F-actin redistribution, loss of epithelial adherent (E-cadherin, Cadherin-16, β-catenin) and tight junction (ZO-1) proteins, and re-expression of the mesenchymal protein Vimentin. The transcriptional repressors Snail and HNF1β were also modulated by TWEAK. In a murine model of obstructive renal pathology, TWEAK expression correlated with the appearance of the mesenchymal marker αSMA in kidney tubular cells. Mechanistically, the epithelial changes induced by TWEAK, including loss of epithelial integrity and EMT, via Fn14 were TGF-β1 independent, but mediated by several intracellular signaling systems, including the canonical NF-κB, ERK activation and the vitamin D receptor modulation. These results highlight potential contributions of TWEAK-induced inflammatory mechanisms that could unveil new pathogenic effects of TWEAK starting tubulointerstitial damage and fibrosis.