ABSTRACT
Klotho is an anti-aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down-regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1-inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF-kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down-regulation of Klotho in TEC in vitro that was NF-kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre-implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down-regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down-regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti-senescence and anti-fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF-associated chronic allograft dysfunction.
Subject(s)
Complement C5a/pharmacology , Delayed Graft Function/etiology , Glucuronidase/metabolism , Graft Rejection/etiology , Kidney Transplantation/adverse effects , Postoperative Complications , Reperfusion Injury/etiology , Acute Kidney Injury/surgery , Animals , Blotting, Western , Cells, Cultured , Delayed Graft Function/metabolism , Delayed Graft Function/pathology , Glucuronidase/genetics , Graft Rejection/metabolism , Graft Rejection/pathology , Graft Survival , Humans , Immunoenzyme Techniques , Immunologic Factors/pharmacology , Klotho Proteins , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Reverse Transcriptase Polymerase Chain Reaction , Swine , Transplantation, HomologousABSTRACT
NADPH oxidase plays a central role in mediating oxidative stress during heart, liver, and lung ischemia/reperfusion injury, but limited information is available about NADPH oxidase in renal ischemia/reperfusion injury. Our aim was to investigate the activation of NADPH oxidase in a swine model of renal ischemia/reperfusion damage. We induced renal ischemia/reperfusion in 10 pigs, treating 5 of them with human recombinant C1 inhibitor, and we collected kidney biopsies before ischemia and 15, 30, and 60 min after reperfusion. Ischemia/reperfusion induced a significant increase in NADPH oxidase 4 (NOX-4) expression at the tubular level, an upregulation of NOX-2 expression in infiltrating monocytes and myeloid dendritic cells, and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis along with a marked upregulation of NADPH-dependent superoxide generation. This burden of oxidative stress was associated with an increase in tubular and interstitial expression of the myofibroblast marker α-smooth muscle actin (α-SMA). Interestingly, NOX-4 and NOX-2 expression and the overall NADPH oxidase activity as well as α-SMA expression and 8-oxo-7,8-dihydro-2'-deoxyguanosine synthesis were strongly reduced in C1-inhibitor-treated animals. In vitro, when we incubated tubular cells with the anaphylotoxin C3a, we observed an enhanced NADPH oxidase activity and α-SMA protein expression, which were both abolished by NOX-4 silencing. In conclusion, our findings suggest that NADPH oxidase is activated during ischemia/reperfusion in a complement-dependent manner and may play a potential role in the pathogenesis of progressive renal damage in this setting.
Subject(s)
Complement System Proteins/metabolism , Dendritic Cells/physiology , Kidney Tubules/blood supply , NADPH Oxidases/metabolism , Reperfusion Injury/enzymology , Actins/genetics , Actins/metabolism , Animals , Cells, Cultured , Complement C1 Inactivator Proteins/administration & dosage , Complement C1 Inhibitor Protein , Complement C3a/metabolism , Deoxyadenosines/biosynthesis , Deoxyadenosines/genetics , Enzyme Activation/drug effects , Enzyme Activation/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Kidney Tubules/metabolism , Kidney Tubules/pathology , Models, Animal , Oxidative Stress , RNA, Small Interfering/genetics , Reperfusion Injury/immunology , Sus scrofaABSTRACT
Acute rejection is still a common complication of kidney transplantation. IL-17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL-17 graft expression in renal transplant recipients with acute antibody-mediated rejection (ABMR), acute T-cell-mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin-inhibitor toxicity (CNI). In acute ABMR, tubular IL-17 protein expression was significantly increased compared to TCMR, where most of the IL-17⺠cells were CD4⺠graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL-17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL-17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL-17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a-induced IL-17 expression. The use of steroids and monoclonal antibodies reduced IL-17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL-17 in ABMR and this event might be mediated by the complement system activation featuring this condition.
