Toll-Like Receptor-4 Coordinates the Innate Immune Response of the Kidney to Renal Ischemia/Reperfusion Injury

Abstract

Toll-like receptors (TLRs) can detect endogenous danger molecules released upon tissue injury resulting in the induction of a proinflammatory response. One of the TLR family members, TLR4, is constitutively expressed at RNA level on renal epithelium and this expression is enhanced upon renal ischemia/reperfusion (I/R) injury. The functional relevance of this organ-specific upregulation remains however unknown. We therefore investigated the specific role of TLR4 and the relative contribution of its two downstream signaling cascades, the MyD88-dependent and TRIF-dependent cascades in renal damage by using TLR4−/−, MyD88−/− and TRIF-mutant mice that were subjected to renal ischemia/reperfusion injury. Our results show that TLR4 initiates an exaggerated proinflammatory response upon I/R injury, as reflected by lower levels of chemokines and infiltrating granulocytes, less renal damage and a more preserved renal function in TLR4−/− mice as compared to wild type mice. In vitro studies demonstrate that renal tubular epithelial cells can coordinate an immune response to ischemic injury in a TLR4-dependent manner. In vivo we found that epithelial- and leukocyte-associated functional TLR4 contribute in a similar proportion to renal dysfunction and injury as assessed by bone marrow chimeric mice. Surprisingly, no significant differences were found in renal function and inflammation in MyD88−/− and TRIF-mutant mice compared with their wild types, suggesting that selective targeting of TLR4 directly may be more effective for the development of therapeutic tools to prevent I/R injury than targeting the intracellular pathways used by TLR4. In conclusion, we identified TLR4 as a cellular sentinel for acute renal damage that subsequently controls the induction of an innate immune response.

A Tissue-Specific Role for Nlrp3 in Tubular Epithelial Repair after Renal Ischemia/Reperfusion

“…9 Briefly, both renal pedicles were clamped for 35 minutes using microaneurysm clamps through a midline abdominal incision under general anesthesia [1.25 mg/mL midazolam (Actavis, Dublin, Ireland), 0.08 mg/mL fentanyl citrate, and 2.5 mg/mL fluanisone (VetaPharma Limited, Leeds, UK)]. After clamp removal, kidneys were inspected for restoration of blood flow.…”

“…Proximal TECs were isolated from kidneys, as described previously, 9 and cultured using HK2 medium supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 100 IU/mL penicillin, and 100 mg/mL streptomycin (Invitrogen) until cells became 70% to 80% confluent. Hypoxia was induced after a protocol described previously.…”

RAGE Does Not Contribute to Renal Injury and Damage upon Ischemia/Reperfusion-Induced Injury

“…The degree of necrosis was scored by a pathologist in a blinded fashion on a 5-point scale: 0 = no damage, 1 = 10% necrosis of the corticomedullary junction, 2 = 10-25%, 3 = 25-50%, 4 = 50-75%, 5 = more than 75%. For immunostaining, 4-m tissue sections were incubated with specific antibodies for granulocytes (FITC-labeled anti-mouse Ly6G mAb; BD Biosciences-Pharmingen), apoptosis (rabbit anti-human active caspase-3; Cell Signaling Technology), GFP (rabbitanti GFP IgG; Molecular Probes), HMGB1 (rabbit-anti mouse HMGB1; Abcam), S100B (rabbit anti-S100B; Sigma-Aldrich) or CML ( N -epsilon carboxymethyl lysine) which has been identified as a major structure in advanced glycation end-products (AGE), mouse IgG, Biologo) followed by appropriate secondary antibodies as described before [15,16,[20][21][22][23]. To determine percentage of positive staining on kidney tissue slides, approximately 10 pictures of tissue section were taken under light microscopy (magnification !…”

L’ischémie reperfusion

“…Il a été montré que le prolongement de la durée d'isché-mie accroît l'immunogénicité du greffon [16,17]. En effet, les lésions d'IR, comme le relargage des DAMP et l'activation endothéliale, favorisent le recrutement et l'activation des monocytes et des cellules dendritiques, ainsi que l'expression des molécules de classe II du complexe majeur d'histocompatibilité (CMH) sur les cellules présentatrices d'antigènes (CPA) [18].…”

“…Il s'y associe un infiltrat inflammatoire du greffon composé aussi SYNTHÈSE REVUES transplant et les présentent aux lymphocytes T médiateurs d'une réac-tion immunitaire adaptative [1,18]. Ainsi, l'IR constitue un obstacle à l'immunotolérance du greffon via les ligands endogènes libérés par les tissus lésés qui vont activer les TLR (Toll-like receptor) et la réponse immunitaire innée [16]. Cette activation des voies des TLR inhiberait la fonction des cellules lymphocytaires régulatrices (Treg) [19].…”