HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p.

Ischemia/reperfusion (I/R) leads to Acute Kidney Injury. HIF-1α is a key factor during organ response to I/R. We previously demonstrated that HIF-1α is induced during renal reperfusion, after ischemia. Here we investigate the role of HIF-1α and the HIF-1α dependent mechanisms in renal repair after ischemia. By interference of HIF-1α in a rat model of renal I/R, we observed loss of expression and mis-localization of e-cadherin and induction of α-SMA, MMP-13, TGFß, and collagen I. Moreover, we demonstrate that HIF-1α inhibition promotes renal cell infiltrates by inducing IL-1ß, TNF-α, MCP-1 and VCAM-1, through NFkB activity. In addition, HIF-1α inhibition induced proximal tubule cells proliferation but it did not induce compensatory apoptosis, both in vivo. In vitro, HIF-1α knockdown in HK2 cells subjected to hypoxia/reoxygenation (H/R) promote cell entry into S phase, correlating with in vivo data. HIF-1α interference leads to downregulation of miR-127-3p and induction of its target gene Bcl6 in vivo. Moreover, modulation of miR-127-3p in HK2 cells subjected to H/R results in EMT regulation: miR127-3p inhibition promote loss of e-cadherin and induction of α-SMA and collagen I. In conclusion, HIF-1α induction during reperfusion is a protector mechanism implicated in a normal renal tissue repair after I/R.

miR-127 protects proximal tubule cells against ischemia/reperfusion: identification of kinesin family member 3B as miR-127 target.

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.

Hypoxia inducible factor 1-alpha (HIF-1 alpha) is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

Differential resolution of inflammation and recovery after renal ischemia-reperfusion injury in Brown Norway compared with Sprague Dawley rats.

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury.

ERK1/2 mediates cytoskeleton and focal adhesion impairment in proximal epithelial cells after renal ischemia.

ERK1/2 has been reported to be activated in the postischemic kidney but its precise role in ischemia/reperfusion (I/R) injury remains unclear. Therefore, we have studied the expression of ERK1/2 and its contribution to cytoskeleton organization and cell adhesion structures in proximal tubular cells, all affected during I/R. We observe ERK1/2 activation at 24 hours of reperfusion in an in vivo model of I/R, when acute tubular necrosis (ATN) is most prominent. In addition, by means of an in vitro model of hypoxia/reoxygenation (H/R) in rat proximal NRK-52E cells we show that p-ERK1/2 is strongly induced early during reoxygenation. Moreover, we also demonstrate that ROS generation contributed to this induction. ERK1/2 activation is contemporary with cell-cell adhesion disruption during reoxygenation but the use of U0126 did not have effect on adherens junctions (AJ) and tight junctions (TJ) disassembly, neither on epithelial monolayer permeability. On the contrary, ERK1/2 affects cytoskeleton organization and focal complexes assembly during H/R, since U0126 improved actin and tubulin cytoskeleton structure, reduced cell contraction and prevented paxillin redistribution. In summary, ERK1/2 signalling plays an essential role in I/R induced injury, mediating proximal cell adhesive alterations which lead to tubular damage and ultimately might compromise renal function.