In the aftermath of acute renal injury (AKI), proximal tubular cells manifest dramatic hyper-responsiveness to Toll receptor ligands, most notably, endotoxin. This exaggerates renal cytokine (e.g., TNF-1) / chemokine (e.g., MCP-1) production which can worsen the severity of acute renal failure (ARF). With renal venous cytokine efflux, extra-renal injury may also result. This LPS hyper-responsiveness is transcriptionally regulated, and mediated, in part, by gene activating chromatin events. The overall goal of this proposal is to: i) further define the pathways which induce the underlying chromatin changes;and ii) delineate how they mediate an exaggerated LPS responsive state. To focus the application, one ARF model (ischemia/reperfusion) 1 exposure to one Toll ligand (LPS) will be tested in three Specific Aims:
Aim#1 : Define the temporal sequence of chromatin changes along the TNF-1 gene in AKI. We will characterize chromatin changes, including CpG methylation/demethylation, histone acetylation, methylation, histone variants, and alterations in nucleosome positions. Both renal injury, and proximal tubule-specific injury (in cultured tubular HK-2 cells), will be studied following reversible ischemia + LPS. These in vivo and in vitro experiments will set the stage for mechanistic assessments (Aim 3).
Aim #2 : Ascertain which transcription and chromatin remodeling factors / enzymes are recruited to the TNF-1 gene in AKI. We have already shown that the chromatin remodeler BRG1 is recruited to the TNF-1 gene and is required for injury-induced TNF-1 transcription. We have also demonstrated that NF-:B and AP-1 transcription factors are recruited to the TNF-1 gene in AKI. Using Matrix ChIP, we will more fully define the spatiotemporal sequence of chromatin and transcription events at the TNF-1 gene in response to reversible ischemia + LPS. This will help to ascertain which factors are hyper-recruited to the TNF-1 gene and may thus drive the LPS hyper-responsive state.
Aim #3 Test the mechanistic relevance of the above defined changes in the induction of the LPS hyper-responsive state. The mechanistic relevance of the above defined changes in establishing LPS hyper-responsiveness will be tested both in vitro and in vivo using siRNA targeting (as we have already done to knock-down BRG1 in HK-2 cells). We will complement these in vivo and in vitro siRNA approaches with the use of pharmacologic reagents directed at the putative molecular mediators of the hyper-responsive chromatin state. The latter approach may lead to therapeutically relevant translational approaches for modulating experimental AKI and associated multiorgan failure. The proposed studies are highly novel in that, with the exception of data obtained by the PIs, virtually no information exists as to the nature, and consequences of, chromatin alterations in response to AKI. Hence, new perspectives on mechanisms of AKI and its downstream consequences should result.
Acute kidney injury (AKI) leads to renal production of inflammatory mediators and sensitizes/primes the kidney to bacterial toxins. These renal inflammatory mediators gain access to the systemic circulation and can induce extra-renal tissue damage, a common cause of mortality in patients with AKI, due to infections, trauma and other illness. We propose to define the chromatin/transcriptional mechanisms for these renal responses with the goal to develop preventive treatments in the future.
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