Zager RA, Johnson ACM, Lund S, Randolph-Habecker J. Toll-like receptor (TLR4) shedding and depletion: acute proximal tubular cell responses to hypoxic and toxic injury. Am J Physiol Renal Physiol 292: F304 -F312, 2007. First published August 1, 2006; doi:10.1152/ajprenal.00237.2006.-Acute renal failure (ARF) induces tubular hyperresponsiveness to TLR4 ligands, culminating in exaggerated renal cytokine/chemokine production. However, the fate of TLR4 protein during acute tubular injury remains unknown. The study sought new insights into this issue. Male CD-1 mice were subjected to 1) unilateral ischemia-reperfusion (I/R), 2) cisplatin (CP) nephrotoxicity, or 3) glycerol-induced myohemoglobinuric ARF. Renal cortical TLR4 protein (Western blotting, immunohistochemistry) and TLR4 mRNA levels (RT-PCR) were determined thereafter (90 min-4 days). Urinary TLR4 excretion post-I/R or CP injection was also assessed. To gain proximal tubule-specific results, TLR4 protein and mRNA were quantified in posthypoxic or oxidant (Fe)-challenged isolated mouse tubules. Finally, TLR4 mRNA was determined in antimycin A-injured cultured proximal tubular (HK-2) cells. Acute in vivo renal injury reduced proximal tubule TLR4 content. These changes corresponded with the appearance of TLR4 fragment(s) in urine and a persistent increase in renal cortical TLR4 mRNA. Isolated proximal tubules responded to injury with rapid TLR4 reductions, dramatic extracellular TLR4 release, and increases in TLR4 mRNA. Glycine blocked these processes, implying membrane pore formation was involved. HK-2 cell injury increased TLR4 mRNA, but not protein levels, suggesting intact transcriptional, but not translational, pathways. Diverse forms of acute tubular injury rapidly reduce proximal tubular TLR4 content. Plasma membrane TLR4 release through glycine-suppressible pores, possibly coupled with a translation block, appears to be involved. Rapid postinjury urinary TLR4 excretion suggests its potential utility as a "biomarker" of impending ARF. acute renal failure; iron; oxidant stress; cisplatin; biomarkers IT IS WELL ACCEPTED that renal inflammation significantly contributes to the pathogenesis of nephrotoxic and postischemic acute renal failure (ARF) (e.g., 3,7,12,14,15,22). The innate immune response/Toll-like receptor (TLR) pathway may be intimately involved in this process. TLRs are a family of ϳ11 plasma membrane glycoproteins that bind a variety of pathogen (e.g., endotoxins 3 TLR4; lipoteichoic acids 3 TLR2)-and nonpathogen (e.g., damaged RNA/DNA; heat shock proteins)-associated molecules (1,2,10,11,13,17,18,21). Once ligation occurs, downstream adaptor proteins (MyD88; Trif; Traf, NF-B) are recruited, culminating in increased cytokine and chemokine production (4,8,9). This process is hyperactive in the acutely damaged kidney (25-27). For example, when mice with diverse forms of ARF [ischemia-reperfusion (I/R), cisplatin (CP), myoglobinuria, urinary tract obstruction] are challenged with specific TLR ligands (endotoxin; lipoteichoic acid), greatly exagge...

Localization ability using phase-preserving amplification does not differ from localization ability using traditional non-phase-preserving amplification after just 3 weeks of use. Listeners quickly acclimated to altered spatial cues. Phase-preserving amplification provided a 2.3% advantage for speech intelligibility in noise after 16 weeks.

DNA templates for protein production remain an unexplored source of variability in the performance of cell-free expression (CFE) systems. To characterize this variability, we investigated the effects of two common DNA extraction methodologies, a postprocessing step, and manual versus automated preparation on protein production using CFE. We assess the concentration of the DNA template, the quality of the DNA template in terms of physical damage, and the quality of the DNA solution in terms of purity resulting from eight DNA preparation workflows. We measure the variance in protein titer and rate of protein production in CFE reactions associated with the biological replicate of the DNA template, the technical replicate DNA solution prepared with the same workflow, and the measurement replicate of nominally identical CFE reactions. We offer practical guidance for preparing and characterizing DNA templates to achieve acceptable variability in CFE performance.