Endotoxin Uptake by S1 Proximal Tubular Segment Causes Oxidative Stress in the Downstream S2 Segment

Abstract

Gram-negative sepsis carries high morbidity and mortality, especially when complicated by acute kidney injury (AKI). The mechanisms of AKI in sepsis remain poorly understood. Here we used intravital two-photon fluorescence microscopy to investigate the possibility of direct interactions between filtered endotoxin and tubular cells as a possible mechanism of AKI in sepsis. Using wild-type (WT), TLR4-knockout, and bone marrow chimeric mice, we found that endotoxin is readily filtered and internalized by S1 proximal tubules through local TLR4 receptors and through fluid-phase endocytosis. Only receptor-mediated interactions between endotoxin and S1 caused oxidative stress in neighboring S2 tubules. Despite significant endotoxin uptake, S1 segments showed no oxidative stress, possibly as a result of the upregulation of cytoprotective heme oxygenase-1 and sirtuin-1 (SIRT1). Conversely, S2 segments did not upregulate SIRT1 and exhibited severe structural and functional peroxisomal damage. Taken together, these data suggest that the S1 segment acts as a sensor of filtered endotoxin, which it takes up. Although this may limit the amount of endotoxin in the systemic circulation and the kidney, it results in severe secondary damage to the neighboring S2 segments.

Quantitative intravital microscopy of hepatic transport

“…Results presented extend previous studies of cultured cells, demonstrating that rifampin inhibits uptake of organic anions by hepatocytes in vivo and provide support for an iterative model of hepatic transport. Although used here to evaluate the effects of chronic rifampin treatment, the ability to analyze processes occurring in the timescale of seconds to minutes also provides intravital microscopy with the unique ability to detect acute, or transient drug effects which, when combined with additional readouts of injury, 43,60,[62][63][64][65][66] provides the potential to characterize the chronology of cholestatic liver injury. Since is both properly sedated and physiologically supported.…”

p53 Is Renoprotective after Ischemic Kidney Injury by Reducing Inflammation

“…Approximately 1 million bone marrow cells obtained from the long bones of donor mice were transplanted via the lateral tail vein as described previously. 31 Eight weeks after bone marrow transfer, the degree of chimerism was assessed by flow cytometry using CD45.1 (BoyJ) and CD45.2 (p53 2/2 ) antibodies (Becton Dickinson; Supplemental Figure 2). …”

New frontiers in intravital microscopy of the kidney

“…Several groups have recently utilized intravital microscopy to try to answer these questions. First, Dagher et al have shown that fluorescently labelled endotoxin [lipopolysaccharide (LPS)] is filtered by the kidney and taken up in early (S1) proximal tubule segments [37]. However, rather than directly causing damage in this segment, evidence of oxidative stress was surprisingly observed downstream in later S2 segments, perhaps due to some kind of crosstalk process, involving paracellular communication.…”