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

Proximal tubular uptake of low dose endotoxin in WT and TLR4 KO mice. Alexa 568-labeled endotoxin (red color) was injected systemically (1 mg/Kg) and its appearance in the kidney detected with live 2-photon microscopy. Green represents tubular autofluorescence. Nuclei are stained blue with Hoechst. Panel A shows 20x views of endotoxin uptake in WT and TLR4 KO mice over a period of 90 min. 60x views are shown in B at the 90-min time point for both mice strains and reveal that endotoxin uptake in WT mice is localized to S1 tubules. S2 tubules of WT and all tubules of KO showed minimal endotoxin uptake. Arrowhead points to concentrated endotoxin in a distal segment or collecting duct. Images are representative of n = 4 per group.

Preexposure to endotoxin enhances endotoxin uptake in S1 segments of WT mice. WT and TLR4 KO mice were pre exposed to vehicle or 0.25 mg/Kg unlabeled endotoxin 24 h before live 2-photon imaging. At the time of imaging, 1 mg/Kg Alexa 568-labeled endotoxin (red) was injected systemically. Preexposure to endotoxin caused enhanced uptake in S1 tubules of WT (B) but not KO mice (D), as early as 5 min after injection. Quantitation of endotoxin uptake in S1 tubules is shown at various time points for WT and KO mice, with and without preexposure to endotoxin (E, data are means ± SD and represent the average S1 tubular fluorescence per field. At least 20 fields were examined per mouse kidney, n = 3 mice per group. #P<0.01compared to WT and both KO groups, *P < 0.01compared to both KO groups).

Proximal tubular uptake of high-dose endotoxin in WT and TLR4 KO mice. Alexa 568-labeled endotoxin (red, 5 mg/Kg) was injected systemically 4 h before 2-photon live imaging of the kidney. 20x views of the WT kidney (A) reveals markedly heterogeneous endotoxin uptake and tubular collapse as opposed to KO mice which showed more homogeneous uptake and normal tubular morphology (C). 60x views localize coarse granular uptake of endotoxin to S1 tubules of WT mice (B). S2 tubules of WT mice (recognized by their high autofluorescence in the green channel) and all tubules of TLR4 KO mice (D) showed less-intense endotoxin uptake that had a fine granular appearance.

TLR4-mediated uptake and fluid-phase endocytosis result in differential intracellular sorting of internalized endotoxin. Cascade blue 4 KDa dextran, a marker of fluid-phase endocytosis, was injected systemically 16 h before imaging (A, E). Four hours before imaging, Alexa 568-labeled endotoxin 3 mg/Kg was injected systemically (B, F). Arrowhead in B points to red endotoxin that does not co localize with blue dextran in a S1 segment of WT mouse. S2 of WT and all tubules of TLR4 KO showed purple color indicating colocalization of red endotoxin with blue dextran. TLR4-mediated uptake of endotoxin in S1 of WT (but not KO) mice is even more evident when TLR4 receptors are upregulated with preexposure to 0.25 mg/Kg unlabeled endotoxin (C, G). Panels D and H show the distribution of red and blue fluorescence in each pixel from panels C and G, respectively. A two-compartment model is evident in WT but not TLR4 KO mice.

Endotoxin-induced oxidative stress measured with carboxy-DCFDA occurs in S2 segments of WT mice. Animals were injected systemically with Alexa 568-labeled endotoxin (red) 5 mg/Kg, 4 h before live imaging. Twenty min before imaging, carboxy-DCFDA (green) was injected systemically. 20x views reveal heterogeneous distribution of oxidative stress among tubules (A). 60x views localize oxidative stress predominantly to S2 tubules which exhibit minimal endotoxin uptake (B, arrow head points to an intermediate segment between S1 and S2). Twelve hours after endotoxin injection, carboxy-DCFDA fluorescence is fully cellular in S2 (C, arrows). TLR4 KO mice showed minimal oxidative stress at all time points (D, E, and F). Arrow in D points to concentrated carboxy-DCFDA in distal segment or collecting duct. Chimera mice generated through bone marrow transfer from TLR4 KO into WT recipient (KO/WT) exhibited oxidative stress similar to WT mice (G, inset is 60x view of one S2 tubule). The reverse chimera with bone marrow from WT into TLR4 KO mice (WT/KO) showed minimal oxidative stress (H). Panel I shows a scatterplot of carboxy-DCFDA fluorescence in S1 and S2 tubules of all groups (n = 4 per group, each point represents average fluorescence per tubule with bars indicating means. *P<0.01 compared with S1 same group, #P<0.01 compared with WT/KO and KO).

Endotoxin-induced oxidative stress measured with DHE occurs predominantly in S2 segments of WT mice. Animals were injected systemically with Alexa 568-labeled endotoxin (red, cytoplasmic) 5 mg/Kg 4 h before 2-photon live imaging. One hour before imaging, the oxidative stress probe DHE (nuclear red stain) was injected systemically. 20x views reveal oxidative stress (red orange nuclei) in some tubules of WT and KO/WT chimeras (A, C). KO mice and WT/KO chimeras showed no nuclear DHE fluorescence indicating lack of oxidative stress (B, D). Because imaging of nuclei done at the basal aspect of tubules does not allow full visualization of apical endotoxin, we took 60x planes at the basal (0 μm) and the apical (15 μm) aspects of tubules with and without nuclear DHE fluorescence (E). These show that tubules with strong nuclear DHE are indeed S2, with fine granular apical endotoxin uptake. S1 tubules with intense and coarse granular endotoxin uptake show no nuclear DHE staining (compare with Figure 4). Panel F shows a scatterplot of nuclear DHE fluorescence in all tubules of all groups (n = 4 animals per group. In each animal, at least 5 fields were examined. Each point represents total DHE fluorescence per nucleus with bars indicating means. *P < 0.01 compared with WT/KO and KO).

CD14 is involved in endotoxin uptake and is essential for the induction of oxidative signaling. In A, CD14 KO mice were injected with 1 mg/Kg Alexa 568-labeled endotoxin and imaged over 90 min. In the preexposure group, the animals were treated with 0.25 mg/Kg unlabeled endotoxin 16 h before imaging. Values represent means ± SD of LPS fluorescence intensity in S1 tubules. The graph of CD14 was superimposed on that of WT and TLR4 KO from Figure 3E. In B, endotoxin was co localized with cascade blue 3KDa dextran as described in Figure 5. In C, oxidative stress was measured with carboxy-DCFDA in CD14 KO, as described in Figure 6. Arrow in C points to concentrated carboxy-DCFDA in distal segment or collecting duct. (*P < 0.05 compared with CD14 KO).

Effect of endotoxin on the expression of HO-1, SIRT1, and peroxisomal markers. Fixed kidney sections from WT mice were exposed to 5 mg/Kg unlabeled LPS and harvested at 4 or 12 h. Sections were stained for HO-1, SIRT1, PMP70, or catalase. After treatment with fluorescence secondary antibodies, they were imaged with a confocal microscope and pseudocolored for clarity. Green color is FITC-phalloidin staining of the apical brush border of proximal tubules. G denotes Glomeruli and * denotes S2, recognized by their thin brush border compared with S1. Fields shown are representative of at least 10 fields per section, taken from n = 3 animals per group. Panel M shows quantitation of fluorescence of HO-1 and SIRT1 in S1 tubules. Only S1 tubules seen to emanate directly from Bowman space were used for quantitation. Panel N shows fluorescence quantitation of PMP70 and catalase in S2 tubules. Values are means ± SD from n = 3 animals per group (#, P < 0.01 when compared with control group).