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Supplemental Videos, Figures and Tables.
Supplemental videos.
Two-photon intravital imaging of rhodamine-labeled poly (I:C) in the kidney (videos 1 - 6).
(Videos 1 – 4) CX3CR1-EGFP mouse was injected with 0.25 mg/kg LPS 24 h before live imaging. Rhodamine-labeled poly (I:C) (red) was injected intravenously 20 min before imaging. The kidney was imaged every 9 seconds or 18 seconds for a total of 20 minutes to 105 minutes. Blue color is Hoechst. Green and brown colors in proximal tubules are autofluorescence.1 Poly (I:C) was localized to mobile macrophages expressing intermediate GFP. These cells are traced with red dots. Mobile macrophages expressing high GFP did not take up poly (I:C) and these cells are traced with green dots. Dendritic cells expressing high GFP are stationary and did not take up poly (I:C).
In video 1, computer-generated track marks of red and green mobile cells are shown. In video 2 (the same as video 1), original video without track marks is shown.
(Videos 3 and 4) Additional fields of the preconditioned kidneys are provided.
(Videos 5 and 6) Non-preconditioned CX3CR1-EGFP mouse was injected with rhodamine-labeled poly (I:C) (red) intravenously 20 min before imaging. Poly (I:C) positive cells were rarely observed.
Supplemental Figure 1. Imaging of freshly dissected kidneys after LPS and H2DCFDA injection.
Because glomeruli and S3 segments of mice are located at depths beyond the reach of 2-photon microscopy, kidney tissues were freshly dissected to image the deeper segments. Mice were injected with Alexa 568-labeled LPS (red) 5 mg/kg ip 4 h before imaging. H2DCFDA was injected intravenously 20 min before harvesting the kidneys. The kidneys were freshly dissected and immediately imaged without tissue fixation using 2-photon microscopy with the same settings applied for intravital imaging.
(A-C) Alexa 568-labeled LPS is internalized predominantly by S1 proximal tubules and tubular oxidative stress (green) is observed in downstream S2 and S3 segments, confirming our previous findings.1 Note that proximal tubules in the outer stripe are exclusively S3. S2 and S3 tubules exhibit only minimal fluid-phase LPS uptake comparable to TLR4-/- mice. G denotes glomerulus. (B) The rim of cortex. (C) A low power cross-sectional view composite of multiple separate fields. Nuclei are labeled blue with Hoechst.
(D - E) We have previously validated our measurements of reactive oxygen species with H2DCFDA by confirming probe loading into all subsegments of proximal tubules as well as duplication of the results with dihydroethidium.1 Here we show that oxidized
H2DCFDA in S1 can be induced by mercury lamp phototoxicity, which further confirms probe loading. The images were obtained live and red color is Alexa 568-labeled LPS.
H2DCFDA, carboxy-2’,7’-dichlorodihydrofluorescein diacetate; IM, inner medulla; IS, inner stripe; OS, outer stripe.
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Supplemental Figure 2. Kidney and serum two-dimensional difference gel electrophoresis (2DIGE).
(A - B) Kidney tissue 2D in-gel electrophoresis images for each condition (A) and two conditions combined (B; color coded as indicated) are shown.
(C - D) Serum 2DIGE images are shown.
(E) Regulation of free hemoglobin and free heme is shown.2, 3 As shown in the main Figure 9, serum haptoglobin (Hp), hemopexin (Hpx), and transferrin (Tf) had at least 2 fold increases in the preconditioned animals (LPS 0.25/5) as compared to non-
preconditioned animals (LPS 5).
Supplemental Figure 3. Hematopoietic TLR4 is essential for protective tubular preconditioning.
KIM-1 mRNA expression levels of kidney tissues were measured 4 hrs after 5 mg/kg LPS. KIM-1 expression levels were normalized to GAPDH. In the absence of LPS preconditioning, single-dose 5 mg/kg LPS caused significant tubular injury in TLR4KO/WT but not in WT/TLR4KO chimeras. After LPS preconditioning, TLR4KO/WT chimeric mice failed to mount tubular protection.
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Supplemental Figure 4. NGAL mRNA expression levels of kidney tissues. Wild-type animals were treated with liposomal
clodronate or vehicle control. Clodronate treatment abrogated tubular protection after preconditioning. *p<0.05 versus LPS 0.25/5 + clodronate.
Supplemental Figure 5. LPS preexposure exacerbates tubular oxidative stress in CD14-/- mice.
CD14-/- mice were injected with 5 mg/kg or 10 mg/kg LPS ip 4 h before live imaging with or without preconditioning.
(A and B) CD14-/- mice exhibited dose-dependent response to a single-dose LPS challenge; 5 mg/kg resulted only in minimal-to- mild oxidative stress whereas 10 mg/kg resulted in moderate oxidative stress. (C and D) LPS preconditioning exacerbated oxidative stress. *p<0.05 versus CD14-/- animals with LPS 5.
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Supplemental Figure 6. CD14-/- mice fail to mount LPS tubular protection in various preconditioning protocols.
Conflicting data exist as to the role of CD14 in response to smooth- versus rough-LPS challenges. In vitro studies have shown that rough LPS activates TLR4 signaling with a higher efficiency compared to smooth LPS, whereas in vivo studies have shown comparable responses.4-6 Here we examined various combinations of smooth and rough LPS using CD14-/- mice. E-coli 0111:B4 (smooth strain, denoted as S) and Salmonella Minnesota R595 (rough strain, denoted as R) were used and comparable results were obtained under all combinations of the LPS. n=3 per group. *p<0.05 versus WT 1.25/5.
Supplemental Figure 7. (A and B) TRIF-/- mice were injected with 5 mg/kg endotoxin with or without 0.25 mg/kg preexposure. The single-dose 5 mg/kg endotoxin resulted only in minimal-to-mild oxidative stress similar to 5 mg/kg into CD14-/- mice. Preconditioning failed to confer full protection in TRIF-/- mice. TRIF, TIR-domain-containing adapter-inducing interferion-β.
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Supplemental Figure 8. Gating strategy
Gating strategy used to analyze macrophage phenotype is shown. To ensure the threshold for CD206 positivity, cells were stained with all reagents except for CD206 antibody (Fluorescence-minus-one control).
Supplemental Figure 9. Effect of LPS on neutrophil infiltration and apoptosis.
(A – C) Representative Gr-1 staining (brown, arrowheads; a marker of neutrophils) 24 hrs after 5 mg/kg LPS.
(D) Quantitation of Gr-1 positive cells per field. No statistical difference was found between the preconditioned and non- preconditioned groups.
(E – G) Representative images of TUNEL staining (red, arrowheads). Magnified areas are shown in the insets of each panel.
(H) Quantitation of TUNEL positive cells per filed. No statistical difference was found between the preconditioned and non- preconditioned groups.
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Supplemental Figure 10. (A – D) Flowcytometric analysis of kidney leukocytes isolated under indicated conditions 4h after 5 mg/kg LPS. Representative dot plots of CD4+ T and CD8+ T cells, B cells (B220+), and NK cells (CD3-NK1.1+). n=3, *p<0.05 versus the other experimental group(s).
Supplemental Figure 11. Effect of LPS on the expression of HO1 and SIRT1 in TLR4KO/WT chimeric mice. (A - C) The live images of oxidative stress in TLR4KO/WT chimeric mice and its experimental strategy are shown again as a reference (Main Figure 5). (D) Fluorescence microscopy of fixed kidney sections from TLR4KO/WT mice and immunostained for HO1 (red). Green color is FITC-phalloidin. TLR4KO/WT mice with 1-day preconditioning (0.25/5) exhibited moderate upregulation of proximal tubular HO1 but lacked myeloid HO1 expression. (E) TLR4KO/WT chimeric mice with 3-day preconditioning (1.25/5) exhibited minimal upregulation of HO1 in both tubules and myeloid cells. (F) Immunostaining for SIRT1 (red). Green color is FITC-phalloidin. TLR4KO/WT mice with 1-day preconditioning (0.25/5) exhibited moderate upregulation of tubular SIRT1 but only weak upregulation in interstitial cells.
(G) TLR4KO/WT mice with 3-day preconditioning (1.25/5) exhibited minimal upregulation of SIRT1 in both tubules and interstitial cells.
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Supplemental Figure 12. Preconditioning results in macrophage clustering around the S1 proximal tubules.
(A - B) Preconditioned and non-preconditioned kidney tissues were harvested 72 hrs after LPS 5 mg/kg IP and stained for F4/80 (red). Green color is FITC-phalloidin. F4/80+ macrophages are clustered around the upstream S1 proximal tubules of preconditioned mice (A), but not non-preconditioned mice (B).
(C) To clarify S1 and S2 subsegments, preconditioned kidney tissues were co-stained for catalase (magenta; a marker of S2 and S3) and F4/80 (red).
Table 1.
Protein ID summary (kidney). Proteins with protein Score Confidence Interval % (C.I.%; calculated from MS data) or Total Ion C.I.%
(calculated from MS/MS data) greater than 95 are considered high confidence matches. The best match was selected based on C.I.% and isoelectric point/molecular weight location of the spot in the gel. F6 (MALDI well number) corresponds to spot number 49 on the figure 3G.
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Table 2.
Protein ID summary (serum). Proteins with protein Score C.I.% (calculated from MS data) or Total Ion C.I.% (calculated from MS/MS data) greater than 95 are considered high confidence matches. The best match was selected based on C.I.% and pI/Mw location of the spot in the gel. B8, B9, and B14 (MALDI well numbers) correspond to spot numbers 8, 10, and 48 respectively in Figure 9.
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Table 3. Cell Tracking Plugin ‘Trk_PP’ Source Text.
Source code is available as a Maven project at https://github.com/icbm-iupui/track-processing.git.
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Cell Tracking Analysis and Development of a Custom Plugin for ImageJ.
