Supplemental Table of Contents
Supplemental Table 1. Primers for RT-PCR experiments.
Supplemental Figure 1. Representative congestion scale for scoring.
Supplemental Figure 2. Effect of pretreatment with low dose LPS on systemic hemodynamics.
Supplemental Figure 3. Effect of low dose LPS on red blood cell aggregation.
Supplemental Figure 4. Markers of renal injury 24 hours following bilateral ischemia-reperfusion.
Supplemental Figure 5. Relative mRNA expression of iCAM and vCAM.
Supplemental Figure 6. Effect of pentoxifylline on vascular congestion.
Supplemental Figure 7. Renal hypo-perfusion on vascular congestion.
Supplemental Figure 8. Reperfusion blood flow and congestion in the outer medulla following bilateral and unilateral ischemia.
Supplemental Figure 9. Effect of LPS pretreatment on the return of blood flow in the cortex and outer medulla during 30 minutes of reperfusion.
Supplemental Figure 10. Outer medullary baseline blood flow between saline and LPS pretreated rats.
Supplemental Figure 11. Relative mRNA expression of vasoactive mechanisms for low dose LPS pretreatment.
Supplemental Figure 12. Outer medullary plexus congestion occurs within minutes of reperfusion.
Supplemental Figure 13. Image of ischemia-reperfusion kidney with regional congestion.
Supplemental Figure 1.
Supplemental Figure 3.
Supplemental Figure 5.
Supplemental Figure 7.
Supplemental Figure 9.
Supplemental Figure 11.
Supplemental Figure 13.
Representative congestion scale for scoring. Trichrome stained kidney sections are scored blinded on a scale of 0-5. A score of 0 indicated no vessels in the region are congested. A score of 5 indicates all vessels within the region appear to be congested.
Supplemental Figure 2.
Effect of pretreatment with low dose LPS on systemic hemodynamics. Mean arterial pressure (A, mmHg) and heart rate (B, beats per minute, bpm) were measured via radiotelemetry. Following 3 days of baseline measurements (-72 to -12 hours), rats were pretreated with either saline (control, n=4, open circles) and LPS (1 mg/kg, n=4, closed triangles) once daily for 3 days:
injection 1 at 0h, injection 2 at 24h, injection 3 at 48h (black arrows). Gray bars indicate rat active hours at night (6pm-6am). Values are expressed as mean ± SEM. Two-way repeated measures ANOVA, Sidak’s post hoc analysis saline to LPS. *p<0.05, #p<0.01. ntotal= 8 male WKY rats per group. Note: these data are the same as baseline (B1 to B3) and injection (-3 to -1) days separated from Figure 7 to highlight the injection period.
Supplemental Figure 3.
Effect of low dose LPS on red blood cell aggregation. Critical sheer stress (A, marker of red blood cell aggregation) was measured in blood samples following 3 days of pretreatment with saline
D-300. Representative blood smears from saline (B) and LPS (C, 1 mg/kg) pretreated rats. Black arrows indicate examples of rouleaux. Values are expressed as mean ± SEM. Unpaired t-test,
#p<0.01. ntotal= 12 male WKY rats.
Supplemental Figure 4.
Markers of renal injury 24 hours following bilateral ischemia-reperfusion. Renal injury following 45 minute bilateral ischemia and 24 hours of reperfusion in rats pretreated with saline (control, open circles) or low dose LPS (1 mg/kg, closed triangles) once daily for 1 (left, n=6/6)), 3 (center, n=7/9), or 7 (right, n=7/10) days. Tubular injury scores in the renal cortex as a percent of tubules with tubular necrosis, loss of brush border, tubular dilation and formation of protein casts (A), plasma creatine (B), blood urea nitrogen (BUN) (C), relative mRNA expression (normalized to saline) of neutrophil gelatinase-associated lipocalin (NGAL) (D) and Kidney injury marker-1 (KIM-1) (E) in the renal outer medulla (OM). Values are expressed as mean ± SEM. Unpaired t-test. ntotal= 45 male WKY rats from Figure 1.
Supplemental Figure 5.
Relative mRNA expression of iCAM and vCAM. RT-PCR for iCAM was measured in the renal cortex (A) and outer medulla (C). RT-PCR for vCAM was measured in the renal cortex (B) and outer medulla (D). mRNA was measured in rats following pretreatment for 3 days of saline (control, open circles) or low dose LPS (1 mg/kg, closed triangles) (Pre-IR). Also measured in pretreatment with 3 days of saline or low dose LPS with 45 minutes of bilateral ischemia with no reperfusion (0h IR). Sham rats with no injections or IR were used as a negative control (open squares). A single injection of high dose LPS (10 mg/kg) was used as a positive control (closed squares). High power imaging of congested capillaries in the outer medulla of the rat kidney following IR using transmission electron microscopy (E). Images indicated tightly packed red
VR) and plexus (OM plexus) 24 hours following a 45 minute bilateral ischemia in rats pretreated (i.p.) with or saline (open circles, IR) or pentoxifylline (100 mg/kg, closed circles, IR + Pento) daily for 3 days. Trichrome stained kidney sections are scored blinded on a scale of 0-5. A score of 0 indicated no vessels in the region are congested. A score of 5 indicates all vessels within the region appear to be congested. Values are mean ± SEM. Unpaired t-test. Male WKY rats (ntotal= 11, 5-6 per group).
Supplemental Figure 7.
Renal hypo-perfusion on vascular congestion. Congestion of the outer medullary vasa recta (OM VR) was scored following 1 hour of renal perfusion pressure of 60mmHg (n=6), 40mmHg (n=6), or sham control (n=6) (A). Trichrome stained kidney sections are scored blinded on a scale of 0- 5. A score of 0 indicated no vessels in the region are congested. A score of 5 indicates all vessels within the region appear to be congested. Representative images of the VR of the outer medulla at 40x (B) and 10x (C).Values are mean ± SEM. One-way ANOVA, #p<0.01. ntotal= 18 Sprague- Dawley rats (8-10 weeks old).
Supplemental Figure 8.
Reperfusion blood flow and congestion in the outer medulla following bilateral and unilateral ischemia. Laser Doppler flux (raw perfusion units, AU) of the renal cortex during the first 30 minutes of reperfusion following a 45 minute bilateral (open circles) or unilateral (closed circles) ischemia in control rats (A). Vascular congestion scores of the outer-medullary capillary plexus (OM plexus) following 45 minutes of bilateral or unilateral ischemia and 24 hours of reperfusion in rats pretreated with saline (control, open circles) or low dose LPS (1 mg/kg, closed triangles) once daily for 3 days (B). Trichrome stained kidney sections are scored blinded on a scale of 0- 5. A score of 0 indicated no vessels in the region are congested. A score of 5 indicates all vessels within the region appear to be congested. Values are mean ± SEM. Two-way ANOVA (R.M. for A only), *p<0.05, #p<0.01. ntotal= 22, 5-6 male WKY rats per group.
Supplemental Figure 9.
Effect of LPS pretreatment on the return of blood flow in the cortex and outer medulla during 30 minutes of reperfusion. Delta change in laser Doppler flux (Δ perfusion arbitrary units, AU) over 30 minutes of reperfusion following 45 minutes of left renal artery clamp in the cortex (A) and outer medulla (B) in saline (control, n=7) and LPS (1 mg/kg, n=6) pretreated once daily for 3 days.
Open circles represent saline controls and closed triangles represent LPS. Values are mean ± SEM. Two-way ANOVA repeated measures was utilized to compare treatment over time. Male WKY rats, ntotal=13.
Supplemental Figure 10.
Outer medullary baseline blood flow between saline and LPS pretreated rats. Laser doppler flux (raw perfusion units, AU) over 10 minutes of baseline perfusion following instrumentation, prior to
circles) or low dose LPS (1 mg/kg, closed triangles) (Pre-IR). Also measured in pretreatment with 3 days of saline (open circles) or low dose LPS (1 mg/kg, closed triangles) with 45 minutes of bilateral ischemia with no reperfusion (0h IR). Sham rats with no injections or IR were used as a negative control (open squares). A single injection of high dose LPS (10 mg/kg) was used as a positive control (closed squares). Values are mean ± SEM. One-way ANOVA, with Tukey’s multiple comparisons to compare each group. *p<0.05, #p<0.01. Male WKY rats (ntotal= 23, 3- 4/group).
Abbreviations: Real time polymerase chain reaction (RT-PCR), Inducible nitric oxide synthase (iNOS), Endothelial nitric oxide synthase (eNOS), Cyclooxygenase 1 (COX-1), Cyclooxygenase 2 (COX-2), Ischemia-reperfusion (IR).
Supplemental Figure 12.
Outer medullary plexus congestion occurs within minutes of reperfusion. Vascular congestion scores in the outer medulla (OM Plexus) following 45 minute bilateral ischemia and 3 minutes of reperfusion in saline and LPS pretreated rats (A). A score of 0 indicates all visible vessels are open and 5 indicates all visible vessels are congested. Open circles represent saline controls and closed triangles represent LPS pretreatment (1 mg/kg, 3 day). Representative images (10x and
(B). Values are mean ± SEM. Unpaired t-test, #p<0.01. n=10 saline, 9 LPS from ntotal=10 male WKY rats.
Supplemental Figure 13.
Image of ischemia-reperfusion kidney with regional congestion. The image depicts a rat kidney at 24 hours post-reperfusion following 45 minutes of warm ischemia. In this kidney, the right side has become congested, but the left side did not (center image, 5x). This split kidney phenotype is common in rats after ischemia-reperfusion (IR). Despite the entire kidney undergoing the same ischemic time (by renal artery clamp), only half the medulla was congested. This phenotype is most easily explained by a hemodynamic event effecting the large renal vessels rather than direct ischemic injury to the tubules. This is because the latter would be expected to be uniform throughout the medulla. This phenotype is also consistent with our hypothesis that the degree of RBCs in the renal medullary circulation during early reperfusion may act as a ‘switch’ by which relatively small differences in hemodynamics during reperfusion may result in failure of the medulla to congest in some animals while severe congestion occurs in others, or even between regions of the same kidney (as depicted here). While tubules on both sides of this kidney demonstrate signs of injury, tubular injury is more severe on the congested side (40x images).
Almost 100% of tubules on the congested (right) side demonstrate tubular cell detachment/sloughing. Conversely, swelling of tubular cells and tubular casts are observed on the uncongested side (left). These findings are consistent with vascular congestion being a major determinant of tubular injury following IR in rodents.
