Extravasation of Blood and Blood Toxicity Drives Tubular Injury from RBC Trapping in Ischemic AKI

Abstract Red blood cell (RBC) trapping is common in ischemic acute kidney injury (AKI) and presents as densely packed RBCs that accumulate within and engorge the kidney medullary circulation. In this study, we tested the hypothesis that “RBC trapping directly promotes tubular injury independent of extending ischemia time.” Studies were performed on rats. Red blood cell congestion and tubular injury were compared between renal arterial clamping, venous clamping, and venous clamping of blood-free kidneys. Vessels were occluded for either 15 or 45 min with and without reperfusion. We found that RBC trapping in the medullary capillaries occurred rapidly following reperfusion from renal arterial clamping and that this was associated with extravasation of blood from congested vessels, uptake of blood proteins by the tubules, and marked tubular injury. To determine if this injury was due to blood toxicity or an extension of ischemia time, we compared renal venous and arterial clamping without reperfusion. Venous clamping resulted in RBC trapping and marked tubular injury within 45 min of ischemia. Conversely, despite the same ischemia time, RBC trapping and tubular injury were minimal following arterial clamping without reperfusion. Confirming the role of blood toward tubular injury, injury was markedly reduced in blood-free kidneys with venous clamping. Our data demonstrate that RBC trapping results in the rapid extravasation and uptake of blood components by tubular cells, causing toxic tubular injury. Tubular toxicity from extravasation of blood following RBC trapping appears to be a major component of tubular injury in ischemic AKI, which has not previously been recognized.


Introduction
3][4][5][6][7][8] Red blood cell trapping is also present in animal models following ischemia r e perfusion injur y (IRI) fr om arterial clamping, including in pigs, dogs, rats, and mice, [9][10][11][12] where it significantly contributes to the decline in kidney function. 1 , 13ur la borator y has r ecentl y r e ported that RBC trapping is str ongl y associated with sev er e tubular injury in the renal OM region and that this injury is evident in as little as 1 h following r e perfusion following arterial clamping. 14It has been speculated that RBC trapping promotes kidney injury by delaying r e perfusion and extending ischemic time to the RBC-congested areas of the kidney, thereby prolonging the period of tissue hypoxia.This period is commonly referred to as the extension phase of renal ischemia. 15 , 16Opposing this concept, however, blood flow is rapidly restored to the OM following reperfusion of the kidney, 14 and there is little evidence of hypoxia in the r enal OM r egion in the initial hours after experimental IR. 17 , 18 Further, early tubular injury in congested areas of the kidney does not r esemb le typical ischemic/hypoxic injury, which is normally not evident in histological sections until 10-12 h post-ischemia. 19 , 20e and others have found that RBC trapping occurs following arterial IRI due to obstruction of the venous vessels that drain the renal medulla. 14 , 21-23The continued influx of blood into the medullary microcirculation while the venous vessels remain occluded, results in increased blood volume and v ascular pr essur es within the medullar y micr ocirculation.The incr easing intrav ascular pr essur e then r esults in the extrav asation of the plasma. 14 , 21 , 22 , 24Together, this explains the accumulation of ∼10 fold more RBCs in the OM microcirculation than ar e normall y pr esent, which expand the OM capillaries and form tightly packed aggregates with little or no plasma separating them. 2 , 14 , 21 , 23 , 25 The goal of the current study was to determine whether RBC trapping dir ectl y causes toxic tubular injur y inde pendent of extending warm isc hemia time .To do this, we compared kidney injury following a period of warm ischemia time without RBC trapping to kidney injury following the same warm ischemia time with RBC trapping.To promote the RBC trapping that normally occurs in the OM during the reperfusion phase during the earlier clamp phase, we utilized r enal v enous clamping.During r enal v enous clamping, the arterial vessels of the kidney remain unobstructed.As such, blood can enter the kidney, but it is una b le to exit due to the venous clamp.This mimics what happens with the collapse of the venous vessels that drain the OM during r e perfusion fr om arterial clamp ischemia. 14We hypothesized that this would result in RBC trapping during the ischemic period.Conv ersel y, during the ischemic phase of arterial clamping, blood cannot enter the kidney, and RBC trapping is limited.Only when the arterial clamp is released does RBC significant trapping normally occur.As such, by comparing tubular injury between arterial and venous clamping without allowing reperfusion, we were able to examine the effects of RBC trapping on tubular injury while maintaining equal warm ischemia time.We hypothesized that RBC trapping during the ischemic phase would cause direct tubular injury.

Animals
All experiments were conducted in accordance with the National Institutes of Health "Guide for the Care and Use of La borator y Animals" and wer e appr ov ed and monitored by the Augusta Uni v ersity Institutional Animal Car e and Use Committee .Age matc hed male and female Wistar Kyoto (WKY) and Sprague-Da wle y rats from Charles River Laboratories were used in all experiments.Initial studies were performed on WKY rats.As w e w er e a b le to demonstrate similar responses in Sprague-Da wle y rats, later studies were performed in these animals as they were less costly.Rats were housed in temperature (20-26 • C) and humidity (30%-70%) controlled, 12:12 h light-cycled conventional animal quarters.Rats were provided ad libitum access to water and standard 18% protein rodent chow (Envigo Teklad, 2918).

Warm Bila ter al Ischemia-Reperfusion Surgery and Tissue Harvest
Ischemia-r e perfusion w as performed as pr eviousl y described. 14 , 26 , 27Briefly, animals were anesthetized with ∼3% isoflurane and 95% oxygen.Body temperatur e (r ectal pr obe) w as maintained at ∼37 • C for the duration of the surgery by a serv o-contr olled heating ta b le and infrared heat lamp (R40, Satco S4998).The renal pedicles were accessed via left and right dorsal flank incisions.In some experiments, both renal arteries were clamped with Schwartz Micro Serrefines (Fine Science Tools #18052-03, Foster City, CA, USA) followed by 2 h of r e perfusion.In other studies, the r enal arter y for one kidney and the renal vein for the other kidney were clamped for either 15 or 45 min without r e perfusion.The kidney (left or right) that r ecei v ed arterial or venous clamping was alternated between animals.At the end of the clamp period, the kidneys were excised prior to the r emov al of the vascular clamp.The kidneys were fixed with VIP-Fixati v e (Fisher #23-730-587) or electron micr oscopy fixati v e (see below) for histological anal ysis.Animals that r ecov er ed fr om anesthesia wer e gi v en bupr enorphine for analgesia.

Saline-P erfused, Blood-F ree P erfusion Studies
Sprague-Da wle y rats were prepared as above except, a midline incision was performed and the abdominal aorta cannulated.The mesenteric artery and vessels of the right kidney were ligated, and a loose tie placed around the abdominal aorta superior to the renal arteries.The renal vein was separated from the renal artery to allow clamp placement.The tie around the aorta was then retracted to prevent blood flow to the left kidney, and the kidney flushed of blood via retrograde perfusion of the abdominal aorta using warm ∼37 • C saline.In blood-free animals, stab le r enal perfusion pr essur e at ∼100 mmgH w as achiev ed using a purpose-made perfusion system consisting of a pressurized saline r eserv oir connected to a smaller in-line r eserv oir in which saline could be heated to 37 • C before being administered.In animals in the b lood-perfused gr oup, perfusion pr essur e w as maintained either at the level of arterial pressure (normal pressure group) or at less than 50 mmHg by adjusting a vascular occluder around the abdominal aorta to lower arterial perfusion pressure to the left kidney (low pr essur e gr oup).Perfusion pr essur e to the r enal arter y w as maintained acr oss the 45-min venous clamp period.At the end of the 45-min clamp period, kidneys were immediatel y harv ested and placed in a fixati v e solution, as per the first study.

Assessment of RBC Congestion
Paraffin-embedded kidney sections were stained with Gomori's trichrome (Thermo Scientific, Cat.No. 87020), according to the man ufactur er's instructions.Congestion of OM vasa recta (VR) and OM plexus capillaries was assessed using a semiquantitati v e scoring method as pr eviousl y described. 14Briefly, a score of 0-5 was given to reflect the extent of RBC congestion inde pendentl y in each region.A score of 0 represents conditions in which all vessels appear open (0% congestion), and a score of 5 r e pr esents congestion in all v essels visualized (100% congestion).

Assessment of Tubular Injury
Both trichrome and hematoxylin and eosin (H&E)-stained kidney sections were scored for tubular injury by a pathologist blinded to the hypothesis of the study and sample identifiers.Percent injur y w as scor ed for the entir e cortex and medulla.Two to three sections from each animal were scored.For trichromestained sections, tubular injury (cell swelling/necrosis) and tubular cast formation were scored in both the cortex and OM.Tubular injury and cast formation are reported as a score of 0-5 with a score of 0 indicating 0%-5%, 1, indicating 5%-20%, 2, indicating 20%-40%, 3, indicating 40%-60%, 4, indicating 60%-80%, and 5, indicating 80%-100% of all tubules demonstrating that trait.For H&E-stained sections, 10-20 fields were scored covering the cortex and OM at magnification 20 ×, and the number of injur ed tubules di vided by the total number of tubules scored and multiplied by 100.

Immunostaining
Imm unostaining w as performed as pr eviousl y described. 14For CD235a staining, slides were incubated with anti-CD 235a mouse monoclonal antibody (Invitrogen cat# MA5-12484) at 1:200 dilution in 10% goat serum in 0.1% phosphate-buffered saline with Tween (PBST) overnight at 4 o C. The next day, slides wer e w ashed 3 times in 0.1% PBST for 5 min each time before being incubated with a secondary antibody [goat anti-mouse IgG-HRP (Santa-Crus cat# sc-2005)] at 1:400 dilution in 10% goat serum in 0.1% PBST for 50 min at r oom temperatur e. Slides were then washed three times in 0.1% PBST for 5 min each time befor e chr omogen staining with 3,3'-Diaminobenzidine (DAB) for 5 min.Slides were then rinsed 3 times in distilled water before being counterstained with hematoxylin for 1 min and washed in running tap water.Slides were covered using Cytoseal XYL medium (Thermo Scientific cat# 8312-4).Hemoglobin (Hb) staining was performed as per CD235a except, Hb alpha recombinant rabbit monoclonal antibody from ThermoFisher, cat# MA5-32328, 1 mg/mL was used at a dilution of 1:200.No primary antibody was used as a control (Supplementary Figure S1).

Western Blots
To examine the specificity of anti-Hb and anti-CD235a antibodies, 3 male WKY were utilized for RBC and kidney cortex tubule protein extraction.For the collection of RBC, rats were anesthetized with isoflurane (3%-5%), and blood was collected by cardiac puncture using a heparinized syringe.Blood was then spun at 400 g, and the pellet collected and re-suspended in saline.This w as r e peated thr ee times and the r emaining RBC pellet w as used for membrane protein isolation.For proximal tubular cells, the left and right kidneys were perfused with ∼10 mL 200 unit/mL collagenase IV in HEPES buffered Hanks' Balanced Salt Solution (HBSS) pH 7.4.The kidneys were then excised and the cortex isolated by dissection and cut into small pieces by a razor blade.Cortical pieces were then placed into the same collagenase solution and incubated at 37 • C for 4 min.The supernatant was then collected into a glass pipette and filtered consecutively through 250 μm and then 75 μm metal sieves.The 75-μm sieve was used to collect proximal tubule fragments.Once proximal tubule fragments collected, the 75-μm sieve was flipped and the underside washed using ice chilled 1% bovine serum albumin in HBSS solution pH 7.4 to stop the collagenase digestion and collect the tubules.The resulting fluid was collected into a 50-mL conical tube and spun at 200 g for 5 min at 4 • C. The resulting pellet was confirmed to contain primarily tubular fragments by inspection under a light-microscope.The pellet was then washed in 15 mL of HBSS before being spun at 200 g for 5 min at 4 • C, and the final pellet collected.We used the Mem-Per Plus Membrane Protein Extraction Kit (Thermo Scientific cat# 89842) for membrane protein and cytoslic protein extraction following the manufacturer's instructions.Protein concentration was determined (Bradford cat# 5000205, BioRad protein assay), and 50 μg RBC and kidney cortex tubule membrane protein was loaded per lane for the CD235a blot.A concentration of 30 μg of RBC and proximal tubular cytosolic protein was loaded per lane for the Hb blot.Pr otein w as loaded onto 4%-20% mini-pr otein TGX gel for electr ophor esis befor e tr ansfer to a Immobilon-FL membr ane (Millipore Cat# IPFL00010).The blot was first incubated with a primary antibody (CD235a 1:100, Hb 1:1500, overnight at 4 • C), followed by a secondary antibody (goat anti-mouse IRDye 680RD, Invitrogen cat# 926-68070, 1:5000 for CD235a, goat anti-r abbit IRdy e 680RD, Invitrogen cat# 926-68071, 1:5000 for Hb).The blot was scanned by a Li-Cor Odyssey CLx.The b lot w as then washed with 1xTBS before sequential incubation with anti-B-actin rabbit antibody for Hb cytosolic protein.As RBC did not show a band for β-actin, we used Ponceau staining as our loading control.We use 0.1% Ponceau Red dye 5% glacial acetic acid to stain our polyvinylidene difluoride membranes saturated in ∼15 min, then rinsed with distilled H 2 O before capturing images (Supplementary Figures S1 and S2).

Evans Blue Imaging
Ev ans b lue (EB) binds str ongl y to plasma pr oteins cr eating a large molecular weight tracer that can been used to investigate plasma extravasation in tissues. 24 , 28 , 29To confirm the extravasation of blood proteins, we injected 50 mg/kg of EB in 1 mL of saline into the tail vein of rats 20 min prior to arterial ( n = 3) or venous clamping ( n = 3) without r e perfusion.Following 45 min of clamp ischemia, kidneys were harvested without removing the clamps, bisected and placed in a fixati v e solution.In addition, we also compared EB staining between control rats ( n = 3, no IRI) and rats following 2 h of r e perfusion fr om 45 min of clamp ischemia.In these animals, EB was injected into the tail vein during the clamp period and allowed to circulate until tissue harvest.A concentration of 5-μm thick, unstained paraffinembedded kidney sections wer e ima ged.Slides wer e ima ged using an Olympus FLUOVIEW TM FV3000 Confocal Laser Scanning Microscope using a 40 × oil objective at 1024 × 1024 resolution.A 488 nm laser (0.6% power) with emission detected between 500 and 540 nm was used to image green autofluorescence from the kidney.Evans blue was imaged using a 640 nm laser for excitation (0.07%) with emission light detected between 620 and 720 nm.The same microscope settings were used for all images.

Endogenous DAB Staining
3,3'-DAB staining was used to visualize the endogenous peroxidase activity of Hb. [30][31][32] Four male WKY rats were used for DAB staining.A 45-min warm unilater al arterial clamp isc hemia was performed followed by 2 h of r e perfusion befor e harv esting the right (ischemic) and left (control) kidneys and placing in fixati v e solution.Paraffin-embedded sections of both control and IR tissue were cut at 5 μm and mounted on the same slide.Slides were then deparaffinized and hydrated to deionized water.Re-hydrated sections were then placed in a dark chamber and incubated with DAB solution (Biocare Medical Betazoid DAB Kit cat# BDB2004L) for 20 min, followed by rinsing in distilled water.Stained sections were counterstained with hematoxylin for 1 min, dehydrated in an ethanol series, and cleared in xylene before mounting.N = 3 slides each from rats undergoing either 45 min of either arterial or venous clamping without r e perfusion or 45 min of venous clamping with saline perfusion were also stained for DAB.

Prussian Blue Staining
Sections from the same animals stained for DAB following reperfusion from arterial clamping were also stained with Prussian  A), the lumen of many tubular cells contains electron-dense material ( * ).Large electron-dense dr oplets of v ar ying density can be found within tubular cells (arro ws).(P anel B), Electron-dense material ( * ) is found in the interstitial space between RBC-congested capillaries and tubular cells.Often the basolateral membrane inv a ginations ar e darkened, and electr on-dense material can be seen to accumulate within these channels (arro ws).(P anel C), The c ytosol of tubular cells surr ounded by electr on-dense material is often darkened (arr ows).Note the m uc h low er density of the cytosol of nearby tubular cells.(Panel D), Often, mitochondria (arrow) and nuclei ( * ) were found to be darkened in cells with electron-dense material within basolateral inv a ginations.Note the much lower density of mitochondria and nucleus of adjacent cells.Significant cell sloughing/blebbing into the lumen is also observed in this image.(Panel E), Tubular injury is prominent.Note the cellular sloughing/blebbing into the lumen filled with electron-dense material.Darkened cell cytosol and cellular vacuolization (arro ws).(P anel F), Electr on-dense material appears contin uous between v essel, interstitial space , and the basolater al side of tubular cell sloughing of basement membr ane .Tubular cells contain darkened mitochondria (arrows).Tubular lumen is filled with electron-dense material.
blue to detect ferrous, non-heme bound iron.ABCAM Iron Stain Kit (Prussian Blue Stain, cat# ab150674) was used for Prussian Blue staining per the man ufactur er's instructions.In brief, the slides wer e de paraffinized and hydrated in distilled water.Equal volumes of potassium ferrocyanide solution and hydrochloric acid solution were mixed to make the iron stain solution.Slides were then incubated with the iron staining solution for 3 min before being rinsed slide in distilled water.The slides were then counterstained with a nuclear fast red solution for 5 min.Slides were then rinsed in 4 changes of distilled water, dehydrated, and clear ed befor e mounting.

Tr anscutaneous Clear ance Measurements
Glomerular filtration rate (GFR) was determined using transcutaneous FITC-Sinistrin clearance 33 in 11 male WKY rats.Transdermal mini-GFR monitors and measurement software (Medibeacon Inc., St. Louis, MO, USA) were used to determine GFR over a 2-h window of time.Rats were anesthetized with isoflurane (2%-5%), and transdermal mini-GFR monitors were placed between the shoulder blades using a double-sided adhesi v e patch.A v olume 20 mg/kg of FITC sinistrin w as then injected into the tail vein.The ½ life of FITC-Sinistrin clearance was determined using the MBLAB software [(Medibeacon Inc., St. Louis, MO, USA] and converted to GFR in mL/min using a 1-compartment model, according to the man ufactur er's instructions.Glomerular filtration rate was determined this way in 6 control rats without ischemia surgery and in 5 rats 0-2 h and ∼24-26 h (4 of 5) into r e perfusion following 45 min of warm bilateral arterial clamping.At the time of harvest, thin (1-2 mm) sections of the kidney were cut and visualized on a fluorescent microscope (Ex500nm and Em515nm) to visualize FITC sinistrin.

Transmission Electron Microscopy
Pr e paration and imaging of kidney tissue were performed by the Augusta Uni v ersity Histology Cor e Facility.Tissue w as fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.1 m sodium cacodylate (NaCac) buffer, pH 7.4, postfixed in 2% osmium tetroxide in NaCac, stained en bloc with 2% uranyl acetate, dehydrated with a graded ethanol series, and embedded in Epon-Araldite resin.Thin 75-nm sections were cut with a diamond knife on a Leica EM UC6 ultramicrotome (Leica Microsystems, Bannockburn, IL, USA), collected on copper grids, and stained with uranyl acetate and lead citr ate .Tissue w as observ ed in a JEM 1400 Flash transmission electr on micr oscope (JEOL USA, P eabod y, MA, USA) at 120 kV and imaged with a Gatan 1095 OneView camera (Gatan, Pleasanton, CA, USA).

Statistics
Graphpad Prizm was utilized for all data analysis.The n numbers and the specific statistical test utilized for individual experiments are listed in each figure legend.No data were excluded for any reason other than a failed experiment, the criteria for which was pre-defined prior to analyzing the data.

Results
We first examined whether sev er e OM tubular injur y following 2 h of r e perfusion fr om r enal arterial clamping and the dev elopment of RBC trapping in the OM is associated with extravasation and tubular uptake of b lood.Trichr ome-stained sections of rat kidneys following 2 h of r e perfusion fr om w arm bilateral arterial clamping confirmed sev er e tubular injur y in the RBCcongested OM, with almost all tubular cells having sloughed from the basement membr ane ( F igure 1 A and B).Many of these injured tubular cells appeared to turn red or contain red droplets that wer e largel y indistinguisha b le in color fr om RBCs ( Figur e 1 C and D).Tubular de gener ation and luminal cast formation were also pr ominent ( Figur e 1 E and F).Vasa r ecta contained congested RBCs and pigmented material ( Figure 1 A and B).
Electr on micr oscopy confirmed detachment of tubular cells from the basolateral membrane, cellular vacuolization, and membrane blebbing ( Figure 2 ).Many cells contained large electr on-dense dr oplets of v arious density ( Figur e 2 A).Often, the basolateral inv a ginations of tubular cells appeared engorged with electron-dense material ( Figure 2 B).The cytosol of many cells was darkened ( Figure 2 C).Many cells also contained electron-dense intraluminal casts ( Figure 2 A and E).In a number of cells, the mitochondria or nuclei were darkened, similar to that r e ported by Janike following cell-free Hb toxicity ( Figure 2 D and F). 34he identity of the electron-dense material within tubular cells and intraluminal casts was further investigated by imm unohistochemistr y.CD235a, or glycophorin A, is a protein that is highly expressed in the cell membrane of er ythr ocytes. 35D235a staining localized primarily to blood vessels; however, droplets within tubular cells and the electron-dense tubular casts in the OM also stained str ongl y positi v e ( Figur e 3 ).Some cortical tubules demonstrated CD235a-positi v e dr oplets; howev er, this w as uncommon ( Figur e 3 ).Hemoglobin staining followed a similar pattern with both the tubular casts and injured tubular cells of the OM staining positi v e ( Figur e 4 ).Western blot confirmed the specificity of the anti-Hb antibody, demonstrating a single band at the correct molecular weight in homogenates of RBCs and no significant signal from salineflushed whole kidney homogenates (Supplementary Figure S1).Western b lot anal ysis of the CD235a antibody r ev ealed a single band at the correct molecular weight for CD235a in membrane fractions from RBCs.However, a second band was also observed in membr ane fr actions fr om isolated pr oximal tubular cells (Supplementar y Figur e S2).The localization of Hb staining w as confirmed using 3,3'-DAB.Hemoglobin acts as an endogenous peroxidase.Oxidized DAB resulted in the deposition of a brown precipitate that localized to RBC and OM tubules in areas of RBC tr apping [30][31][32] ( F igure 5 ).Consistent with both anti-CD235a and Hb staining, the cortical tubules were mostly negative for DAB staining ( Figure 5 A).Both the tubular cytosol of OM tubules and tubular casts stained positi v e for endogenous Hb peroxidase activity using DAB ( Figure 5 C and E).
To determine the direct effect of RBC trapping on tubular injury, we needed a model in which we could promote RBC trapping prior to the r e perfusion phase during the clamp period.As suc h, w e tested the hypothesis that "renal venous clamping results in greater OM RBC trapping during ischemia than arterial clamping."Moderate RBC trapping was present in the cortical peritubular capillaries and veins following both 15 and 45 min of ischemia from either renal arterial or venous clamping without r e perfusion.Howev er, RBC trapping w as significantl y gr eater following 45 min of venous clamping compared with 45 min of arterial clamping ( Figure 6 ).In the OM capillary plexus, renal v enous occlusion r esulted in significantl y gr eater RBC trapping than arterial clamping at both 15 ( Figure 6 B, P < 0.0001) and 45 min ( Figure 6 D, P < 0.0001) of clamping.The larger VR vessels were congested following both arterial and venous clamping; however, the VR were more engorged with RBCs following venous clamping ( Figure 6 G and H).
As r enal v enous clamping pr omoted significant RBC trapping during the clamp period, we next tested the hypothesis that "tubular injury would be greater following renal venous clamping without r e perfusion, compar ed with r enal arterial clamping without r e perfusion, inde pendent of w arm isc hemia time ."Following 15 min of renal venous clamping without reperfusion, there was significant tubular injury in the renal OM.This included cell swelling and tubular cast formation.In contrast, few tubules in the OM re gion demonstr ated significant tubular injury following 15 min of arterial clamping without reperfusion ( Figure 7 A, P < 0.0001).When ischemic time was increased to 45 min, tubular injury following venous clamping without r e perfusion incr eased.This included e vidence of p yknotic nuclei, loss of tubular structure, cellular swelling, and cast formation ( Figure 7 B and F).In contrast, the percentage of injured tubular cells remained lower, and tubular morphology looked r elati v el y normal following 45 min of arterial clamping without r e perfusion ( Figur e 7 B and E).
Ther e w as also evidence of tubular injur y in the kidney cortex.Following 15 min of venous or arterial clamping without r e perfusion cortical injury was greater with renal venous compar ed with r enal arterial clamping ( Figur e 8 A, P = 0.003).Tubular cast formation in the renal cortex was exclusive to animals in which the r enal v ein w as clamped ( Figur e 8 C-F).Tubular injur y scor es wer e similar when injur y w as assessed using H&Estained sections ( Figure 9 ).Injured tubular cells were often observed to contain red pigment and this was enhanced by venous clamping.Trichromestained sections r ev ealed the pr esence of n umer ous r ed droplets within tubular cells following venous clamping without r e perfusion ( Figur e 10 A).This finding was similar to that observed following RBC trapping from arterial clamping with r e perfusion.These r ed dr oplets wer e pr esent in cortical tubules following both arterial and venous clamping without reperfusion; however, these droplets were much more prominent following venous clamping ( Figure 10 A, B, and D; P < 0.002).In trichrome-stained sections, following venous clamping without r e perfusion, some cells appeared to turn bright red and were almost indistinguisha b le in color fr om the RBCs in the surrounding v asculatur e ( Figur e 10 A).This r eddening of the tubular cells was less prominent in the OM; however, injured, sloughed cells generall y appear ed a darker r ed ( Figur e 10 E).This r ed color w as absent in tubules of the inner stripe of the outer-medulla following arterial clamping ( Figure 10 C).
Transmission electr on micr oscopy ima ges fr om the OM of the kidneys of rats following 45 min of r enal v enous clamping without r e perfusion confirmed electr on-dense dr oplets within tubular cells ( Figure 11 A).These droplets often appeared to be in different states of decay, with varying electron density often observ ed ( Figur e 11 B).Electr on-dense material w as also found within the basolateral inv a ginations of tubular cells and within the tubular lumen ( Figure 11 A-C).These electron-dense structur es wer e completel y a bsent fr om sections, and the lumen clear of electron-dense material, in blood-free kidneys following 45 min of venous clamping ( Figure 11 D).
Following venous clamping without reperfusion, many tubules in both the cortex and medulla had CD235a-positi v e droplets within their cytoplasm ( Figure 12 C and E).In the renal OM following ischemia from venous clamping, there was also diffuse CD235a staining within tubular segments ( Figure 12 D-F).Despite the same ischemic clamp time, tubular CD235a staining was minimal in both the cortex and medulla following ischemia from arterial clamping without reperfusion ( Figure 12 A and B).DAB staining of arterial and venous clamped kidneys without r e perfusion followed a similar pattern to anti-CD235.Absorption droplets in cortical tubules from venous clamping, but not arterial clamped kidneys, stained positi v e for endogenous peroxidase activity with DAB ( Figure 13 ).Similarly, tubular casts in the OM of venous clamp kidneys stained positive for endogenous peroxidase activity ( Figure 13 D).Staining of OM tubules following 45 min of venous clamping was observed as punctate areas of positive staining ( Figure 13 C).In order to confirm the uptake of extrav asated b lood pr oteins by both cortical and medullary tubules in areas of RBC trapping, we used confocal microscopy to image EB.In rats not injected with EB, both cortical and OM staining were negati v e ( Figur e 14 A and B).In control rats injected with EB that did not undergo kidney ischemia r e perfusion, RBCs and v ascular structur es stained str ongl y positi v e for EB but staining tubular structures in both the cortex and OM was low ( Figure 14 C and D).Following 2 h of r e perfusion fr om 45 min of warm arterial clamping, tubular staining for EB in the cortical tubules was similar to that of non-ischemic controls ( Figure 14 E).In contrast, the OM tubules stained str ongl y positi v e for EB ( Figur e 14 F).Red b lood cell trapping does not occur following 45 min of arterial clamping without r e perfusion.In these animals, both the cortical and OM tubules remained largely negative for EB ( Figure 14 G and H).Following 45 min of venous clamping without r e perfusion, in whic h RBC tr apping is present in both the cortex and OM, both the cortical and OM tubules stained positi v e for EB ( Figur e 14 J).The intensity of staining, however, was less than that of the OM following 2 h of r e perfusion fr om arterial clamping ( Figur e 14 F).
The cellular and sub-cellular distribution of EB was similar to that observed with anti-CD235a and DAB staining.Following venous clamping, large absorption droplets within the proximal tubules of the cortex were positive for EB ( Figure 15 A   and C).Evans blue staining in the OM tubules w as mor e diffuse ( Figure 15 B).
Ther e w as a close association between ar eas of tubular injury and blood protein uptake.Following 2 h of reperfusion from arterial clamping, injured tubular cells in the OM demonstrated marked uptake of EB, often staining as positi v e as RBCs ( Figure 16 C and E).Almost all tubular cells demonstrated red fluor escent dr oplets within their cytoplasm ( Figure 16 C).Similarly, OM tubular casts were strongly positive for EB ( Figure 16 E and  F).Our data are consistent with extravasation of blood proteins fr om ar eas of RBC tr apping as the sour ce of tubular EB.
Ther e w as no evidence of significant leaka ge of EB into the Bowman's space of the kidneys following 2 h of r e perfusion fr om arterial clamping ( Figures 15 A and 16 A).In line with this, there was little evidence of significant EB uptake by cortical tubules following 2 h of r e perfusion fr om 45 min of w arm r enal arterial clamping ( Figure 16 B).Further excluding filtration as a major source of EB uptake in the medullary tubules, we found that GFR w as markedl y r educed or completel y a bsent in kidneys between 0 and 2 h of r e perfusion fr om 45 min of w arm bilateral arterial clamping when OM tubular EB uptake was observed ( Figure 17 A).We confirmed the absence of significant sinistrin clearance was due to loss of glomerular filtr ation r ather than tubular back leak across this time period.Sinistrin fluorescence was most often completely absent in the glomeruli and tubules of thin sections of kidneys harvested 2 h post-reperfusion from 45 min of arterial clamping ( Figure 17 B and C).Only following 24 h of r e perfusion w as filtration in some glomeruli observed, although this remained patchy ( Figure 17 D and E).
We then tested the hypothesis that "tubular injury from venous clamping is mediated by blood."Tubular injury, including OM tubular cast formation and cell swelling was almost exclusi v e to the b lood-perfused kidney ( Figur e 18 A-C; P < 0.0001).In kidneys that were perfused with saline to remove the blood prior to placing the venous clamp, the tubules were mostl y uninjur ed ( Figur e 18 A and B).Finall y, we tested the hypothesis that reducing renal arterial perfusion pressure in the blood-perfused kidney would limit injury from venous clamping.Renal perfusion pr essur e w as significantl y lo wer in the lo w perfusion pr essur e gr oup v erses the normal perfusion pr essur e group being 38 ± 3 and 75 ± 2 mmHg, r especti v el y ( P = 0.0016).While tubular injury scores tended to be lower in the low renal perfusion pr essur e gr oup compar ed to the normal renal perfusion pr essur e gr oup, this did not reach statistical significance ( Figure 18 A, P = N.S).

Discussion
The major finding of this study is that blood toxicity rather than warm ischemia time is likely to be responsible for much of the kidney injur y observ ed in ischemic AKI.The localization of injur y primaril y to the outer medulla, along with evidence of a cortical-medullary oxygen gradient, led to the hypothesis that medullary hypoxia was largely responsible for kidney injury in AKI. 368][39][40] Trueta first demonstrated that a marked restriction or complete cessation of flow to the outer two-thirds of the kidney cortex may be associated with the maintenance of a normal or increased medullary circulation. 41This phenomenon has been documented in a variety of experimental situations 42 , 43 and in kidney ischemia in men. 37-40 , 44This is consistent with the typical gross appearance of the AKI kidney, in which the cortex is pale and cortical vessels are collapsed, while the outer-medulla appears dark red and the outer-medullary vessels are dilated. 3 , 4 , 8 , 25 , 45-47While initially it was thought that the dusky red color of the outermedulla r e pr esented medullar y hyper emia, it has since become evident that the continued inflow of blood into the medulla during periods of kidney ischemia results in intense RBC congestion (RBC trapping) in the outer-medullar y v asculatur e due to the failure of these medullary vessels to drain. 14 , 25Why then, if cortical ischemia is sev er e and the medullary circulation continues to be perfused (at least initially as RBC trapping develops), is ischemic/hypoxic kidney injury largely restricted to the OM? Ischemic/hypoxic injury also does not well explain the type of injur y observ ed in human AKI.Ischemic injury is known to r esult in coa gulati v e necr osis and the formation of ghost cells; how ever, suc h injury is rare in human AKI. 42Ischemic tubular injury also does little to explain the presence of heme casts in the distal ne phr on segments or the strong link between cell-free heme and ischemic AKI following shock. 45 , 48-51Our finding that extrav asation of b lood fr om the RBC-congested OM capillaries in AKI results in toxic injury to the tubular cells, likely provides an answer to these questions.
Much evidence supports a role of heme toxicity in ischemic AKI. 4 , 45 , 48 , 50 , 52 , 53 In fact, transient heme in the urine is such a pr ominent featur e of isc hemic AKI following shoc k in humans 54 that the syndrome was once referred to as "hemoglobinuric ne phr osis." 4 , 45 , 55 The source of these heme pr oteins, howev er, has remained a mystery for over 70 yr. 45 , 56We hypothesized that extravasation of Hb or other blood proteins out of the RBC-congested OM capillaries may be the primary source of heme in AKI.We pr eviousl y r e ported that RBC trapping is associated with marked tubular injury to the OM within 1-2 h of r e perfusion fr om arterial clamping. 14As such, in the current study, we first examined rat kidneys following 2 h of r e perfusion from 45 min of ischemia from warm bilateral arterial clamping.At this time point, we found evidence of extravasation of blood, including RBCs and their contents from congested vessels and uptake by nearby tubular cells.Evidence supporting the extravasation of blood proteins and RBC material includes: (1) transmission electron microgr aphs demonstr ating the presence of electron-dense material (presumably free Hb from damaged RBC) within and surrounding RBC-congested vessels ( Figure 19 ), and ( 2 ) the presence of blood proteins (EB) and Hb peroxidase activity (DAB) outside the vascular space in areas where RBC trapping w as pr esent.As we and others have found that the v asculatur e endothelium r emains largel y intact at this time, 13 , 14 we speculate that the extravasation of this fluid is likely due to incr eased intrav ascular pr essur es associated with RBC trapping and venous obstruction ( Figure 19 ).Our data suggests this extravasated material is taken up nearb y kidne y tubules, a process that may explain toxic tubular injury in this setting.Evidence for the uptake of this extravasated material by the tubular cells includes: (1) electron microscopy images demonstrating that there is electron-dense material surrounding the RBC congested v asculatur e and that this was often continuous with similar electron-dense material filling the basolateral inv a ginations of tubular cells ( Figure 19 ), ( 2 ) electron-dense material within absorption droplets within tubular cells, which w as r estricted to ar eas of RBC trapping and stained positi v e for CD235a, Hb, DAB, and EB.While we were unable to confirm the specificity of the anti-CD235a antibody we utilized for RBCs, CD235a staining was restricted to areas of RBC trapping.The cellular and sub-cellular distribution of CD235a staining was also similar to our other blood markers.Further supporting the extrav asation of b lood pr oteins into the tubular lumen, following both venous clamping and arterial clamping with reperfusion, what looked like fibrin deposits were also often present in the tubular lumen ( Figure 19 F).Taken together, our data are highly consistent with the uptake of extrav asated b lood and RBC material by the tubules in areas where RBC trapping occurs.
While the source of tubular heme and protein casts in AKI has traditionally been thought to be through filtration, our data are not consistent with this.Both the Ev ans-b lue albumin complex and Hb are large molecules that are not generally filtered.While it is possible that glomerular injury following ischemia results in increased permeability to large molecules, we found no evidence of significant EB filtration within 2 h of r e perfusion when OM tubular uptake was occurring.In rats 2 h following r e perfusion fr om 45 min of arterial clamping, both the Bowman's capsule and proximal tubules were negative for EB staining ( Figure 16 ).Furthermore, our data using transcutaneous sinistrin clearance indicate that there is little or no filtration of even freely filtered molecules in the early hours of r e perfusion ( Figur e 17 ).Despite this, the distal and the OM tubules stain str ongl y positi v e for EB.Bright red fluorescent casts fill the many distal tubules and injured tubular cells stain almost as positi v e as b lood.Extrav asation of b lood fr om the OM vessels has previously been reported following AKI. 24Extravasation of blood from areas of RBC trapping not only explains the localization of this material to RBC-congested areas in the absence of filtration but also the almost complete absence of plasma in much of the congested vasculature ( Figure 19 B-D).
To determine whether tubular injury was directly due to blood toxicity or was secondary to prolonged isc hemia, w e compar ed r enal arterial and v enous clamping without r e perfusion.OM RBC congestion is minimal during the clamp period with arterial clamping.Upon r e perfusion fr om arterial clamping, RBC congestion forms due to obstruction of the venous vessels that drain the medulla. 14 , 22 , 23Using renal venous clamping, we wer e a b le to mimic this venous obstruction during the clamp period, promoting early RBC congestion prior to reperfusion.We found that despite the same 45-min warm ischemia time, tubular injury was much more prominent, and tubular cast formation exclusi v e to v enous clamping when compar ed with arterial clamping without r e perfusion.Tubular injury with 45 min of venous clamping, although milder (presumably due to reduced time of exposure to RBC congestion), r esemb led that observed at 2 h post-reperfusion from arterial clamping, with tubular cells demonstrating evidence of heme uptake and toxic tubular injury.This injury could not have been due to increased intra-r enal pr essur es alone.Confirming the role of b lood tow ard injury, this type of injury was absent in saline-perfused kidneys following venous clamping.Together, our data indicate that extravasation of blood and blood toxicity, rather than extension of isc hemia time , is r esponsib le for the early devastating tubular injury occurring in RBC-congested areas of the kidney OM following r e perfusion fr om arterial clamping in the rat IRI model. 14While extension of ischemic time to the OM does not appear to be r esponsib le for early injury to the OM, our data do not exclude a role of RBC trapping and delayed r e perfusion to promote further injury or delayed recovery of the medullary tubules at later time points.
Red blood cell congestion and tubular RBC toxicity likely explain differences in kidney injury with venous compared with arterial clamping.8][59][60][61] As with venous clamping, congestion forms during the ischemic period, even with short periods of venous clamping, toxic tubular injury develops.Our observations with venous clamping indicate that RBC congestion often r esolv es within min utes of r eleasing the v enous clamp.As RBC congestion often rapidly resolves following removal of the venous clamp, the time in which tubular RBC toxicity occurs with venous clamping is limited to the clamp period.In contrast, while OM vascular congestion commonly develops following r e perfusion fr om w arm arterial clamping, this r esponse is highl y v aria b le, particularl y with shorter ischemic periods. 1 , 14 When congestion does develop, however, it is prolonged, lasting 24-48 h. 2 As such, when the ischemic period is short, vascular congestion from arterial clamping is unlikely to dev elop, r esulting in r educed toxic kidney injur y compar ed with similar lengths of venous clamping.When the ischemic period is prolonged, RBC congestion is more likely to develop, resulting in an overall much longer period of congestion and greater tubular toxicity than that of venous clamping.While we did observe RBC trapping in both the cortex and OM following venous clamping, and this was associated with proximal tubular injury, in humans, RBC trapping onl y rar el y occurs in the kidney cortex, such as following renal venous thrombosis.As such, the focus of our studies is on RBC trapping and injury in the OM, which commonly occurs after severe renal ischemia or shock. 25While our data suggest that RBC trapping-associated tubular injur y occurs secondar y to the tubular uptake of extrav asated b lood pr oteins, the b lood pr otein(s) r esponsib le for injur y r emain unclear.A likely source of tubular injury may be free Hb, which can cause cellular iron accumulation and ferroptosis of tubular cells. 62 , 63Fr ee Hb, r eleased fr om dama ged RBCs, has been r e ported to enter both proximal tubular cells 64 and tubules in the distal ne phr on betw een the membr ane inv a ginations on their surface. 34When the transport capacity of the cell is overwhelmed, this Hb then accumulates in electron-dense droplets within the cell, similar to those observed in our study. 64In our study, we observed evidence of electron-dense material filling the basolateral inv a ginations of tubular cells, suggesting free Hb from RBCs de gener ating in the congested v asculatur e is being absorbed.Consistent with the release and breakdown of Hb from RBCs, we also observed evidence of free iron accumulation in the OM.This was most prominent around congested VR, indicating RBC breakdown as the likely source ( Figure 20 ).Inter estingl y, high circulating levels of free Hb produce tubular injury remarkably similar to that observed in the congested renal OM.This includes the formation of heme-positi v e casts in the distal ne phr on, 19 , 34 , 45 , 48 , 54 cell sloughing, 34 , 65 darkened cell c ytosol, 34 , 65-67 c ytoplasmic vacuolization, 34 , 68 and the invasion of mononuclear cells into the vasa-recta. 19 , 34 , 45 , 69Critically, our new data indicate that the RBC congested medullary vasculature may be the primary source of this toxic heme in ischemic AKI.Further studies are needed to determine whether RBC trappingassociated injury is primarily mediated by Hb and/or other blood proteins.
Our data indicate that RBC trapping occurs and causes toxic tubular injury even at low renal perfusion pressures.Venous clamping, b locka g e, or physiolog ical b locka ge of the vessels that drain the medulla following renal arterial ischemia, results in an increase in intravascular pressure within the kidney. 70 , 71To examine the role of renal perfusion pressure on RBC trapping and injury, we compared blood-perfused kidneys with venous clamping at normal and low renal perfusion pressures.As RBC trapping and tubular injury were similar at both normal and low renal perfusion pressures, this suggests that RBC trapping and tubular blood toxicity may occur even at low arterial pressures, such as in shock.Our data do not exclude a role of high r enal perfusion pr essur es in worsening toxic tubular injur y fr om RBC trapping.Wei et al. have reported that high renal perfusion pr essur e r esults in worsening r enal injur y with v enous clamping. 72Trapped RBCs are so tightly packed together that they form polygonal shapes. 2 , 22 , 23 It is possible that higher renal perfusion pr essur es, by increasing both the physical compression of the RBC membrane and subsequent RBC lysis and the rate of fluid extravasation of this fluid, may worsen tubular toxicity when there is RBC trapping.Our finding that blood is extravasated from areas of RBC trapping leading to toxic tubular injury can explain much of the pathology observed following ischemic AKI in humans.The clinical syndrome and histopathology of AKI ar e r emarka b l y similar across AKI of diverse etiology. 45 , 73-75Histopathological injury observed in AKI from various pathologies incudes lipid degeneration or vacuolization of the thick ascending limb of the Loop of Henle, 2 , 17 , 18 large numbers heme casts in the distal ne phr ons, de gener ation and desquamation of the lower ne phr ons, and ruptur e and non-occlusi v e thr ombosis of the thin-w alled v eins and VR. 2 , 3 , 17 , 21 Remarka b l y, the r enal cortex most often appears largel y uninjur ed. 2 , 19 , 21 , 23 In line with our previous study, 14 we found sev er e tubular injur y in the OM within 2 h of r e perfusion from arterial clamping.Severe tubular sloughing in the OM has often been viewed as post-mortem autol ysis; howev er, evidence suggests this may be incorrect.Lerrolle et al .found that RBC trapping along with cytoplasmic de gener ation and detac hment of the tubular cells from their basement membrane wer e pr esent in kidney biopsies from humans with AKI taken immediately following death, where autolysis would not yet have occurred. 76ur data indicate that the rapid sloughing of the medullary tubules upon r e perfusion of the kidney 14 is secondary to toxic injur y fr om extrav asation of b lood fr om the RBC congested medullar y v asculatur e.The timing of this, early in r e perfusion, corresponds with the period in which there is transient excretion of Hb in the urine early in the course of the development of oliguria in patients with ischemic AKI. 4 Remarka b l y, in rats, many of these sev er el y injur ed OM tubules appear to re gener ate by 24 h of r e perfusion. 14While we speculate that similar regeneration of the tubular epithelium may be r esponsib le for obscuring the most sev er e injur y fr om RBC trapping in humans, our data suggests that many of the key pathological features of ischemic AKI found in humans at later time points can likely be attributed RBC trapping and extravasation of blood proteins from the OM capillaries.A number of studies have reported electron-dense dr oplets and heme-positi v e luminal casts in the distal ne phr on segments following r e perfusion fr om ischemia. 4 , 42 , 45 , 65 , 68 , 77-798][79] Opposing this assumption and consistent with our observations in rats, studies of human kidneys following AKI demonstrate little evidence that blood or heme proteins traversed the lumen of the upstream nephron segments. 48 , 54 , 56urther, heme casts ar e pr esent in the distal ne phr on following ischemic AKI, even in cases in which a source of filtered heme , suc h as hemolysis or crush injury, is absent. 45 , 66Our data provides explanation to these seemingly disparate findings.That is, rather than being filtered, RBC material in the lumen of distal tubular cells was extrasavated from the RBC congested medullar y capillar y circulation befor e being taken up by tubular cells and secreted or leaked into the tubular lumen.This mechanism would explain the presence of heme casts in the lumen of uninjured tubules as well as evidence that hemoglobinuria absent shock/kidney ischemia, such as paroxysmal nocturnal hemoglobinuria, does not cause renal failure. 48 , 49 , 56That is, the  luminal heme itself is not highly toxic, but rather r e pr esents the product of a process that overwhelmed many tubular cells (cellular uptake and secretion of extravasated RBC material across the basolateral membrane).A number of authors have also noted the proximity of tubular injury to microvessels. 4 , 68This r elationship, wher e injur y is gr eatest in tubules with close pr oximity to vessels, is consistent with extravasation of toxins from the v asculatur e and toxic injur y to nearby tubular cells, with cells more distant to the extravasated capillary material being spar ed.Finall y, m uddy br own urinar y casts ar e pathognomonic of tubular injury in ischemic AKI.These casts are known to contain heme; however, the origin of this heme is unknown. 80Our findings suggest that these m uddy br own heme casts are likely to be made up of sloughed, de gener ated cells that took on heme from RBC material extravasated from the congested medullary v asculatur e.
Red blood cell trapping has traditionally been thought to promote tubular injury by extending ischemia time to the OM.Our data contest this assumption, demonstrating that RBC trapping in the kidney OM v asculatur e r esults in r apid extr avasation and uptake of blood material by tubular cells, resulting in toxic injury to the tubules early in the reperfusion period.Further, our study identifies RBC trapping and extravasation of b lood fr om the OM as the source of distal tubular heme casts in ischemic AKI, the source of which has remained a mystery for over 70 yr. 4 , 48 , 56Blood toxicity from RBC trapping appears to be a major component of tubular injury in ischemic AKI, which has not pr eviousl y been r ecognized.As injur y fr om b lood toxicity closely mimics that observed in human kidneys following ischemic AKI, this suggests blood toxicity may be the primary cause of outer-medullary tubular injury in ischemic AKI.If confirmed, this would explain the failure of interventions targeting medullary hypoxia to improve outcomes in ischemic AKI [81][82][83] and open the door for new approaches targeting RBC trapping and tubular blood toxicity to prevent nephron loss in ischemic AKI.

Figure 1 .
Figure 1.Arterial clamping with r e perfusion: Re pr esentati v e ima ges of trichr ome-stained sections of the rat outer-medulla 2 h following r e perfusion fr om 45 min of warm, bilateral, and arterial clamp ischemia.(Panel A), 20 × image of the outer-medulla (OM).There is significant RBC vascular congestion (dark red).Almost all tubule cells in the inner stripe of the OM appear detached from the basement membrane.(Panel B), 20 × image of the OM of another rat.There is marked RBC congestion of the VR and peritubular capillaries with RBCs.Almost all tubule cells in the congested inner stripe of the OM have sloughed into the lumen.(Panel C), 100 × image of the outer stripe of the outer-medulla.Many tubular cells and their nuclei appear bright red.(arrows) There is prominent blebbing (#) and red-cast material in the tubular lumen ( * ).(Panel D), 100 × image of the inner stripe of OM of the rat shown in Panel B. Almost all tubules appear to have de gener ated and have large numbers of cells sloughed into the tubular lumen (#).Injured tubular cells take on a bright red appearance with darkened nuclei (arrows).(Panel E), 100 × image of the inner stripe of OM of the rat shown in Panel A. Many tubular cells appear swollen with blebbing and intraluminal casts ( * ).Note the r ed dr oplets within and dark red nuclei of some cells (arrows) compared with surrounding cells.(Panel F), 100 × image of inner stripe of OM.Note prominent intraluminal casts ( * ), cell blebbing, and swelling.The kidneys of 3 rats were examined, all demonstrating similar morphology.Scale bars are shown in each image.

Figure 2 .
Figure 2. Arterial clamping with r e perfusion: Re pr esentati v e , high-pow er tr ansmission electr on micr oscopy ima ges of the rat outer-medulla 2 h following r e perfusion from 45 min of warm, bilateral, and arterial clamp ischemia.(Panel A), the lumen of many tubular cells contains electron-dense material ( * ).Large electron-dense dr oplets of v ar ying density can be found within tubular cells (arro ws).(P anel B), Electron-dense material ( * ) is found in the interstitial space between RBC-congested capillaries and tubular cells.Often the basolateral membrane inv a ginations ar e darkened, and electr on-dense material can be seen to accumulate within these channels (arro ws).(P anel C), The c ytosol of tubular cells surr ounded by electr on-dense material is often darkened (arr ows).Note the m uc h low er density of the cytosol of nearby tubular cells.(Panel D), Often, mitochondria (arrow) and nuclei ( * ) were found to be darkened in cells with electron-dense material within basolateral inv a ginations.Note the much lower density of mitochondria and nucleus of adjacent cells.Significant cell sloughing/blebbing into the lumen is also observed in this image.(Panel E), Tubular injury is prominent.Note the cellular sloughing/blebbing into the lumen filled with electron-dense material.Darkened cell cytosol and cellular vacuolization (arro ws).(P anel F), Electr on-dense material appears contin uous between v essel, interstitial space , and the basolater al side of tubular cell sloughing of basement membr ane .Tubular cells contain darkened mitochondria (arrows).Tubular lumen is filled with electron-dense material.

Figure 3 .
Figure 3. Arterial clamping with r e perfusion: RBC membrane protein CD235a is concentrated in tubular casts and tubular cells in the outer-medulla following 45 min of arterial clamping with 2 h of r e perfusion.Re pr esentati v e 100 × ima ges are shown of the cortex (Panel A) and outer-medulla (OM) (Panel B) of rats following 2 h of r e perfusion fr om 45 min of warm, bilateral, and arterial clamp ischemia.CD235a is shown in brown.Tissue is counterstained with hematoxylin.Note the luminal cast material stains positi v e for CD235a (P anel B , * ).Tubular cells in the OM also stain diffusely positive for CD235a (Panel B, arrow).In contrast, tubular cells in the renal cortex are mostly negative for CD245a staining.Only mild CD235a staining is observed within RBCs contained within capillaries (Panel A).

Figure 4 .
Figure 4. Arterial clamping with r e perfusion: Hb is found in tubular cells and tubular casts of outer-medulla following 2 h of r e perfusion fr om arterial clamping.(Panel A), lo w-po wer (20 × original magnification) representative image of the rat kidney cortex 2 h after r e perfusion fr om 45 min of warm bilateral arterial clamping (AC).Only RBCs in the v asculatur e stain positi v e for Hb .(P anel B), No-primary antibod y control section of the kidne y cortex.P ositi v e staining is a bsent.(P anel C), high-po wer (100 × original magnification) representative image of the rat kidney cortex 2 h after r e perfusion fr om 45 min of w arm bilateral AC.Onl y RBCs in the v asculatur e stain positi v e for Hb.Tubular cells are ne gative .(Panel D), low-power (20 × original ma gnification) r e pr esentati v e ima ge of the rat kidney outer medulla (OM) 2 h after r e perfusion from 45 min of warm bilateral AC.Red blood cells in the v asculatur e stain positi v e for Hb.Tubular cells in the OM and luminal casts also stain positi v e for Hb.(Panel E), No-primary antibody control section of kidney OM.Positi v e staining is absent.(Panel F), High-power (100 × original magnification) representative image of the rat kidney OM 2 h after r e perfusion fr om 45 min of w arm bilateral arterial clamping.Red b lood cells in the v asculatur e stain positi v e for Hb.Many tubular cells ar e lightl y positi v e. Luminal casts are also positi v e for Hb (arr o w).(P anel G), Same as for Panel F. In this example, a single tubular cell in a tubular cross section stains positi v e for Hb (arrow).(Panel H), High-power (100 × original magnification) representative image of the inner stripe of the rat kidney OM 2 h after reperfusion from 45 min of warm bilateral arterial clamping.Red blood cells in the congested VR stain positive for Hb.There is evidence of free Hb in the VR without RBCs ( * ).Tubular cell staining is observed (arrow).(Panel I), Same as for Panel H.In this example, sloughed tubular cells stain positi v e for Hb (arrows).

Figure 5 .
Figure 5. Arterial clamping with r e perfusion and no ischemia control kidneys: DAB staining for endogenous Hb peroxidase acti vity.Re pr esentati v e ima ges of 3,3'-DAB staining for endogenous peroxidase activity from male WKY rats.Images in the left panels (Panels A, C, and E) are from kidney that underwent 45 min of arterial clamp ischemia with 2 h of r e perfusion (AC with Re p).Ima ges in the right panels (Panels B, D, and F) ar e fr om contr ol kidneys fr om the same animal that did not undergo ischemia r e perfusion (no IRI contr ol).Ima ges ar e r e pr esentati v e of n = 4 animals observed.Tubular DAB staining is minimal in the cortex, with only RBCs within the v asculatur e staining positi v e (Panel A).Red b lood cells stain positi v e due to the endogenous per oxidase acti vity of Hb.In the outer-medulla (OM), injured tubular cells contain diffuse DAB staining (Panels C and E).Tubular casts also stain positi v e for DAB (Panels C and E).In controls, tubular DAB staining is absent in the cortex (Panel B) and OM (Panels D and E).All sections were stained at the same time, and sections from both ischemic and control kidneys from the same animal were mounted and stained on the same slide.Original magnification 100 ×.

Figure 6 .
Figure 6.Arterial and venous clamping without reperfusion: The effect of arterial versus venous clamping on RBC trapping in the cortex and outer-medulla.Red blood cell trapping scores of the cortex or outer-medullary (OM) capillary plexus following 15 (Panel A and B, r especti v el y) or 45 min (Panel C and D, r especti v el y) occlusion of the r enal arter y (AC) or renal vein (VC) with no reperfusion in male ( n = 7) and female ( n = 6) WKY rats.Vascular congestion scores: 0 = 0%, 1 = 20%, 2 = 40%, 3 = 60%, 4 = 80%, and 5 = 100%.Values are expressed as mean ± SEM.Mann-Whitney test was used to compare clamp position, * P < 0.05.Note: for each rat, the renal v ein w as clamped on one kidney and the r enal arter y w as clamped on the other kidney.(Panel E), Re pr esentati v e ima ges of trichr ome-stained sections of the cortex following 45-min occlusion of the r enal arter y with no r e perfusion.Ther e is onl y moderate congestion of the cortical capillaries.(Panel F), Re pr esentati v e ima ges of trichrome-stained sections of the cortex following a 45-min occlusion of the r enal v ein with no r e perfusion.Cortical capillaries are congested with RBCs (dark red between tubules).(Panel G), Re pr esentati v e ima ges of trichr ome-stained sections of the OM following a 45-min occlusion of the r enal arter y with no r e perfusion.Onl y the VR bundles appear congested.(Panel H), Re pr esentati v e ima ges of trichrome-stained sections of the OM following a 45-min occlusion of the renal vein with no r e perfusion.Vasa recta bundles and capillary plexus are packed with RBCs.Images were taken at 40 ×.Scale bar denotes 20 μm.

Figure 7 .
Figure 7. Arterial and venous clamping without r e perfusion: The effect of arterial versus venous clamping on outer-medullary tubular injury.Tubular injury scores of the outer medulla (OM) following 15 (Panel A) or 45 min (Panel B), of occlusion of the renal artery (AC) or renal vein (VC) with no reperfusion in male ( n = 7) and female ( n = 6) WKY rats.Tubular cast formation scores of OM following 15 (Panel C) or 45 min (Panel D) occlusion of the renal artery or renal vein with no reperfusion.Tubular injury or cast formation is reported as 0-5, representing the % of tubules demonstrating tubular injury with a score of 0 indicating 0%-5%, 1, indicating 5%-20%, 2, indicating 20%-40%, 3, indicating 40%-60%, 4, indicating 60%-80%, and 5, indicating 80%-100% of all tubules demonstrating that trait.Values are expressed as mean ± SEM.Mann-Whitney test was used to compare clamp position, * P < 0.05.Note: for each rat, the renal vein was clamped on one kidney and the renal artery was clamped on the other kidney.(Panel E), Re pr esentati v e ima ge of tubular morphology in trichrome-stained sections of the OM following 45 min of occlusion of the r enal arter y with no r e perfusion.Tubular morphology r emains r elati v el y normal follo wing arterial clamping.(P anel F), Re pr esentati v e ima ge of tubular morphology in trichrome-stained sections of the OM following 45 min of occlusion of the renal vein with no reperfusion.Swollen pale cells are common, often with blue or red casts within their lumen.Following 45 min of venous clamping, tubular necrosis is also observed, as evidenced by darkened pyknotic nuclei, loss of cellular cytoplasmic area, and tubular structure.Images taken at 40 ×.

Figure 8 .
Figure 8. Arterial and venous clamping without reperfusion: The effect of arterial versus venous clamping on cortical tubular injury.Tubular injury scores of the kidney cortex following 15 (Panel A) or 45 min (Panel B) of occlusion of the renal artery (AC) or renal vein (VC) with no reperfusion in male ( n = 7) and female ( n = 6) WKY rats.Tubular cast formation scores of the cortex following 15 (Panel C) or 45 min (Panel D), occlusion of the renal artery or renal vein with no reperfusion.Tubular injury or cast formation is r e ported as 0-5, r e pr esenting the % of tubules demonstrating injury with a score of 0 indicating 0%-5%, 1, indicating 5%-20%, 2, indicating 20%-40%, 3, indicating 40%-60% 4, indicating 60%-80%, and 5, indicating 80%-100% of all tubules demonstrating that trait.Values are expressed as mean ± SEM.Two-way ANOVA comparing clamp position and sex, * P < 0.05.Note: for each rat, the renal vein was clamped on one kidney and the renal artery was clamped on the other kidney.(Panel E), r e pr esentati v e trichr ome-stained 20 × ima ge of the kidney cortex following 45 min of arterial clamping without r e perfusion.(Panel F), r e pr esentati v e trichrome-stained 20 × image of the kidney cortex following 45 min of venous clamping without reperfusion.Casts were almost exclusive to the venous clamped kidne y.F ollowing 15 min of venous clamping, casts often appeared as discr ete dr oplets within the tubular lumen rather than the continuous casts filling the entire lumen observed at 45 min of clamping ( Figure 4 F).Images were taken at 40 ×.Casts appeared more developed following 45 min of venous clamping when compared with 15 min of clamping.

Figure 9 .
Figure 9. Arterial and venous clamping without reperfusion: H&E injury scoring.Injury was scored using H&E-stained slides as % of injured tubules [aggregate score of cortex and outer-medulla (OM)] by a resear c her unaware of the hypothesis being tested or the sample identifiers.(For Panels A and D), Y -axis, % of tubules in the cortex and OM demonstrating signs of injury; X -axis, clamp position; males are shown as open circles; females are shown as closed circles.Data are mean ± SEM.Statistics are a result of a 2-way ANOVA comparing the effect of clamp position and sex.(Panel A), Aggregate tubular injury score (% of tubules with injury) in cortex and outer-medulla (OM) following 15 min of arterial (AC) or venous (VC) clamping without r e perfusion.(Panel D), Aggregate tubular injury score (% of tubules with injury) in cortex and OM following 45 min of arterial or venous clamping without r e perfusion.(P anels B , C, E, and F), Re pr esentati v e 100 × ima ges of the inner stripe of the outer-medulla and cortex of H&E-stained kidney sections.(Panel B), r e pr esentati v e ima ge of OM of rat following 45 min of arterial clamping without r e perfusion.Red blood cell congestion of the plexus vasculature is minimal, and tubular ar c hitecture remains relatively normal.(Panel C), representative image of OM of rat following 45 min of venous clamping without reperfusion.The plexus vasculature is congested with RBCs (dark red between tubules).Tubular architecture is disrupted.There is cell swelling and blebbing.Casts are observed in the tubular lumen.(Panel E), r e pr esentati v e ima ge of the cortex of rat following 45 min of arterial clamping without r e perfusion.Much of the peritubular v asculatur e is congested with RBCs.Tubular architecture is disrupted.Some tubular cells are swollen.There is blebbing/sloughing of tubular cell material into the tubular lumen.(Panel F), r e pr esentati v e ima ge of the cortex of rat following 45 min of venous clamping without r e perfusion.Similar to the cortex following arterial clamping, much of the peritubular v asculatur e is congested with RBC.Tubular ar c hitecture is disrupted.Some tubular cells are swollen.There is blebbing/sloughing of tubular cell material into the tubular lumen.

Figure 10 .
Figure 10.Arterial and venous clamping without reperfusion: The effect of arterial versus venous clamping on tubular appear ance .(Panel A), Re pr esentati v e ima ge of a trichrome-stained section of the kidney cortex following 45 min of renal venous clamping (VC).Red droplets (heme loaded mitochondria?)can be observed to have accumulated within the tubular structures, causing them to appear bright red in trichrome-stained sections (arrow).(Panel B), Re pr esentati v e ima ge of a trichromestained section of the cortex following a 45-min renal artery clamping (AC) with no reperfusion.Some red droplets are observed in cortical tubules; however the density of these is much less than that following venous clamping.(Panel C), Re pr esentati v e ima ge of a trichr ome-stained section of the kidney outer-medulla (OM) following 45 min of arterial occlusion.Tubular cells have little to no evidence of RBC uptake.(Panel D), Re pr esentati v e ima ge of a trichr ome-stained section of the cortex following 45-min occlusion of the renal vein with no reperfusion.Tubular cells become deeply red (arrow) and red casts can be observed in the lumen.Note the color change in the cytosol of tubular cells when comparing 45 min of arterial or venous clamping as in Panels B and D. (Panel E), Re pr esentati v e ima ge of a trichrome-stained section of the kidney OM following 45 min of venous occlusion.Cells a purple with trichrome staining with little to no evidence of RBC uptake.Degenerated tubular structure with cells with deep red pigment are common (arrow).All images taken at 100 × magnification.

Figure 11 .
Figure 11.Venous clamping with and without b lood: Electr on-dense material is taken up and secreted/leaked into the tubular lumen.(Panels A, B, and C) are electron micrographs of the outer-medulla (OM) of a rat kidneys 45 min after venous clamping (VC) without r e perfusion in b lood-perfused kidneys.Asterisk ( * ) identifies electron-dense material within tubular cells that appears to be in different states of decay.Arrows point tow ard electr on-dense material, which appears to be secreted into the tubular lumen.Electron-dense material can also be seen to fill the interstitial space.Suggesting these electr on-dense structur es ar e deri v ed fr om RBCs.(Panel D), These electron-dense structures are absent and the lumen clear of electron-dense material in the OM of b lood-fr ee kidneys following the same 45 min of venous clamping.Some blood cells remain.The interstitial space is also clear of electron-dense material.

Figure 12 .
Figure 12.Venous clamping with blood: RBC membrane protein CD235a is concentrated in tubular casts and tubular cells following 45 min of venous clamping.(Panel A), CD235a staining in the cortex of a kidney following 45 min of ischemia from arterial clamping without reperfusion.Image is 40 × magnification.CD235a staining (brown) is minimal and localized to within vascular structures.(Panel B), CD235a staining in the outer-medulla (OM) of a kidney following 45 min of ischemia from arterial clamping without r e perfusion.Ima ge is 40 × magnification.CD235a staining is localized to within vascular structures.(Panel C), CD235a staining in the cortex of a kidney following 45 min of ischemia from venous clamping without reperfusion.Image is 40 × magnification.Tubular casts stain strongly positive for CD235a.Most tubules also stain positi v e for CD235a.(Panel D), CD235a staining in the OM of a kidney following 45 min of ischemia from venous clamping without r e perfusion.Ima ge is 40 × magnification.Tubular casts again stain strongly positive for CD235a with a diffuse staining pattern observed in many tubules.(Panel E), Higher magnification (100 ×) images of cortical tubules following 45 min of ischemia from venous clamping without reperfusion.Within cortical tubules, CD235a staining is observed within discr ete dr oplets of v arious sizes.(Panel F), Higher magnification (100 ×) images of OM tubules following 45 min of ischemia from venous clamping without r e perfusion.Within the OM, CD235a staining within tubules is diffuse and not within discrete droplets.

Figure 13 .
Figure 13.Venous clamping with and without blood: DAB staining for endogenous Hb peroxidase activity.Representative images of 3,3'-DAB (brown) staining for endo genous pero xidase acti vity fr om male WKY rats.(Panel A), Re pr esentati v e ima ge of cortical tubules fr om rat b lood-perfused kidney following 45 min of v enous clamping (VC) with without r e perfusion.Red blood cells stain positive due to the endogenous peroxidase activity of Hb.Absorption droplets (arrows) within the cytosol also stain positi v e for DAB.(Panel B), Re pr esentati v e ima ge of cortical tubules from rat saline-perfused kidney following 45 min of venous clamping without r e perfusion.Cortical tubules ar e negati v e for DAB-positi v e a bsorption dr oplets.(Panel C), Re pr esentati v e ima ge of outer-medullar y (OM) tubules fr om rat b lood-perfused kidney following 45 min of venous clamping without r e perfusion.The v essels ar e congested with tightl y packed RBC that stain positi v e for DAB.Punctate areas of DAB staining can be observed within the tubules (arro ws).(P anel D), Re pr esentati v e ima ge of OM tubules from saline-perfused rat kidney following 45 min of venous clamping without r e perfusion.The v essels ar e congested with tightly packed RBCs that stain positi v e for D AB.A D AB-positi v e tubular cast can be seen ( * ).All sections were stained together.Original magnification 100 ×.

Figure 14 .
Figure 14.Control, arterial clamping with reperfusion and arterial and venous clamping without reperfusion: Comparison of EB fluorescence in cortex and outermedulla.(Panel A), Re pr esentati v e ima ge of kidney cortex fr om rat following 45 min of v enous clamping (VC) without r e perfusion that did not r ecei v e EB.No r ed fluor escence is pr esent; howev er, gr een autofluor escence of the tubules is observed.(Panel B), Re pr esentati v e ima ge of the kidney outer-medulla (OM) from rat following 45 min of VC without r e perfusion that did not r ecei v e EB.No r ed fluor escence is pr esent; howev er, gr een autofluor escence of the tubules is observ ed.Gr een autofluorescence is less than that of the cortical tubules.(Panel C), Re pr esentati v e ima ge of kidney cortex fr om r at kidney without isc hemic clamping (control), in whic h EB was cir culated for 2 h and 45 min.Red b lood cells and v essels ar e positi v e for EB r ed fluor escence.Ther e is light positi vity in the pr oximal tubules for EB.(Panel D), Re pr esentati v e ima ge of kidney OM fr om rat kidney without ischemic clamping (control), in which EB was circulated for 2 h and 45 min.Red blood cells are positi v e for EB red fluorescence.The tubules are negative for EB.(Panel E), Re pr esentati v e ima ge of kidney cortex fr om rat kidney following 2 h of r e perfusion fr om 45 min of arterial clamp (AC) ischemia in which EB was administered.Red blood cells and vessels are positive for EB.The proximal tubules are ne gative .(Panel F), Re pr esentati v e ima ge of kidney OM fr om rat kidney following 2 h of r e perfusion fr om 45 min of arterial clamp kidney isc hemia in whic h EB w as administer ed.Almost all tubules and vascular structures are strongly positive for EB red fluorescence.(Panel G), Representative image of kidney cortex from rat kidney following 45 of arterial clamping without r e perfusion (AC, no r e perfusion), in which EB was administered prior to clamping.Red blood cells and vessels are positive for EB.The proximal tubules are mostly ne gative .(Panel H), Representative image of OM from rat kidney following 45 of arterial clamping without reperfusion (AC, no reperfusion), in which EB w as administer ed prior to clamping.Red blood cells and v essels ar e positi v e for EB.The outer-medullar y tubules ar e mostl y negati v e. (Panel I), r e pr esentati v e ima ge of kidney cortex from rat kidney following 45 min of VC without r e perfusion (VC, no r e perfusion), in which EB was administered prior to clamping.Red blood cells and v essels ar e positi v e for EB.The pr oximal tubules ar e lightl y positi v e for EB, indicated by r ed fluor escence.(Panel J), Re pr esentati v e ima ge of OM fr om rat kidney following 45 of venous clamping without reperfusion (VC, no reperfusion), in which EB was administered prior to clamping.Red blood cells and vessels are positive for EB.The OM tubules are positive for EB, indicated by red fluorescence.Representative images from n = 3 kidneys in each group that were imaged.The original magnification of all images was 40 ×.Imaging settings (laser pow er/camer a sensitivity) were the same for all images shown.

Figure 15 .
Figure 15.Arterial and venous clamping without r e perfusion: Cellular and sub-cellular distribution of EB fluorescence.(Panel A), High-magnification representative image of kidney cortex from rat kidney following 45 of venous clamping (VC) without reperfusion in which EB was administered prior to clamping.Red blood cells and v essels ar e positi v e for EB.Ev ans b lue-positi v e a bsorption dr oplets can be observ ed in some tubules, indicated by r ed fluor escence .Ther e is no EB in the Bowman's space of the glomerulus.(Panel B), High-ma gnification r e pr esentati v e ima ge of kidney outer-medulla (OM) from rat kidney following 45 of VC without r e perfusion in which EB w as administer ed prior to clamping.Ther e is diffuse EB-positi v e r ed fluor escence within the tubules.(Panel C), High-ma gnification r e pr esentati v e ima ge of kidney cortex from rat kidney following 45 of VC without r e perfusion in which EB was administered prior to clamping.Red blood cells and vessels are positive for EB.Ev ans b lue-positi v e a bsorption dr oplets can be observ ed in some tubules, indicated by r ed fluor escence (arr ows).Tubular casts ar e positi v e for EB ( * ).(Panel D), Re pr esentati v e ima ge of kidney papilla from rat kidney following 45 of VC without r e perfusion in which EB w as administer ed prior to clamping.Only RBCs and vessels ar e positi v e for EB .(P anel E), High-ma gnification r e pr esentati v e ima ge of kidney cortex fr om rat kidney following 45 of arterial clamping (AC) without r e perfusion in which EB w as administer ed prior to clamping.Onl y RBCs and v essels ar e positi v e for EB .(P anel F), High-ma gnification r e pr esentati v e ima ge of kidney OM fr om rat kidney following 45 of AC without r e perfusion in which EB was administered prior to clamping.Only RBCs and vessels are positive for EB.Representative images from n = 3 kidneys in each group that were imaged.Imaging settings (laser pow er/camer a sensitivity) were the same for all images shown.

Figure 16 .
Figure 16.Arterial clamping with 2 h of r e perfusion: EB localization.(Panel A and B), Re pr esentati v e ima ges of kidney cortex fr om rat kidney following 45 min of arterial clamping (AC) with 2 h of r e perfusion in which EB w as administer ed.Red b lood cells and v essels ar e positi v e for EB.Cortical tubules are mostly negative for red EB fluorescence.(Panel C), Representative image of high magnification of the kidney outer-medulla (OM) from rat kidney following 45 min of AC with 2 h of r e perfusion in which EB w as administer ed.Ther e is diffuse EB-positi v e r ed fluor escence within the tubules.Some tubular cells appear bright red (arro ws).(P anel D), Re pr esentati v e image of the kidney papilla from rat kidney following 45 min of AC with 2 h of r e perfusion in which EB was administered.Many bright red tubular casts are present in the distal tubules.(Panel E), Re pr esentati v e ima ge of high magnification of OM from rat kidney following 45 min of AC with 2 h of r e perfusion in which EB was administered.A sloughing tubular cell is bright red (arrow).A tubular cast is also highly positive for red EB fluorescence ( * ).(Panel F), Representative image of high magnification of of OM from rat kidney following 45 min of AC with 2 h of r e perfusion in which EB was administered.A sloughing tubular cell is bright red (arrow).Tubular casts are also strongly positive for EB ( * ).Representative images from n = 3 kidneys in each group that were imaged.Imaging settings (laser pow er/camer a sensiti vity) wer e the same for all ima ges shown.

Figure 17 .
Figure 17.No ischemia-r e perfusion contr ol and arterial clamping with 2 and 24 h of r e perfusion: GFR.(Panel A), Glomerular filtration rate (mL/min) measured by tr anscutaneous FITC-sinistrin clear ance in male WKY r ats.no isc hemia controls (no IRI, cir cles, n = 6), 45 min bilater al warm arterial clamp (AC) isc hemia with clearance measured between 0 and 2 h of r e perfusion (squar es, n = 5), and 45 min bilateral w arm AC isc hemia with clear ance measured betw een 24 and 26 h of r e perfusion (triangles, n = 4).Individual rats and mean shown.Statistics are the result of a one-way ANOVA with Tukey post-hoc test.( * * * = P < 0.001).(Panels B and C), Re pr esentati v e ima ges of the kidney cortex and outer-medulla (OM) fr om rats following 2 h of r e perfusion fr om 45 min of w arm bilater al isc hemia from arterial clamping (5 × original magnification).There was no evidence of filtration in kidneys between 0 and 2 h of r e perfusion fr om arterial clamping.Neither the glomeruli nor tubules demonstrated the presence of FITC-sinistrin.(Panels D and E), Re pr esentati v e ima ges of the kidney cortex and OM from rats following 26 h of r e perfusion from 45 min of warm bilateral ischemia from arterial clamping.Some glomeruli and ne phr ons demonstrate the presence of FITC-sinistrin while others were negati v e.

Figure 18 .
Figure 18.Venous clamping with and without blood: Effect of renal perfusion pr essur e on outer-medullar y tubular injury in b lood-fr ee, saline-perfused and bloodperfused kidneys with venous clamping.(Panel A), Tubular injury scores of the outer-medulla (OM) following 45 min of renal venous clamping (VC) with no reperfusion in b lood-fr ee , saline-perfused (Saline , n = 4), normal renal perfusion pr essur e (normal pr essur e, n = 4), and low r enal perfusion pr essur e (low pr essur e, n = 4) male Sprague-Da wle y rats (11-13 wk of age).Tubular injury is reported as a score of 0-10 with cell swelling, tubular cast formation, and tubular injury scored.Values are expressed as mean ± SEM.Statistics r e pr esent a 2-w ay ANOVA with Sidaks multiple comparison test, * P < 0.05.Re pr esentati v e ima ges of trichr ome-stained sections of the OM following 45 min of venous occlusion with no reperfusion in saline-perfused "blood free" (Panel B) and blood-perfused (Panel C) kidneys are shown.Images taken at 100 × magnification.Tubular casts, tubular de gener ation, and cell swelling are prominent in the blood-perfused kidney but absent in saline-perfused kidneys following venous clamping.

Figure 19 .
Figure 19.Evidence of tubular leakage of electron-dense material from congested micr ov essels following arterial clamping with r e perfusion and v enous clamping without r e perfusion.(Panel A), Electr on micr ograph fr om rat kidney following 45 min of arterial clamping with 2 h of r e perfusion (AC Re p), demonstrating electr ondense material (pr esuma b l y Hb) filling the lumen of a capillary.Adjacent ar e injur ed tubular cells with electron-dense cytoplasm and a bsorption dr oplets.(Panel B), Electr on micr ograph fr om rat kidney following 45 min of v enous clamping without r e perfusion (VC), demonstrating electr on-dense material (pr esuma b l y Hb) filling the interstitial space around a capillary containing tightly packed RBCs with almost no plasma separating them.Note the v essel w all appears largel y intact.(Panel C), Electr on micr ograph fr om rat kidney following 45 min of arterial clamping with 2 h of r e perfusion (AC r e p), demonstrating electr on dense b lobs (pr esuma b l y fr om RBC) in interstitial space.Asterisk ( * ) denotes adjacent collagen fibers.(Panel D), Electron micrograph from rat kidney following 45 min of venous clamping (VC) without r e perfusion, demonstrating electron-dense material filling the interstitial space surrounding RBC-congested capillaries.In places this material can been seen filling the basolateral inv a ginations of nearby tubular cells.(Panel E), Electr on micr ograph fr om rat kidney following 45 min of VC without r e perfusion, demonstrating electr on dense b lobs (pr esuma b l y fr om RBC) in the interstitial space.Asterisk ( * ) denotes adjacent collagen fibers.(Panel F), Electron micrograph from rat kidney following 45 min of VC without r e perfusion, demonstrating electr on-dense material in the space between a sloughing tubular cell and a congested capillary.Lower-density material fills the tubular lumen containing what looks like fibrin (#), suggesting a leak of plasma into the luminal space.(Panel G), Electron micrograph from rat kidney following 45 min of VC without r e perfusion, demonstrating electr on-dense material in the interstitial space between a RBC-congested v essel and tubular cell.Electr on-dense material appears to fill and expand the basolateral inv a ginations of the tubule (arrow).(H) Electron micrograph from rat kidney following 45 min of AC with 2 h of r e perfusion (AC Re p), demonstrating electr on-dense material within the basolateral inv a ginations of the tubule (arr ow).Similarl y dense material fills the interstitial space between the tubular cell and a RBC-congested vessel.(Panel I), Electron micrograph from rat kidney following 45 min of arterial clamping with 2 h of r e perfusion (AC Re p), demonstrating electr on-dense material within the basolateral inv a ginations of the tubule (arr ow).This w as seen in tubular cells without any evidence of similar electron-dense material in the lumen or apical invaginations, suggesting the source was basolateral.Yellow scale bar = 5 μm .White scale bar = 2 μm .

Figure 20 .
Figure 20.Arterial clamping with r e perfusion and no ischemia control kidneys: Prussian blue staining for non-heme bound iron.Representative images of Prussian blue staining staining for non-heme bound iron from male WKY rats.(Panel A), Re pr esentati v e ima ge of cortex fr om contr ol kidney that did not undergo ischemia r e perfusion (no IRI contr ol).No ir on de posits ar e observ ed.(Panel B), Re pr esentati v e ima ge of the outer-medulla (OM) fr om contr ol kidney that did not undergo ischemia r e perfusion (no IRI contr ol).No ir on de posits ar e observ ed.(Panel C), Re pr esentati v e ima ge of the cortex from kidney that was subject to 45 min of warm arterial clamp ischemia with 2 h of r e perfusion (45 AC 2 h Rep).No ir on de posits ar e observ ed.(Panel D), Re pr esentati v e ima ge of the OM fr om kidney that was subject to 45 min of warm arterial clamp ischemia with 2 h of r e perfusion (45 AC 2 h Re p).Ir on de posits ar e observ ed both in v ascular and tubular structures (dark specs).(Panel E), Re pr esentati v e ima ge of the OM from kidney that was subject to 45 min of warm arterial clamp ischemia with 2 h of reperfusion (45 AC 2 h Rep).Image demonstrates localization of iron deposits (dark specs) to RBC congested VR bundles.This is consistent with RBC and Hb breakdown and the release of Hb-free iron from RBC.