Ischemia-reperfusion injury (IRI) after surgical procedures is a common in-hospital event that can result in AKI.1 2 3
Renal tubular injury is a key pathophysiologic outcome of IRI, however, it is known that AKI is a complex disorder resulting from inflammation and microvascular changes in addition to tubular injury.4 5 6 7 7 8
Integrins comprise a large family of membrane receptors that bind extracellular matrix proteins as well as other secreted proteins and membrane-tethered ligands. The RGD integrins are one class of integrins that bind this motif (arginine-glycine-aspartic acid), found in several extracellular matrix proteins. Integrin α vβ 5 is an RGD-binding integrin that binds to several extracellular ligands, including vitronectin9 10 α vβ 5 has been reported to be expressed in multiple cell types in vitro , including endothelial cells, epithelial cells, fibroblasts, and various leukocyte lineages.11–15 α vβ 5, ranging from tumor angiogenesis16 17 18 19 20
Several studies have also demonstrated a role of α vβ 5 in regulating vascular barrier integrity. α vβ 5-deficient mice have attenuated VEGF-induced vascular leak in the brain and skin,21 22 α vβ 5-deficient mice or mice treated with an α vβ 5 function-blocking antibody have attenuated vascular leak in acute lung injury.23 , 24 α vβ 5 inhibition could prevent stress fiber formation in response to stimuli (e.g. , VEGF, IL1β , and LPS) and subsequently attenuate endothelial cell permeability.22 , 23
Given the role of α vβ 5 in regulating vascular permeability and its previously described function in models of acute injury, we sought to explore the role of α vβ 5 in the pathogenesis of IRI-induced AKI. Here we demonstrate that treatment with a function-blocking mAb specific for α vβ 5 attenuates AKI in a rat model of renal IRI. We further show that α vβ 5 inhibition attenuates microvascular permeability in this model. Analysis of α vβ 5 immunostaining reveals expression in perivascular cells of the kidney, including vascular smooth muscle cells (VSMCs) and pericytes. We demonstrate that blocking α vβ 5 can attenuate vascular leak in a three-dimensional microfluidic coculture system in a pericyte-dependent manner. Our data support a novel mechanism for α vβ 5 in mediating vascular permeability in AKI.
Results
Inhibition of α vβ 5 Integrin Reduces Renal Injury after IRI
A rat model of renal IRI was used to investigate the role of α vβ 5 integrin in AKI. Rats were treated with two doses of a function-blocking α vβ 5 antibody (ALULA, previously described by Su et al .23 α vβ 5 antibody-treated rats (Figure 1A ). Because no significant differences among the 10, 3, and 1 mg/kg doses of α vβ 5 antibody were detected, further experiments were conducted with lower doses of antibody and under these conditions a dose-response was observed (Figure 1B ).
Figure 1.: Treatment with α vβ 5 mAb protects kidneys in a rat model of renal IRI. To establish the dose-response efficacy of an α vβ 5-blocking antibody (α vβ 5 antibody), rats were treated with (A) 10, 3, or 1 mg/kg or (B) 3, 1, 0.3, or 0.1 mg/kg at 18 hours pre- and 30 hours post-clamp. Doses of 10 and 3 mg/kg were equally effective in attenuating ischemia-induced serum creatinine increases at 24, 48, and 72 hours post-injury. *P <0.05; **P <0.01 versus control antibody; error bars = SEM.
A single dose of α vβ 5 antibody administered 6 hours preclamp also significantly reduced serum creatinine levels at 24, 48, and 72 hours postinjury (Figure 2A ). Effects of this single antibody dose were further evaluated using histopathologic end points. Composite lesion scores combining features of injury and regeneration were assessed by stereological analysis of hematoxylin and eosin–stained kidney sections. These correlated well with the serum creatinine results, with significant inhibition of lesions in the α vβ 5 antibody group observed at 72 hours post-ischemia (Figure 2B ).
Figure 2.: Single dose of α vβ 5 antibody improves kidney function and reduces tubular injury. (A) Rats were treated with a single dose (3 mg/kg) of α vβ 5 antibody 6 hours before ischemic injury. This single dose was effective in attenuating ischemia-induced serum creatinine increases at 24, 48, and 72 hours postinjury. *P <0.05; **P <0.01 versus control antibody; error bars = SEM. (B) Kidneys were harvested at 24 and 72 hours postclamp from the study in A. Histopathologic examination of hematoxylin and eosin–stained kidney sections utilized several morphologic features to generate a composite lesion score (see Concise Methods for details). Rats treated with α vβ 5 antibody had a significantly lower score at 72 hours post-IRI. *P <0.05 compared with control antibody.
A major consequence of renal ischemia is tubular epithelial cell death.25 α vβ 5 inhibition modulates ischemia-induced epithelial cell loss. Activated caspase-3 levels in the renal medulla were found to be significantly reduced at 24 hours in the kidneys of α vβ 5 mAb-treated rats (Figure 3 ). The levels of additional injury markers α -smooth muscle actin and PDGFRβ were also substantially reduced 72 hours post-ischemia with α vβ 5 inhibition, further supporting the protective effect of α vβ 5 inhibition during IRI (Supplemental Figures 1 and 2
Figure 3.: α vβ 5 inhibition decreases ischemia-induced apoptosis. Kidneys were harvested at 24 and 72 hours postclamp after a single dose (3 mg/kg) of α vβ 5 antibody 6 hours preclamp. Activated capsase-3 was used as a measure of apoptosis in post-IRI kidney sections. The degree of caspase-3 immunoreactivity was quantified and calculated as a percent area of medulla. Rats treated with α vβ 5 antibody had significantly reduced apoptosis in the renal medulla at 24 hours postinjury. *P <0.05 compared with control antibody.
Expression Profiling Reveals Biomarkers of Renal Injury Are Decreased with α vβ 5 Blockade
To evaluate the effects of α vβ 5 inhibition on translatable biomarkers, we tested urine and serum collected post-IRI with the proteomic Rat KidneyMAP to evaluate the presence of proteins associated with renal injury. We identified two well established biomarkers, urinary KIM-1 and serum MCP-1 (Supplemental Figure 3 α vβ 5 antibody treatment.
Microarray analysis was performed on kidneys from control and α vβ 5 mAb-treated rats at 24 and 72 hours post-IRI, to explore the impact of α vβ 5 inhibition on global gene expression. Blockade of α vβ 5 resulted in 1460 and 604 transcripts differentially regulated at 24 and 72 hours compared with control antibody treatment, respectively. The differential expression of a subset of these genes was confirmed by quantitative PCR (Supplemental Table 1 α vβ 5 inhibition, including increased migration/differentiation of endothelial cells and increased proliferation of kidney cells with a concomitant decrease in immune cell recruitment and renal epithelial cell death (Table 1 ). In addition, markers of ischemic injury previously described in either rodent models of AKI or in human AKI, including KIM-1 (havcr1 ), Ngal (lcn2 ), and uromodulin (umod ), were also altered by α vβ 5 inhibition (Figure 4 ). Ingenuity Upstream Regulator Analysis evaluated this gene expression dataset and predicted the inhibition of three signaling pathways involved in vascular permeability (IL1β , thrombin, and VEGF) as possible α vβ 5-mediated molecular mechanisms in IRI (Supplemental Figure 4
Table 1. -
Selected pathways regulated by
α v
β 5 inhibition
Function Annotation
P Value
Z -Score
Predicted Activation
Angiogenesis
3.11E-05
2.574
Increased
Migration of endothelial cells
1.66E-04
2.335
Increased
Differentiation of endothelial cells
3.34E-05
1.893
Increased
Kidney development
9.52E-03
1.676
Increased
Proliferation of kidney cells
2.57E-02
1.580
Increased
Apoptosis of renal epithelial cells
1.10E-03
−2.011
Decreased
Failure of kidney
7.16E-06
−2.126
Decreased
Adhesion of immune cells
2.10E-04
−2.298
Decreased
Atrophy of renal tubule
1.62E-04
−2.414
Decreased
Fibrosis
2.23E-06
−2.498
Decreased
Recruitment of immune cells
1.12E-05
−2.555
Decreased
Hydronephrosis
6.94E-07
−2.600
Decreased
Ingenuity Pathway Analysis was performed on transcripts differentially expressed at 24 and 72 hours post-IRI with α vβ 5 antibody compared with control antibody (fold change >1.5; false discovery rate <0.05). Ingenuity Pathway Analysis Z -scores >1.5 or <−1.5 predicted increased or decreased activation of biologic functions with α vβ 5 inhibition.
Figure 4.: Profiling of kidney reveals previously identified genes in rodent and human AKI are altered with α vβ 5 inhibition. Heat map depicts transcripts differentially expressed at 24 or 72 hours post-IRI with α vβ 5 antibody treatment compared with control antibody treatment. Table includes genes previously identified as altered in rodent IR models and in human AKI that were oppositely regulated with α vβ 5 antibody treatment.
Integrin α vβ 5 Modulates Injury-Induced Vascular Permeability
The described role for α vβ 5 in vascular leak led us to examine the ability of α vβ 5 antibody treatment to attenuate vascular permeability after IRI. Intravital imaging was performed in rats 24 hours post-IRI using a rhodamine-conjugated albumin and a high molecular weight dextran to evaluate the microvascular permeability over time. We observed leakage of both the albumin and dextran from the renal microvasculature in the control antibody-treated animals. Leakage of both markers was also observed in the α vβ 5 mAb-treated animals. However, the extent of leakage in the α vβ 5 mAb-treated animals was significantly less than that observed in the control antibody-treated animals (Figure 5 ).
Figure 5.: α vβ 5 inhibition reduces vascular leak after injury. (A) Texas Red-conjugated albumin and a 150 kD fluorescein-dextran were intravenously coinjected into rats at 24 hours post-ischemia to evaluate microvascular permeability after injury. Intravital imaging of the cortex revealed leakage of both the albumin and the high molecular weight dextran from the renal microvasculature in the control antibody-treated animals (A and B), and this leak was substantially reduced in the α vβ 5 antibody-treated animals (C and D). The images were scored for degree of extravasation and the average leak scores for albumin (E) and the 150 kD dextran (F) were calculated. *P <0.05; **P <0.01 versus control antibody; error bars = SEM; image dimensions = 212×212 μ m.
In an effort to understand whether changes in renal blood flow would also contribute to the protection observed from α vβ 5 antibody treatment, we conducted additional intravital microscopy to evaluate red blood cell (RBC) flow rates. At 24 hours post-IRI there was no significant difference in RBC flow rates between control and α vβ 5 antibody-treated animals (Supplemental Figure 5 α vβ 5 inhibition does not alter cortical blood flow in IRI at this time point. This study does not rule out the possibility that α vβ 5 inhibition is altering blood flow at an earlier time point or altering flow in the outer medulla. Future studies should comprehensively address the possibility of α vβ 5-mediated changes in blood flow and its contribution to the protection from vascular leak and injury observed with α vβ 5 antibody treatment.
Integrin α vβ 5 Expression in the Kidney
Previous studies report expression of α vβ 5 in multiple cell types in vitro 11–15 in vivo . To address the localization of α vβ 5 in the kidney, we generated an affinity-purified rabbit polyclonal antibody that could detect integrin β 5 in formalin-fixed paraffin-embedded tissue. Staining mouse kidneys with this antibody revealed broad expression of α vβ 5 in multiple regions of the kidney, including the glomerulus and cortical interstitium, and specificity of the staining was confirmed using β 5 null mouse tissue and competition with soluble α vβ 5 protein (Supplemental Figure 6, B and C
To assess if α vβ 5 is present in renal perivascular and endothelial cells, we conducted immunofluorescence for α vβ 5, PDGFRβ , and CD31 on frozen tissue sections from mouse (Figure 6 ) and rat (Supplemental Figure 7 α vβ 5 is generally present on PDGFRβ -expressing cells and is largely absent from CD31-expessing endothelial cells (Figure 6 ). The PDGFRβ immunostaining also colocalized with NG2 (a proteoglycan expressed on pericytes and VSMCs), providing further evidence for α vβ 5 expression in perivascular cells (Supplemental Figure 8
Figure 6.: Characterization of α vβ 5 integrin expression in the kidney. Frozen sections of mouse kidney were stained for α vβ 5 (red), CD31 (green), and PDGFRβ (white) with nuclei in blue. Representative images (63× magnification) from the glomerulus, cortex, medulla as well as an artery are shown. α vβ 5 is expressed in podocytes, mesangial, epithelial, and perivascular cells of the kidney and within interstitial cells, and appears to be present within PDGFRβ -positive cells, but not in CD31-positive endothelial cells.
To identify any changes that injury may induce in the expression or localization of α vβ 5, we immunostained rat kidneys 24 hours post-IRI. No substantial changes were detected in the levels of α vβ 5 expression or in its localization pattern post-IRI compared with an uninjured kidney (Supplemental Figure 7
Integrin α vβ 5 Attenuates Vascular Leak in a Pericyte-Dependent Manner
To evaluate α vβ 5 antibody treatment in human kidney cells, we performed three-dimensional vascular permeability assays. Human renal endothelial cells were cultured in two different conditions: in the absence and presence of human kidney pericytes in a three-dimensional microfluidic device. As expected, treatment of the microvessels with thrombin significantly increased vascular permeability (P d ) in both the absence and presence of pericytes. Treatment with the α vβ 5 antibody had no significant effect on thrombin-induced permeability in endothelial cells alone (Figure 7A ) but had a profound inhibitory effect on vascular leak in the coculture system (Figure 7B ).
Figure 7.: α vβ 5 regulates vascular permeability in a pericyte-dependent manner. (A) Human kidney endothelial cells were seeded into a three-dimensional microfluidic device and allowed to form a tube in the presence of flow. Cells were treated with control or α vβ 5 antibody 1 hour before challenge with thrombin (3 U/ml). 70 kD Texas Red albumin was perfused into the vessel and the diffusion was measured by confocal microscopy to calculate the diffusive permeability coefficient (P d ). No significant difference was observed with α vβ 5 antibody with endothelial cells alone (n =6). (B) All conditions are the same as described above except human kidney pericytes are first seeded into the microfluidics device, followed by seeding with the endothelial cells. In this condition, the α vβ 5 antibody significantly inhibited the thrombin-induced vascular permeability. *P <0.05 versus thrombin + control antibody; error bars = SEM.
Quantitative PCR on cells from the microfluidic devices revealed substantially higher levels of itgb5 mRNA in the kidney pericytes and in the coculture compared with endothelial cells alone, indicating that β 5 integrin expression is very low or absent in endothelial cells (Supplemental Figure 9
Additional permeability studies were performed with LPS to demonstrate a broad role for α vβ 5 in regulating vascular permeability. As with thrombin, α vβ 5 inhibition also significantly inhibited LPS-induced permeability (Supplemental Figure 10A α vβ 5 inhibition (Supplemental Figure 10B α vβ 5 regulates vascular permeability in a pericyte-dependent manner.
Integrin α vβ 5 Is Important for Pericyte Adhesion and Migration
Integrins, including α vβ 5, have a well defined role in regulating adhesion and migration in numerous cell types, and these processes could contribute to the function of pericytes in response to injury. To examine the contribution of α vβ 5 to pericyte adhesion we plated human kidney pericytes on either vitronectin (a known α vβ 5 ligand) or collagen (not an α vβ 5 ligand) in the presence or absence of antibody treatment. The α vβ 5 mAb led to a nearly 90% reduction in cell adhesion to vitronectin, but had no significant impact on adhesion to collagen (Figure 8A ).
Figure 8.: α vβ 5 regulates pericyte adhesion and migration on vitronectin. Human kidney pericytes were seeded on plates coated with collagen or vitronectin. (A) Fluorescently labeled cells are seeded in the presence of the α vβ 5 antibody, an α vβ 3 antibody, or a control antibody and were counted after washing the adherent cells. The α vβ 5 antibody significantly reduced adhesion of cells on vitronectin compared with control antibody treatment. ***P <0.001 versus control antibody; error bars = SEM. (B) Cells were treated with antibody 1 hour before scratch wound, and images were taken every 2 hours for 2 days and wound confluence was calculated. The α vβ 3 antibody had no significant effect on migration, the JNK inhibitor SP600125 (included as a positive control) inhibited migration on both substrates and the α vβ 5 antibody significantly inhibited migration of pericytes on vitronectin. *P <0.05 versus control antibody.
Migration studies were also performed using a scratch wound assay on human kidney pericytes on either collagen or vitronectin. Antibody treatment did not impact migration on collagen; however, on vitronectin, α vβ 5 antibody treatment significantly inhibited pericyte migration (Figure 8B ). These studies demonstrate a ligand-dependent function for α vβ 5 in pericyte adhesion and migration, properties which are likely to contribute to the α vβ 5-dependent function of pericytes in vascular permeability.
Postclamp Treatment with α vβ 5 Antibody Attenuates Injury Response
In order to evaluate the translational capacity of α vβ 5 inhibition, we employed a therapeutic dosing strategy. Rats were administered α vβ 5 antibody 8 hours postclamp release. This single therapeutic dose significantly reduced serum creatinine levels at 48 hours postinjury (Figure 9A ). The effects of this dosing strategy were further evaluated using quantitative PCR of AKI biomarkers in kidneys isolated 72 hours post-IRI. α vβ 5 inhibition significantly reduced the expression of two biomarkers induced by injury (havcr1 , lcn2 ) and increased the expression of two biomarkers diminished by injury (umod , egf ). Together, these data demonstrate that treatment even several hours after ischemia may have therapeutic benefit.
Figure 9.: Treatment with α vβ 5 antibody after ischemia provides protection from injury. (A) Rats were treated with a single dose (3 mg/kg) of α vβ 5 antibody 8 hours postclamp release. This single dose was effective in attenuating ischemia-induced serum creatinine increases at 48 hours postinjury. *P <0.05 versus control antibody; error bars = SEM. ( B) Kidneys were harvested at 72 hours postclamp from the study in A for quantitative PCR analysis. At 8 hours postclamp, treatment significantly reduced the expression of two biomarkers induced by injury (havcr1 , lcn2 ) and increased the expression of two biomarkers diminished by injury (umod , egf ). *P <0.05; **P <0.01 versus control antibody; error bars = SEM.
Discussion
Here, we have shown that inhibition of integrin α vβ 5, with a selective mAb, attenuates AKI in an IRI rat model. We further show that this antibody reduces vascular permeability, suggesting a mechanistic role for α vβ 5 in the pathogenesis of AKI. Importantly, we show that α vβ 5 is predominantly expressed in perivascular cells in the kidney, including pericytes. Using a novel three-dimensional microfluidic culture system we demonstrate for the first time a role for α vβ 5 function in pericytes leading to regulation of vascular leak. Importantly, inhibition of α vβ 5 hours after ischemia was still able to provide renal protection, indicating that this antibody could be a promising therapy for AKI.
The demonstration that α vβ 5 plays a key role in the kidney upon acute injury is consistent with previous data in other tissues, including protection from cerebral ischemia in β 5 null mice21 α vβ 5 inhibition resulting in reduced vascular leak in pulmonary ischemia.24 in vivo studies, reports indicate that α vβ 5 is expressed in monolayer cultures of endothelial cells and that anti-α vβ 5 antibody treatment attenuates endothelial permeability.22 , 23 , 26 α vβ 5 in vascular leak using a three-dimensional microfluidic system that better mimics the in vivo structural and physiologic environment of the intact microvasculature. In this system, we observed that α vβ 5 does indeed play a key role in modulating vascular leak, but that this effect is largely dependent on pericytes. The reason for the experimental discrepancy between the three-dimensional system and the previous two-dimensional endothelial culture systems is uncertain, but there are numerous examples of three-dimensional culture systems better mimicking in vivo biology.27 28 , 29 30
Consistent with a pericyte-dependent role for α vβ 5 in modulating vascular leak, we observe expression of α vβ 5 in perivascular cells of the kidney, including VSMCs and pericytes, whereas no detectable expression was observed in endothelial cells. Similarly, stromal cell-restricted expression of α vβ 5 in lung has also been observed (data not shown). Consistent with our results, previous studies evaluating in situ expression of α vβ 5 in the kidney have reported predominant mesangial cell expression in the glomerulus.31
Improved renal blood flow post-IRI is likely to have a protective effect on kidney function. The observed expression of α vβ 5 in VSMCs suggests there may be a mechanism whereby α vβ 5 inhibition ameliorates injury-induced vasoconstriction and improves blood flow. Our initial study using intravital microscopy to evaluate RBC flow in renal cortical vasculature post-IRI did not detect a significant difference in blood flow with α vβ 5 inhibition. However, it is important to note that this study does not rule out the possibility that α vβ 5 inhibition is altering blood flow at an earlier time point or altering flow in the outer medulla. Future studies that include a detailed time course analysis of blood flow throughout all phases of the injury could reveal another novel function for α vβ 5 and expand on the protective effects of α vβ 5 antibody treatment.
The in vivo immunostaining and ex vivo microfluidics studies suggest a role for α vβ 5 in pericytes, which are known to be critical for proper barrier function in the microvasculature.32 33 , 34 α SMA-positive myofibroblasts and play a key role in contributing to the fibrotic response.35 36 α vβ 5 in pericytes that contribute to its role in AKI.
To begin to understand this we have evaluated some functions of α vβ 5 in pericytes in vitro . We have shown that α vβ 5 is instrumental for pericyte adhesion as well as migration on a vitronectin substrate, a known α vβ 5 ligand. Vitronectin has been associated with the pathogenesis of several disease processes including neointimal formation in blood vessels,37 38 39 40
It has been proposed that pericytes may migrate from endothelial cells after ischemic injury1 8 , 35 , 41 α vβ 5 inhibition could maintain pericyte–endothelial interactions, resulting in reduced vascular leak and diminished myofibroblast differentiation. This is supported by the reduced myofibroblast expansion observed with α vβ 5 inhibition. This suggests that α vβ 5 antibody could potentially protect from longer-term damage like the renal fibrosis that occurs in the weeks and months after AKI (reviewed in Ferenbach and Bonventre1
Overall, our data highlight an important role for α vβ 5 in the pathogenesis of AKI and suggests that targeting this integrin to attenuate vascular permeability could provide an effective therapy, one that is mechanistically highly differentiated from other therapeutic approaches that have failed in the clinic setting.
Concise Methods
Unilateral Clamp Ischemia Model
Animal experiments were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. Male Sprague Dawley rats (Harlan Laboratories) were anesthetized with isoflurane and placed on a warming pad for temperature maintenance. The right kidney was removed and the renal artery and vein sutured off. Ischemia of the left kidney was initiated by clamping the renal artery and vein for 30 minutes using nontraumatic clamps (Fine Scientific Tools). At the conclusion of the ischemic period, the clamps were removed and the kidneys were observed to insure rapid re-establishment of blood flow. The α vβ 5 blocking antibody (ALULA) used in these studies was previously described.23 μ l by subcutaneous injection at defined times before clamping.
Immunostaining of Fixed Kidneys
To detect α vβ 5 in formalin-fixed, paraffin-embedded tissues, a human β 5 integrin-specific polyclonal antiserum was prepared by immunizing a rabbit with purified soluble human α vβ 5 ectodomain protein generated with recombinant constructs in CHO cells. The total IgG fraction was purified from the antiserum using protein-A affinity chromatography, and subjected to affinity depletion using Sepharose beads conjugated to soluble human α vβ 6 protein, in order to remove human α v-reactive components. The human β 5-integrin specific rabbit antibodies were then affinity purified using Sepharose beads conjugated to soluble human α vβ 5 protein. The final isolated rabbit IgG fraction was only reactive to human β 5-containing integrin but not any other integrin subunits, as tested by ELISA (data not shown). Formalin-fixed tissues were embedded in paraffin, sectioned at 5 µ M, and processed on the Ventana Discovery XT platform (Roche) using the purified anti-human β 5 polyclonal antibody. Immunohistochemistry of additional markers was performed as above, using the following antibodies: PDGFRβ (Thermo Fisher), α -smooth muscle actin (α SMA; Abcam, Inc.), and activated caspase-3 (Cell Signaling Technology). Quantification of caspase-3 immunoreactive area was accomplished by custom-designing algorithms in Visiopharm (Denmark) software.
Additional immunostaining for α vβ 5 was performed on frozen tissue from rat (Sprague–Dawley strain; Harlan/ENVIGO) and mouse (Itgb5 tm1Des β 5 null strain and wild-type 129 SvJ strain; Jackson Laboratories). Tissues were fixed in 4% PFA and embedded in OTC (Electron Microscopy Sciences). Frozen blocks were sectioned on a cryostat at 10–20 µ m thickness and slides were stored at −80°C. Primary and secondary antibodies were as follows: α vβ 5 antibody ALULA23 z -axis resolution. Three-dimensional image stacks were pseudocolored and reconstructed with Volocity (Perkin-Elmer).
Histologic Evaluation
Hematoxylin and eosin–stained sections from formalin-fixed, paraffin-embedded kidneys were examined by a blinded, board certified veterinary pathologist for injury and regeneration using a scoring method modified from Kelleher et al.42 1 ) injury score: tubular necrosis, calcific debris in tubular lumina, interstitial inflammation, casts, juxta-glomerular apparatus prominence, interstitial edema, brush border loss, tubular proteinosis, and pelvic congestion; and (2 ) regeneration score: neutrophils in vasa recti, tubular regeneration, and tubular cell mitoses. Lesions were scored on a scale of 0–2 (0, none; 0.5, minimal; 1, mild; 1.5 moderate; and 2, marked). Composite lesion scores presented are a combination of the injury and regeneration scores.
Serum and Urine Biomarker Measurements
Serum creatinine was measured at baseline and at days 1, 2, and 3 postclamp (animals euthanized at 24 hours postclamp have only baseline and day 1 serum creatinine measurements). Levels were measured using an AU480 Chemistry Analyzer (Beckman) and reported as milligram per deciliter. Profiling of serum and urine biomarkers of renal injury was conducted using the Luminex Bead assay platform with the multianalyte panel Rat KidneyMAP v1.0 (Myriad RBM).
RNA Isolation, Microarray Analysis, and Quantitative PCR
Total RNA from the kidney was isolated in QIAzol (Qiagen) using mechanical disruption on the FastPrep system (MP Biomedical), and purified using the RNeasy Mini Kit (Qiagen). RNA was quantitated (NanoDrop; Thermo Fisher) and was evaluated for integrity using the RNA 6000 Nano Assay on a Bioanalyzer 2100 (Agilent).
Gene expression microarray experiments were performed at Expression Analysis (a Quintiles company) as follows. Total RNA was labeled and hybridized using the Ambion WT Expression kit according to the manufacturer’s instructions. After hybridization, arrays were washed and stained using standard Affymetrix procedures before scanning on the Affymetrix GeneChip Scanner. For differential expression analysis, a two-group analysis was performed using the comparative method Permutation Analysis for Differential Expression to develop false discovery rate-based estimates of groups of genes using a shrunken t-statistic. A false discovery rate of <0.05 and a fold change of >1.5 (control antibody-treated versus α vβ 5 antibody-treated) was considered significant.
For quantitative PCR confirmation of microarray results, cDNA was synthesized from 1 µ g of RNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher) and quantitative PCR was performed using Taqman Gene Expression assays run on the QuantStudio 7 (Thermo Fisher). Ct values of target genes were normalized to the eif1a control gene to generate Δ Ct values.
Intravital Microscopy to Evaluate Vascular Permeability and RBC Flow In Vivo
Rat serum albumin was conjugated to Texas Red as previously described (TR-RSA).43
Images were acquired using an Olympus FV-1000 microscope, with an original magnification of ×60, 1.2NA water immersion objective, and highly sensitive GaAsP nondescanned (external) detectors. Ten random fields were marked and imaged at 5, 15, and 30 minutes postinfusion of the dual TR-RSA/150 kD fluorescein dextran bolus. All of the images were taken approximately 5–10 μ m below the kidney capsule where interstitial space is prevalent.
The degree of extravasation of the two different molecular mass markers (approximately 66 kDa for the TR-RSA and 150 kDa for the fluorescein-dextran) was used examine the severity of damage to vascular integrity. TR-RSA was used to rank mild to moderate vascular damage and the 150 kD fluorescein-dextran was used to rank more severe damage (see Supplemental Material
RBC velocity was calculated as previously described.44 y -axis) and distance (the x -axis).
Microfluidic Platform for Vascular Permeability
To determine the permeability of endothelial monolayers and endothelial-pericyte co-cultures in vitro , microvessels were formed in microfluidic devices as described previously.45 μ m diameter tubes formed in collagen type I hydrogels to create lumenized microvessels, and 70 kD dextran was perfused through the vessel lumens to quantify the diffusive permeability.46
In Vitro Pericyte Adhesion AssayCostar 96-well high binding plates were coated overnight at 4°C with 0.1% collagen (Sigma) or 10 μ g/ml vitronectin in PBS (R&D Systems). Plates were washed then blocked for 1 hour at 37°C and washed with buffer again. Antibodies (10 µ g/ml) 2H6, anti-α Vβ 5, and anti-α Vβ 3 (MAB1976; Millipore) were added to wells prior to cell seeding. Human fetal kidney pericytes were fluorescently labeled with Calcein-AM (Molecular Probes) and seeded into antibody-containing wells. After incubation, plates were washed then read on a Synergy 1 H microplate reader (BioTek) to quantify adherent cells via fluorescent intensity.
Scratch Wound Migration Assay
Essen Bioscience 96-well ImageLock plates were coated overnight at 4°C with 0.1% collagen (Sigma) or 10 μ g/ml vitronectin in PBS (R&D Systems), then washed with PBS. Human fetal kidney pericytes were seeded in Opti-MEM (Thermo Fisher) and incubated at 37°C for 6 hours prior to scratch using the Woundmaker (Essen Bioscience). Antibodies 2H6, anti-α Vβ 5, anti- α Vβ 3 (MAB1976; Millipore), or a JNK inhibitor (Selleckchem SP600125) were added to the wells 1 hour before scratch. Plates were imaged in the Incucyte ZOOM system (Essen Bioscience) every 2 hours for 48 hours. An analysis was run on each plate and wound confluence was calculated at each individual time point for each treatment condition.
Statistical Analyses
GraphPad Prism 6 (GraphPad Inc.) was used to analyze the data. Data were analyzed by two-tailed t test or two- or one-way ANOVA with post hoc analysis as appropriate. P <0.05 was used to indicate significance.
Disclosures
J.E.C., C.Q., I.G.G., J.D., B.M.D., S.M., J.W.M, J.S.D., and M.A.C. are former employees of Biogen, and A.T.M., T.L.R., G.M., A.H., F.Q., P.H.W. and S.M.V. are currently employees of and stockholders in Biogen. This does not alter the authors' adherence to all the policies of the Journal of the American Society of Nephrology on sharing data and materials.
This work was supported by the following National Institutes of Health grants (to C.S.C.): EB00262, EB017103, HL115553, HL119346, and the Biogen Fibrosis Consortium.
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