Acute kidney injury (AKI, as defined in Mehta et al. 1) occurs frequently in critically ill patients and is a crucial contributor to morbidity and mortality in both adults and children (2-4). Severe kidney injury is associated with increased intensive care unit mortality and length of stay (5) and often occurs in the setting of multiple organ dysfunction. In particular, there appears to be a clinical relationship between kidney injury and acute lung injury (ALI) and/or the acute respiratory distress syndrome (ARDS). In the intensive care setting, AKI is associated with a significantly higher need for mechanical ventilation (6). In addition, oliguria and increases in serum creatinine (SCr) level greater than 85% above baseline have been associated with more difficult weaning from mechanical ventilation in adults (7). Studies have demonstrated that acute lung injury is often associated with AKI and that the presence of both conditions predicts mortality (8, 9).
Despite this frequent association, little is known about the mechanistic relationships between AKI and ALI. Historically, the pathogenesis of ALI was attributed to alterations in mechanical and physiologic factors such as decreased urine output leading to pulmonary edema. More recent studies, however, have explored the cellular and molecular mechanisms of pulmonary injury after an insult remote to the lungs. For example, AKI promotes activation of proinflammatory and proapoptotic molecules, and the development of lung injury after AKI can be attenuated with anti-inflammatory compounds (10-12). In addition, experimental AKI leads to alterations in pulmonary vascular permeability and inflammation as well as sodium and water transporter expression (13, 14). Finally, extensive genomic changes occur in both lung and kidney tissue after AKI, particularly in inflammatory and apoptotic processes (15).
All previous studies, however, have only evaluated changes in whole lung tissue after kidney injury (15, 16). We hypothesized that the complex interactions between the lung and kidney might be better delineated by evaluating cell-specific changes after AKI. We identified the pulmonary vascular endothelium, in particular, as a potential mediator between kidney and lung injury. During AKI, circulating soluble factors or inflammatory cells might lead to changes in lung endothelial cell (EC) structure and function, leading to microvascular permeability, alveolar inflammation, and ultimately acute lung injury.
In the current study, AKI resulted in significant changes in pulmonary EC gene expression of multiple factors involved in inflammation, vascular reactivity, and apoptosis. In addition, in vitro studies revealed that pulmonary microvascular cells treated with AKI serum underwent functional changes consistent with initiation of apoptosis, including actin rearrangement and phosphatidylserine translocation. This is the first study detailing the pulmonary EC response to AKI; future studies will continue to investigate the underlying mechanisms responsible for deleterious kidney-lung crosstalk in the critically ill.
MATERIALS AND METHODS
All procedures were approved by the Johns Hopkins Animal Care and Use Committee, and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male 6- to 8-week-old mice (C57/BL6), 25 to 30 g, were obtained from Jackson Laboratory (Bar Harbor, Maine) and male 6- to 8-week-old Sprague-Dawley rats (200-250 g) were obtained from Charles River (Wilmington, Mass). All animals were housed under pathogen-free conditions at least 5 days before operative procedures. All procedures were performed using sterile techniques under general anesthesia with pentobarbital (50-75 mg/kg intraperitoneally). Assessment of adequate anesthesia was obtained by paw and tail pinch.
After anesthesia, animals were placed on a heating table to maintain a rectal temperature of 37°C and underwent midline laparotomy with isolation of bilateral renal pedicles. For rodents assigned to experimental ischemia-reperfusion injury (IRI), nontraumatic microvascular clamps were applied across both renal pedicles for 60 min. After placement of clamps, 1 mL warm sterile saline was administered intraperitoneally. We used 60 min of ischemia because previous experiments in our laboratory demonstrated that 30 min of kidney ischemia did not lead to significant changes in the lung transcriptome compared with sham surgery alone (15). After the allotted ischemia time, clamps were gently removed, a second intraperitoneal 1 mL saline bolus was given, and the incision closed in one layer with 4-0 silk suture. The animals were then allowed to recover with free access to food and water. The rodents assigned to bilateral nephrectomy (BNx) underwent similar procedures except both renal pedicles were ligated with 5-0 silk suture, and the kidneys were removed. Sham animals underwent the identical procedure without placement of the vascular clamps or ligature. At 4 or 24 h after the experimental procedure, the animals were euthanized by exsanguination under pentobarbital anesthesia, and tissue and blood were collected for analysis.
Blood samples (0.2 mL) were obtained from each animal before surgery and at sacrifice, centrifuged for 10 min at 8000 rpm to obtain serum and stored at −20°C until analysis. Serum creatinine levels were measured as a marker of renal function, using a 557A Creatinine kit (Sigma Diagnostics, St. Louis, Mo) and analyzed on a Cobas Mira S Plus automated analyzer (Roche Diagnostics Corp., Indianapolis, Ind).
Pulmonary vascular EC isolation
Pulmonary vascular EC isolation was adapted from previously published protocols (17, 18). Unless otherwise noted, all steps were performed on ice/at 4°C and under sterile conditions. Pulmonary tissue was dissected from animals after exsanguination. After gross mechanical dissociation with razor blades, tissue was incubated for 45 min at 37°C in a type-1 collagenase solution. Tissue was then homogenized into a single cell suspension by disruption via a Seward Stomacher 80 Biomaster for 240 s on high, and then passage through a 70-μm cell strainer. Endothelial cells (CD45-/CD31+) were then sorted using a magnetic microbead sorting system (MACS, Miltenyi Biotec). Nonspecific antibody binding was prevented by preincubation with 5% normal rabbit serum for 10 min before sorting.
After isolation, ECs were suspended in 1 mL of TriZol reagent and then snap frozen in liquid nitrogen. Total RNA was later extracted using the TriZol reagent method (Invitrogen, catalog number 15596-026). The quality of total RNA samples was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif).
SuperArray quantitative reverse transcription polymerase chain reaction analysis
Reverse transcription was performed on total RNA isolated from mouse pulmonary microvascular ECs and processed with Applied Biosystems (Foster City, Calif) High-capacity cDNA archive kit first-strand synthesis system for reverse transcription polymerase chain reaction (RT-PCR) according to the manufacturer's protocol. Quantitative RT-PCR (qRT-PCR) was performed using the RT2 Profiler PCR array from SuperArray (Gaithersburg Md). The RT2 Profiler PCR arrays are designed for relative qRT-PCR based on SybrGreen detection and performed on a one sample/one plate 96-well format using primers for a preset list of genes corresponding to a particular biological pathway. The specific array type included here was the EC biology PCR arrays (PAMM-015). In brief, cDNA volumes were adjusted to about 2.5 mL with SuperArray RT2 real-time SYBR Green/ROX PCR 2× Master Mix (PA-012). A total of 25 μL of cDNA mix was added to all wells. The PCR plate was sealed, spun at 1500 rpm × 4 min, and real-time PCR was performed on an Applied Biosystem (Foster City, Calif) 7300 Real Time PCR System. ABI instrument settings include setting reporter dye as "SYBR," passive reference is "ROX." Delete UNG Activation, and add Dissociation Stage.
Relative gene expressions were calculated using the 2-ΔΔCt method, in which Ct indicates cycle threshold, the fractional cycle number where the fluorescent signal reaches detection threshold (19). The normalized ΔCt value of each sample is calculated using up to a total of five endogenous control genes (18S rRNA, HPRT1, RPL13A, GAPDH, and ACTB). Fold change values are presented as average fold change = 2-(average ΔΔCt) for genes in treated relative to control samples.
Cluster and heat map analysis was performed using the Cluster and Treeview software from the Eisen Lab (http://rana.lbl.gov/EisenSoftware.htm).
Rat lung microvascular ECs (RLMVEC) were purchased from Vec Technologies (Rensselaer, NY; http://www.vectechnologies.com/). Cells were cultured at 37°C, 5% carbon dioxide on fibronectin-coated flasks in MCDB-131 complete media according to the recommendations of Vec Technologies. All experiments were performed on early passage cells (p4-p9) to preserve true EC phenotype.
The RLMVECs (passage 4) were plated on fibronectin-coated sides and allowed to reach confluence. They were treated with rat serum from animals that underwent either sham surgery or IRI at 5% and 10% for 24 h and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with 3% Triton X-100 and blocked with 10% normal goat serum for 1 h before staining with both AlexaFluor 488 phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) staining. All images were taken at 20× magnification.
Annexin V/propidium iodide analysis
The RLMVECs (at passage 4) were plated in 12-well plates on fibronectin and allowed to reach confluence. They were treated with rat serum from animals that underwent either sham surgery or IRI at varying concentrations (5%, 10%, or 25%). At 24 h, apoptosis was assessed by AnnexinV/propidium iodide (PI) staining. Briefly, both floating and adherent cells were collected from each well and stained with Annexin V/PI for 15 min at RT. Cells were fixed in 1% paraformaldehyde, and FACS analysis was performed the next day. Results are expressed as percentage of cells that were Annexin V (+)/PI(-) as a marker of apoptosis. n = 3 wells per group, and experiments were repeated three times.
Intercellular adhesion molecule 1 Western blot
The RLMVECs (at passage 4) were plated in 12-well plates on fibronectin and allowed to reach confluence. They were treated with 10% rat serum from animals that underwent either sham surgery or IRI. At 24 h, the cells were homogenized immediately after isolation in buffer containing 20 mM HEPES, 51.5 mM MgCl2, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1× Protease Inhibitor Cocktail Set I (Calbiochem, no. 539131), 500 μM 4-(2-aminoethyl) benzenesulfonyl fluoride, 150 nM aprotinin, 1 μM E-64, 500 μM EDTA, and 1 μM leupeptin. Homogenates were centrifuged at 10,000g for 15 min, and the supernatants stored at -80°C until used for Western immunoblot.
Aliquots from cellular homogenates prepared as described above were assayed for protein measurement using the Bradford protein assay. Equal amounts of protein (50 μg) were then loaded in each well of 12% Tris-glycine gels (BioRad, Hercules, Calif). After electrophoresis for 90 min at 125 V of constant voltage, the gel was blotted onto a nitrocellulose membrane by electrophoretic transfer at 70 V of constant voltage for 1 to 2 h. The membrane was then washed, blocked with 5% blocking solution, and probed with intercellular adhesion molecule 1 (ICAM-1) antibody (Cell Signaling, Danvers, Mass). The immunoreactive bands were visualized using a secondary antibody conjugated to horseradish peroxidase and a chemiluminescent detection system (Pierce, Rockford, Ill). Nitrocellulose blots were visualized using a BioRad Gel doc 1000 (BioRad, Hercules, Calif).
Data are expressed as mean ± SD and were analyzed via one-way analysis of variance, with pairwise multiple comparison procedures performed via the Holm-Sidak method. Values of P < 0.05 were considered statistically significant.
Renal function during experimental AKI
Mice (∼25 g) underwent 60 min of bilateral kidney IRI, BNx, or sham surgery, and were then killed at either 4 or 24 h postprocedure. Serum creatinine level was measured before surgery and at the time of sacrifice. Both bilateral IRI and nephrectomy resulted in a significant increase in SCr level at both time points, whereas sham surgery did not (Fig. 1).
Microbead sorting leads to isolation of a pure EC population
Whole lung tissue was digested and homogenized into a single cell suspension and pulmonary ECs were isolated using a microbead sorting system (Miltenyi Biotec). Cells were first incubated with antibodies against CD31 and CD45 (Fig. 2B). Cells were then negatively sorted to remove CD45+ cells, and then a second positive sort enriched the CD31+ pool. This resulting in a homogenous population (∼90% pure) of CD31+ ECs (Fig. 2C). For each experiment, lung tissue from five mice was pooled to isolate enough cells/RNA for analysis.
AKI-induced changes in pulmonary vascular endothelial gene expression
To investigate the effects of AKI on pulmonary ECs, we performed either kidney IRI, BNx, or sham surgery on mice as previously described. At either 4 or 24 h after injury, mice were killed, and lung tissue was pooled and harvested. After isolation of pulmonary ECs, total mRNA was extracted and analyzed for endothelial-specific gene expression by a novel qRT-PCR array (SABiosciences).
Figure 3 is a heatmap representation of mRNA expression in pulmonary ECs after AKI. Fold changes compared with sham surgery are represented by color and intensity, with red indicating increase in gene expression and green signifying a decrease. In addition, hierarchal cluster analysis reveals functional groupings for several genes, including those associated with inflammation, apoptosis and stress response, vascular reactivity, and angiogenesis. Notably, there was no significant difference in gene expression levels between normal mice that did not undergo surgery and those that underwent sham surgeries (data not shown). Specific genes exhibiting fold changes greater than 1.5× (increased or decreased) are presented in Table 1.
We identified prominent early and sustained activation of genes related to leukocyte activation and trafficking including E-selectin, P-selectin, and ICAM-1. We also noted activation of early injury response genes IL-6 and inducible nitric oxide synthase (NOS-2) and later activation of genes related to cell motility and angiogenesis such as plasminogen activator inhibitor-1 (Serpine-1). At 24 h, there was significant downregulation of the prominent proapoptotic gene Fas ligand. Interestingly, we have previously demonstrated a prominent role for the TNF receptor-1 (TNFR1) in AKI-induced lung apoptosis, and these data may lend further support that TNFR1 is the primary death signaling receptor involved in this model (20).
AKI induces actin reorganization and apoptosis in pulmonary microvascular ECs
To further investigate the apoptosis-specific effects of AKI on pulmonary ECs, an in vitro system was developed. Rat lung microvascular ECs were obtained and cultured in recommended conditions. At early passage, cells were allowed to reach confluence and then grown for 24 h in serum from rats that had undergone either IRI or sham surgery. Cells were then fixed and double stained with fluorescent-conjugated phalloiden and DAPI to evaluate actin cytoskeletal organization as well as nuclear morphology. Exposure to IRI serum for 24 hours led to noticeable changes in actin and nuclear morphology (Fig. 4). Compared with sham serum, actin stress fiber formation was evident in cells grown in IRI serum. In addition, there were notable perinuclear actin fibers in the IRI group. Finally, by DAPI staining, the IRI-exposed cells exhibit nuclei that are large and abnormally shaped compared with sham cells (4B).
Apoptosis was then assessed and quantified by Annexin V/PI analysis (Fig. 5). Twenty-four hours after cells were exposed to either IRI or sham serum at varying concentrations (5%, 10%, or 25%), they were stained with fluorescein-tagged Annexin V and PI and then analyzed by FACS. Cells exposed to 10% and 25% IRI serum had significantly more apoptosis (Annexin V positive, PI negative) than those exposed to sham serum in a dose-dependent fashion.
Using this novel in vitro system, we further validated our findings of specific EC transcriptional changes by measuring ICAM-1 protein expression by Western blot in RLMVECs after 24 h exposure to rat serum from untreated (i.e., normal), sham-, and IRI-treated rats. Cells exposed to IRI serum demonstrated increased protein expression compared with normal or sham-treated rats (Fig. 6).
Clinical data suggest that AKI contributes to the development and exacerbation of multiorgan failure, although the physiological and molecular mechanisms responsible are largely unknown. The link between AKI and pulmonary dysfunction is particularly important, given the significant clinical morbidity and mortality associated with the presence of both pathologies. Understanding the ways in which AKI influences pulmonary pathophysiology is a crucial step in improving outcomes during multiple organ dysfunction syndrome.
Previous animal studies have shown that there are derangements in the innate immune response, inflammatory cascade, and the response to oxidative stress during AKI (reviewed in 21). For example, AKI in several models (60 min bilateral IRI, BNx, or bilateral pedicle ligation) induced pulmonary injury and neutrophil infiltration that was similar in nature to that induced by sepsis (22). Additional studies have revealed that proinflammatory and anti-inflammatory cytokines appear in some fashion to mediate AKI-associated lung damage (10, 20).
Given that interactions between the kidney and the lung in the setting of renal injury have so far been shown to be mediated by either serum factors such as interleukins or serum cellular components such as neutrophils or macrophages, we hypothesized that one likely target for such factors would be the pulmonary endothelium. The pulmonary microvascular endothelium is central to the development of both acute lung injury (ALI) and ARDS (reviewed in Maniatis and Orfanos 23). Cytoskeletal rearrangement, changes in intracellular signaling, and interactions with circulating inflammatory cells promote the breakdown of the endothelial barrier and development of pulmonary edema. There is also evidence that early EC activation is both necessary and sufficient for the further development of multiple organ dysfunction syndrome (24).
Many alterations in the pathophysiology of pulmonary vascular ECs should be reflected by changes in mRNA expression, particularly in those genes responsible for cellular adhesion, intracellular signaling and initiation of apoptosis. We therefore developed a strategy to isolate murine pulmonary ECs after AKI and to analyze mRNA expression of various genes using novel RT-PCR arrays. Unlike cDNA arrays, qRT-PCR arrays allow for reliable analysis of expression of a focused panel of genes, with amplification of various gene-specific products simultaneously under uniform cycling conditions. This provides the PCR array with high-specificity and high-amplification efficiencies. Housekeeping genes are included in the experiment to control for variability in PCR performance.
Pulmonary ECs were isolated 4 or 24 h after injury by a magnetic sorting protocol using a negative/positive sort strategy (CD45-/CD31+). One limitation of this study is that even after magnetic bead sorting, our population of pulmonary ECs was, at best, approximately 90% pure by flow cytometry (Fig. 2). We found pulmonary ECs to be fragile and attempted to maintain an appropriate balance between purity of population and loss of cells during the isolation process. Although our populations were not 100% pure, however, we felt that this still represented a major improvement over previous experiments using whole lung tissue and was warranted given the opportunity to examine cell response in an in vivo model.
We performed differential RT-PCR on total RNA from these pulmonary ECs at both early and late time points. Key genes involved in inflammation, such as E-selectin, revealed small fold increases (∼2×) 4 h after injury and much larger increases in expression 24 h after injury when compared with sham. The ICAM-1 expression also increased 24 h after AKI, approximately 5-fold after IRI and 10-fold after BNx. E-selectin and ICAM-1 expression are known to correlate with inflammatory damage in pulmonary endothelium and surrounding tissues by promoting inflammatory cell recruitment (25, 26); recent studies have also revealed increased levels of membrane-bound and circulating E-selectin and ICAM-1 in patients with sickle cell disease, with elevated protein levels associated with increased morbidity and mortality (27). Upregulation of leukocyte adhesion factors is one of the hallmarks of EC activation (28).
There were also several changes in gene expression related to angiogenesis, vascular reactivity, and thrombosis. Plasminogen activator inhibitor 1 (PAI-1 or Serpine 1) was noted to have increased expression 24 h after AKI. Notably, elevated levels of PAI-1 in pulmonary edema fluid have been associated with increased mortality (29). The PAI-1 has also recently been shown to inhibit phagocytosis of neutrophils and apoptotic cells (30); increased expression of PAI-1 in the lung could, therefore, perpetuate and promote the inflammatory cascade and subsequent tissue injury. Reverse transcription PCR array analysis also revealed differential expression of endothelin-1 (ET-1) and ET-2, as well as inducible NOS (iNOS). Endothelin-2 changed most appreciably at both early and late time points, whereas changes in the expression of ET-1 and iNOS were most notable 4 h after injury. Endothelin-2 is a potent chemoattractant for macrophages (31); its upregulation may allow for macrophage infiltration into lung parenchyma, contributing to inflammation and subsequent pulmonary injury. These changes in gene expression suggest a transition to a prothrombotic state, another feature of EC activation (32).
Clearly, AKI induces distant organ changes in apoptosis-related gene expression; to investigate whether functional apoptosis might be occurring in pulmonary endothelium after IRI, we turned to an in vitro cell system. Rat pulmonary microvascular ECs were cultured in the presence of serum from animals that had undergone IRI or sham surgery. Lung microvascular ECs are very difficult to culture, and at the time of this study, the only cells available were rat instead of mouse. We first evaluated actin architecture to look for qualitative changes in cytoskeletal arrangement indicative of early apoptosis. Classically, actin reorganization and nuclear disorganization/breakup are hallmarks of apoptosis (33). Compared with cells treated with sham serum, cells exposed to IRI serum exhibited actin rearrangement, including thick stress fiber formation, as well as intercellular gap formation. Apoptosis was confirmed by Annexin V/PI staining and analysis via FACS. Annexin V is used to detect phosphatidylserine translocation from the inner to the outer plasma membrane, a hallmark of early apoptosis. We found that cells exposed to IRI serum showed significant increases in apoptosis in a dose-dependent fashion compared with those exposed to sham serum. Endothelial cell apoptosis has been postulated to be another pathway by which lung injury evolves in both sepsis and emphysema (34, 35); it may be possible that it also promotes ALI in the setting of AKI given these findings.
This is the first study to demonstrate detailed pulmonary endothelial-specific downstream consequences of AKI, and it builds on our previous work that identified whole lung proinflammatory and proapoptotic changes during ischemic AKI. We used a newly developed method to isolate pulmonary ECs ex vivo after AKI and analyzed differential mRNA expression to help elucidate interactions between AKI and pulmonary injury. Acute kidney injury leads to specific changes in the EC phenotype consistent with activation. These include upregulation of leukocyte adhesion factors as well as changes that may indicate a transition to a more prothrombotic state. In addition, we used an in vitro model to further reveal the functional pulmonary endothelial apoptotic events that occur after AKI. Future studies can focus on the specific AKI regulated genes and pathways we have identified in pulmonary ECs to investigate the mechanisms by which AKI predisposes to pulmonary dysfunction and ARDS.
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