1 Departments of Medicine, Physiology, and Biophysics and Program in Bioengineering, State University of New York at Stony Brook, Stony Brook, New York 11794-8152; 2 Departments of Urology and Medical Engineering, Kawasaki Medical School, Okayama 701-0114; and 3 Department of Electrical Engineering, Okayama University of Science, and 4 Department of Cardiovascular Physiology, Okayama University School of Medicine and Dentistry, Okayama 700-0005, Japan
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ABSTRACT |
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There is accumulating circumstantial evidence suggesting that endothelial cell dysfunction contributes to the "no-reflow" phenomenon in postischemic kidneys. Here, we demonstrated the vulnerability of in vitro, ex vivo, and in vivo endothelial cells exposed to pathophysiologically relevant insults, such as oxidative and nitrosative stress or ischemia. All of these stimuli compromised the integrity of the endothelial lining. Next, we performed minimally invasive intravital microscopy of blood flow in peritubular capillaries, which provided direct evidence of the existence of the no-reflow phenomenon, attributable, at least in part, to endothelial injury. In an attempt to ameliorate the hemodynamic consequences of lost endothelial integrity, we transplanted endothelial cells or surrogate cells expressing endothelial nitric oxide synthase into rats subjected to renal artery clamping. Implantation of endothelial cells or their surrogates expressing functional endothelial nitric oxide synthase in the renal microvasculature resulted in a dramatic functional protection of ischemic kidneys. These observations strongly suggest that endothelial cell dysfunction is the primary cause of the no-reflow phenomenon, which, when ameliorated, results in prevention of renal injury seen in acute renal failure.
endothelial cell transplantation; renal ischemia; intravital videomicroscopy; erythrocyte velocity; peritubular capillaries
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INTRODUCTION |
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THE PATHOPHYSIOLOGY OF ACUTE renal failure (ARF) has been tightly linked to tubular epithelial cell injury. In fact, most theories are based on this pathological feature, i.e, back-leakage, tubular obstruction, calcium overload, loss of cytoskeletal integrity, and loss of cell-matrix adhesion, all consider tubular epithelial cells as the main culprit (1, 17, 19, 29, 30, 34, 35, 37, 40, 41, 46, 52). Ischemic or nephrotoxic insults are believed to target principally and primarily the epithelium of the tubules, relegating to the endothelium a functional role in maintaining vasoconstriction (5).
However, three decades ago, Flores et al. (13) demonstrated that endothelial cells in the renal vasculature undergo an early swelling, leading to the narrowing of the lumen, an observation conceptualized in the "no-reflow" hypothesis (23, 43). This hypothesis has been supported by the observation that ischemic renal vasculature is characterized by a profound loss of acetylcholine-induced vasorelaxation (25). Conger et al. (7, 8) have demonstrated that vasorelaxation in response to stimuli generating endothelium-derived relaxing factor was inhibited in ARF. In addition, nitric oxide (NO) production in response to bradykinin was found to be suppressed in ischemic kidneys (33). Overexpression of intracellular adhesion molecule-1 (ICAM-1) by the vascular endothelium of the ischemic kidney has been demonstrated to play a major pathophysiological role in the development of renal dysfunction, and neutralizing anti-ICAM-1 antibodies significantly improved the outcome of renal ischemia (21). Furthermore, we have previously demonstrated that the endothelium of renal microvessels in ischemic kidneys show a loss of polarity in the expression of Arg-Gly-Asp-binding integrins, similar to that seen in the tubular epithelium (39). Most recently, permanent damage to peritubular capillaries has been discovered in rats subjected to renal ischemia (2). Collectively, these observations may be indicative of endothelial cell activation and dysfunction in ARF. If so, it becomes important to examine whether endothelial cell dysfunction is a cause of the tubular epithelial cell injury, its consequence, or an independent variable not affecting the course of ARF.
Here, we demonstrated the vulnerability of in vitro, ex vivo, and in vivo endothelial cells exposed to pathophysiologically relevant insults, such as oxidative and nitrosative stress, or to ischemia. These stimuli distorted the integrity of endothelial layers by desquamating or retracting cells. Next, we performed minimally invasive intravital microscopy of blood flow in peritubular capillaries, which directly demonstrated the existence of the no-reflow phenomenon. Finally, we showed that injection of endothelial cells or surrogate cells expressing endothelial NO synthase into rats subjected to renal artery clamping resulted in the implantation of these cells in the renal microvasculature. This was associated with a dramatic functional protection of ischemic kidneys. These observations strongly suggest that endothelial cell dysfunction is the primary cause of the no-reflow phenomenon, which, when ameliorated, results in prevention of tubular epithelial cell injury seen in ARF.
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MATERIALS AND METHODS |
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Cell culture. Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics and maintained in EBM-2 medium under conditions of 37°C and 95% air-5% CO2. HUVEC were used between passages 3 and 5. Before injection, cells were loaded with CellTracker (Molecular Probes) according to the manufacturer's protocol. Briefly, confluent HUVEC were lifted with trypsin-EDTA and suspended in EBM-2 medium. CellTracker was added (10 µM), and cells were incubated for 5 min at 37°C and then for an additional 15 min at 4°C. Wild-type and human embryonic kidney cells (HEK-293) stably expressing human endothelial NO synthase (eNOS), established by Liu et al. (26), were kindly provided by Dr. S. S. Gross (Cornell Medical College).
After being washed with PBS, cells were resuspended in EBM-2 (serum and growth factor free) at a final concentration of 5 × 106 cell/ml. The cell suspension was kept on ice until transplantation, but not longer than 30 min. Before injection, a small volume (5 µl) of cell suspension was aliquoted and replated on 35-mm dishes, and cell viability was tested 2 h later: >90% of cells excluded trypan blue. Simultaneously, fluorescence images of labeled cells were obtained to confirm the completeness of cell labeling.Surgical procedure. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and approved by the Institutional Animal Care and Use Committee. Male athymic nude rats weighing 160-180 g were allowed food and water ad libitum for 2 wk of recovery after transportation. After an overnight fast, animals were anesthetized with a combination of ketamine hydrochloride (6.0 mg/100 g) and xylazine hydrochloride (0.77 mg/100 g). The animals were placed on a heated surgical pad, and rectal temperature was maintained at 37°C. A subcutaneous injection of 250 units/kg heparin was given 15 min before the operation. A 2.5-cm midlaparotomy was performed, the right kidney was exposed, and two 3-0 sutures were passed under the renal pedicle in all rats. The left kidney was exposed, the renal artery was separated from the renal vein, and underpassed with a 3-0 suture. Renal ischemia (ischemic group) was initiated by clamping the left renal artery with microserrefines (Fine Science Tools, Forster City, CA). After 45 min, the left renal artery was released, while the right renal pedicle and the right ureter were ligated, and a right nephrectomy was performed. After release of the clamp, 106/0.2 ml HUVEC labeled with CellTracker (Molecular Probes) were injected into the right jugular vein or the aorta (through a catheter inserted into the left carotid artery). The incision was closed with a 3-0 suture and surgical staples. Blood was drawn for determination of plasma creatinine concentration, using a Raichem kit (San Diego, CA) before and 24 h after the surgery. Sham-operated rats (right nephrectomy only) receiving intra-aortic infusion of the same dose of HUVEC served as controls. Tissue specimens (kidney, lung, liver, and spleen) were collected 24 h after clamp release for fluorescence and histochemical analyses. The scoring of renal injury was performed in a blinded fashion, as previously described (33).
Intravital microscopy and analysis of peritubular blood flow. Experiments were performed in animals surgically prepared as described above. Renal cortical peritubular capillaries were visualized by using a charge-coupled device videomicroscope, with a pencil-lens probe having a tip diameter of 1 mm. The probe has a magnification of ×520 and spatial resolution of 0.86 µm, permitting identification of individual erythrocytes (53, 54). Peritubular capillary blood flow was recorded on digital videocassette tapes before and after renal artery clamping. After 45-min renal artery occlusion, the clamp was removed, and reflow was initiated. The consecutive images of blood flow were collected at a rate of 30 frames/s for 60 min and at 24 h postoperatively. Images were analyzed using the freeze-frame mode. The velocity of red blood cells in individual segments of the peritubular capillaries was analyzed using a point-tracking method and motion-detection program (see Fig. 3C) written specifically for the study of peritubular capillary blood flow.
Cell detachment assay. HUVEC were cultured in 35-mm culture dishes. After cells were washed with PBS, 2 ml of Krebs-HEPES buffer with or without H2O2 or peroxynitrite, at final concentrations of 50 µM each, were added to each dish. Cells were incubated at room temperature on a Speci-Mix rocker shaker (Thermolyne, IA) at 12 rocks/min, 30° incline, and 10-µl aliquots were collected at specified times. The number of detached cells was counted using a Levy-Hausser counting chamber (Electron Microscopy Sciences). All experiments were repeated three times.
Histochemical techniques and scanning electron microscopy. Silver nitrate staining of endothelial cells was performed according to the previously described technique (12, 28). Briefly, anesthetized rats were perfused (through a catheter inserted into the left carotid artery) with 350 ml of fixative [1% paraformaldehyde and 0.5% glutaraldehyde in 75 mmol/l of cacodylate buffer (pH 7.4)] at a pressure of 120-140 mmHg for 5 min, followed by 80 ml of 0.9% NaCl for 2 min, 25 ml of 5% glucose for 10 s, 20 ml of 0.2% silver nitrate for 7 s, 25 ml of 5% glucose for 10 s, and 50 ml of fixative for 1 min.
After perfusion, the kidneys were removed, postfixed, and sectioned (30 µm thickness) using M-1 embedding matrix for frozen sections (LiPSHAW, Pittsburgh, PA). The silver halide was developed by a 15-min exposure to light emitted by a 150-W bulb. Finally, the kidney sections were dehydrated in alcohol, cleared in toluene, mounted in Permount, and observed with a Nikon Diaphot microscope. For counting of implanted CellTracker-labeled cells, HUVEC or HEK, kidneys were analyzed using confocal microscopy (Odyssey) with a 2-µm distance between consecutive focal planes. The number of cells in 50 separate areas was calculated for each group. Scanning electron microscopy was carried out on critical point-dried cells, as previously described (18).Statistical analysis. Statisical analysis was performed using a paired or unpaired t-test and/or ANOVA followed by Tukey's posttest, with P < 0.05 considered significant. Comparison of the renal injury score among different groups was carried out using nonparametric Kolmogorov-Smirnov analysis. Correlation analysis among the parameters of cell implantation, renal injury score, and plasma creatinine concentration was performed using nonparametric Spearman's correlation.
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RESULTS |
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Oxidative and nitrosative stress impair the integrity of
endothelial layers in vitro and in vivo.
Confluent HUVEC were exposed to hydrogen peroxide (50 µM) or
peroxynitrite (50 µM). Peroxynitrite resulted in a rapid detachment of endothelial cells (Fig.
1A), leading to the gradual
loss of integrity of HUVEC monolayers. The latter was confirmed using scanning electron microscopy of endothelial monolayers subjected to
peroxynitrite (50 µM), which showed frequent gaps in its integrity (Fig. 1B). This finding is similar to that described by
Nakamura et al. (31), who have observed a
"peeling-off" phenomenon in endothelial cells subjected to the
stimulated neutrophils or the neutrophil-derived oxidant
NH2Cl.
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Intravital microscopy of the blood flow in peritubular capillaries.
Cortical blood flow in peritubular capillaries was visualized by
intravital minimally invasive microscopy using a pencil-lens probe. In
animals subjected to 45-min renal ischemia, the initially robust capillary blood flow partially recovered immediately after the
release of the renal artery clamp, although many capillary loops
remained nonperfused. However, within a minute blood flow again became
stagnant for the next 3.6 ± 1.6 min (n = 11),
after which time red blood cell velocity gradually recovered to
approximately one-third of the initial speed. During this recovery
process, many capillaries remained nonperfused for longer periods (Fig. 3a), probably explaining the
characteristic patchiness of renal injury in acute renal
ischemia. Recovery of the blood flow was nonlinear, displaying
an oscillating pattern. During the recovery phase, the blood flow in
individual capillaries exhibited both the orthograde and retrograde
flow (not shown). Twenty-four hours after the release of the renal
artery clamp, average red blood cell velocity in the peritubular
capillaries was 227.5 ± 113 µm/s (see also Fig. 7). These
findings directly demonstrate the existence of a profound defect in the
perfusion of peritubular capillaries of ischemic kidney.
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In vivo injection of HUVEC improves renal function
after renal artery cross-clamping.
We argued that the loss of integrity of the endothelial layer,
occurring in vivo after acute renal ischemia, may lead to the paradoxical vasoconstriction in response to endothelium-dependent vasorelaxing stimuli, similar to that observed by Furchgott and Zawadzki (14) in vascular segments with denuded
endothelium. Given the possibility that the circulating endothelial
cells may become implanted at the sites of endothelial denudation,
athymic nude rats received a single injection of HUVEC (106
cells in 0.2 ml of serum-free culture medium) after a sham operation or
after the release of the renal artery clamp. In rats that received 0.2 ml of a vehicle alone (serum-free cell culture medium), renal artery
cross-clamping resulted in a significant elevation of plasma creatinine
concentration (1.36 ± 0.2 vs. 0.38 ± 0.05 mg/dl in control,
Fig. 4A). Intravenous infusion
of HUVEC after the release of the renal artery clamp prevented the
elevation of plasma creatinine concentration (0.66 ± 0.09 mg/dl,
P < 0.01). The injection of HUVEC was also associated
with a lesser degree of renal injury (Fig. 4B), scored in a
blinded fashion, according to the previously described criteria
(33). These data suggested that circulating exogenous
endothelial cells could protect the kidney against ischemic injury.
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In vivo injection of HEK cells stably expressing
human eNOS improves renal function after renal artery
cross-clamping.
To elucidate the functional role of defective eNOS generation of NO
(23) and its improvement by implantation of intact
endothelial cells, in the next series of experiments we utilized HEK
cells stably expressing human eNOS (HEK/eNOS) as surrogates for
endothelial cells. In vivo intra-aortic infusion of HEK/eNOS cells
after release of the renal artery clamp resulted in the amelioration of
renal dysfunction (Fig. 7A).
In contrast, wild-type HEK cells, devoid of eNOS, did not affect the
degree of renal dysfunction 24 h postischemia. Similar
results were obtained in rats injected with HEK cells stably expressing
the palmitoylation-deficient eNOS (HEK/G2A), which has been previously
demonstrated to abolish the ability to produce NO (26).
Despite these functional differences, the density of implantation of
wild-type and eNOS-expressing HEK cells was not significantly different
in ischemic kidneys and was again significantly lower in
kidneys from sham-operated animals (Fig. 7B).
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DISCUSSION |
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The data presented herein demonstrate the vulnerability of endothelial cells to nitrosative and oxidative stress in vitro and renal artery cross-clamping in vivo, suggesting that the vascular endothelium may represent one of the targets in renal ischemia. Furthermore, animals injected with a suspension of intact endothelial cells showed that these cells implanted in the renal microvasculature, a phenomenon which was associated with the improved renal function. These data lend support to the idea that endothelial cell dysfunction represents a proximal pathophysiological trigger for ensuing epithelial cell damage in acute renal ischemia.
The previous paradoxical observation that nonselective NOS inhibitors exaggerate renal damage after renal artery clamping, whereas selective inhibition of inducible NOS (iNOS) protected renal function against ischemia, alluded to the critical role of endothelial function in determining the outcome of renal ischemia (33). Together with the demonstration of reduced NO production by ischemic kidneys stimulated with bradykinin (33), these findings implicated eNOS as an important contributor to the loss of kidney function after renal artery cross-clamping.
It has been demonstrated that tissue ischemia and cytokines mobilize bone marrow-derived endothelial progenitor cells (45) and that circulating endothelial cells of hematopoietic origin are recruited to ischemic areas to form adult blood vessels (10). Hence, one may argue that the observed amelioration of renal dysfunction after renal artery clamping could be explained by the above mechanism. Both processes, however, occur on a time scale of 3-7 days to several weeks, respectively, thus effectively precluding de novo angiogenic mechanisms as major contributors to the observed dramatic improvement in postischemic renal function. We therefore concluded that pathophysiologically relevant events should take place in the early postischemic period. This conclusion was further supported by the previous observation of Bird et al. (3) indicating successful amelioration of renal ischemic injury on improvement of microcirculation using the antioxidant probucol.
Minimally invasive intravital microscopy of peritubular capillary blood flow in control and postischemic kidneys provided the direct characterization of renal microvascular hemodynamics and confirmed the existence of the no-reflow phenomenon. This was characterized by a sudden cessation of the peritubular capillary flow within 1-3 min after the removal of the clamp, followed by a gradual and partial recovery of the microcirculatory blood flow. During the recovery process, restoration of blood flow was not uniform but rather sporadic capillaries showed stagnation or cessation of flow. Twenty-four hours postischemia, blood flow in the peritubular capillaries remained severely impaired in nontreated or vehicle-treated animals but improved significantly in animals transplanted with HUVEC and HEK/eNOS. The question arises, Is NO generated by a relatively small number of engrafted cells sufficient for improving renal microcirculation? The observed chaotic distribution of endothelial injury and/or denudation scattered in renal microvasculature is in concert with the apparently chaotic involvement of peritubular capillaries in no-reflow. Assuming that a significant proportion of transplanted cells engraft in the areas of denudation, it is possible that, by preventing no-reflow at these scattered sites, even a relatively small number of engrafted NO-producing cells could improve microcirculation.
The salient finding that endothelial cells can rescue the function of an ischemic organ consisting of different cell types illustrates a more general principle of cell-cell interaction, in which the dysfunction of one cell type affects the functions of other cells. For instance, abdominal radiation injury, known to produce severe diarrhea and damage to the crypts of Lieberkuhn, was previously attributed to the lethal damage of epithelial stem cells. However, recent evidence indicates that the primary lesion in intestinal radiation syndrome occurs in microvascular endothelial cells (36). Epithelial cell exfoliation triggered by abnormalities in the vasculature have been shown in the gastric mucosa. Injury to the gastric microvasculature or severe vasoconstriction induced by endothelin-1 caused an almost complete exfoliation of the interpit cells and apoptosis of superficial cells of gastric mucosa, with the eventual formation of ulcers (42). Exposing gastric mucosa to aspirin resulted in exfoliation of surface epithelium and deep mucosal necrosis, which was preceded by microvascular injury manifesting itself as the rupture of capillary walls, necrosis of the endothelium, deposition of fibrin, and platelet adhesion (47). Similar findings were reported after intragastric administration of ethanol in healthy volunteers (48). Together with our findings, these data may allude to a more general principle: endothelial dysfunction leads to injury of nonendothelial cells located within the basin of a feeding capillary.
Therapeutic strategies based on the use of endothelial progenitor cells have been described. In rats with acute myocardial ischemia induced by ligation of the left anterior descending coronary artery, transplantation of endothelial progenitor cells partially rescued left ventricular function (20). This effect was attributed to improved angiogenesis in the ischemic myocardium, although the possible role of improved vascular function was not addressed in the study. In diabetic mice, but not in control animals, transplantation of blood-derived angioblasts accelerated the restoration of blood flow to an ischemic hindlimb (40). This differential response in diabetic and control animals may be related to the preexisting endothelial dysfunction in animals that benefited from angioblast transplantation (reviewed in Ref. 5). However, in acute renal ischemia, we reasoned that the use of endothelial progenitor cells did not appear to represent the strategy of choice due to the protracted period of differentiation of transplanted cells (10, 45) and gave preference to the use of fully differentiated HUVEC for transplantation.
Recently accumulated evidence suggests that many aspects of endothelial dysfunction are intimately linked to the expression and function of eNOS and/or bioavailability of NO (27). In particular, NO generation inhibits platelet aggregation and adhesion of leukocytes to the vascular endothelium (11, 22, 24, 32, 44, 49). Endothelial regulation of vascular smooth muscle relaxation, proliferation, and migration is, in part, governed by the integrity of the L-arginine-eNOS-NO system (15, 38, 51). In addition, vascular/endothelial permeability and some synthetic functions of endothelial cells have been linked to the activity of eNOS (reviewed in Ref. 6). Hence, NO production or availability can regulate diverse functions in endothelial cells per se and their interaction with circulating formed elements (both inflammatory and thrombogenic interactions) and vascular smooth muscle cells. Despite the fact that NO generation in epithelia of ischemic kidneys is excessive due to the induction of iNOS, endothelial generation of NO appears to be compromised (33), thus participating in the pathogenesis of no-refow. The previous suggestion that the lack of endothelium-dependent vasorelaxation in postischemic kidneys is due to the maximal stimulation of eNOS (9), mainly on the basis of the increased expression of eNOS 1 wk after ischemic insult, perhaps reflects on "uncoupled" eNOS generating superoxide but has no direct relevance to the present findings.
On the basis of these considerations, we performed an additional series of experiments utilizing HEK cells stably expressing eNOS or its palmitoylation-deficient mutant. The results of these experiments demonstrated that the implantation of cells expressing the functional eNOS, not necessarily endothelial cells per se, was sufficient to ameliorate renal dysfunction accompanying acute ischemia. These findings further implicate eNOS and endothelial cell dysfunction in the initiation of pathophysiological cascades leading to ischemic ARF.
The studies presented herein were not designed to define a therapeutic approach to prevention of ARF with injected endothelial cells. Rather, they provide a proof of the principle that endothelial cell dysfunction triggers the pathophysiological cascade of events resulting in ischemic renal injury. Endothelial cell dysfunction should be seen in a broader context of the "fight-or-flight" cellular reaction to stress (16). This reaction has been proposed to serve as a default mechanism, leading to cell detachment under stress situations. Therefore, it is possible that endothelial cell dysfunction is not an isolated feature of ischemic acute renal injury but may be a companion of renal injury initiated by other etiological factors.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. Leon Moore (Dept. of Physiology, SUNY Stony Brook) for critically reading the manuscript.
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FOOTNOTES |
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* S. V. Brodsky and T. Yamamoto contributed equally to this study.
These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45462, DK-54602, and DK-52783 (M. S. Goligorsky), American Heart Association Fellowship 0120200T (S. V. Brodsky), and sabbatical support from the Catholic University of Seoul, South Korea (B. Kim).
Address for reprint requests and other correspondence: M. S. Goligorsky, Dept. of Medicine, New York Medical College, Valhalla, NY 10595 (E-mail: Michael_Goligorsky{at}NYMC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 18, 2001;10.1152/ajprenal.00329.2001
Received 30 October 2001; accepted in final form 15 December 2001.
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