Minocycline reduces renal microvascular leakage in a rat model of ischemic renal injury
http://www.100md.com
《美国生理学杂志》
Division of Nephrology, Department of Medicine and the Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis, Indiana
ABSTRACT
Tetracyclines exhibit significant anti-inflammatory properties, inhibit matrix metalloproteinases (MMPs), and are protective in models of ischemia-reperfusion injury (IRI). Both inflammatory cascades and MMP activation have been demonstrated to modulate microvascular permeability. Because increased microvascular permeability occurs during IRI in a variety of organ systems including the kidney, we hypothesized that minocycline, a semisynthetic tetracycline, would diminish microvascular leakage during renal IRI. To test this hypothesis, we used intravital 2-photon microscopy to examine leakage of fluorescent dextrans from the vasculature in a rodent model of IRI. Minocycline significantly reduced the extent of dextran (500 kDa) leakage from the renal microvasculature 24 h after ischemia. Although minocycline diminished leukocyte accumulation in the kidney following ischemia, areas of leukocyte accumulation did not correlate with areas of microvascular permeability in either the saline- or minocycline-pretreated animals. Minocycline diminished the perivascular increase in MMP-2 and MMP-9, as well as the increase in MMP-2 activity 24 h after ischemia. ABT-518, a specific inhibitor of MMP-2 and MMP-9, also significantly reduced the extent of dextran (500 kDa) leakage from the renal microvasculature 24 h after ischemia. Our results indicate that minocycline mitigates the renal microvascular permeability defect following IRI. This effect is spatially distinct from the effect of minocycline on leukocyte accumulation and may be related to diminished activity of MMPs on the integrity of the perivascular matrix.
kidney; tetracycline; vascular permeability; matrix metalloproteinase
THE DEVELOPMENT OF ACUTE RENAL failure (ARF) in the hospitalized patient portends a significant increase in morbidity and mortality (25). Recent therapeutic interventions demonstrated to be successful in experimental models of ARF have failed to translate into successful clinical interventions in human ARF (19). Consequently, current therapeutic strategies remain primarily preventative and/or supportive. However, continued progress into understanding the pathophysiology of ARF provides a source of optimism toward future clinical advancements.
Ischemia-reperfusion injury (IRI) alone, or as a contributor in the setting of sepsis and/or multiorgan failure, is the most common cause of human ARF (24). Tubular epithelial cell injury has been of central importance in explaining the decrement in glomerular filtration rate that is the hallmark of ARF; however, the pathophysiology of ischemic ARF has evolved to include a complex interplay between tubular injury, inflammation, and altered renal microvascular function (5). Functional and morphological alterations of the renal vasculature have played a conceptual role in the pathophysiology of ARF for a number of years (9, 31). Recent studies have provided additional evidence for the role that vascular injury, in particular endothelial cell injury, plays in the pathophysiology of ischemic ARF (3, 6, 32, 39). Endothelial cell swelling, disorganization of the actin-cytoskeleton in vascular smooth muscle and endothelial cells, and disruption of endothelial cell attachments are some of the morphological alterations that have been observed in the renal microvasculature following ischemic injury (6, 13, 23, 32). Functional consequences of these morphological alterations include disordered vascular reactivity, imbalanced intravascular hemostatic mechanisms, increased leukocyte adherence, and increased vascular permeability (31). Preventing or ameliorating the microvascular injury that occurs during ARF appears to be an increasingly important component in a multiprong therapeutic approach to ARF.
Tetracyclines are a class of compounds that have been well characterized for their antibiotic properties. Additionally, tetracyclines have recently been demonstrated to have protective effects during IRI in skeletal muscle, liver, heart, brain, and kidney (2, 7, 8, 20, 27, 30, 33, 37, 40, 41). The protective mechanisms of tetracyclines in IRI are not fully understood, although a variety of potential mechanisms have been studied including inhibition of matrix metalloproteinases (MMPs), modulation of inflammatory cascades, and inhibition of apoptosis (2, 20, 27, 33, 37, 41). The potential protective effect of tetracyclines on the microvasculature following IRI has not been investigated in any organ. Consequently, we hypothesized that minocycline, a semisynthetic, second-generation tetracycline, would have a protective effect on the renal microvasculature in ischemic ARF. In this paper, we used intravital 2-photon microscopy to demonstrate that minocycline ameliorates renal microvascular leakage in a rat model of ischemic ARF and that this effect is mediated, in part, by modulation of MMPs.
MATERIALS AND METHODS
Animal model of renal ischemia. 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 Sprague-Dawley rats weighing 180–220 g (Harlan, Indianapolis, IN) were administered minocycline (Sigma, St. Louis, MO) 45 mg/kg in 0.9% NaCl or an equal volume of 0.9% NaCl (placebo) via intraperitoneal injection. This was given 36 h before surgery and was followed by 22.5 mg/kg ip every 12 h for a total of four doses. The dosing regimen for minocycline was derived, in part, from prior in vivo studies demonstrating a beneficial effect of minocycline in IRI (41). In separate experiments, male Sprague-Dawley rats were administered ABT-518 (kind gift of Dr. D. H. Albert, Abbott Laboratories, Abbott Park, IL), an orally active inhibitor of MMP-2 and MMP-9 (15, 36), at a dose of 100 mg/kg in olive oil or an equal volume of olive oil (placebo) by gavage. This dose was given daily for 2 days before surgery and on the day of surgery. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/kg) and placed on a homeothermic table to maintain core body temperature at 37°C. A midline incision was made, the renal pedicles were isolated, and bilateral renal ischemia was induced by clamping the renal pedicles for 45 min followed by reperfusion. Sham surgery consisted of an identical procedure with the exception of immediate release of the microaneurysm clamps. For experiments involving live 2-photon microscopic imaging of rat kidneys, a flank incision was made over the left kidney, the renal pedicle was isolated, and unilateral renal ischemia was induced by clamping the left renal pedicle for 45 min as previously described (12).
Intravital 2-photon microscopy. For studies examining renal microvascular permeability, 100 μl of rhodamine-conjugated dextran (3,000 Da, 20 mg/ml in 0.9% saline; Molecular Probes, Eugene, OR), 500 μl of FITC-conjugated dextran (500,000 Da, 7.5 mg/ml in 0.9% saline; Molecular Probes), and 400 μl of Hoechst 33342 (1.5 mg/ml in 0.9% saline; Molecular Probes) were injected via the tail vein into anesthetized rats just before imaging. The left kidney of the anesthetized rat was imaged through a retroperitoneal window via a left-flank incision using a Bio-Rad MRC-1024MP laser-scanning confocal/multiphoton scanner (Hercules, CA) with an excitation wavelength of 800 nm attached to a Nikon Diaphot inverted microscope (Fryer, Huntley, IL) as described by Dunn et al. (12). Image processing was performed using Metamorph software (Universal Imaging, West Chester, PA). Images obtained were analyzed in a 4 x 4 grid, and each grid section (16 per image) was scored for the presence or absence of dextran extravasation for each dextran. The extravasation score for each dextran was calculated by summing the number of grid segments with extravasation of the dextran of interest and dividing by the total number of grid segments scored. Approximately 30 images were collected for each animal examined.
For studies examining the spatial relationship of leukocyte infiltration to renal microvascular permeability, leukocytes were isolated from two normal rats and red blood cells were lysed in an ammonium chloride solution. Leukocytes were labeled with acridine orange (30 μg/ml; Molecular Probes) for 10 min at 37°C. Rhodamine was conjugated to dextran (500,000 Da; Molecular Probes) using 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (Molecular Probes). Leukocytes (107) labeled as above were injected intravenously 24 h after renal ischemia. Approximately 30 min after leukocyte injection, 500 μl of the rhodamine-conjugated dextran (500,000 Da, 7.5 mg/ml in 0.9% saline) were injected. This time differential was chosen because accumulation of labeled leukocytes reached a steady state by 20 min. Imaging was performed following injection of dextrans as described in the preceding paragraph.
Western blotting. Kidneys were removed without fixation, washed with ice-cold saline, minced, and rapidly transferred into 400 μl of ice-cold PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgSO4, pH 6.9) containing 0.5% Triton X-100, 10 μg/ml chymostatin, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1 mM 1,4-dithiothreitol (DTT). Samples were sonicated and then allowed to extract on ice for 10 min. Subsequently, the samples were centrifuged at 4°C and the supernatants were carefully removed for protein determinations and Western blotting. Proteins were measured by a Coomassie blue assay (Coomassie Plus; Pierce Chemical, Rockford, IL) and resolved on a 15% Tris·HCl gel by electrophoresis. An equal amount of protein was loaded in each lane for a given experiment. After electrophoresis, proteins were transferred to a PVDF filter membrane and probed with either mouse monoclonal anti-rat MMP-9 (Oncogene, San Diego, CA), mouse monoclonal anti-rat MMP-2 (Oncogene), or rabbit polyclonal anti-human MMP-1 (Calbiochem, San Diego, CA) followed by horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse IgG or HRP-conjugated polyclonal goat anti-rabbit IgG (Bio-Rad). Immunoreactive bands were detected by chemiluminescence (SuperSignal West Dura Extended, Pierce Chemical). Blots were scanned on a Bio-Rad Fluor-S Multimager to determine band densities.
Gelatin zymography. Kidneys were removed without fixation and proteins were extracted using standard techniques. Equal amounts of protein were loaded and resolved on 10% Tris·HCl gel containing 1 mg/ml gelatin (Type A from porcine skin; Sigma). The gels were then processed and stained as previously described (17, 35) to detect gelatinolytic activity. Blots were scanned on a Bio-Rad Fluor-S Multimager to determine band densities.
Fluorescence confocal microscopy. Kidney sections from anesthetized mice were fixed ex vivo in 4% paraformaldehyde, and 50-μm vibratome sections were obtained. Sections were stained with DAPI (Molecular Probes), mouse monoclonal anti-rat MMP-9, or mouse monoclonal anti-rat MMP-2 followed by polyclonal Texas red-labeled sheep anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Images were collected with an LSM-510 Zeiss confocal microscope (Heidelberg, Germany) equipped with argon and helium/neon lasers. Image processing was performed using Metamorph software.
Statistical analysis. All results represent an n = 3 unless noted otherwise. Results are expressed as means ± SE. Homogeneity of dextran extravasation from images within the same animal and from animals under the same experimental condition was analyzed by a 2- test. Subsequently, pooled dextran extravasation data from experimental and control animals were analyzed for significance by a 2-test. Correlation of the spatial relationship between leukocyte extravasation and dextran leakage was analyzed by Spearman's rank order correlation. Band density data were analyzed for significance by unpaired Student's t-test and ANOVA. A P value of 0.05 was considered to be statistically significant.
RESULTS
Minocycline diminishes the increase in microvascular permeability following renal ischemia. To investigate the effect of minocycline on the integrity of the renal microvasculature during IRI, we used intravital 2-photon microscopy to examine changes in renal microvascular permeability. We chose to examine renal microvascular permeability at 24 h following ischemia because this is when the defect has been demonstrated to be most extensive (32). We observed no increase in renal microvascular permeability in the sham-operated rats irrespective of treatment with minocycline. Twenty-four hours after ischemia, we observed leakage of both the low-molecular-weight (3,000 Da) dextran (LMWD) and the high-molecular-weight (500,000 Da) dextran (HMWD) from the renal microvasculature in the saline-treated animals (Fig. 1, A–C). The extent of LMWD leakage was greater than that of the HMWD. Extravasation scores following ischemia in the saline-treated animals were 2.71 ± 0.36 and 1.21 ± 0.25, respectively, for LMWD and HMWD. Leakage of both dextrans was also observed following ischemia in the minocycline-treated animals (Fig. 1, D–F). However, the extravasation score (0.03 ± 0.01) and the extent of leakage of the HMWD in the minocycline-treated animals ischemia were significantly less than that observed in the saline-treated animals following ischemia (n = 2 animals and 30 images per animal, P < 0.001). There was no significant difference in the extravasation score (2.47 ± 0.27) and the extent of LMWD leakage in the minocycline-treated animals compared with the saline-treated animals following ischemia.
Leukocyte accumulation is spatially distinct from areas of increased microvascular permeability in the rat kidney following renal ischemia. Tetracyclines have been demonstrated to have generalized anti-inflammatory properties (22, 26). Because leukocytes interacting with the microvascular endothelium have been demonstrated to play a role in enhancing endothelial permeability in IRI (11, 14), modulating leukocyte accumulation in the kidney may be a potential mechanism by which minocycline imparts a protective effect on the integrity of the renal microvasculature following ischemia. To investigate the effect of minocycline on leukocyte accumulation and its relationship with microvascular permeability during renal IRI, we used intravital 2-photon microscopy to examine the spatial relationship of leukocyte accumulation to areas of increased renal microvascular permeability following ischemia. As we previously reported (20), fewer leukocytes per field were observed in the minocycline-treated animals compared with the saline-treated animals following ischemia; however, we did not observe a spatial correlation (r = 0.05) of labeled leukocytes with areas of HMWD leakage in either the minocycline-treated or the saline-treated animals following ischemia (Fig. 2).
Minocycline diminishes the increase in MMP-2 and MMP-9 following renal ischemia. Inhibition of MMPs is a well-recognized function of tetracyclines (18). Activation of MMP-2 and MMP-9 following IRI has been implicated in the disruption of microvascular integrity leading to increased permeability in the brain (1, 28). To further elucidate the mechanism by which minocycline may preserve renal microvascular integrity, we used Western blotting, zymography, and immunofluorescence confocal microscopy to examine the effect of minocycline on MMPs in the kidney during IRI. After ischemia, protein levels of both MMP-2 and MMP-9 increased above the levels observed in the sham-operated animals (Fig. 3A). This change was most pronounced in the renal medulla, where on average a greater than 10-fold increase in both MMP-2 and MMP-9 was observed. There was no change in the level of MMP-1 following renal ischemia. Treatment with minocycline diminished the increase in MMP-2 and MMP-9 protein levels following ischemia to 48 ± 6 (n = 3) and 51 ± 8% (n = 3), respectively, of that observed in the saline-treated animals. Minocycline had no effect on MMP-1 levels. Evaluation of MMP activity by zymography revealed a 340 ± 8% (n = 3) increase in a 62-kDa band consistent with the active form of MMP-2 following ischemia (Fig. 3B). Treatment with minocycline diminished the increase in MMP-2 activity following ischemia to 46 ± 4% (n = 3) of that observed in the saline-treated animals. Interestingly, an increase in MMP-9 activity was not observed 24 h after ischemia.
Consistent with the changes in MMP-2 and MMP-9 observed with Western blotting, examination of immunostained kidney sections by confocal microscopy revealed an increase in MMP-2 and MMP-9 following ischemia compared with the sham-operated animals (Fig. 4). This increase was much less pronounced in the minocycline-treated animals following ischemia compared with the saline-treated animals. Both MMP-2 and MMP-9 immunostaining were most pronounced in the outer medullary region of the ischemic kidney and both demonstrated an interstitial pattern of staining consistent with perivascular localization. Outside of an overall decrease in the extent of immunostaining, minocycline treatment did not appear to alter the general localization of MMP-2 or MMP-9 in the ischemic kidney.
To further examine the role that attenuation of MMP-2 activity has on the renal microvascular integrity following ischemia, we examined the effect of ABT-518, an orally active inhibitor of MMP-2 and MMP-9, on HMWD extravasation following renal ischemia. The extravasation score (0.05 ± 0.03) and the extent of leakage of the HMWD in the ABT-518-treated animals were significantly less than that observed in the saline-treated animals following ischemia (n = 3 animals and 20 images per animal, P < 0.001).
DISCUSSION
Alteration of microvascular permeability can be a critical component of tissue injury during IRI. Our finding that minocycline exerts a protective effect on the renal microvasculature during renal IRI by ameliorating the leakage of high-molecular-weight substances from the microvasculature provides a novel effect of minocycline and an additional therapeutic mechanism by which minocycline may exert a protective effect in ischemic ARF and IRI in general. The implication of selective protection against leakage of higher molecular weight substances is not fully elucidated. However, in a previous study, we observed that diminished renal microvascular flow following ischemia was most often adjacent to areas where leakage of the higher molecular weight dextran occurred (32). Leakage of the higher molecular weight dextran may indicate areas where microvascular barrier function is most compromised and thus where the renal microvasculature has sustained the greatest damage during IRI. Although our observations were limited to the cortical area of rats due to technical considerations, presumably the permeability defect in the corticomedullary area would be even more pronounced than what we observed in the cortical microvasculature (16). Altered microvascular permeability may have important functional and/or mechanistic significance in the overall pathophysiology of ischemic ARF. Increased microvascular permeability can contribute to extending ischemic injury through compressing peritubular capillaries thereby further compromising medullary blood flow (21). Additionally, leakage of plasma from the vascular space can contribute to hemoconcentration that can lead to stasis and serve to further diminish perfusion as has been observed in other organs (34). Furthermore, leakage of plasma proteins into the interstitium may have important consequences for the modulation of renal injury, renal recovery, and, ultimately, renal function (4).
Our intravital imaging of fluorescent-labeled leukocytes following ischemia provided a dynamic method for the spatial comparison of leukocyte accumulation and microvascular permeability defects in the kidney during IRI. The interaction of activated leukocytes and the endothelium has been demonstrated to play a role in enhancing endothelial permeability (11, 14). As anticipated, we did not observe accumulation of leukocytes or permeability defects in the kidney of the sham-operated animals despite the possibility of activating leukocytes during the harvesting procedure. Interestingly, we also did not observe colocalization of leukocytes with areas of dextran leakage in kidneys rendered ischemic regardless of treatment with minocycline. Our finding suggests that direct, ongoing interaction of leukocytes with the renal microvascular endothelium is not a prerequisite for the increase in microvascular permeability at 24 h. In addition, our finding also suggests the converse: that vascular permeability is not a prerequisite for leukocyte accumulation 24 h after renal ischemia. Although increased vascular permeability during IRI has been documented to occur in the absence of leukocytes (29), our observation does not exclude the possibility that inflammatory cascades contribute to renal microvascular permeability during IRI. Initiation of paracrine inflammatory cascades by accumulating leukocytes in the kidney may result in spatially distinct areas of increased microvascular permeability during IRI. Furthermore, the temporal interval of our study was limited due to the nature of intravital imaging. Consequently, we chose to examine leukocyte accumulation at the peak of microvascular permeability. Certainly, a temporal lag between the accumulation of leukocytes, inflammatory injury to the microvasculature, and initiation of microvascular leakage could exist which our study would not have captured. In addition, our observation does not exclude modulation of inflammatory pathways as a mechanism by which minocycline ameliorates renal microvascular permeability during IRI. Minocycline could indirectly alter paracrine inflammatory cascades initiated by accumulating leukocytes by decreasing the overall number of accumulating leukocytes or by directly inhibiting paracrine inflammatory cascades. Finally, our results do not exclude the possibility that activation of unlabeled, endogenous leukocytes could be spatially related to areas of HMWD extravasation, although it would seem unlikely that there would be a complete segregation of the effects of endogenous vs. exogenous leukocytes. Overall, further studies are needed to clarify the full extent that leukocytes play in increasing renal microvascular permeability during IRI.
Another potential mechanism by which minocycline may protect microvascular integrity is through modulation of MMPs. Indeed, recent data suggest that MMPs play an important role in the disruption of cerebral microvascular integrity following ischemia (1, 28). Critical constituents of the vascular matrix, including collagen IV, are known to be substrates of MMP-2 and MMP-9. In the brain, degradation of the perivascular matrix has been demonstrated to weaken vessels and lead to vascular leakage. Our observation in this study that both minocycline and ABT-518 mitigate the renal microvascular permeability defect following ischemia provides evidence that MMPs are important regulators of renal microvascular integrity following renal ischemia. A very recent study demonstrated that MMP-2 and MMP-9 protein levels increased 24 h following renal ischemia and that this increase was localized to the interstitium and thereby in close proximity to the renal microvasculature (4). This study also demonstrated an increase in MMP-2 activity 24 h after ischemia but a delay in the increase in MMP-9 activity until 48–72 h after ischemia. Although we only examined MMP protein levels, localization, and activity 24 h after ischemia to correspond with the maximal microvascular permeability defect, our findings are consistent with this study and suggest that MMP-2 may play a more important role in renal microvascular permeability following ischemia. In addition to breaking down the perivascular matrix, MMPs may be involved in cleaving endothelial cell-cell contacts, providing an additional mechanism by which MMPs could promote an increase in microvascular leakage (38). Interestingly, we observed that minocycline treatment diminished MMP-2 activity and both MMP-2 and MMP-9 protein levels following renal ischemia. We did not observe an effect of ischemia or minocycline on MMP-1 protein levels, suggesting that our observation is not a generalized effect for all MMPs. Although tetracyclines are well recognized for their effect as direct MMP antagonists, the ability of tetracyclines to transcriptionally and posttranslationally regulate MMPs, including MMP-2 and MMP-9, has just recently been appreciated (10). This combination of regulatory mechanisms may be a particularly effective strategy for inhibiting MMPs following ischemia.
In summary, we demonstrated that minocycline mitigates the renal microvascular permeability defect following IRI. This protective effect appears to be spatially distinct from the effect of minocycline on leukocyte accumulation and may be related to diminished activity of MMPs on the integrity of the perivascular matrix. In our previous study (20), minocycline has been demonstrated to inhibit tubular cell apoptosis, diminish inflammation, and provide an overall protective effect on renal function following ischemia. Our finding that minocycline exerts an additional protective effect on the renal microvasculature following ischemia is likely to be an important component of the overall protection provided by minocycline during renal IRI.
GRANTS
This work was supported by the following: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; Grants 60621 and 61594), Ralph W. and Grace M. Showalter Research Trust, and National Kidney Foundation (NKF) of Indiana grants to T. A. Sutton; John Bower, MD Clinical Scientist Award of the NKF, American Heart Association Midwest Affiliate (0255990Z), Clarian Health Values Fund, Paul Teschan Research Fund of Dialysis Clinics, and NIDDK Grant 61594 to K. J. Kelly; and NIDDK Grants 60495 and 61594 to P. C. Dagher.
ACKNOWLEDGMENTS
The authors acknowledge D. H. Albert (Abbott Laboratories, Abbott Park, IL) for the kind gift of ABT-518, B. Molitoris for support and valuable discussions, and K. Dunn for valuable discussions.
FOOTNOTES
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.
REFERENCES
Aoki T, Sumii T, Mori T, Wang X, and Lo EH. Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical vs. embolic focal ischemia in spontaneously hypertensive rats. Stroke 33: 2711–2717, 2002.
Arvin KL, Han BH, Du Y, Lin SZ, Paul SM, and Holtzman DM. Minocycline markedly protects the neonatal brain against hypoxic-ischemic injury. Ann Neurol 52: 54–61, 2002.
Basile DP, Donohoe D, Roethe K, and Osborn JL. Renal ischemic injury results in permanent damage to peritubular capillaries and influences long-term function. Am J Physiol Renal Physiol 281: F887–F899, 2001.
Basile DP, Fredrich K, Weihrauch D, Hattan N, and Chilian WM. Angiostatin and matrix metalloprotease expression following ischemic acute renal failure. Am J Physiol Renal Physiol 286: F893–F902, 2004.
Bonventre JV and Weinberg JM. Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 14: 2199–2210, 2003.
Brodsky SV, Yamamoto T, Tada T, Kim B, Chen J, Kajiya F, and Goligorsky MS. Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells. Am J Physiol Renal Physiol 282: F1140–F1149, 2002.
Cheung PY, Sawicki G, Wozniak M, Wang W, Radomski MW, and Schulz R. Matrix metalloproteinase-2 contributes to ischemia-reperfusion injury in the heart. Circulation 101: 1833–1839, 2000.
Clark WM, Lessov N, Lauten JD, and Hazel K. Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat. J Mol Neurosci 9: 103–108, 1997.
Conger J. Vascular alterations in ARF: roles in initiation and maintenance. In: Acute Renal Failure: A Companion to Brenner and Rector's The Kidney (1st ed.), edited by Molitoris BA and Finn WF. Philadelphia, PA: Saunders, 2001, p. 535.
Curci JA, Mao D, Bohner DG, Allen BT, Rubin BG, Reilly JM, Sicard GA, and Thompson RW. Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinases in patients with abdominal aortic aneurysms. J Vasc Surg 31: 325–342, 2000.
Demertzis S, Langer F, Graeter T, Dwenger A, Georg T, and Schafers HJ. Amelioration of lung reperfusion injury by L- and E-selectin blockade. Eur J Cardiothorac Surg 16: 174–180, 1999.
Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ, Bacallao RL, and Molitoris BA. Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am J Physiol Cell Physiol 283: C905–C916, 2002.
Flores J, DiBona DR, Beck CH, and Leaf A. The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J Clin Invest 51: 118–126, 1972.
Gudemez E, Turegun M, Porvasnik S, Carnavale K, Zins J, and Siemionow M. Determination of hindlimb transplantation-induced vascular albumin leakage and leukocyte activation during the acute phase of rejection. J Reconstr Microsurg 15: 133–141, 1999.
Gum RJ, Hickman D, Fagerland JA, Heindel MA, Gagne GD, Schmidt JM, Michaelides MR, Davidsen SK, and Ulrich RG. Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes. Biochem Pharmacol 62: 1661–1673, 2001.
Hellberg PO, Kallskog OT, Ojteg G, and Wolgast M. Peritubular capillary permeability and intravascular RBC aggregation after ischemia: effects of neutrophils. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1018–F1025, 1990.
Heussen C and Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102: 196–202, 1980.
Hidalgo M and Eckhardt SG. Development of matrix metalloproteinase inhibitors in cancer therapy. J Natl Cancer Inst 93: 178–193, 2001.
Kelly KJ and Molitoris BA. Acute renal failure in the new millennium: time to consider combination therapy. Semin Nephrol 20: 4–19, 2000.
Kelly KJ, Sutton TA, Weathered N, Ray N, Caldwell EJ, Plotkin Z, and Dagher PC. Minocycline inhibits apoptosis and inflammation in a rat model of ischemic renal injury. Am J Physiol Renal Physiol 287: F760–F766, 2004.
Klingebiel T, von Gise H, and Bohle A. Morphometric studies on acute renal failure in humans during the oligoanuric and polyuric phases. Clin Nephrol 20: 1–10, 1983.
Kloppenburg M, Brinkman BM, de Rooij-Dijk HH, Miltenburg AM, Daha MR, Breedveld FC, Dijkmans BA, and Verweij C. The tetracycline derivative minocycline differentially affects cytokine production by monocytes and T lymphocytes. Antimicrob Agents Chemother 40: 934–940, 1996.
Kwon O, Phillips CL, and Molitoris BA. Ischemia induces alterations in actin filaments in renal vascular smooth muscle cells. Am J Physiol Renal Physiol 282: F1012–F1019, 2002.
Liano F and Pascual J. Outcomes in acute renal failure. Semin Nephrol 18: 541–550, 1998.
Mehta RL and Chertow GM. Acute renal failure definitions and classification: time for change J Am Soc Nephrol 14: 2178–2187, 2003.
Nieman GF and Zerler BR. A role for the anti-inflammatory properties of tetracyclines in the prevention of acute lung injury. Curr Med Chem 8: 317–325, 2001.
Roach DM, Fitridge RA, Laws PE, Millard SH, Varelias A, and Cowled PA. Upregulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury; protection by the MMP inhibitor, doxycycline. Eur J Vasc Endovasc Surg 23: 260–269, 2002.
Rosenberg GA, Estrada EY, and Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain. Stroke 29: 2189–2195, 1998.
Sakuma T, Takahashi K, Ohya N, Kajikawa O, Martin TR, Albertine KH, and Matthay MA. Ischemia-reperfusion lung injury in rabbits: mechanisms of injury and protection. Am J Physiol Lung Cell Mol Physiol 276: L137–L145, 1999.
Smith JR and Gabler WL. Doxycycline suppression of ischemia-reperfusion-induced hepatic injury. Inflammation 18: 193–201, 1994.
Sutton TA, Fisher CJ, and Molitoris BA. Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int 62: 1539–1549, 2002.
Sutton TA, Mang HE, Campos SB, Sandoval RM, Yoder MC, and Molitoris BA. Injury of the renal microvascular endothelium alters barrier function after ischemia. Am J Physiol Renal Physiol 285: F191–F198, 2003.
Villarreal FJ, Griffin M, Omens J, Dillmann W, Nguyen J, and Covell J. Early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling. Circulation 108: 1487–1492, 2003.
Vollmar B, Glasz J, Leiderer R, Post S, and Menger MD. Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion. Am J Pathol 145: 1421–1431, 1994.
Waas ET, Lomme RM, DeGroot J, Wobbes T, and Hendriks T. Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer. Br J Cancer 86: 1876–1883, 2002.
Wada CK, Holms JH, Curtin ML, Dai Y, Florjancic AS, Garland RB, Guo Y, Heyman HR, Stacey JR, Steinman DH, Albert DH, Bouska JJ, Elmore IN, Goodfellow CL, Marcotte PA, Tapang P, Morgan DW, Michaelides MR, and Davidsen SK. Phenoxyphenyl sulfone N-formylhydroxylamines (retrohydroxamates) as potent, selective, orally bioavailable matrix metalloproteinase inhibitors. J Med Chem 45: 219–232, 2002.
Wang J, Wei Q, Wang CY, Hill WD, Hess DC, and Dong Z. Minocycline upregulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279: 19948–19954, 2004.
Wu WB and Huang TF. Activation of MMP-2, cleavage of matrix proteins, and adherens junctions during a snake venom metalloproteinase-induced endothelial cell apoptosis. Exp Cell Res 288: 143–157, 2003.
Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F, and Goligorsky MS. Intravital videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol Renal Physiol 282: F1150–F1155, 2002.
Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, and Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci USA 95: 15769–15774, 1998.
Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, and Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci USA 96: 13496–13500, 1999.(Timothy A. Sutton, K. J. )
ABSTRACT
Tetracyclines exhibit significant anti-inflammatory properties, inhibit matrix metalloproteinases (MMPs), and are protective in models of ischemia-reperfusion injury (IRI). Both inflammatory cascades and MMP activation have been demonstrated to modulate microvascular permeability. Because increased microvascular permeability occurs during IRI in a variety of organ systems including the kidney, we hypothesized that minocycline, a semisynthetic tetracycline, would diminish microvascular leakage during renal IRI. To test this hypothesis, we used intravital 2-photon microscopy to examine leakage of fluorescent dextrans from the vasculature in a rodent model of IRI. Minocycline significantly reduced the extent of dextran (500 kDa) leakage from the renal microvasculature 24 h after ischemia. Although minocycline diminished leukocyte accumulation in the kidney following ischemia, areas of leukocyte accumulation did not correlate with areas of microvascular permeability in either the saline- or minocycline-pretreated animals. Minocycline diminished the perivascular increase in MMP-2 and MMP-9, as well as the increase in MMP-2 activity 24 h after ischemia. ABT-518, a specific inhibitor of MMP-2 and MMP-9, also significantly reduced the extent of dextran (500 kDa) leakage from the renal microvasculature 24 h after ischemia. Our results indicate that minocycline mitigates the renal microvascular permeability defect following IRI. This effect is spatially distinct from the effect of minocycline on leukocyte accumulation and may be related to diminished activity of MMPs on the integrity of the perivascular matrix.
kidney; tetracycline; vascular permeability; matrix metalloproteinase
THE DEVELOPMENT OF ACUTE RENAL failure (ARF) in the hospitalized patient portends a significant increase in morbidity and mortality (25). Recent therapeutic interventions demonstrated to be successful in experimental models of ARF have failed to translate into successful clinical interventions in human ARF (19). Consequently, current therapeutic strategies remain primarily preventative and/or supportive. However, continued progress into understanding the pathophysiology of ARF provides a source of optimism toward future clinical advancements.
Ischemia-reperfusion injury (IRI) alone, or as a contributor in the setting of sepsis and/or multiorgan failure, is the most common cause of human ARF (24). Tubular epithelial cell injury has been of central importance in explaining the decrement in glomerular filtration rate that is the hallmark of ARF; however, the pathophysiology of ischemic ARF has evolved to include a complex interplay between tubular injury, inflammation, and altered renal microvascular function (5). Functional and morphological alterations of the renal vasculature have played a conceptual role in the pathophysiology of ARF for a number of years (9, 31). Recent studies have provided additional evidence for the role that vascular injury, in particular endothelial cell injury, plays in the pathophysiology of ischemic ARF (3, 6, 32, 39). Endothelial cell swelling, disorganization of the actin-cytoskeleton in vascular smooth muscle and endothelial cells, and disruption of endothelial cell attachments are some of the morphological alterations that have been observed in the renal microvasculature following ischemic injury (6, 13, 23, 32). Functional consequences of these morphological alterations include disordered vascular reactivity, imbalanced intravascular hemostatic mechanisms, increased leukocyte adherence, and increased vascular permeability (31). Preventing or ameliorating the microvascular injury that occurs during ARF appears to be an increasingly important component in a multiprong therapeutic approach to ARF.
Tetracyclines are a class of compounds that have been well characterized for their antibiotic properties. Additionally, tetracyclines have recently been demonstrated to have protective effects during IRI in skeletal muscle, liver, heart, brain, and kidney (2, 7, 8, 20, 27, 30, 33, 37, 40, 41). The protective mechanisms of tetracyclines in IRI are not fully understood, although a variety of potential mechanisms have been studied including inhibition of matrix metalloproteinases (MMPs), modulation of inflammatory cascades, and inhibition of apoptosis (2, 20, 27, 33, 37, 41). The potential protective effect of tetracyclines on the microvasculature following IRI has not been investigated in any organ. Consequently, we hypothesized that minocycline, a semisynthetic, second-generation tetracycline, would have a protective effect on the renal microvasculature in ischemic ARF. In this paper, we used intravital 2-photon microscopy to demonstrate that minocycline ameliorates renal microvascular leakage in a rat model of ischemic ARF and that this effect is mediated, in part, by modulation of MMPs.
MATERIALS AND METHODS
Animal model of renal ischemia. 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 Sprague-Dawley rats weighing 180–220 g (Harlan, Indianapolis, IN) were administered minocycline (Sigma, St. Louis, MO) 45 mg/kg in 0.9% NaCl or an equal volume of 0.9% NaCl (placebo) via intraperitoneal injection. This was given 36 h before surgery and was followed by 22.5 mg/kg ip every 12 h for a total of four doses. The dosing regimen for minocycline was derived, in part, from prior in vivo studies demonstrating a beneficial effect of minocycline in IRI (41). In separate experiments, male Sprague-Dawley rats were administered ABT-518 (kind gift of Dr. D. H. Albert, Abbott Laboratories, Abbott Park, IL), an orally active inhibitor of MMP-2 and MMP-9 (15, 36), at a dose of 100 mg/kg in olive oil or an equal volume of olive oil (placebo) by gavage. This dose was given daily for 2 days before surgery and on the day of surgery. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (65 mg/kg) and placed on a homeothermic table to maintain core body temperature at 37°C. A midline incision was made, the renal pedicles were isolated, and bilateral renal ischemia was induced by clamping the renal pedicles for 45 min followed by reperfusion. Sham surgery consisted of an identical procedure with the exception of immediate release of the microaneurysm clamps. For experiments involving live 2-photon microscopic imaging of rat kidneys, a flank incision was made over the left kidney, the renal pedicle was isolated, and unilateral renal ischemia was induced by clamping the left renal pedicle for 45 min as previously described (12).
Intravital 2-photon microscopy. For studies examining renal microvascular permeability, 100 μl of rhodamine-conjugated dextran (3,000 Da, 20 mg/ml in 0.9% saline; Molecular Probes, Eugene, OR), 500 μl of FITC-conjugated dextran (500,000 Da, 7.5 mg/ml in 0.9% saline; Molecular Probes), and 400 μl of Hoechst 33342 (1.5 mg/ml in 0.9% saline; Molecular Probes) were injected via the tail vein into anesthetized rats just before imaging. The left kidney of the anesthetized rat was imaged through a retroperitoneal window via a left-flank incision using a Bio-Rad MRC-1024MP laser-scanning confocal/multiphoton scanner (Hercules, CA) with an excitation wavelength of 800 nm attached to a Nikon Diaphot inverted microscope (Fryer, Huntley, IL) as described by Dunn et al. (12). Image processing was performed using Metamorph software (Universal Imaging, West Chester, PA). Images obtained were analyzed in a 4 x 4 grid, and each grid section (16 per image) was scored for the presence or absence of dextran extravasation for each dextran. The extravasation score for each dextran was calculated by summing the number of grid segments with extravasation of the dextran of interest and dividing by the total number of grid segments scored. Approximately 30 images were collected for each animal examined.
For studies examining the spatial relationship of leukocyte infiltration to renal microvascular permeability, leukocytes were isolated from two normal rats and red blood cells were lysed in an ammonium chloride solution. Leukocytes were labeled with acridine orange (30 μg/ml; Molecular Probes) for 10 min at 37°C. Rhodamine was conjugated to dextran (500,000 Da; Molecular Probes) using 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (Molecular Probes). Leukocytes (107) labeled as above were injected intravenously 24 h after renal ischemia. Approximately 30 min after leukocyte injection, 500 μl of the rhodamine-conjugated dextran (500,000 Da, 7.5 mg/ml in 0.9% saline) were injected. This time differential was chosen because accumulation of labeled leukocytes reached a steady state by 20 min. Imaging was performed following injection of dextrans as described in the preceding paragraph.
Western blotting. Kidneys were removed without fixation, washed with ice-cold saline, minced, and rapidly transferred into 400 μl of ice-cold PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgSO4, pH 6.9) containing 0.5% Triton X-100, 10 μg/ml chymostatin, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1 mM 1,4-dithiothreitol (DTT). Samples were sonicated and then allowed to extract on ice for 10 min. Subsequently, the samples were centrifuged at 4°C and the supernatants were carefully removed for protein determinations and Western blotting. Proteins were measured by a Coomassie blue assay (Coomassie Plus; Pierce Chemical, Rockford, IL) and resolved on a 15% Tris·HCl gel by electrophoresis. An equal amount of protein was loaded in each lane for a given experiment. After electrophoresis, proteins were transferred to a PVDF filter membrane and probed with either mouse monoclonal anti-rat MMP-9 (Oncogene, San Diego, CA), mouse monoclonal anti-rat MMP-2 (Oncogene), or rabbit polyclonal anti-human MMP-1 (Calbiochem, San Diego, CA) followed by horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse IgG or HRP-conjugated polyclonal goat anti-rabbit IgG (Bio-Rad). Immunoreactive bands were detected by chemiluminescence (SuperSignal West Dura Extended, Pierce Chemical). Blots were scanned on a Bio-Rad Fluor-S Multimager to determine band densities.
Gelatin zymography. Kidneys were removed without fixation and proteins were extracted using standard techniques. Equal amounts of protein were loaded and resolved on 10% Tris·HCl gel containing 1 mg/ml gelatin (Type A from porcine skin; Sigma). The gels were then processed and stained as previously described (17, 35) to detect gelatinolytic activity. Blots were scanned on a Bio-Rad Fluor-S Multimager to determine band densities.
Fluorescence confocal microscopy. Kidney sections from anesthetized mice were fixed ex vivo in 4% paraformaldehyde, and 50-μm vibratome sections were obtained. Sections were stained with DAPI (Molecular Probes), mouse monoclonal anti-rat MMP-9, or mouse monoclonal anti-rat MMP-2 followed by polyclonal Texas red-labeled sheep anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Images were collected with an LSM-510 Zeiss confocal microscope (Heidelberg, Germany) equipped with argon and helium/neon lasers. Image processing was performed using Metamorph software.
Statistical analysis. All results represent an n = 3 unless noted otherwise. Results are expressed as means ± SE. Homogeneity of dextran extravasation from images within the same animal and from animals under the same experimental condition was analyzed by a 2- test. Subsequently, pooled dextran extravasation data from experimental and control animals were analyzed for significance by a 2-test. Correlation of the spatial relationship between leukocyte extravasation and dextran leakage was analyzed by Spearman's rank order correlation. Band density data were analyzed for significance by unpaired Student's t-test and ANOVA. A P value of 0.05 was considered to be statistically significant.
RESULTS
Minocycline diminishes the increase in microvascular permeability following renal ischemia. To investigate the effect of minocycline on the integrity of the renal microvasculature during IRI, we used intravital 2-photon microscopy to examine changes in renal microvascular permeability. We chose to examine renal microvascular permeability at 24 h following ischemia because this is when the defect has been demonstrated to be most extensive (32). We observed no increase in renal microvascular permeability in the sham-operated rats irrespective of treatment with minocycline. Twenty-four hours after ischemia, we observed leakage of both the low-molecular-weight (3,000 Da) dextran (LMWD) and the high-molecular-weight (500,000 Da) dextran (HMWD) from the renal microvasculature in the saline-treated animals (Fig. 1, A–C). The extent of LMWD leakage was greater than that of the HMWD. Extravasation scores following ischemia in the saline-treated animals were 2.71 ± 0.36 and 1.21 ± 0.25, respectively, for LMWD and HMWD. Leakage of both dextrans was also observed following ischemia in the minocycline-treated animals (Fig. 1, D–F). However, the extravasation score (0.03 ± 0.01) and the extent of leakage of the HMWD in the minocycline-treated animals ischemia were significantly less than that observed in the saline-treated animals following ischemia (n = 2 animals and 30 images per animal, P < 0.001). There was no significant difference in the extravasation score (2.47 ± 0.27) and the extent of LMWD leakage in the minocycline-treated animals compared with the saline-treated animals following ischemia.
Leukocyte accumulation is spatially distinct from areas of increased microvascular permeability in the rat kidney following renal ischemia. Tetracyclines have been demonstrated to have generalized anti-inflammatory properties (22, 26). Because leukocytes interacting with the microvascular endothelium have been demonstrated to play a role in enhancing endothelial permeability in IRI (11, 14), modulating leukocyte accumulation in the kidney may be a potential mechanism by which minocycline imparts a protective effect on the integrity of the renal microvasculature following ischemia. To investigate the effect of minocycline on leukocyte accumulation and its relationship with microvascular permeability during renal IRI, we used intravital 2-photon microscopy to examine the spatial relationship of leukocyte accumulation to areas of increased renal microvascular permeability following ischemia. As we previously reported (20), fewer leukocytes per field were observed in the minocycline-treated animals compared with the saline-treated animals following ischemia; however, we did not observe a spatial correlation (r = 0.05) of labeled leukocytes with areas of HMWD leakage in either the minocycline-treated or the saline-treated animals following ischemia (Fig. 2).
Minocycline diminishes the increase in MMP-2 and MMP-9 following renal ischemia. Inhibition of MMPs is a well-recognized function of tetracyclines (18). Activation of MMP-2 and MMP-9 following IRI has been implicated in the disruption of microvascular integrity leading to increased permeability in the brain (1, 28). To further elucidate the mechanism by which minocycline may preserve renal microvascular integrity, we used Western blotting, zymography, and immunofluorescence confocal microscopy to examine the effect of minocycline on MMPs in the kidney during IRI. After ischemia, protein levels of both MMP-2 and MMP-9 increased above the levels observed in the sham-operated animals (Fig. 3A). This change was most pronounced in the renal medulla, where on average a greater than 10-fold increase in both MMP-2 and MMP-9 was observed. There was no change in the level of MMP-1 following renal ischemia. Treatment with minocycline diminished the increase in MMP-2 and MMP-9 protein levels following ischemia to 48 ± 6 (n = 3) and 51 ± 8% (n = 3), respectively, of that observed in the saline-treated animals. Minocycline had no effect on MMP-1 levels. Evaluation of MMP activity by zymography revealed a 340 ± 8% (n = 3) increase in a 62-kDa band consistent with the active form of MMP-2 following ischemia (Fig. 3B). Treatment with minocycline diminished the increase in MMP-2 activity following ischemia to 46 ± 4% (n = 3) of that observed in the saline-treated animals. Interestingly, an increase in MMP-9 activity was not observed 24 h after ischemia.
Consistent with the changes in MMP-2 and MMP-9 observed with Western blotting, examination of immunostained kidney sections by confocal microscopy revealed an increase in MMP-2 and MMP-9 following ischemia compared with the sham-operated animals (Fig. 4). This increase was much less pronounced in the minocycline-treated animals following ischemia compared with the saline-treated animals. Both MMP-2 and MMP-9 immunostaining were most pronounced in the outer medullary region of the ischemic kidney and both demonstrated an interstitial pattern of staining consistent with perivascular localization. Outside of an overall decrease in the extent of immunostaining, minocycline treatment did not appear to alter the general localization of MMP-2 or MMP-9 in the ischemic kidney.
To further examine the role that attenuation of MMP-2 activity has on the renal microvascular integrity following ischemia, we examined the effect of ABT-518, an orally active inhibitor of MMP-2 and MMP-9, on HMWD extravasation following renal ischemia. The extravasation score (0.05 ± 0.03) and the extent of leakage of the HMWD in the ABT-518-treated animals were significantly less than that observed in the saline-treated animals following ischemia (n = 3 animals and 20 images per animal, P < 0.001).
DISCUSSION
Alteration of microvascular permeability can be a critical component of tissue injury during IRI. Our finding that minocycline exerts a protective effect on the renal microvasculature during renal IRI by ameliorating the leakage of high-molecular-weight substances from the microvasculature provides a novel effect of minocycline and an additional therapeutic mechanism by which minocycline may exert a protective effect in ischemic ARF and IRI in general. The implication of selective protection against leakage of higher molecular weight substances is not fully elucidated. However, in a previous study, we observed that diminished renal microvascular flow following ischemia was most often adjacent to areas where leakage of the higher molecular weight dextran occurred (32). Leakage of the higher molecular weight dextran may indicate areas where microvascular barrier function is most compromised and thus where the renal microvasculature has sustained the greatest damage during IRI. Although our observations were limited to the cortical area of rats due to technical considerations, presumably the permeability defect in the corticomedullary area would be even more pronounced than what we observed in the cortical microvasculature (16). Altered microvascular permeability may have important functional and/or mechanistic significance in the overall pathophysiology of ischemic ARF. Increased microvascular permeability can contribute to extending ischemic injury through compressing peritubular capillaries thereby further compromising medullary blood flow (21). Additionally, leakage of plasma from the vascular space can contribute to hemoconcentration that can lead to stasis and serve to further diminish perfusion as has been observed in other organs (34). Furthermore, leakage of plasma proteins into the interstitium may have important consequences for the modulation of renal injury, renal recovery, and, ultimately, renal function (4).
Our intravital imaging of fluorescent-labeled leukocytes following ischemia provided a dynamic method for the spatial comparison of leukocyte accumulation and microvascular permeability defects in the kidney during IRI. The interaction of activated leukocytes and the endothelium has been demonstrated to play a role in enhancing endothelial permeability (11, 14). As anticipated, we did not observe accumulation of leukocytes or permeability defects in the kidney of the sham-operated animals despite the possibility of activating leukocytes during the harvesting procedure. Interestingly, we also did not observe colocalization of leukocytes with areas of dextran leakage in kidneys rendered ischemic regardless of treatment with minocycline. Our finding suggests that direct, ongoing interaction of leukocytes with the renal microvascular endothelium is not a prerequisite for the increase in microvascular permeability at 24 h. In addition, our finding also suggests the converse: that vascular permeability is not a prerequisite for leukocyte accumulation 24 h after renal ischemia. Although increased vascular permeability during IRI has been documented to occur in the absence of leukocytes (29), our observation does not exclude the possibility that inflammatory cascades contribute to renal microvascular permeability during IRI. Initiation of paracrine inflammatory cascades by accumulating leukocytes in the kidney may result in spatially distinct areas of increased microvascular permeability during IRI. Furthermore, the temporal interval of our study was limited due to the nature of intravital imaging. Consequently, we chose to examine leukocyte accumulation at the peak of microvascular permeability. Certainly, a temporal lag between the accumulation of leukocytes, inflammatory injury to the microvasculature, and initiation of microvascular leakage could exist which our study would not have captured. In addition, our observation does not exclude modulation of inflammatory pathways as a mechanism by which minocycline ameliorates renal microvascular permeability during IRI. Minocycline could indirectly alter paracrine inflammatory cascades initiated by accumulating leukocytes by decreasing the overall number of accumulating leukocytes or by directly inhibiting paracrine inflammatory cascades. Finally, our results do not exclude the possibility that activation of unlabeled, endogenous leukocytes could be spatially related to areas of HMWD extravasation, although it would seem unlikely that there would be a complete segregation of the effects of endogenous vs. exogenous leukocytes. Overall, further studies are needed to clarify the full extent that leukocytes play in increasing renal microvascular permeability during IRI.
Another potential mechanism by which minocycline may protect microvascular integrity is through modulation of MMPs. Indeed, recent data suggest that MMPs play an important role in the disruption of cerebral microvascular integrity following ischemia (1, 28). Critical constituents of the vascular matrix, including collagen IV, are known to be substrates of MMP-2 and MMP-9. In the brain, degradation of the perivascular matrix has been demonstrated to weaken vessels and lead to vascular leakage. Our observation in this study that both minocycline and ABT-518 mitigate the renal microvascular permeability defect following ischemia provides evidence that MMPs are important regulators of renal microvascular integrity following renal ischemia. A very recent study demonstrated that MMP-2 and MMP-9 protein levels increased 24 h following renal ischemia and that this increase was localized to the interstitium and thereby in close proximity to the renal microvasculature (4). This study also demonstrated an increase in MMP-2 activity 24 h after ischemia but a delay in the increase in MMP-9 activity until 48–72 h after ischemia. Although we only examined MMP protein levels, localization, and activity 24 h after ischemia to correspond with the maximal microvascular permeability defect, our findings are consistent with this study and suggest that MMP-2 may play a more important role in renal microvascular permeability following ischemia. In addition to breaking down the perivascular matrix, MMPs may be involved in cleaving endothelial cell-cell contacts, providing an additional mechanism by which MMPs could promote an increase in microvascular leakage (38). Interestingly, we observed that minocycline treatment diminished MMP-2 activity and both MMP-2 and MMP-9 protein levels following renal ischemia. We did not observe an effect of ischemia or minocycline on MMP-1 protein levels, suggesting that our observation is not a generalized effect for all MMPs. Although tetracyclines are well recognized for their effect as direct MMP antagonists, the ability of tetracyclines to transcriptionally and posttranslationally regulate MMPs, including MMP-2 and MMP-9, has just recently been appreciated (10). This combination of regulatory mechanisms may be a particularly effective strategy for inhibiting MMPs following ischemia.
In summary, we demonstrated that minocycline mitigates the renal microvascular permeability defect following IRI. This protective effect appears to be spatially distinct from the effect of minocycline on leukocyte accumulation and may be related to diminished activity of MMPs on the integrity of the perivascular matrix. In our previous study (20), minocycline has been demonstrated to inhibit tubular cell apoptosis, diminish inflammation, and provide an overall protective effect on renal function following ischemia. Our finding that minocycline exerts an additional protective effect on the renal microvasculature following ischemia is likely to be an important component of the overall protection provided by minocycline during renal IRI.
GRANTS
This work was supported by the following: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; Grants 60621 and 61594), Ralph W. and Grace M. Showalter Research Trust, and National Kidney Foundation (NKF) of Indiana grants to T. A. Sutton; John Bower, MD Clinical Scientist Award of the NKF, American Heart Association Midwest Affiliate (0255990Z), Clarian Health Values Fund, Paul Teschan Research Fund of Dialysis Clinics, and NIDDK Grant 61594 to K. J. Kelly; and NIDDK Grants 60495 and 61594 to P. C. Dagher.
ACKNOWLEDGMENTS
The authors acknowledge D. H. Albert (Abbott Laboratories, Abbott Park, IL) for the kind gift of ABT-518, B. Molitoris for support and valuable discussions, and K. Dunn for valuable discussions.
FOOTNOTES
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.
REFERENCES
Aoki T, Sumii T, Mori T, Wang X, and Lo EH. Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical vs. embolic focal ischemia in spontaneously hypertensive rats. Stroke 33: 2711–2717, 2002.
Arvin KL, Han BH, Du Y, Lin SZ, Paul SM, and Holtzman DM. Minocycline markedly protects the neonatal brain against hypoxic-ischemic injury. Ann Neurol 52: 54–61, 2002.
Basile DP, Donohoe D, Roethe K, and Osborn JL. Renal ischemic injury results in permanent damage to peritubular capillaries and influences long-term function. Am J Physiol Renal Physiol 281: F887–F899, 2001.
Basile DP, Fredrich K, Weihrauch D, Hattan N, and Chilian WM. Angiostatin and matrix metalloprotease expression following ischemic acute renal failure. Am J Physiol Renal Physiol 286: F893–F902, 2004.
Bonventre JV and Weinberg JM. Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 14: 2199–2210, 2003.
Brodsky SV, Yamamoto T, Tada T, Kim B, Chen J, Kajiya F, and Goligorsky MS. Endothelial dysfunction in ischemic acute renal failure: rescue by transplanted endothelial cells. Am J Physiol Renal Physiol 282: F1140–F1149, 2002.
Cheung PY, Sawicki G, Wozniak M, Wang W, Radomski MW, and Schulz R. Matrix metalloproteinase-2 contributes to ischemia-reperfusion injury in the heart. Circulation 101: 1833–1839, 2000.
Clark WM, Lessov N, Lauten JD, and Hazel K. Doxycycline treatment reduces ischemic brain damage in transient middle cerebral artery occlusion in the rat. J Mol Neurosci 9: 103–108, 1997.
Conger J. Vascular alterations in ARF: roles in initiation and maintenance. In: Acute Renal Failure: A Companion to Brenner and Rector's The Kidney (1st ed.), edited by Molitoris BA and Finn WF. Philadelphia, PA: Saunders, 2001, p. 535.
Curci JA, Mao D, Bohner DG, Allen BT, Rubin BG, Reilly JM, Sicard GA, and Thompson RW. Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinases in patients with abdominal aortic aneurysms. J Vasc Surg 31: 325–342, 2000.
Demertzis S, Langer F, Graeter T, Dwenger A, Georg T, and Schafers HJ. Amelioration of lung reperfusion injury by L- and E-selectin blockade. Eur J Cardiothorac Surg 16: 174–180, 1999.
Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ, Bacallao RL, and Molitoris BA. Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am J Physiol Cell Physiol 283: C905–C916, 2002.
Flores J, DiBona DR, Beck CH, and Leaf A. The role of cell swelling in ischemic renal damage and the protective effect of hypertonic solute. J Clin Invest 51: 118–126, 1972.
Gudemez E, Turegun M, Porvasnik S, Carnavale K, Zins J, and Siemionow M. Determination of hindlimb transplantation-induced vascular albumin leakage and leukocyte activation during the acute phase of rejection. J Reconstr Microsurg 15: 133–141, 1999.
Gum RJ, Hickman D, Fagerland JA, Heindel MA, Gagne GD, Schmidt JM, Michaelides MR, Davidsen SK, and Ulrich RG. Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes. Biochem Pharmacol 62: 1661–1673, 2001.
Hellberg PO, Kallskog OT, Ojteg G, and Wolgast M. Peritubular capillary permeability and intravascular RBC aggregation after ischemia: effects of neutrophils. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1018–F1025, 1990.
Heussen C and Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102: 196–202, 1980.
Hidalgo M and Eckhardt SG. Development of matrix metalloproteinase inhibitors in cancer therapy. J Natl Cancer Inst 93: 178–193, 2001.
Kelly KJ and Molitoris BA. Acute renal failure in the new millennium: time to consider combination therapy. Semin Nephrol 20: 4–19, 2000.
Kelly KJ, Sutton TA, Weathered N, Ray N, Caldwell EJ, Plotkin Z, and Dagher PC. Minocycline inhibits apoptosis and inflammation in a rat model of ischemic renal injury. Am J Physiol Renal Physiol 287: F760–F766, 2004.
Klingebiel T, von Gise H, and Bohle A. Morphometric studies on acute renal failure in humans during the oligoanuric and polyuric phases. Clin Nephrol 20: 1–10, 1983.
Kloppenburg M, Brinkman BM, de Rooij-Dijk HH, Miltenburg AM, Daha MR, Breedveld FC, Dijkmans BA, and Verweij C. The tetracycline derivative minocycline differentially affects cytokine production by monocytes and T lymphocytes. Antimicrob Agents Chemother 40: 934–940, 1996.
Kwon O, Phillips CL, and Molitoris BA. Ischemia induces alterations in actin filaments in renal vascular smooth muscle cells. Am J Physiol Renal Physiol 282: F1012–F1019, 2002.
Liano F and Pascual J. Outcomes in acute renal failure. Semin Nephrol 18: 541–550, 1998.
Mehta RL and Chertow GM. Acute renal failure definitions and classification: time for change J Am Soc Nephrol 14: 2178–2187, 2003.
Nieman GF and Zerler BR. A role for the anti-inflammatory properties of tetracyclines in the prevention of acute lung injury. Curr Med Chem 8: 317–325, 2001.
Roach DM, Fitridge RA, Laws PE, Millard SH, Varelias A, and Cowled PA. Upregulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury; protection by the MMP inhibitor, doxycycline. Eur J Vasc Endovasc Surg 23: 260–269, 2002.
Rosenberg GA, Estrada EY, and Dencoff JE. Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain. Stroke 29: 2189–2195, 1998.
Sakuma T, Takahashi K, Ohya N, Kajikawa O, Martin TR, Albertine KH, and Matthay MA. Ischemia-reperfusion lung injury in rabbits: mechanisms of injury and protection. Am J Physiol Lung Cell Mol Physiol 276: L137–L145, 1999.
Smith JR and Gabler WL. Doxycycline suppression of ischemia-reperfusion-induced hepatic injury. Inflammation 18: 193–201, 1994.
Sutton TA, Fisher CJ, and Molitoris BA. Microvascular endothelial injury and dysfunction during ischemic acute renal failure. Kidney Int 62: 1539–1549, 2002.
Sutton TA, Mang HE, Campos SB, Sandoval RM, Yoder MC, and Molitoris BA. Injury of the renal microvascular endothelium alters barrier function after ischemia. Am J Physiol Renal Physiol 285: F191–F198, 2003.
Villarreal FJ, Griffin M, Omens J, Dillmann W, Nguyen J, and Covell J. Early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling. Circulation 108: 1487–1492, 2003.
Vollmar B, Glasz J, Leiderer R, Post S, and Menger MD. Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion. Am J Pathol 145: 1421–1431, 1994.
Waas ET, Lomme RM, DeGroot J, Wobbes T, and Hendriks T. Tissue levels of active matrix metalloproteinase-2 and -9 in colorectal cancer. Br J Cancer 86: 1876–1883, 2002.
Wada CK, Holms JH, Curtin ML, Dai Y, Florjancic AS, Garland RB, Guo Y, Heyman HR, Stacey JR, Steinman DH, Albert DH, Bouska JJ, Elmore IN, Goodfellow CL, Marcotte PA, Tapang P, Morgan DW, Michaelides MR, and Davidsen SK. Phenoxyphenyl sulfone N-formylhydroxylamines (retrohydroxamates) as potent, selective, orally bioavailable matrix metalloproteinase inhibitors. J Med Chem 45: 219–232, 2002.
Wang J, Wei Q, Wang CY, Hill WD, Hess DC, and Dong Z. Minocycline upregulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279: 19948–19954, 2004.
Wu WB and Huang TF. Activation of MMP-2, cleavage of matrix proteins, and adherens junctions during a snake venom metalloproteinase-induced endothelial cell apoptosis. Exp Cell Res 288: 143–157, 2003.
Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F, and Goligorsky MS. Intravital videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol Renal Physiol 282: F1150–F1155, 2002.
Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, and Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci USA 95: 15769–15774, 1998.
Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, and Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci USA 96: 13496–13500, 1999.(Timothy A. Sutton, K. J. )