High-density lipoproteins (HDL) represent a broad group of mostly spheroidal plasma lipoproteins that exhibit considerable diversity in their size and apolipoprotein and lipid composition. HDL particles fall into the density range of 1.063 to 1.21 g/ml (1); as they are smaller than other lipoproteins, HDL can penetrate between endothelial cells more readily, allowing relatively high concentrations to accumulate in tissue fluids (2). The major apolipoprotein (apo) of almost all plasma HDL is apo A-I, which, in association with phospholipids and cholesterol, encloses a core of cholesteryl esters (1). Nascent (i.e., newly synthesized) HDL secreted by the liver and intestine contain no cholesteryl esters and are discoidal in shape (1). A negative association of plasma HDL concentration with the risk of developing atherosclerosis and coronary heart disease has been documented from numerous epidemiologic studies (3,4) and animal experiments (5,6), demonstrating that HDL have direct anti-atherogenic activity (7). The mechanisms by which HDL provides these cardioprotective actions are not clearly understood but may include a role for HDL in reverse transport of cholesterol from peripheral tissues to the liver, inhibition of the oxidation of low-density lipoproteins, or modulation of vasodilatation and platelet activation mediated by changes in the production of prostacyclin (8,9). HDL can also activate endothelial nitric oxide synthase subsequent to its interaction with scavenger receptor-B1 (SR-B1) (10).
Although HDL are involved in the removal of cholesterol from extra-hepatic tissues, this subset of lipoproteins has recently been reported to possess functions unrelated to their role in plasma cholesterol transport. Almost 10 yr ago, it was reported that in transgenic mice in which plasma levels of HDL were twofold higher, the increase in plasma levels of TNF-α as well as mortality caused by bacterial lipopolysaccharide (LPS) were significantly reduced (11). We and others have subsequently demonstrated that administration of HDL significantly reduces organ injury and levels of mortality in animal models of endotoxic (LPS-mediated) and hemorrhagic shock (12–14). The beneficial actions observed in these models are mediated by the ability of HDL to bind and inactivate LPS (11,15,16), directly inhibit expression of adhesion molecules on endothelial cells and via modulation of the expression of pro-inflammatory cytokines (11,17–19). In human volunteers, systemic administration of HDL also downregulates the LPS ligand CD14 on monocytes and attenuates the release of TNF-α, IL-6, and IL-8 caused by small doses of intravenously administered LPS (20). HDL has also been shown to directly inhibit TNF-α-induced expression of P-selectin on human endothelial cells (11,17–19), which clearly suggest that HDL exerts anti-inflammatory effects. However, in view of these antiinflammatory actions, there are no studies investigating the potential beneficial effects of HDL against the renal dysfunction and injury caused by I/R of the kidney with a view to reducing the severity of ischemic acute renal failure (ARF).
Renal ischemia, whether caused by shock or during surgery or transplantation, is a major cause of ischemic ARF, which despite significant advances in critical care medicine, remains a major clinical problem, producing grave morbidity and mortality rates that have not decreased significantly over the past 50 yr (21). The prognosis of ARF is complicated by the fact that reperfusion, although essential for the survival of ischemic renal tissue, causes additional damage (reperfusion-injury) (22). The mechanisms involved in the development of the renal dysfunction and injury associated with reperfusion-injury and ARF have been thoroughly investigated, and it is now clear that the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and selectins, such as P-selectin and E-selectin, play an important role (23–25), followed by the adhesion, activation, and transmigration of polymorphonuclear leukocytes (PMN) into renal tissues and subsequent production of reactive oxygen species (ROS) and nitric oxide (NO) (24,26). Although we have reported the beneficial effects of HDL against renal dysfunction caused by endotoxic or hemorrhagic shock (12,13), the ability of HDL to protect the kidney against I/R injury has not been investigated. To achieve this goal, we have investigated the effects of reconstituted human HDL on the renal injury (serum and urinary markers of renal reperfusion-injury and histology) and dysfunction (serum creatinine, FENa) caused by bilateral clamping of both renal arteries (for 45 min) followed by reperfusion (for up to 48 h). To gain a better insight into the mechanism(s) of action of the observed beneficial effects of HDL, we have also investigated the effects of HDL on PMN infiltration into renal tissues and the expression of adhesion molecules (specifically ICAM-1 and P-selectin) associated with renal I/R.
Materials and Methods
Renal Ischemia/Reperfusion (Short-Term Model)
In vivo studies were carried out using 42 male Wistar rats (Tuck, Rayleigh, Essex, UK) weighing 220 to 330 g. Rats received a standard diet and water ad libitum and were cared for in accordance with both the Home Office Guidance in the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty’s Stationery Office, London, U.K. and the Guiding Principles in the Care and Use of Animals published by the American Physiological Society. Rats were subjected to bilateral renal ischemia for 45 min followed by reperfusion for 6 h as described previously (27,28). Upon completion of surgical procedures, rats were randomly allocated into the following four groups: (1) I/R + saline group: rats were subjected to renal ischemia followed by reperfusion for 6 h (n = 12); (2) I/R + HDL group: rats were administered HDL (80 mg/kg intravenous bolus) 30 min before commencement of I/R (n = 12); (3) Sham + saline group: rats were subjected to identical surgical procedures, except for renal I/R, and were maintained under anesthesia for the duration of the experiment (45 min + 6 h, n = 12); (4) Sham + HDL group: identical to Sham-operated rats, except for the administration of HDL (80 mg/kg intravenous bolus) 30 min before commencing I/R (n = 6).
The time course and doses of HDL used were based on those previously shown by us to provide beneficial actions against renal dysfunction in models of endotoxic and hemorrhagic shock in the rat (12,13). All rats received an intravenous infusion of saline (2 ml/kg per hour) throughout the I/R period.
Renal Ischemia/Reperfusion-Injury (Long-Term Model)
To assess the effects of HDL administration on renal function and injury in the later stages of ischemic ARF, further studies were performed using 25 male Wistar rats (Charles River, Milan, Italy) weighing 200 to 260 g. Rats were allowed access to food and water ad libitum and were cared for in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (DM 116192), as well as with the European Economic Community regulations (OJ of EC L358/1 12/18/1986).
Rats were subjected to bilateral renal ischemia for 45 min followed by reperfusion for up to 72 h as described previously (29). Rats were anesthetized using chloral hydrate (400 mg/kg intraperitoneally), and core body temperature was maintained at 37°C using a homoiothermic blanket. Rats were then divided into the following three groups: (1) I/R + saline group: rats were subjected to renal ischemia (45 min) followed by reperfusion for 48 h (n = 5); (2) I/R + HDL group: rats were administered HDL (80 mg/kg intravenous bolus via tail vein) 30 min before commencement of I/R and after 12-h reperfusion (n = 10); (3) Sham + saline group: rats were subjected to identical surgical procedures except for renal I/R (n = 10). Rats that did not receive HDL were administered 2 ml/kg saline (vehicle for HDL), intravenous bolus via tail vein at equivalent time points (30 min before Sham operation and after 12 h).
After performing flank incisions, rats from the I/R group were subjected to bilateral renal ischemia for 45 min, during which the renal arteries and veins were occluded using microaneurysm clamps. After the renal clamps were removed, the kidneys were observed for an additional 5 min to ensure reflow, after which 1 ml of saline at 37°C was injected into the abdomen and the incision was sutured in two layers. Rats were then returned to their cages, where they were allowed to recover from anesthesia and observed for 48 h. Sham-operated rats underwent identical surgical procedures to I/R rats except that microaneurysm clamps were not applied.
Measurement of Biochemical Parameters
At the end of the reperfusion period (short-term model) or after 24-h and 48-h reperfusion (long-term model), blood samples were collected via the carotid artery or tail vein into tubes containing serum gel. The samples were centrifuged (6000 rpm for 3 min) to separate serum from which biochemical parameters were measured (Vetlab Services, Sussex, UK, or by DB, University of Teramo, Italy). Serum creatinine concentrations were used as indicators of renal (glomerular) function (27–29). As discussed previously (28) and in the Discussion section, aspartate aminotransferase (AST), an enzyme located in the PT (28), was used as an indicator of reperfusion-injury. Urine samples were collected throughout the reperfusion period, and the volume of urine production was recorded. Urine concentrations of Na+ were measured (Vetlab Services) at the end of the reperfusion period and were used in conjunction with serum Na+ concentrations to estimate fractional excretion of Na+ (FENa), which was used as an indicator of tubular function (27,28). FENa was calculated using standard formulae on the basis that the amount of Na+ given to each animal was the same, i.e., rats were administered saline solution at a rate dependent on their weight (i.e., 2 ml/kg per hour intravenously) and, as rats remained anesthetized throughout the experimental period, did not have any additional Na+ intake. Activity of urinary N-acetyl-β-D-glucosaminidase (NAG), a specific indicator of tubular damage (28), was also measured (Clinica Medica e[Combining Acute Accent] Diagno[Combining Acute Accent]stico Dr Joaquim Chaves, Lisbon, Portugal).
Histologic Evaluation
Histologic imaging of renal sections was performed as described previously (28). Briefly, kidneys were removed from rats at the end of the experimental period after tying the renal pedicle and were cut in a sagittal section into two halves, which were fixed by immersion in 10% (wt/vol) formaldehyde in phosphate-buffered saline (PBS; 0.01 M; pH 7.4) at room temperature for 1 d. After dehydration using graded ethanol, pieces of kidney were embedded in Paraplast (Sherwood Medical, Mahwah, NJ) and cut in fine (8 μm) sections and mounted on glass slides. Sections were then deparaffinized with xylene, counterstained with hematoxylin and eosin, and viewed under a light microscope (Dialux 22, Leitz, Milan, Italy).
For histologic scoring, renal sections were prepared as described previously and used for the assessment of renal I/R injury (28). Briefly, 100 intersections were examined for each kidney, and a score from 0 to 3 was given for each tubular profile involving an intersection: 0 = normal histology; 1 = tubular cell swelling, brush border loss, nuclear condensation, with up to one third of tubular profile showing nuclear loss; 2 = as for score 1, but more than one third and less than two thirds of tubular profile shows nuclear loss; 3 = more than two thirds of tubular profile shows nuclear loss. The total score for each kidney was calculated by addition of all 100 scores with a maximum score of 300.
Analysis of Polymorphonuclear Leukocyte Influx into Renal Tissues
As it has now become apparent that the myeloperoxidase and naphthol-AS-D-chloracetatesterase assays can crossreact with monocytes and macrophages (30), standard hematoxylin-eosin staining was performed to estimate the presence of PMN, based on the morphology of the nucleus. The total number of infiltrating leukocytes (e.g., neutrophils and mononuclear cells) in cortical interstitial spaces was assessed quantitatively by counting the number of PMN in 20 high-power fields.
Measurement of Myeloperoxidase Activity
Myeloperoxidase (MPO) activity in kidneys was also used as an indicator of PMN infiltration into renal tissues using a method previously described (12,28). Briefly, at the end of the experiments, kidney tissue was weighed and homogenized in a solution containing 0.5% (wt/vol) hexadecyltrimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.4) and centrifuged for 30 min at 20,000 × g at 4°C. An aliquot of supernatant was removed and added to a reaction mixture containing 1.6 mM tetramethylbenzidine and 0.1 mM hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme required to degrade 1 μmol of hydrogen peroxide at 37°C and was expressed in mu/100 mg wet tissue.
Determination of Malondialdehyde Levels
Levels of malondialdehyde (MDA) in kidneys were determined as an indicator of lipid peroxidation following a protocol described previously (12,28). Briefly, kidney tissue was weighed and homogenized in a 1.15% (wt/vol) KCl solution. A 100-μl aliquot of homogenate was then removed and added to a reaction mixture containing 200 μl of 8.1% (wt/vol) lauryl sulfate, 1.5 ml 20% (vol/vol) acetic acid, 1.5 ml 0.8% (wt/vol) thiobarbituric acid, and 700 μl of distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3000 × g for 10 min. The absorbance of the supernatant was measured spectrophotometrically at 650 nm. MDA levels were expressed as μM MDA/100 mg wet tissue.
Immunohistochemical Localization of ICAM-1 and P-Selectin
Immunohistochemical localization of ICAM-1 and P-selectin in kidney sections was performed as described previously (12). Sections were incubated overnight at 4°C with primary anti-ICAM-1 (CD54) or anti-P-selectin antibody (1:500 [vol/vol] in PBS) (DBA, Milan, Italy). Controls included kidney sections incubated with buffer alone or nonspecific purified IgG (DBA). After blocking endogenous avidin and biotin, specific labeling of antigen-antibody complex was visualized using an avidin-biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine.
Materials
Unless otherwise stated, all compounds used in this study were purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK). HDL was supplied by ZLB-Bioplasma, Bern, Switzerland. All stock solutions were prepared using non-pyrogenic saline (0.9% [wt/vol] NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK).
Statistical Analyses
All values described in the text and figures are expressed as mean ± SEM for n observations. Each data point represents biochemical measurements obtained from up to 12 separate animals. For histologic scoring and PMN counts, each data point represents analysis of kidneys taken from six individual animals. For immunohistochemical analysis, the figures shown are representative of at least three experiments performed on different experimental days on six individual animals. Statistical analysis was carried out using GraphPad Prism 3.02/Instat 1.1 (GraphPad Software, San Diego, CA). Data were analyzed using one-way ANOVA followed by Dunnett post hoc test, and a P value of less than 0.05 was considered to be significant.
Results
The mean ± SEM for the weights of the rats used in the short-term study was 262 ± 5 g, n = 42 (Sham + saline group: 253 ± 4 g, n = 12; Sham + HDL group: 264 ± 11 g, n = 6; I/R + saline: 262 ± 5 g, n = 12; I/R + HDL group: 270 ± 3, n = 12) and for the long-term study, was 227 ± 2 g, n = 25 (Sham + saline group: 227 ± 3 g, n = 5; I/R + saline: 224 ± 4 g, n = 10; I/R + HDL: 231 ± 2, n = 10). On comparison with Sham animals, renal I/R produced significant increases in serum, urinary, and histologic markers of renal dysfunction and injury as described in detail below (Figures 1 to 4). When compared with Sham-operated rats given saline only, administration of HDL to rats used as Shams did not have any effect on renal function or markers of renal function (serum creatinine levels, FENa; Figure 1, A and B), tubular or reperfusion-injury (urinary NAG activity and serum AST; Figure 2, A and B), histologic scoring of renal injury (Figure 4A), or on renal MPO activity and MDA levels (Figure 5, A and B). When compared with rats used as Shams, renal I/R (in the presence or absence of HDL or its vehicle), did not have a significant effect on urine flow (Sham + saline group: 0.017 ± 0.002 ml/min, n = 12; Sham + HDL group: 0.015 ± 0.003 ml/min, n = 6; I/R + saline group: 0.014 ± 0.003 ml/min, n = 12; I/R + HDL group: 0.012 ± 0.001, n = 12; overall mean: 0.015 ± 0.002 ml/min, n = 42).
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Figure 1. :
Effect of high-density lipoprotein (HDL) on renal dysfunction caused by ischemia/reperfusion (I/R). Serum creatinine levels (A) and fractional excretion of Na+ (FENa) (B) were measured as respective biochemical markers of glomerular and tubular dysfunction subsequent to Sham-operation (Sham + saline, n = 12 for both determinations) or renal I/R (I/R only, n = 12 for both determinations). HDL (80 mg/kg intravenous bolus) was administered 30 min before renal I/R (I/R + HDL, n = 12 for both determinations) or to Sham-operated rats (Sham + HDL, n = 6 for both determinations). Data represent mean ± SEM for n observations. ★ P < 0.05 versus I/R only group.
Figure 2. :
Effect of HDL on tubular and reperfusion-injury caused by renal I/R. (A) and urinary N-acetyl-β-D-glucosaminidase (NAG) activity and (B) serum aspartate aminotransferase (AST) levels were measured as biochemical markers of tubular and reperfusion-injury, respectively, subsequent to Sham-operation (Sham + saline: n = 6 for urinary NAG, n = 12 for serum AST) or renal I/R (I/R only: n = 6 for urinary NAG, n = 12 for serum AST). Rats were administered HDL (80 mg/kg intravenous bolus) 30 min before renal I/R (I/R + HDL: n = 6 for urinary NAG, n = 12 for serum AST) or Sham-operation (Sham + HDL: n = 6 for both determinations). Data represent mean ± SEM for n observations. ★ P < 0.05 versus I/R only group.
Figure 3. :
Histologic analysis of renal I/R — effect of HDL. A renal section taken from a Sham-operated rat (A) is compared with that taken from a rat subjected to renal I/R (B). Infiltrating polymorphonuclear leukocytes (PMN) were observed in the renal cortex of rats subjected to I/R of the kidney (B1). A renal section taken from a rat subjected to renal I/R after administration of HDL (80 mg/kg intravenous) is also represented (C). Hematoxylin and eosin; original magnification, ×150; figures are representative of at least three experiments performed on different days (n = 6 for all groups).
Figure 4. :
Effect of HDL on renal-injury and PMN infiltration into renal tissues. (A) Renal sections were scored for characteristic histologic signs of renal injury out of a total of 300 (see Materials and Methods section). (B) The total number of infiltrating leukocytes (e.g., neutrophils and mononuclear cells) in cortical interstitial spaces was assessed quantitatively by counting the number of PMN in 20 high-power fields. Measurements were performed subsequent to Sham-operation (Sham + saline, n = 6 for both determinations) or renal I/R (I/R only, n = 6 for both determinations). Rats were administered HDL (80 mg/kg intravenous bolus) 30 min before renal I/R (I/R + HDL, n = 6 for both determinations) or Sham-operation (Sham + HDL, n = 6 for both determinations). Data represent the mean ± SEM for 20 counts obtained from the kidneys of each treatment group, n = 6. ★ P < 0.05 versus I/R-only group.
Figure 5. :
Effect of HDL on (A) PMN infiltration (kidney myeloperoxidase activity) and (B) lipid peroxidation (kidney malondialdehyde levels). MPO activity (A) and MDA levels (B) were measured subsequent to Sham-operation (Sham + saline, n = 6 for both determinations) or renal I/R (I/R only, n = 6 for both determinations). Rats were administered HDL (80 mg/kg intravenous bolus) 30 min before renal I/R (I/R + HDL, n = 6 for both determinations) or Sham-operation (Sham + HDL, n = 6 for both determinations). Data represent mean ± SEM for n observations. ★ P < 0.05 versus I/R only group.
Effect of HDL on Renal Dysfunction Mediated by I/R
Rats that underwent renal I/R exhibited significant increases in the serum concentrations of creatinine compared with Sham-operated animals (Figure 1A), suggesting a significant degree of glomerular dysfunction. Compared with control animals (I/R + saline), administration of HDL produced a modest but significant reduction in serum levels of creatinine (Figure 1A). Renal I/R produced significant increases in FENa (Figure 1B), suggesting a marked increase in tubular dysfunction. On comparison with FENa measured in control (I/R + saline) rats, administration of HDL produced a significant reduction of FENa (Figure 1B), suggesting marked improvement in tubular function.
Effect of HDL on Tubular and Reperfusion-Injury Caused by Renal I/R
On comparison with values obtained from Sham-operated animals, renal I/R produced a significant increase in urinary NAG activity (Figure 2A), suggesting tubular injury. Renal I/R also produced significant increases in serum levels of AST, suggesting significant reperfusion-injury (Figure 2B). Administration of HDL caused a significant reduction in urinary NAG activity and serum AST levels (Figure 2, A and B), suggesting a marked reduction in the tubular and reperfusion-injury associated with renal I/R, respectively.
Effects of HDL on Histologic Alterations Caused by Renal I/R
On comparison with the renal histology observed in kidneys taken from Sham-operated rats (Figure 3A), rats that underwent renal I/R demonstrated the recognized features of renal injury (Figure 3B). Specifically, this included degeneration of tubular structure, tubular dilatation, swelling and necrosis, luminal congestion, and the presence of eosinophilia. A marked infiltration of PMN was observed in renal tissues obtained from rats subjected to I/R (Figure 3B). In contrast, renal sections obtained from rats administered 80 mg/kg HDL before renal I/R demonstrated marked reduction of the histologic features of renal injury on comparison with kidneys obtained from rats subjected to I/R only (Figure 3C).
Effects of HDL on Scoring of Renal Histopathology
On comparison with the histology score measured from kidneys obtained from Sham-operated animals, renal I/R produced a significant increase in histology score (Figure 4A). Administration of HDL significantly reduced the histology score when compared with that obtained from rats subjected to renal I/R only (Figure 4A), indicating a reduction in renal injury.
Effect of HDL on Polymorphonuclear Leukocyte Infiltration into Renal Tissues
Quantitation of infiltrating PMN into renal tissues showed that there was only a minimal number of PMN in non-ischemic kidneys obtained from Sham-operated rats (Figure 4B). However, a large number of infiltrating PMN were observed in the renal cortex of rats subjected to I/R of the kidney (Figures 3B and 4B). HDL administration significantly reduced the numbers of PMN infiltrating into renal tissues by approximately 50% (Figure 4B).
Effect of HDL on Kidney Myeloperoxidase Activity and Malondialdehyde Levels
Rats subjected to renal I/R exhibited a substantial increase in kidney MPO activity (Figure 5A), also suggesting increased PMN infiltration into renal tissues. However, administration of HDL produced a significant reduction of MPO activity on comparison with that obtained from control rat kidneys (Figure 5A). Rats subjected to renal I/R exhibited a substantial increase in kidney MDA levels (Figure 5B), suggesting increased lipid peroxidation subsequent to oxidative stress. However, HDL administration produced a significant reduction in MDA levels on comparison with levels obtained from control rat kidneys (Figure 5B).
Effect of HDL on ICAM-1 and P-Selectin Expression Subsequent to Renal I/R
When compared with kidneys obtained from Sham-operated rats (Figures 6A and 7A), kidneys obtained from rats subjected to I/R demonstrated marked staining for ICAM-1 (Figure 6B) and P-selectin (Figure 7B), suggesting adhesion molecule expression during reperfusion. Kidneys obtained from rats administered HDL demonstrated markedly reduced staining for both ICAM-1 and P-selectin (Figures 6C and 7C) when compared with kidneys obtained from rats subjected to renal I/R only, suggesting a reduction in the expression of these adhesion molecules during reperfusion.
Figure 6. :
Immunohistochemical localization of intercellular adhesion molecule-1 (ICAM-1) in rat kidney sections. (A) Sham-operated group, (B) I/R only group, (C) I/R after administration of HDL (80 mg/kg intravenous bolus) 30 min before renal I/R. Sections were incubated overnight at 4°C with primary mouse anti-rat monoclonal antibody directed at ICAM-1 (CD54) (1:500 [vol/vol] in PBS). Specific labeling of antigen-antibody complex was visualized using an avidin-biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine. Magnification: ×125, figures are representative of at least three experiments performed on different experimental days (n = 6 for all groups).
Figure 7. :
Immunohistochemical localization of P-selectin in rat kidney sections. (A) Sham-operated group, (B) I/R only group, (C) I/R after administration of HDL (80 mg/kg intravenous bolus) 30 min before renal I/R. Sections were incubated overnight at 4°C with primary mouse anti-rat monoclonal antibody directed at P-selectin (1:500 [vol/vol] in PBS). Specific labeling of antigen-antibody complex was visualized using an avidin-biotin peroxidase complex immunoperoxidase technique using chromogen diaminobenzidine. Magnification: ×125, figures are representative of at least three experiments performed on different experimental days (n = 6 for all groups).
Effect of HDL on the Course of Renal Ischemia/Reperfusion Injury
In the long-term model of ischemic ARF, renal function (assessed by serum creatinine levels) was significantly improved after administration of HDL to rats subjected to renal ischemia followed by reperfusion for 24 and 48 h (Figure 8A). Histologic examination of kidney sections prepared from rats subjected to ischemia followed by reperfusion for 48 h and treated with saline (I/R + saline, Figure 8B) demonstrated greater renal injury than rats subjected to I/R but which were administered HDL (I/R + HDL, Figure 8C).
Figure 8. :
Effect of HDL on the course of renal I/R injury. (A) Serum creatinine levels were measured 24 and 48 h after renal ischemia in rats subjected to I/R and which were administered either saline (• = I/R + saline: n = 6 at 24 h, n = 5 at 48 h) or 80 mg/kg HDL (• = I/R + HDL: n = 9 at 24 h, n = 7 at 48 h) 30 min before ischemia and after 12-h reperfusion). Data represent mean ± SEM for n observations; ★ P < 0.05 versus I/R only group. Histologic examination of kidney sections prepared from rats subjected to ischemia followed by reperfusion for 48 h and treated with saline in place of HDL (n = 5, Figure 8B) displayed more severe renal injury than rats subjected to I/R but which were administered HDL (n = 7, Figure 8C). Hematoxylin and eosin; original magnification, ×125; figures are representative of at least three experiments performed on different days.
Discussion
In the present study, we have shown that renal I/R of the rat kidney results in a reduction in renal function as demonstrated by increased serum levels of creatinine. This glomerular dysfunction correlated with increased FENa, indicating dysfunction of the PT. Tubular injury was also confirmed by increased NAG enzymuria. As previously discussed, increased serum levels of AST confirmed reperfusion-injury caused by renal I/R (28). Increased levels of AST in serum or plasma usually denote hepatic injury; however, this enzyme is not only specific to the liver and is also found in other organs such as kidney and smooth muscle. Although in many cases of human ischemic ARF, serum AST levels do not rise, levels of this enzyme are elevated after renal tubular injury in the rat (31). As AST is present within the PT (32) and is regarded as a nonspecific marker of extensive cellular disruption or necrosis (33), we have used serum AST in this study as a marker of reperfusion-injury. All these data, together with the expression of the adhesion molecules ICAM-1 and P-selectin, confirmed a well-known pattern of renal dysfunction and injury associated with I/R of the kidney (21,22) and are also in agreement with the notion that reperfusion causes both glomerular and tubular dysfunction (22). Characteristic histologic signs of marked tubular injury were observed in kidneys subsequent to renal I/R, in keeping with the observation that the S3 segment of the PT is particularly susceptible to renal I/R injury (22).
Although we have recently reported the beneficial effects of HDL against renal dysfunction in different models of shock (12,13), the ability of HDL to reduce renal dysfunction and injury caused by I/R of the kidney has not been investigated. We demonstrate here, for the first time, that administration of HDL before I/R produces a significant reduction of renal dysfunction and injury caused by I/R of the kidney of the rat in vivo. The effects of HDL on renal dysfunction and injury were also investigated in a longer-term model of ARF in which renal function was assessed after 24 and 48 h after ischemia. The data obtained indicate that administration of HDL can improve renal function at the later stages of reperfusion following ischemia, which was reflected by a marked reduction in the histologic evidence of renal injury after 48-h reperfusion.
It is interesting to consider the mechanism(s) that may be involved in the beneficial actions provided by HDL against the renal dysfunction and injury caused by I/R. As the antiinflammatory properties of HDL in vivo and in vitro have already been demonstrated (17–19), we postulated that elevation of plasma HDL could provide beneficial actions by reducing the inflammatory components of renal I/R. Several aspects of the effects of HDL on endothelium and PMN could have contributed to their protective effects in our model.
In this study, HDL (1) significantly reduced the numbers of PMN infiltrating into renal tissues during reperfusion and (2) attenuated renal MPO activity to levels that were not significantly different from those measured from the kidneys of Sham-operated rats, together suggesting an almost total reduction in PMN infiltration into renal tissues. Although measurement of MPO activity has been used as an indicator of PMN infiltration into renal tissues (34), PMN counts were also performed as it has recently been reported that the myeloperoxidase assay, chloroacetate esterase, and HIS48 can crossreact with monocytes and macrophages (30,35). The PMN counts obtained in this study were in keeping with those obtained in another renal study investigating interventions against renal I/R in the rat and demonstrating reduction of PMN infiltration into renal tissues (36). Furthermore, our findings are in keeping with previous studies reporting that depletion of PMN activity or numbers reduces renal I/R injury (24,26).
Activated PMN are generally considered to be the principal effectors of renal I/R-injury, as they can release (1) superoxide, which can be converted to hydroxyl radicals, and (2) NO, which can combine with superoxide to form peroxynitrite (22,26,37). Hydroxyl radicals and peroxynitrite are highly reactive and cause tissue injury, e.g., via lipid peroxidation, DNA damage, and activation of poly (ADP-ribose) polymerase (22,26–28). PMN also release myeloperoxidase, which catalyzes the formation of another potent oxidant, hypochlorous acid (37), and also generate cytokines, which interact with the renal endothelium, leading to further pathophysiology (38). In this study, the beneficial role of attenuation of PMN infiltration into renal tissues was reflected by a significant reduction in MDA levels in the kidneys of rats administered HDL before I/R (almost to levels measured in renal tissues obtained from Sham-operated rats), suggesting significant reduction in lipid peroxidation (subsequent to a reduction in the production of ROS).
The results presented in this study also demonstrate that administration of HDL can modulate the expression of adhesion molecules known to be induced during renal I/R. HDL markedly reduced the expression of ICAM-1 and P-selectin, the expression of which, among other adhesion molecules (e.g., VCAM-1, E-selectin), have been associated with renal I/R-injury (23–25). For example, antibodies directed against ICAM-1 and ligands that block P-selectin can significantly reduce renal I/R injury (39,40). Adhesion molecule expression is a fundamental requirement for the recruitment of PMN into renal tissues during renal reperfusion (23–26), and studies using specific markers of PMN indicate their accumulation in postischemic kidneys (26). The data presented here demonstrate that HDL causes a marked attenuation of both the renal expression of adhesion molecules and infiltration of PMN into renal tissues during reperfusion of the rat kidney.
As the pathophysiologic processes involved in renal I/R and the development of ARF are known to be influenced greatly by ROS production (22,41), the antioxidant properties of HDL may have contributed to the observed beneficial effects obtained using HDL in this study. However, the antioxidant properties of HDL that have been documented (42) are related partly to the presence of paraoxonase in some particles (43) and partly to an independent property of apo A-I (44). The beneficial actions of HDL presented here are unlikely to have been due to an effect on paraoxonase, as reconstituted HDL (unlike native HDL) contains apo A-I as the sole protein component (12). Nevertheless, some degree of antioxidant activity of apo A-I cannot be discounted, which may have accounted for part of the effect of HDL in the present model. This possibility is supported by the ability of HDL, in this study, to reduce lipid peroxidation in the kidneys of rats subjected to renal I/R.
HDL has been shown to bind endotoxin in vitro, thereby preventing the pathophysiologic consequences of endotoxin infusion in vivo (20). Although endotoxemia is a known consequence of bowel and liver ischemia (45,46), which can cause renal injury (46), there is no evidence that renal I/R causes a direct increase in plasma levels of endotoxin. It is therefore unlikely that the ability of HDL to bind endotoxin contributed to the renoprotective effects observed in this study.
Lipid metabolism is altered in the early stages of ARF (47), leading to a large decrease in serum HDL levels (48). Furthermore, the protective effects of HDL against oxidative stress are known to be impaired in patients receiving hemodialysis (49). These factors could contribute to the increased incidence of premature cardiovascular disease in patients suffering CRF (50). There are, however, no reports investigating the beneficial effects of administration of HDL during renal I/R or in the development of ARF. In conclusion, this study demonstrates for the first time that administration of HDL significantly reduces renal dysfunction and injury in rats subjected to I/R of the kidneys. The mechanisms underlying the observed protective effects appear to involve reduction of PMN infiltration into renal tissues and oxidative stress subsequent to an attenuation of the expression of adhesion molecules. We propose that HDL, or compounds utilizing similar mechanisms of action, may be useful in enhancing the tolerance of the kidney (or other organs) against I/R injury, e.g., during aortovascular surgery or before renal transplantation. Furthermore, these data suggest that clinical studies could be considered for the evaluation of the therapeutic potential of HDL in improving the course of ischemic ARF.
We thank Dr. Tiziana Genovese and Dr. Rosanna Di Paola, Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Italy, for expert technical assistance. We are also grateful to ZLB-Bioplasma, Bern, Switzerland, for providing HDL. PKC is funded by the National Kidney Research Fund (R41/2/2000). This work was supported in part by the Cli[Combining Acute Accent]nica Me[Combining Acute Accent]dica e[Combining Acute Accent] Diagno[Combining Acute Accent]stico Dr Joaquim Chaves, Lisbon, Portugal.
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