Increased blood levels of platelet-activating factor in insulin-dependent diabetic patients with microalbuminuria
Paolo Cavallo-Perin1,
Enrico Lupia1,
Gabriella Gruden1,
Carla Olivetti1,
Antonella De Martino,
Maurizio Cassader1,
Daniela Furlani1,
Luigi Servillo2,
Lucro Quagliuolo2,
Eugenio Iorio2,
Marcia Rosario Boccellino2,
Guiseppe Montrucchio1 and
Giovanni Camussi1,
1 Department of Internal Medicine and Department of Clinical Pathophysiology, University of Turin and
2 Department of Biochemistry and Biophysics, II University of Neaples, Italy
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Abstract
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Background. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, may induce an enhanced size- and charge-dependent glomerular permeability in experimental animals. Studies on the role of PAF in enhanced glomerular permeability in the early phase of diabetic nephropathy are still lacking.
Methods. We evaluated the intravascular levels of PAF and its main catabolic enzyme, the PAF-specific plasma acetyl-hydrolase (PAF-AH), in basal conditions and after exercise, in normo- or micro-albuminuric insulin-dependent diabetic (IDD) patients and in normal subjects.
Results. The results obtained indicate that the concentration of PAF in whole blood was significantly enhanced in basal conditions, during and after exercise in all microalbuminuric IDD patients, but not in normoalbuminuric IDD or in control subjects. The increased concentration of PAF did not correlate with changes in the activity of PAF-AH, suggesting an enhanced production rather than a decreased catabolism of PAF.
Conclusions. These results indicate an association between increased production of PAF and enhanced glomerular permeability in microalbuminuric IDD patients.
Keywords: PAF; diabetes; microalbuminuria; exercise
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Introduction
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In insulin-dependent diabetic (IDD) patients microalbuminuria is considered an expression of widespread vascular injury possibly indicating micro- and macro-angiopathy [1]. In particular, the increased albumin excretion rate (AER) is an early predictor for the development of overt diabetic nephropathy [2,3]. Although the exact mechanism of microalbuminuria is still unknown, several factors that may contribute to its development have been identified [4]. Intraglomerular haemodynamic alterations [4] and changes in size- and charge-dependent permeability selectivity of the glomerular basement membrane (GBM) [5,6] have been implicated in the pathogenesis of the enhanced albumin excretion. Recently, it has been suggested that endothelial dysfunction precedes the development of microalbuminuria [7]. Endothelial dysfunctions include an increase in vascular resistance due to reduced synthesis of nitric oxide [8] and enhanced production of endothelin by the endothelium [9], a decrease in endothelial anticoagulant and profibrinolytic properties [10], and an altered barrier function of the endothelium [1]. Several mechanisms of inflammation have been studied as potential candidates for the endothelial alterations [810] and the changes in permaselectivity of GBM [5,6]. Due to its potent biological actions on endothelial cells, leukocytes and platelets (PLTs), platelet-activating factor (PAF) is a potential candidate for the enhanced vascular and glomerular permeability observed in diabetes [11]. Previous studies on experimental animals have shown that PAF, a phospholipid mediator of inflammation, may induce an enhanced size- and charge-dependent permeability of GBM [12,13]. PAF may act either directly on glomerular permeability or indirectly, via the release of cationic mediators from PLTs [12,13]. Acute physical exercise is able to induce an increase in AER [14] in IDD patients.
The aim of the present study was to evaluate whether the intravascular levels of PAF and of PAF-specific plasma acetyl-hydrolase (PAF-AH) vary in normo- or micro-albuminuric IDD patients with respect to normal subjects. The study was performed under basal conditions and after an increase in AER induced by exercise.
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Subjects and methods
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Reagents
1-O-Hexadecyl-2-sn-lyso-glyceryl-3-phosphorylcholine (2- lysoPAF C16 : O) and 1-O-hexadecyl-2-sn-acetyl-glyceryl-3-phosphorylcholine (PAF C16 : O) were from Bachem Feinchemikalien (Bubendorff, Switzerland). WEB 2170 (Boehringer Ingelheim KG, Germany) was used as PAF-receptor antagonist [15]. Silica gel 60F254 thin-layer chromatography (TLC) plates were obtained from Merck (Darmstadt, Germany). µPorasil high-performance liquid chromatography (HPLC) columns were provided from Millipore Chromatographic Division (Waters, Milford, MA). 7-Diethylamino-coumarin-3-carbonylazide (DEACZ) was purchased from Molecular Probes (Eugene, Oregon, USA). Anhydrous toluene, butanone, chloroform, methanol, and choline chloride were from Fluka Chemika (Buchs, Switzerland). Bovine serum albumin (fraction V, lipid-free) was purchased from Sigma Chemical Company (St Louis, MO, USA).
Subjects
We recruited seven healthy control subjects and 14 IDD patients (seven microalbuminuric and seven normoalbuminuric), without acute or chronic infections, preproliferative or proliferative retinopathy, diabetic neuropathy, cardiovascular disease, hypertension and drug treatment (except insulin in diabetic patients). None of the subjects had positive familial or personal history of non-diabetic renal disease or altered urine sediments and creatinine clearance values. All subjects had a similar degree of physical fitness (regular exercise once or twice per week). All subjects gave their informed consent to participate in the study that was conducted according to the declaration of Helsinki.
Microalbuminuric IDD patients were recruited consecutively, whereas normoalbuminuric patients and control subjects were selected to obtain groups comparable for age, gender, body mass index (BMI), diabetes duration and glycated haemoglobin (HbAlc) (Table 1
). The maximum difference in individual pairs was 11 years for age, 4.7 for BMI, 6 years for diabetes duration, and 3.7% for HbAlc. Patients were considered normoalbuminuric if AER was <20 µg/min and microalbuminuric if AER was 20200 µg/min in two of three overnight urine collections. The AER values were log-transformed to perform statistical analysis. Blood pressure was measured three times on the right arm with a standard clinical sphygmomanometer and the mean value used for comparison. Hypertension and normotension were defined according to Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure criteria. Retinopathy was assessed by fundic examination after pupil dilation. The absence of cardiovascular disease was assessed by history, physical examination, and resting ECG. Somatic or autonomic neuropathy was assayed by standard tests.
Exercise test
At 08 : 00 after an overnight fasting, subjects voided their bladder and rested in sitting position for a 1-h period, immediately preceded and followed by a 500 ml drink of tap water to increase urinary output. During this period, subjects received their regular subcutaneous insulin injection and their breakfast (milk and bread).
Subsequently, 20 min of exercise at the constant workload of 600 kpm/min on a bicycle ergometer (Monark-Crescent AB, Varberg, Sweden) was performed. After exercise, patients rested in a sitting position for 60 min. Blood pressure and heart rate were monitored at the beginning, every 10 min during the exercise, and 30 and 60 min after exercise. Urine was collected over the following three periods, according to the method of Viberti et al. [14]: (i) at 1 h pre-exercise resting period; (ii) at 20 min exercise period and (iii) during 1 h post-exercise period. The total urine volume was measured and stored in aliquots at -80°C.
Venous blood samples for plasma glucose and free insulin determinations, were drawn at the beginning and at the end of the exercise, and at 30 and 60 min of the recovery phase from an indwelling butterfly needle kept open with 0.9% NaCl infusion.
Analytical methods
Plasma glucose was assayed by the glucose oxidase method (Beckman II, Glucose Analyser, Fullerton CA); free insulin was measured by radioimmunoassay (RIA) using the two-antibody method (Eiken Chemical Co. Ltd, Tokyo, Japan); HbAlc was determined by HPLC (DIAMAT, Bio-Rad, Richmond, CA); plasma triglycerides and total cholesterol were determined enzymatically (Boehringer Mannheim, Germany) and high-density lipoprotein (HDL)-cholesterol was measured by precipitation with heparin and MgCl2 on whole plasma. Low-density lipoprotein (LDL)-cholesterol was calculated by Friedewald's formula; and urinary albumin concentration was determined by RIA (Kabi-Pharmacia, Uppsala, Sweden). AER (µg/min) was calculated as the timed urine volume multiplied by the albumin concentration.
Extraction and purification of PAF from blood
Blood (5 ml) was collected into plastic tubes containing 0.125 ml of 0.2 mol/l EDTA. Immediately after withdrawal, blood was acidified to pH 3.03.5 with 1 N HCl (to destroy the acid-labile inhibitor of PAF) and was mixed with 20 ml of methanol to precipitate proteins and extract lipids [16]. After 1 h of incubation at room temperature, the precipitated proteins were removed by centrifugation, and 2 vol. chloroform, 0.1 vol. methanol, and 0.8 vol. water were added to the supernatant to induce phase separation [16]. The lower chloroform-rich phase was evaporated and submitted to the procedure of PAF purification, quantification, and characterization. The extracted lipids were fractionated by TLC with 65 : 35 : 6 (vol./vol./vol.) chloroform:methanol:water as solvent and then by HPLC using a µPorosil column eluted with 60 : 55 : 5 (vol./vol./vol.) chloroform:methanol:water at 1 ml/min flow rate as previously described [16]. The overall recovery of PAF was 96% as measured by addition of 10 nCi of [3H]PAF whole blood before extraction and purification procedure.
PAF assay
PAF extracted and purified was quantified by bioassay on washed rabbits PLTs [16]. PAF bioactivity, tested after extraction [16] and purification by TLC and HPLC [16], was characterized by comparison with synthetic PAF according to the following criteria: (i) induction of platelet aggregation by a pathway independent of both ADP- and arachidonic acid/thromboxane A2-mediated pathways; (ii) specificity of platelet aggregation as inferred from the inhibitory effect of PAF receptor antagonist WEB 2170 (5 µM); (iii) TLC and HPLC chromatographic behaviour and physicochemical characteristics such as inactivation by strong bases and 5 min heating in boiling water. The methods used were previously described in detail [16].
Fluorimetric PAF-AH assay
Enzymatic assay.
PAF-AH was measured by a recently described fluorimetric assay [17]. Briefly, 25 µl of serum were mixed with 25 µl of 1 mg/ml solution of PAF C16 : O, in Tris Tyrode' s buffer (2.6 mM KCl, 1 mM MgCl2, 137 mM NaCl, 6 mM CaCl2, 0.1% glucose, 1 mM Tris, pH 7.4), containing 0.25% lipid free bovine serum albumin.
The incubation was carried out at 37°C for 10 min. In this time, the initial reaction rate was linear for serum volumes from 0 to 30 µl. Then 25 µl of reaction mixture were added with 5 µl of internal standard solution and submitted to extraction and drying procedures in order to quantitate 2-lysoPAF produced by PAF-AH activity [17]. In parallel experiments, the PAF-AH activity was inhibited by boiling serum (10 min) or by acidification to pH 3.5 with 1 N HC1 and this solution was used as a blank.
The enzymatic activity was expressed as pmoles of 2-lysoPAF produced per minute per microlitre of serum.
Extraction procedures.
In a typical assay, 25 µl of reaction mixture, plus 5 µl of internal standard solution, was added with 500 µl of water-saturated butanone, in order to extract 2-lysoPAF produced by the enzymatic reaction [17]. The mixture was then shaken a few minutes and centrifuged (800 g) for 10 min at room temperature to allow phase separation. Samples of 250 µl of the organic phase were dried under vacuum in a 1-ml reacti-vial (Pierce) and derivatized as described below.
Derivatization reaction.
A known volume of organic phase, generally 250 µl, was dried under vacuum in reacti-vials by using a Univapor Concentrator Centrifuge Univapor 100 H (Uni Equip, Martinsried, Germany) [17]. The drying procedure, necessary to completely eliminate water that interferes with the successive derivatization procedure, takes about 30 min at room temperature. One hundred microlitres of a 1 mg/ml solution of DEACZ in anhydrous toluene (derivatizing solution) were then added into the reacti-vials, which were tightly capped and heated at 80°C. After 3 h the vials were cooled and the content directly analysed by HPLC [17].
HPLC.
A model 342 Gradient liquid chromatograph (Beckman) equipped with a Shimadzu model 160 fluorescence spectrometer and a C-R3A Shimadzu Chromatopack integrating system were used [17]. A 3.9x300 mm Nova-Pack C 18 Waters (Milford, USA) reversed-phase column, filled with 4 µm average particle size was used. The mobile phase consisted of a gradient between head solvent (A) composed of methanol-water (80 : 20 vol./vol.) containing 0.25 g/l choline chloride, and chloroform (B). The flow rate of the mobile phase was 1 ml/min. The gradient, starting at the sample injection, was from 0 to 55% solution B in 22 min. At the end of each analysis the column was equilibrated for 10 min with solution A. The fluorescence detector was set at excitation wavelength of 400 nm and at an emission wavelength of 480 nm. The concentrations of derivatized lysophospholipids were calculated by comparing the peak area with that of the internal standard [17].
Statistical analysis
Results are given as mean±SEM. Normoalbuminuric IDD patients and control subjects were matched to microalbuminuric patients for age, gender, BMI, diabetes duration and HbAlc, and one way or two-way variance analysis (ANOVA) for repeated measures was used to compare the patients on pair-wised manner for AER and PAF levels, where appropriate. Differences in plasma PAF-AH activity among the three study groups were evaluated by one-way ANOVA followed by NewmanKeuls multiple-comparison test. Linear regression was used to correlate log-transformed AER values with PAF concentrations. The threshold of statistical significance was taken as P<0.05.
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Results
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AER
AER data in basal conditions as well as during exercise are reported in Figures 1A
and 2A
. Values were significantly higher in microalbuminuric patients compared with normoalbuminuric patients or control subjects both in basal conditions and during exercise.

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Fig. 1. Urinary albumin excretion (A) and intravascular concentration of PAF (B) in seven control subjects, seven normoalbuminuric and seven microalbuminuric IDD patients in basal conditions. Individual values are indicated. Statistical analysis was performed with one-way ANOVA for repeated measures to compare the subjects on pair-wised manner. (*P<0.001: microalbuminuric patients vs controls; P<0.001: microalbuminuric vs normoalbuminuric patients).
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Fig. 2. Urinary albumin excretion (A) and intravascular concentration of PAF (B) before, at the end (20 min) and 60 min after the end of exercise in seven control subjects, seven normoalbuminuric and seven microalbuminuric IDD patients. Data are expressed as mean±SEM. Statistical analysis was performed with two-way ANOVA for repeated measures to compare the subjects on pair-wised manner. (A) Microalbuminuric patients vs controls P<0.0001; microalbuminuric vs normoalbuminuric patients P<0.0001; controls vs normoalbuminuric patients P<0.05). (B) Microalbuminuric patients vs controls P<0.0001; microalbuminuric vs normoalbuminuric patients P<0.0001; controls vs normoalbuminuric patients not significant.
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Intravascular release of PAF
The intravascular release of PAF in micro- or normoalbuminuric IDD patients and normal subjects was evaluated after whole blood extraction. As shown in Figure 1B
, a significant amount of PAF was detected under basal conditions in all microalbuminuric IDD patients. In contrast, PAF was undetectable in normal subjects and all but one of normoalbuminuric IDD patients. During exercise the intravascular concentration of PAF was slightly increased in microalbuminuric IDD patients and was unchanged in the other two groups of subjects (Figure 2B
). Sixty minutes after the end of exercise the concentration of PAF decreased but remained significantly elevated in microalbuminuric IDD patients with respect to normoalbuminuric IDD patients or normal subjects. A significant correlation between log-transformed AER and PAF concentrations was detected (r2=0.3879, P<0.0001). These observations can not be accounted for a different metabolic control, as plasma glucose and free insulin concentrations were comparable in microalbuminuric and in normoalbuminuric IDD patients (data not shown).
Plasma PAF-AH activity
As shown in Figure 3
, no significant variations were observed in the PAF-AH activity in plasma of micro- and normoalbuminuric IDD patients or normal subjects, both in basal conditions and during exercise.
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Discussion
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The results of the present study indicate that microalbuminuric IDD patients have an enhanced intravascular synthesis of PAF. PAF belongs to the structurally related family of acetylated alkyl phosphoglycerides produced by a broad range of cells including neutrophils, macrophages and endothelial cells (reviewed in [11]). It acts through a specific receptor belonging to the family of seven spanning membrane domain receptors [11] and elicits diverse and potent biological properties relevant for the development of inflammatory reaction [11]. In experimental animal models, PAF has been shown to be a potent enhancer of glomerular [12,13] and vascular permeability [11]. Human endothelial cells express PAF receptor [11] and act as a target for PAF produced by themselves as well as by proximal cells [11]. In vitro PAF enhances the permeability of endothelial monolayers by inducing changes in the cell cytoskeleton leading to intercellular gap formation [11]. In vivo PAF was shown to be 100010 000 times more potent on molar bases than histamine in inducing vascular permeability when injected in rabbit skin [11]. The enhanced glomerular permeability induced by PAF was shown to lead to a significant proteinuria in experimental animals [12,13]. The mechanism of such increase in glomerular permeability has been related both to a direct action of PAF on size-selective permeability [13] and to a PAF-induced release of cationic mediators from PLTs [12]. These mediators include a cationic vascular permeability factor, platelet factor 4, PDGF and a cationic heparitinase [18]. The binding of such cationic proteins to glomerular polyanions was shown to induce neutralization and/or proteolytic degradation of glomerular anionic sites leading to changes in charge-selective permaselectivity [18]. Moreover, the bioactive cationic proteins derived from PLTs may elicit changes in the local haemodynamic conditions by acting on glomerular mesangial cells [18]. We have previously demonstrated the release of platelet-derived cationic proteins and their urinary excretion in IDD patients after increased urinary AER induced by exercise [19]. This evidence may suggest a role for platelet-derived cationic proteins in inducing the loss of charge-dependent permaselectivity observed in diabetic patients. In diabetes, a decrease in the ratio between the biosynthesis of heparan sulphates and the rate of the synthesis of several other matrix components may lower the threshold of concentration of cationic mediators capable to neutralize glomerular and vascular anionic sites [1]. Therefore, diabetes, which is associated with inhibition of N-deacylase activity per se, may represent a condition where a sub-threshold intravascular activation of PLTs induces an enhanced vascular and glomerular permeability [1]. Nathan et al. had previously reported elevated PAF concentrations in IDD patients compared to healthy volunteers [20]. However, in this study, microalbuminuria was not evaluated and it was only reported that patients did not present overt diabetic nephropathy. The results of the present study suggest that an enhanced concentration of PAF, a putative mediator of platelet activation and enhanced vascular permeability, was present in basal conditions and during exercise in blood of microalbuminuric IDD patients but not in normoalbuminuric IDD patients and in controls. This suggests a relationship between this mediator and the enhanced glomerular permeability. The increased blood levels of PAF did not correlate with changes in the activity of PAF-AH, thus suggesting an enhanced production rather than a decreased catabolism [21]. As PAF was shown to alter in vivo endothelial cell barrier functions 11] and to induce release of von Willebrand factor into the circulation [22], it is a potential candidate causing the endothelial cell dysfunction that has been shown to precede microalbuminuria in IDD patients [23]. Plasma glucose and free insulin concentrations were comparable in microalbuminuric and in normoalbuminuric IDD patients suggesting that increases in PAF were not due to alteration in metabolic control. A slight, but not statistically significant, increase in blood pressure was detected in the group of microalbuminuric patients that exhibited enhanced PAF production. However, the intravascular release of PAF has been mainly associated with hypotensive conditions due to the potent hypotensive effect of this mediator 11]. Therefore, the increased levels of PAF in microalbuminuric IDD patients reflects most probably an injury of microvascular endothelium, which can synthesize PAF under a variety of stimuli [11].
In conclusion, this study indicates that an enhanced level of PAF was present under basal conditions in blood of microalbuminuric IDD patients, but not in normoalbuminuric IDD patients and in controls. The increased blood levels of PAF may be related to an enhanced production rather than a decreased catabolism, as no differences in the activity of PAF-AH were detected. Further studies are needed to determine the cellular source of PAF and the mechanisms involved in the enhanced synthesis of this mediator in microalbuminuric IDD patients.
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Acknowledgments
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This work was supported by MURST and by the National Research Council (CNR), Targeted Project Biotechnology to GC.
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Notes
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Correspondence and offprint requests to: G. Camussi, Laboratorio di Immunopatologia Renale, Cattedra di Nefrologia, Dipartimento di Medicina Interna, Corso Dogliotti, 14, I-10126 Torino, Italy. 
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Received for publication: 3. 5.99
Revision received 10. 3.00.