Human In Vitro Oxidized Low-Density Lipoprotein (oxLDL) Increases Urinary Albumin Excretion in Rats.

Hypercholesterolemia-associated oxidative stress increases the formation of oxidized low-density lipoprotein (oxLDL), which can affect endothelial cell function and potentially contribute to renal dysfunction, as reflected by changes in urinary protein excretion. This study aimed to investigate the impact of exogenous oxLDL on urinary excretion of albumin and nephrin. LDL was isolated from a patient with familial hypercholesterolemia (FH) undergoing lipoprotein apheresis (LA) and was oxidized in vitro with Cu (II) ions. Biochemical markers of LDL oxidation, such as TBARS, conjugated dienes, and free ε-amino groups, were measured. Wistar rats were treated with a single intraperitoneal injection of PBS, LDL, or oxLDL (4 mg of protein/kg b.w.). Urine was collected one day before and two days after the injection. We measured blood lipid profiles, urinary protein excretion (specifically albumin and nephrin), and markers of systemic oxidative stress (8-OHdG and 8-iso-PGF2α). The results showed that injection of oxLDL increased urinary albumin excretion by approximately 28% (310 ± 27 µg/24 h vs. 396 ± 26 µg/24 h, p = 0.0003) but had no effect on nephrin excretion. Neither PBS nor LDL had any effect on urinary albumin or nephrin excretion. Additionally, oxLDL did not affect systemic oxidative stress. In conclusion, hypercholesterolemia may adversely affect renal function through oxidatively modified LDL, which interferes with the renal handling of albumin and leads to the development of albuminuria.

Redox regulation of hemodynamics response to diadenosine tetraphosphate an agonist of P2 receptors and renal function in diet-induced hypercholesterolemic rats.

Hypercholesterolemia and oxidative stress may lead to disturbances in the renal microvasculature in response to vasoactive agents, including P2 receptors (P2R) agonists. We investigated the renal microvascular response to diadenosine tetraphosphate (Ap4 A), an agonist of P2R, in diet-induced hypercholesteremic rats over 28 days, supplemented in the last 10 days with tempol (2 mM) or DL-buthionine-(S,R)-sulfoximine (BSO, 20 mM) in the drinking water. Using laser Doppler flowmetry, renal blood perfusion in the cortex and medulla (CBP, MBP) was measured during the infusion of Ap4 A. This induced a biphasic response in the CBP: a phase of rapid decrease was followed by one of rapid increase extended for 30 min in both the normocholesterolemic and hypercholesterolemic rats. The phase of decreased CBP was not affected by tempol or BSO in either group. Early and extended increases in CBP were prevented by tempol in the hypercholesterolemia rats, while, in the normocholesterolemic rats, only the extended increase in CBP was affected by tempol; BSO prevented extended increase in CBP in normocholesterolemic rats. MBP response is not affected by hypercholesterolemia. The hypercholesterolemic rats were characterized by increased urinary albumin and 8-isoPGF2α excretion. Moreover, BSO increased the urinary excretion of nephrin in the hypercholesterolemic rats but, similar to tempol, did not affect the excretion of albumin in their urine. The results suggest the important role of redox balance in the extracellular nucleotide regulation of the renal vasculature and glomerular injury in hypercholesterolemia.

Suramin enhances the urinary excretion of VEGF-A in normoglycemic and streptozotocin-induced diabetic rats.

BACKGROUND: Vascular endothelial growth factor A (VEGF-A) and P2-receptors (P2Rs) are involved in the pathogenesis of diabetic nephropathy. The processing of VEGF-A by matrix metalloproteinases (MMP) regulates its bioavailability. Since the ATP-induced release of MMP-9 is mediated by P2Rs, we investigated the effect of suramin on VEGF-A excretion in urine and the urinary activity of total MMP and MMP-9. METHODS: The effect of suramin (10 mg/kg, ip) on VEGF-A concentration in serum and its excretion in urine was investigated in streptozotocin (STZ)-induced diabetic rats over a 21-day period. The rats received suramin 7 and 14 days after a single STZ injection (65 mg/kg, ip). A 24-h collection of urine was performed on the day preceding the administration of STZ and the first administration of suramin and on the day before the end of the experiment. The VEGF-A in serum and urine, albumin in urine, and total activity of MMP and MMP-9 in urine were measured using immunoassays. RESULTS: Diabetic rats are characterized by a sixfold higher urinary excretion of VEGF-A. Suramin potentiates VEGF-A urinary excretion by 36% (p = 0.046) in non-diabetic and by 75% (p = 0.0322) in diabetic rats but it did not affect VEGF-A concentration in the serum of non-diabetic and diabetic rats. Urinary albumin excretion as well as total MMP and MMP-9 activity was increased in diabetic rats, but these parameters were not affected by suramin. CONCLUSION: Suramin increases the urinary excretion of VEGF-A in normoglycemia and hyperglycaemia, possibly without the involvement of MMP-9. Suramin may be used as a pharmacological tool enhancing VEGF-A urinary secretion.

Diabetes Affects the A1 Adenosine Receptor-Dependent Action of Diadenosine Tetraphosphate (Ap4A) on Cortical and Medullary Renal Blood Flow.

Diabetes through adenosine A1 receptor (A1R) and P2 receptors (P2Rs) may lead to disturbances in renal microvasculature. We investigated the renal microvascular response to Ap4A, an agonist of P2Rs, in streptozotocin-induced diabetic rats. Using laser Doppler flowmetry, renal blood perfusion (RBP) was measured during infusion of Ap4A alone or in the presence of A1R antagonist, either DPCPX (8-cyclopentyl-1,3-dipropylxanthine) or 8-cyclopentyltheophylline (CPT). Ap4A induced a biphasic response in RBP: a phase of rapid decrease was followed by a rapid increase, which was transient in diabetic rats but extended for 30 min in nondiabetic rats. Phase of decreased RBP was not affected by DPCPX or CPT in either group. Early and extended increases in RBP were prevented by DPCPX and CPT in nondiabetic rats, while in diabetic rats, the early increase in RBP was not affected by these antagonists. A1R mRNA and protein levels were increased in isolated glomeruli of diabetic rats, but no changes were detected in P2Y1R and P2Y2R mRNA. Presence of unblocked A1R is a prerequisite for the P2R-mediated relaxing effect of Ap4A in nondiabetic conditions, but influence of A1R on P2R-mediated renal vasorelaxation is abolished under diabetic conditions.

The effect of Cistus incanus herbal tea supplementation on oxidative stress markers and lipid profile in healthy adults.

BACKGROUND: Oxidative stress and dyslipidemia play a critical role in the development of cardiovascular disease (CVD). Regular intake of polyphenol-rich diets is associated with a reduced risk of CVDs. METHODS: The present study was a pilot study with 24 healthy volunteers and was designed to determine if a 12-week administration of Cistus incanus herbal tea, containing phenolic acids and flavonoids, reduces cardiovascular risk factors including oxidative stress and dyslipidemia in healthy adults. Phenolic compounds profile and antibacterial activity of Cistus incanus infusion were also measured. RESULTS: Herbal infusion led to improvement in lipid profile by increase (D4%, p = 0.033) high-density lipoprotein cholesterol concentration and decrease triglyceride (D14%, p = 0.013) concentrations. In addition, the Cistus incanus diet was associated with decreased serum concentrations of malondialdehyde (D16%, p < 0.01) and advanced oxidation protein products (D18%, p < 0.001). CONCLUSIONS: Cistus incanus administration decreases cardiovascular risk factors including oxidative stress and dyslipidemia and this action supports the idea of using Cistus incanus tea on a daily basis as an effective dietary component for prevention of atherosclerotic CVD.

Plasma Levels of Preß1-HDL Are Significantly Elevated in Non-Dialyzed Patients with Advanced Stages of Chronic Kidney Disease.

In chronic kidney disease (CKD), the level of high-density lipoprotein (HDL) decreases markedly, but there is no strong inverse relationship between HDL-cholesterol (HDL-C) and cardiovascular diseases. This indicates that not only the HDL-C level, but also the other quantitative changes in the HDL particles can influence the protective functionality of these particles, and can play a key role in the increase of cardiovascular risk in CKD patients. The aim of the present study was the evaluation of the parameters that may give additional information about the HDL particles in the course of progressing CKD. For this purpose, we analyzed the concentrations of HDL containing apolipoprotein A-I without apolipoprotein A-II (LpA-I), preß1-HDL, and myeloperoxidase (MPO), and the activity of paraoxonase-1 (PON-1) in 68 patients at various stages of CKD. The concentration of HDL cholesterol, MPO, PON-1, and lecithin-cholesterol acyltransferase (LCAT) activity were similar in all of the analyzed stages of CKD. We did not notice significant changes in the LpA-I concentrations in the following stages of CKD (3a CKD stage: 57 ± 19; 3b CKD stage: 54 ± 15; 4 CKD stage: 52 ± 14; p = 0.49). We found, however, that the preß1-HDL concentration and preß1-HDL/LpA-I ratio increased along with the progress of CKD, and were inversely correlated with the estimated glomerular filtration rate (eGFR), even after adjusting for age, gender, triacylglycerols (TAG), HDL cholesterol, and statin therapy (ß = -0.41, p < 0.001; ß = -0.33, p = 0.001, respectively). Our results support the earlier hypothesis that kidney disease leads to the modification of HDL particles, and show that the preß1-HDL concentration is significantly elevated in non-dialyzed patients with advanced stages of CKD.

Progression of Chronic Kidney Disease Affects HDL Impact on Lipoprotein Lipase (LPL)-Mediated VLDL Lipolysis Efficiency.

BACKGROUND/AIMS: Hypertriglyceridaemia (HTG) and reduction and dysfunction of high density lipoprotein (HDL) are common lipid disturbances in chronic kidney disease (CKD). HTG in CKD is caused mainly by the decreased efficiency of lipoprotein lipase (LPL)-mediated very low density lipoprotein triglyceride (VLDL-TG) lipolysis. It has not been clarified whether HDL dysfunction in CKD contributes directly to HTG development; thus, the aim of this study was to assess the impact of CKD progression on the ability of HDL to enhance LPL-mediated VLDL-TG lipolysis efficiency. METHODS: VLDL was isolated from non-dialysis patients in CKD stages 3 and 4 and from non-CKD patients. The VLDL was incubated with LPL at the constant LPL:VLDL-TG ratio, in the absence or presence of HDL. After incubation, the VLDL was separated and the percentage (%) of hydrolyzed TG was calculated. RESULTS: HDL presence increased the lipolysis efficiency of VLDL isolated from CKD and non-CKD patients, for the VLDL-TG> 50 mg/dl. Its effect was dependent on the VLDL-TG and HDL-cholesterol concentrations in the reaction mixtures: the higher the concentrations of VLDL-TG and HDL-cholesterol, the greater the effect. The positive impact of HDL on VLDL lipolysis was modified by CKD progression: the percentage of lipolyzed VLDL-TG in the presence of HDL decreased with a reduction in eGFR (r=0.43, p=0.009), and for patients with stage 4 CKD, no positive impact of HDL on lipolysis was observed. The percentage of lipolyzed TG correlated negatively with apoE and apoCs content in VLDL, and positively with HDL-apoCII, as well as with VLDL and HDL apoCII/ apoCIII ratios. The progression of CKD was associated with unfavourable changes in VLDL and HDL composition; apoE and apoCs levels increased in VLDL with a decrease in eGFR whereas the HDL-cholesterol level decreased. CONCLUSION: The progression of CKD affects lipoprotein composition and properties, and modulates the positive impact of HDL on VLDL lipolysis efficiency. In CKD patients, HDL deficiency and dysfunction can directly affect hypertriglyceridaemia development.

HDL subpopulations containing apoA-I without apoA-II (LpA-I) in patients with angiographically proven coronary artery disease.

BACKGROUND: High density lipoproteins (HDL) can be divided into two metabolically distinct fractions, one containing apolipoprotein (Apo) A-I but not ApoA-II [apolipoprotein A-I; lipoprotein (Lp) A-I] and the other containing both ApoA-I and ApoA-II (LpA-I/A-II). LpA-I fraction which, seeming to be more cardioprotective than LpA-I/A-II particles, is itself heterogeneous. Preß1-HDL is a minor subfraction of LpA-I and the initial acceptor of cellular cholesterol in the process of reverse cholesterol transport. The aim of the study was to determine the usefulness of the determination of LpA-I fractions as indicators for the atherosclerotic process. METHODS: The study included 112 patients with angiographically-documented coronary artery disease (CAD+) and 51 patients with negative results of coronary angiography (CAD-). We evaluated LpA-I concentration in serum in HDL2 and HDL3 fractions as well as Preß1-HDL concentration. Furthermore, we analyzed the association of the assessed parameters with the extent and severity of CAD assessed by Gensini score. RESULTS: CAD+ patients were characterized by a lower concentration of serum LpA-I by 19%, LpA-I in HDL2 by 26%, higher level of Preß1-HDL by 27%, and elevated Preß1-HDL/LpA-I values by 62%. Univariate correlation analysis indicated that serum LpA-I and HDL-cholesterol concentrations were negatively correlated with Gensini score (R=-0.279; p=0.002, R=-0.227; p=0.016, respectively) whereas Preß1-HDL/LpA-I values were positively correlated with the severity of CAD (R=0.529; p<0.001). In multiple linear regression, after adjusting for age, gender, preexisting hypertension, diabetes, and statin therapy, only the Preß1-HDL/LpA-I values remained an independent determinant of atherosclerosis severity (ß=-0.499; p<0.001). CONCLUSIONS: Our results show a lower level of LpA-I and higher concentration of Preß1-HDL in the CAD+ patients compared to the CAD- group. We suggest that the distribution of LpA-I is different in CAD and the Preß1-HDL/LpA-I ratio may have additional value in assessing anti-atherogenic potential of HDL particles and it is likely to become a clinically valuable indicator of atherosclerosis development.

Impact of plant-based diet on lipid risk factors for atherosclerosis.

BACKGROUND: The aim of the study was to investigate the effect of a vegan diet on the serum lipid profile with particular regard to the parameters characterizing the high-density lipoprotein (HDL) fractions in subjects without subclinical atherosclerosis, measured by carotid Doppler ultrasonography. METHODS AND RESULTS: Forty-two 23 to 38 year old subjects (21 omnivores and 21 vegans) participated in the study. Compared to the omnivores, the vegan subjects were characterized by lower parameters of lipid profile: total cholesterol (p < 0.001), low-density lipoprotein (LDL)-cholesterol (p < 0.001), non-HDL-cholesterol (p < 0.001), apolipoprotein B (apoB) (p < 0.001) and phospholipids (p < 0.01). Concentration of HDL-cholesterol was apparently similar between groups. Furthermore, the parameters which characterize HDL particles (con-centration of apolipoproteins AI [apoAI] and AII, HDL-phospholipids, LpAI fraction and pre-b1-HDL fraction) were not significantly different between omnivore and vegan subjects. The apoB/apoAI ratio in vegans was lower than in omnivores (p < 0.01). There was no difference between serum concentration of triacylglycerols between omnivores and vegans. The activity of paraoxonase-1 and 8-iso-prostaglandin F2a concentration were also not different between the study groups. CONCLUSIONS: We suggest that a vegan diet may have a beneficial effect on serum lipid profile and cardiovascular protection, but it is not associated with changes in HDL composition.

Impact of phosphatidylcholine liposomes on the compositional changes of VLDL during lipoprotein lipase (LPL)-mediated lipolysis.

Lipoprotein lipase (LPL)-mediated triacylglycerol (TAG) hydrolysis in very low density lipoprotein (VLDL) is accompanied by the release of surface material containing phospholipids (PL), free cholesterol (FC) and apolipoproteins, E (apoE) and Cs (apoCII, apoCIII). The released molecules are accepted by high density lipoprotein (HDL), and new HDL-sized apoE-containing particles are also generated. A decrease in the number of HDL particles or abnormalities in their structure is associated with unfavourable changes in the features of VLDL remnants. Phosphatidylcholine liposomes (PC-L) can also act as acceptors of surface material components released from lipoproteins. Thus, the aim of this study was to assess the impact of liposomes on compositional changes of VLDL during its LPL-mediated lipolysis. VLDL isolated from human sera was incubated with LPL (LPL:VLDLTAG; 24 µg/ml:90 mg/dl) and/or PC-L (VLDLPL:PC-LPL; 1:30 weight ratio). After incubation (2h, 37 °C) VLDL was separated from other reaction products, and VLDL lipid and apolipoprotein content were analysed. Newly generated HDL-sized apoE-containing lipoproteins were separated by two-dimensional non-denaturing gradient gel electrophoresis (2D-PAGGE). The reaction of VLDL with PC-L in the presence or absence of LPL significantly affected the VLDL composition. The ratio of core (TAG+cholesteryl ester) to surface (PL+FC) lipids in VLDL decreased 1.8-fold with PC-L, 1.2-fold with LPL and 3-fold with PC-L+LPL. The reaction with PC-L and PC-L+LPL caused a 3.7-fold and 3.2-fold decrease of apoCs/apoE average weight ratio, respectively. Compositional changes in VLDL under the influence of PC-L were accompanied by an increase in the efficiency of VLDL lipolysis and the generation of apoE-containing HDL-sized particles, heterogeneous in size (from ∼ 9 to ∼ 18.8 nm) and mobility (γ and preß). We conclude that PL-rich particles, similarly to HDL, promote the release of surface material components from VLDL during LPL-mediated lipolysis and positively influence VLDL features which can facilitate VLDL metabolism. Such PC-L activity may impact on its antiatherogenic properties.

PON-1 Activity and Plasma 8-Isoprostane Concentration in Patients with Angiographically Proven Coronary Artery Disease.

The aim of the study was to estimate association of the extent of angiographically proven coronary artery disease (CAD) with plasma 8-isoprostane F2 (8-iso-PGF2α) levels as a reliable marker of lipid peroxidation and serum activity of paraoxonase-1, which demonstrates the ability to protect against lipid oxidation. The study included 105 patients with angiographically documented CAD (CAD+) and 45 patients with negative results of coronary angiography (CAD-). Compared to the control group CAD+ patients were characterized by increased 8-iso-PGF2α levels (P = 0.007) and reduced activity of PON-1 towards paraoxon (PONase, P = 0.002) and phenyl acetate (AREase, P = 0.037). Univariate correlation analysis indicated that 8-iso-PGF2α concentrations were positively associated with the severity of CAD as evaluated by the Gensini score (R = 0.41, P < 0.001) while PONase activity (R = -0.26, P < 0.05) and AREase activity (R = -0.23, P < 0.05) were inversely correlated with CAD severity. PONase activity and 8-iso-PGF2α concentration remained independent determinant of atherosclerosis severity in multiple linear regression after adjusting for age, gender, smoking habits, hypertension, type 2 diabetes, statin therapy, and HDL-C and TAG concentration (ß coefficients -0.267; P < 0.05 and 0.368; P < 0.001, resp.). The results suggest that PON-1 activity and 8-iso-PGF2α concentration are associated with the presence and extent of coronary stenosis and may be considered additional markers of coronary artery disease.