Urinary extracellular vesicle (uEV) proteins may be used as specific markers of kidney damage in various pathophysiological conditions. The nanoparticle-tracking analysis (NTA) appears to be the most useful method for the analysis of uEVs due to its ability to analyze particles below 300 nm. The NTA method has been used to measure the size and concentration of uEVs and also allows for a deeper analysis of uEVs based on their protein composition using fluorescence measurements. However, despite much interest in the clinical application of uEVs, their analysis using the NTA method is poorly described and requires meticulous sample preparation, experimental adjustment of instrument settings, and above all, an understanding of the limitations of the method. In the present work, we demonstrate the usefulness of an NTA. We also present problems encountered during analysis with possible solutions: the choice of sample dilution, the method of the presentation and comparison of results, photobleaching, and the adjustment of instrument settings for a specific analysis. We show that the NTA method appears to be a promising method for the determination of uEVs. However, it is important to be aware of potential problems that may affect the results.

Bicuspid aortic valve (BAV) affects 0.5–2% of the general population and constitutes the major cause of severe aortic valve stenosis (AVS) in individuals ≤70 years. The aim of the present study was to evaluate the parameters that may provide information about the risk of AVS developing in BAV patients, with particular emphasis on lipoprotein(a) (Lp(a)), which is a well-recognized risk factor for stenosis in the general population. We also analyzed the impact of autotaxin (ATX) and interleukin-6 (IL-6) as parameters potentially related to the pathomechanism of Lp(a) action. We found that high Lp(a) levels (>50 mg/dL) occurred significantly more frequently in patients with AVS than in patients without AVS, both in the group below and above 45 years of age (p = 0.036 and p = 0.033, respectively). Elevated Lp(a) levels were also strictly associated with the need for aortic valve replacement (AVR) at a younger age (p = 0.016). However, the Lp(a) concentration did not differ significantly between patients with and without AVS. Similarly, we observed no differences in ATX between the analyzed patient groups, and both ATX activity and concentration correlated significantly with Lp(a) level (R = 0.465, p < 0.001 and R = 0.599, p < 0.001, respectively). We revealed a significantly higher concentration of IL-6 in young patients with AVS. However, this observation was not confirmed in the group of patients over 45 years of age. We also did not observe a significant correlation between IL-6 and Lp(a) or between CRP and Lp(a) in any of the analyzed groups of BAV patients. Our results demonstrate that a high level of Lp(a), greater than 50 mg/dL, may be a significant predictive factor for earlier AVR. Lp(a)-related parameters, such as ATX and IL-6, may be valuable in providing information about the additional cardiovascular risks associated with developing AVS.

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.

Oxidative modifications of lipoproteins play a crucial role in the initiation of atherosclerotic cardiovascular diseases (ASCVDs). Nowadays, the one effective strategy for the treatment of patients with hyperlipoproteinemia(a) is lipoprotein apheresis (LA), which has a pleiotropic effect on reducing the risk of ASCVDs. The significance of oxidative susceptibility of the LDL fraction in ASCVDs has been extensively studied. Whether LA alters the susceptibility of lipoprotein(a) to oxidative modifications remains an unresolved issue. In this study, we isolated lipoprotein fractions by ultracentrifugation in patients with hyperlipoproteinemia(a) undergoing apheresis (LA group) at three time points and patients who were qualified for LA but did not consent to the procedure (non-LA group). We performed copper-mediated oxidation of Lp(a) and LDL fractions and determined autotaxin activity. After apheresis, we observed a lower susceptibility to oxidation of the Lp(a) and LDL fractions as expressed by the extended value of oxidation lag time, decreased slope of the oxidation curve, and decreased final concentration of conjugated dienes. No significant differences were found between these parameters before and 7 days after LA. Additionally, both patients undergoing and not undergoing LA had a significant correlation between autotaxin activity and all parameters characterizing susceptibility to oxidation in the Lp(a) fraction. Our results demonstrate that the pleiotropic effect of apheresis may be related to the reduced oxidative susceptibility of Lp(a) and LDL particles, which may influence the reduction in ASCVD risk in patients undergoing apheresis. The results of the rebound effect 7 days after LA will contribute to a better definition of apheresis frequency guidelines.

Diabetic nephropathy (DN) accounts for approximately 50% of end-stage renal diseases. Vascular endothelial growth factor A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in DN, but its role is unclear. The lack of pharmacological tools to modify renal concentrations further hinders the understanding of its role in DN. In this study, rats were evaluated after 3 weeks of streptozotocin-induced diabetes and two suramin treatments (10 mg/kg, ip). Vascular endothelial growth factor A expression was evaluated by western blot of glomeruli and immunofluorescence of the renal cortex. RT-PCR for receptors Vegfr1 mRNA and Vegfr2 mRNA quantitation was performed. The soluble adhesive molecules (sICAM-1, sVCAM-1) in blood were measured by ELISA and the vasoreactivity of interlobar arteries to acetylcholine was evaluated using wire myography. Suramin administration reduced the expression and intraglomerular localisation of VEGF-A. Increased VEGFR-2 expression in diabetes was reduced by suramin to non-diabetic levels. Diabetes reduced the sVCAM-1 concentrations. Suramin in diabetes restored acetylcholine relaxation properties to non-diabetic levels. In conclusion, suramin affects the renal VEGF-A/VEGF receptors axis and has a beneficial impact on endothelium-dependent relaxation of renal arteries. Thus, suramin may be used as a pharmacological agent to investigate the potential role of VEGF-A in the pathogenesis of renal vascular complications in short-term diabetes.

The improvement in the lifespan of individuals with Down syndrome (DS) has created interest in the context of the development of age-related diseases. Among them is atherosclerosis-based cardiovascular disease (CVD), which seems to be an especially urgent and important issue. The aim of the present study was to evaluate the lipid markers that may clarify cardiovascular risk profiles in individuals with DS. To this end, we analyzed lipid profile parameters, including lipoprotein(a) (Lp(a)) levels, protein composition, and the antioxidative properties of high-density lipoprotein (HDL), in 47 adolescents with DS and 47 individuals without DS. Compared with the control group (C), subjects with DS had significantly increased concentrations of low-density lipoprotein cholesterol (105 ± 31 vs. 90 ± 24 mg/dL, p = 0.014), non-high-density lipoprotein cholesterol (120 ± 32 vs. 103 ± 26 mg/dL, p = 0.006), and triglycerides (72 [55–97] vs. 60 [50–77] mg/dL, p = 0.048). We found that patients with DS were characterized by significantly higher Lp(a) levels (31.9 [21.5–54.3] vs. 5.2 (2.4–16.1) mg/dL, p < 0.001). In fact, 57% of individuals with DS had Lp(a) levels above 30 mg/dL, which was approximately four times higher than those in the control group (DS 57% vs. C 15%). Apart from decreased high-density lipoprotein cholesterol levels in the subjects with DS (53 ± 11 vs. 63 ± 12 mg/dL, p < 0.001), differences in parameters showing the quality of HDL particles were observed. The concentrations of the main proteins characterizing the HDL fraction, apolipoprotein A-I and apolipoprotein A-II, were significantly lower in the DS group (144 ± 21 vs. 181 ± 33 mg/dL, p < 0.001; 33 ± 6 vs. 39 ± 6 mg/dL, p < 0.001, respectively). No significant differences between the groups were observed for the concentration of paraoxonase-1 (DS 779 ± 171 vs. C 657 ± 340 ng/mL, p = 0.063), enzyme activities toward paraoxon (DS 219 [129–286] vs. C 168 [114–272] IU/L, p = 0.949), or phenyl acetate (DS 101 ± 20 vs. C 93 ± 21 kIU/L, p = 0.068). There were no differences in myeloperoxidase activity between the study groups (DS 327 [300–534] vs. C 426 [358–533] ng/mL, p = 0.272). Our results are the first to demonstrate an unfavorable lipid profile combined with higher Lp(a) levels and quality changes in HDL particles in individuals with DS. This sheds new light on cardiovascular risk and traditional healthcare planning for adolescents with DS.