Nephrinuria and podocytopathies.

The discovery of nephrin in 1998 has launched a new era in glomerular diseases research, emphasizing its crucial role in the structure and function of the glomerular filtration barrier. In the past 20 years, substantial advances have been made in understanding podocyte structure and function as well as the discovery of several podocyte-related proteins including nephrin. The glomerular filtration barrier is comprised of podocytes, the glomerular basement membrane and endothelial cells. Podocytes, with their specialized slit diaphragm, form the essential backbone of the glomerular filtration barrier. Nephrin is a crucial structural and functional feature of the slit diaphragm that prevents plasma protein, blood cell and macromolecule leakage into the urine. Podocyte damage results in nephrin release. Podocytopathies are kidney diseases in which podocyte damage drives proteinuria, i.e., nephrotic syndrome. Many kidney diseases involve podocytopathy including congenital nephrotic syndrome of Finnish type, diffuse mesangial sclerosis, minimal change disease, focal segmental glomerulosclerosis, collapsing glomerulonephropathy, diabetic nephropathy, lupus nephropathy, hypertensive nephropathy and preeclampsia. Recently, urinary nephrin measurement has become important in the early detection of podocytopathies. In this chapter, we elaborate the main structural and functional features of nephrin as a podocyte-specific protein, pathomechanisms of podocytopathies which result in nephrinuria, highlight the most commonly used methods for detecting urinary nephrin and investigate the diagnostic, prognostic and potential therapeutic relevance of urinary nephrin in primary and secondary proteinuric kidney diseases.

Urinary Nephrin and Podocalyxin Levels as Predictors of Pre-eclampsia in High-Risk Pregnant Women.

INTRODUCTION: Pre-eclampsia (PE) is characterized by new-onset hypertension and proteinuria. Damage of podocyte cells has been reported in pre-eclamptic women, thus podocyte specific proteins such as nephrin and podocalyxin could be useful biomarkers in PE. AIM: To investigate the role of urinary nephrin (u-nephrin) and urinary podocalyxin (u-PDX) levels in predicting PE in women with a high-risk pregnancy. MATERIALS AND METHODS: We included 101 pregnant women in this study and allocated them into three groups: group 1 included pregnant women at high risk of developing PE (n=41), group 2 - pregnant women with PE (n=30), and group 3 was the controls including healthy pregnant women (n=30). The inclusion criteria for women with PE were de novo hypertension >140/90 mm Hg, proteinuria >300 mg/24 hours, and presence of edema after 20 weeks of gestation, while the exclusion criteria were a history of renal diseases and pregnant women younger than 18. Inclusion criteria for the group of women with a high-risk pregnancy was gestational week >15, a history of PE in a previous pregnancy, pre-existing diabetes type 1 or 2, pre-existing hypertension, multiple gestations, prior placental abruption, obesity women, nulliparity, maternal age >35 years, and a family history of PE. The study was conducted from March 2016 to May 2017 in the Medical Faculty at the Institute of Medical and Experimental Biochemistry in Skopje. Urine samples were used to measure the nephrin and podocalyxin levels using immunoenzyme assay, creatinine and microalbumin. Blood samples were collected for biochemical analyses. RESULTS: U-nephrin levels were elevated in 96.7% of women with PE, and 73% of women with a high-risk pregnancy. U-PDX levels were elevated in 63% of the women with PE and 100% of the women with a high-risk pregnancy. U-nephrin and u-PDX levels were significantly increased in women with a high-risk pregnancy and women with PE compared with a control group (p.

Role of urinary podocalyxin in early diagnosis of diabetic nephropathy.

Introduction. Podocyte injury has been reported as an early feature of DN therefore, the assessment of podocyte injury can be accomplished by estimation of podocalyxin in urine. This study aimed to estimate the urinary podocalyxin levels and to determine the sensitivity and specificity of this biomarker for early detection of DN.Materials and methods. A total of 90 patients with type 2 diabetes mellitus (T2DM) were included in this cross-sectional study. Sixty of them were without diagnosed DN, and 30 with diagnosed DN. A control group consisted of 30 healthy subjects. All patients with T2DM were divided into three subgroups according to urinary microalbumin/creatinine ratio (UM/CR): normoalbuminuric, microalbuminuric and macroalbuminuric patients. Urine samples, were used for measurement of podocalyxin by ELISA, creatinine and microalbumin. Fasting venous blood samples was collected for biochemical analyses.Results. The levels of urinary podocalyxin (u-PDX) were higher in patients with T2DM compared to control subjects and a statistically significant difference among studied subgroups regarding u-PDX was found (p < 0.05). Levels of u-PDX are increasing gradually with the degree of DN (p < 0.029). u-PDX levels were positively correlated with UM/CR (r = 0.227, p = 0.002). A cut-off level of 43.8 ng/ml u-PDX showed 73.3% sensitivity and 93.3% specificity to detect DN in early stage. A cut-off level of 30 mg/g UM/CR showed 41.5% sensitivity and 90% specificity in predicting DN. u-PDX was elevated in 48,2% of normoalbuminuric patients.Conclusion. Urinary podocalyxin be useful and more sensitive and specific marker in early detection of DN than microalbuminuria.

Urinary nephrin is earlier, more sensitive and specific marker of diabetic nephropathy than microalbuminuria.

BACKGROUND: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. Progressive damage and decline in the number of podocytes often occur in the early stages of DN. Thus, nephrin as a podocyte-specific protein may be regarded as a potential biomarker of early detection of DN. The aim of this study is to determine whether urinary nephrin is an earlier marker in DN than microalbuminuria and to test the significance of urinary nephrin as a marker for early detection of DN. METHODS: Our cross-sectional study included 90 patients with type 2 diabetes mellitus (T2DM), 30 patients with diagnosed DN and 60 patients without diagnosed DN. As a control group, we used 30 healthy subjects. All patients with T2DM were classified into three subgroups according to urinary microalbumin/creatinine ratio (UMCR): normoalbuminuric, microalbuminuric and macroalbuminuric patients. Nephrin in urine was measured by immunoenzyme assay, microalbumin with turbidimetric and creatinine with the photometric method. In blood sera, we measured a few standard biochemical parameters. RESULTS: Nephrinuria was found to be present in 100% of patients with T2DM and macroalbuminuria, in 88% with microalbuminuria, as well as 82% of patients with T2DM and normoalbuminuria. A concentration of urinary nephrin was significantly increased in all groups of subjects with T2DM compared to the control group (p<0.05). Nephrinuria correlated statistically negative with eGFR (r=-0.54). ROC analysis showed that nephrin has a total predicted probability of 96% in patients with DN. CONCLUSIONS: Urinary nephrin is earlier, more specific and sensitive marker than microalbumin in early detection of DN.

Lipoprotein(a) - Link between Atherogenesis and Thrombosis.

Lipoprotein(a) - Lp(a) - is an independent risk factor for cardiovascular disease (CVD). Indeed, individuals with plasma concentrations of Lp(a) > 200 mg/l carry an increased risk of developing CVD. Circulating levels of Lp(a) are remarkably resistant to common lipid lowering therapies, currently available treatment for reduction of Lp(a) is plasma apheresis, which is costly and labour intensive. The Lp(a) molecule is composed of two parts: LDL/apoB-100 core and glycoprotein, apolipoprotein(a) - Apo(a), both of them can interact with components of the coagulation cascade, inflammatory pathways and blood vessel cells (smooth muscle cells and endothelial cells). Therefore, it is very important to determine the molecular pathways by which Lp(a) affect the vascular system in order to design therapeutics for targeting the Lp(a) cellular effects. This paper summarises the cellular effects and molecular mechanisms by which Lp(a) participate in atherogenesis, thrombogenesis, inflammation and development of cardiovascular diseases.

Patient's knowledge and awareness about the effect of the over-the-counter (OTC) drugs and dietary supplements on laboratory test results: a survey in 18 European countries.

Background Nowadays over-the-counter (OTC) drugs and dietary supplements are widely used. Their use can have a significant impact on the validity of laboratory results. The aim of this multicenter European study was to determine the frequency of consumption of various dietary products and OTC drugs among patients and explore their level of knowledge and awareness about the potential impact of various products on laboratory test results. Methods Eighteen European countries participated in this study. The survey was carried out anonymously on a subsequent series of outpatients (n=200) in each participating country. Included were patients who were referred to the laboratory for blood sampling and who voluntarily agreed to participate in the study. The survey included questions about the frequency of consumption of various products, awareness of the importance of informing physicians and laboratory staff about it and information about influence of preanalytical factors in general on laboratory test results. Results In total, 68% of patients were regularly taking at least one OTC drug or dietary supplement. The frequency of patients consuming at least one OTC drug or dietary supplement differed between countries (p=0.001). Vitamins (38%), minerals (34%), cranberry juice (20%), acetylsalicylic acid (ASA) (17%) and omega fatty acids (17%) were the most commonly used in our study. Conclusions The use of various OTC drugs and dietary supplements is highly prevalent in Europe and patients are often not willing to disclose this information to the laboratory staff and ordering physician. The education of both patients and healthcare staff is needed.

Cholesteryl ester transfer protein, low density lipoprotein particle size and intima media thickness in patients with coronary heart disease.

Cholesteryl ester transfer protein (CETP) plays a key role in reverse cholesterol transport and high density lipoprotein (HDL) metabolism. Predominance of small, dense LDL particles is associated with an increased risk of atherosclerosis and coronary heart disease (CHD).The aim of the study was to determine the potential relationship between the CETP concentration and low density lipoprotein (LDL) particle size and their association with intima media thickness (IMT) in patients with CHD. Lipid parameters, CETP concentration and LDL particle size were determined in 100 healthy subjects (control group) and in 100 patients with CHD, aged 43 to 77 years. Plasma CETP concentrations were measured by an enzyme-linked immuno-sorbent assay with two different monoclonal antibodies. LDL subclasses were separated by nondenaturing polyacrilamide 3-31% gradient gel electrophoresis. CETP concentration was higher in patients compared to controls (2.02 ± 0.75 mg/ml vs. 1.74 ± 0.63 mg/ml, p<0.01). Mean LDL particle size (nm) was significantly smaller in patients than in controls (24.5 ± 1.1 vs. 26.1 ± 0.9; p<0.001). There was no relation between LDL particle size and CETP concentration (r=-0.1807, p=0.072). Age, diastolic blood pressure, CETP concentration and LDL particle size were independent factors for determing IMT by multiple linear regression analysis. They accounted for 35.2 % of the observed variability in IMT. CETP is not an independent contributor of LDL particle size. CETP might play a role in determining lipoprotein distributions, but did not seem to be the sole factor in the formation of small LDL particles.

Distribution of low density lipoprotein subclasses in Macedonian children.

BACKGROUND: Current reports claim that small and dense LDL particles are more atherogenic than larger LDL particles. There are many studies presenting LDL subclass distribution in adults, but there is not enough data regarding children in the literature on this problem. The aim of our study was to examine LDL subclass distribution in healthy children in the Republic of Macedonia. MATERIALS/METHODS: Plasma LDL subclasses in 100 children aged 9-18 years were analyzed using non-denaturing polyacrilamide gradient (3-31%) gel electrophoresis. Conventional plasma lipid and apoprotein parameters thought to be related to LDL size were determined as well. RESULTS: The results obtained showed the prevalence of large LDL subclasses (phenotype A) in 89% of the children, whereas small LDL subclasses (phenotype B) were observed in 11%. The mean LDL size was 26.37 +/- 0.68 nm, and there was no difference between gender groups. No association was noted between LDL size and plasma lipid and apoprotein levels, age, or BMI. CONCLUSIONS: LDL size and distribution is not gender- or age-dependent, or influenced by plasma lipid and apoprotein concentrations in childhood. This suggests that analysis of LDL subclass phenotype may provide better information on the risk of atherosclerosis development in adulthood.

Apoprotein(a) phenotypes and plasma lipoprotein(a) concentration in patients with diabetes mellitus.

OBJECTIVES: To determine whether apo(a) isoforms and plasma Lp(a) concentrations in association with some lipid parameters increase the relative risk for the development of atherosclerosis in patients with diabetes mellitus (IDDM and NIDDM). DESIGN AND METHODS: Apo (a) isoforms, Lp(a) and plasma lipids were determined in 40 IDDM and 65 NIDDM patients and in 182 healthy individuals. Apo(a) isoforms were separated by 3 to 15% gradient SDS-PAGE followed by immunoblotting. RESULTS: Logistical analysis showed that: Lp(a) levels >30 mg/dL (RR = 0.25, p < 0.000001; RR = 0.18, p < 0.00002), HTA (RR = 0.212, p < 0.00001; RR = 0.30, p < 0.00001), LMW-S1 apo(a) isoform (RR = 6.86, p < 0.0131; RR = 7.04, p < 0.0057) play a significant role in aterogenecity in both groups of patients with DM (IDDM and NIDDM). The 6.50-fold increase in risk was found in NIDDM patients with high Lp(a) levels (>30 mg/dL) and plasma total/HDL cholesterol ratio (4.5-5.8). CONCLUSION: Elevated Lp(a) levels, LMW S1 apo(a) isoform, HTA and combination of increased Lp(a) levels and total/HDL cholesterol ratio increase the risk for the development of atherosclerosis in patients with DM (IDDM and NIDDM).

Frequency distribution of apoprotein(a) isoforms in patients with diabetes mellitus and healthy subjects.

AIM: To determine the frequency distribution of apoprotein(a) isoforms in patients with insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus and healthy subjects. METHOD: We separated and visualized 5 apo(a) isoforms in 40 patients with IDDM (12 men aged 48.00-/+4.59 and 28 women aged 52.37-/+8.21), 65 patients with NIDDM (26 men aged 61.88-/+9.25 and 39 women aged 60.15-/+7.98), and 182 healthy subjects, using 3-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting. RESULTS: The frequency distribution of apo(a) isoforms was very similar in patients with diabetes mellitus and the control group. Atherogenic low molecular weight (LMW) S1 apo(a) isoform was more frequent in patients with IDDM (7.5%) and NIDDM (6.15%) than in the control group (0.78%). LMW S1 apo(a) isoform in patients with IDDM (relative risk [RR], 6.86; 95% confidence interval [CI], 1.19-25.21; p<0.001) and patients with NIDDM (RR, 7.04; 95% CI, 1.40-35.40; p=0.0057) as well as high molecular weight >S4 apo(a) isoform in patients with NIDDM (RR, 2.39; 95% CI, 1.28-5.21; p=0.0067) significantly increased the risk for the development of atherosclerosis. Mean molecular weight of S3, S1, and B apo(a) isoforms was higher in patients with IDDM and NIDDM than in the healthy subjects carriers of the same isoforms, but this difference was not statistically significant. We estimated high inverse statistical correlation between apo(a) size (kDa) and plasma lipoprotein(a) concentration in all study groups, patients with IDDM (p<0.001), patients with NIDDM (p<0.001), and healthy subjects (p<0.01). CONCLUSION: Not only the increased plasma Lp(a) levels, but also apoprotein(a) isoforms may play an important role as a risk factor for the development of atherosclerosis in patients with diabetes mellitus.

LDL and HDL subclass distribution in patients with end-stage renal diseases.

OBJECTIVES: To examine the alterations in LDL and HDL subclass distribution in ESRD patients compared with a control group and to investigate the relationship of LDL particle size to the other plasma lipoproteins levels. DESIGN AND METHODS: Plasma lipids, LDL and HDL subclasses were determined in 63 hemodialysis patients (HD), 42 predialysis patients and 345 control subjects. Lipoprotein subclasses were separated by polyacrylamide 3 to 31% gradient gel electrophoresis. RESULTS: In predialysis group, 88% subjects had small LDL particles compared with 58.5% of hemodialysis patients and 16.5% of control subjects. Mean LDL size particle diameter was significantly smaller in HD and predialysis patients in comparison with controls (p < 0,0005, p < 0,0001; respectively). Significant inverse correlation between LDL particle size and triglyceride level was observed for both patient groups. Decreased levels of the largest HDL2b subclass was found in both predialysis (16.5%) and in HD patients (30%) as compared with controls (50%), and increased levels of the small HDL3a subclass was found only in predialysis group (21%) in comparison with controls (4.5%). CONCLUSIONS: Alterations in LDL and HDL subclass distribution toward smaller particles is the main lipid abnormality associated with atherogensis found in ESRD. ESRD is associated with reduced levels of HDL2b subclass and increased levels of HDL3c subclass, which occurs in coronary artery disease (CAD) as well.

Low density lipoprotein particle size phenotyping in healthy persons and patients with myocardial infarction.

AIM: To determine distribution, size, and phenotype of low density lipoprotein (LDL) subclasses and examine the influence of plasma lipid concentrations on lipoprotein particle size in both healthy population and patients with myocardial infarction. METHOD: Nondenaturing gradient (3-31%) gel electrophoresis for lipoprotein separation was used to determine the distribution and size of LDL subclasses in 132 patients with myocardial infarction and 334 healthy control subjects. RESULTS: Large LDL subclasses (LDL1, LDL2, phenotype A) were dominant in 88.5% of the healthy population, whereas in most patients with myocardial infarction (81%) the dominant subclasses were LDL3 and LDL4 (phenotype B). Only 19% of the patients belonged to the phenotype A (LDL1 and LDL2). Mean LDL subclass size (nm) was significantly smaller in patients with myocardial infarction than in controls (24.381.07 nm vs 25.940.89 nm; p<0.001). In both groups, LDL size was independent of LDL plasma cholesterol but associated with high triglyceride plasma concentrations. CONCLUSION: Coronary artery disease is associated with the predominance of small LDL particles and high plasma triglyceride concentrations. The risk of development of cardiovascular disease can be assessed more accurately by determining lipoprotein subclasses.

Gradient gel electrophoretic separation of LDL and HDL subclasses on BioRad Mini Protean II and size phenotyping in healthy Macedonians.

BACKGROUND: Lipoprotein subclass determinations provide a more detailed reflection of lipoprotein metabolism and an accurate prediction for risk of cardiovascular disease. Gradient gel electrophoresis for lipoprotein separation on Pharmacia electrophoretic apparatus has been most commonly used for many years. METHODS: In this paper, we describe a new method for separating LDL and HDL subclasses by nondenaturing polyacrylamide gradient (3-31%) gel electrophoresis, using BioRad Mini Protean II electrophoretic cells. RESULTS: The mean particle diameters of cholesterol-stained LDL and HDL lipoproteins were estimated after calibrating the gels with size standards, using fractional absorbance profiles. For the first time in the Republic of Macedonia, lipoprotein distribution and size phenotyping were studied in 345 healthy individuals. Large LDL subclasses (phenotype A) were dominant in 88.5% of the population, whereas small LDL subclasses (phenotype B) were dominant in 11.5%. The mean dominant LDL size was 26.08+/-0.8 nm. Five HDL subclasses were separated on the same gels, and HDL2b and HDL2a (larger) were dominant in healthy Macedonians. CONCLUSION: Antiatherogenic, larger LDL and HDL particles are most commonly found in healthy populations in the Republic of Macedonia.