An automated, low-latency environment for studying the neural basis of behavior in freely moving rats.

BACKGROUND: Behavior consists of the interaction between an organism and its environment, and is controlled by the brain. Brain activity varies at sub-second time scales, but behavioral measures are usually coarse (often consisting of only binary trial outcomes). RESULTS: To overcome this mismatch, we developed the Rat Interactive Foraging Facility (RIFF): a programmable interactive arena for freely moving rats with multiple feeding areas, multiple sound sources, high-resolution behavioral tracking, and simultaneous electrophysiological recordings. The paper provides detailed information about the construction of the RIFF and the software used to control it. To illustrate the flexibility of the RIFF, we describe two complex tasks implemented in the RIFF, a foraging task and a sound localization task. Rats quickly learned to obtain rewards in both tasks. Neurons in the auditory cortex as well as neurons in the auditory field in the posterior insula had sound-driven activity during behavior. Remarkably, neurons in both structures also showed sensitivity to non-auditory parameters such as location in the arena and head-to-body angle. CONCLUSIONS: The RIFF provides insights into the cognitive capabilities and learning mechanisms of rats and opens the way to a better understanding of how brains control behavior. The ability to do so depends crucially on the combination of wireless electrophysiology and detailed behavioral documentation available in the RIFF.

Increased Circulating Irisin Levels in Kidney Transplant Patients: Is There a Connection with Glycaemic Control?

Irisin is a myokine with potential effects on glucose metabolism and the development of diabetes in humans. We analysed irisin serum levels (ISL) in 47 patients without diabetes before and 1, 2, 3, 4 and 5 weeks after kidney transplantation (KTx). All measurements of irisin before KTx levels were lower than 25 ng/mL (median 8.4 ng/mL). We found an outstanding increase in ISL measured after KTx, reaching more than 1000 times in 44% of patients (HIL-high irisin level group). The increase appeared at the first measurement (one week after KTx). Factors connected to the large growth of ISL were, i.e., BMI > 30 (p = 0.04) and subsequent KTx-second and third (p < 0.001). The global mean blood glucose level during the first two weeks after KTx was significantly lower in the HIL group (p = 0.002), the same as the day-by-day analysed mean fasting and postprandial serum glucose in the first days after KTx. In 12 months of observation, diabetes requiring insulin therapy occurred in the HIL group at a rate of 19%, while in the rest of the patients, the rate was 27%, p = 0.526. Irisin levels increase significantly in some patients after kidney transplantation, accompanied by lower blood glucose levels in the early post-transplant period. Whether an increase in irisin levels results in better glycaemic control remains questionable and requires further research, as well as the relationship between irisin levels and the occurrence of PTDM.

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.

Closing the loop: autonomous experiments enabled by machine-learning-based online data analysis in synchrotron beamline environments.

Recently, there has been significant interest in applying machine-learning (ML) techniques to the automated analysis of X-ray scattering experiments, due to the increasing speed and size at which datasets are generated. ML-based analysis presents an important opportunity to establish a closed-loop feedback system, enabling monitoring and real-time decision-making based on online data analysis. In this study, the incorporation of a combined one-dimensional convolutional neural network (CNN) and multilayer perceptron that is trained to extract physical thin-film parameters (thickness, density, roughness) and capable of taking into account prior knowledge is described. ML-based online analysis results are processed in a closed-loop workflow for X-ray reflectometry (XRR), using the growth of organic thin films as an example. Our focus lies on the beamline integration of ML-based online data analysis and closed-loop feedback. Our data demonstrate the accuracy and robustness of ML methods for analyzing XRR curves and Bragg reflections and its autonomous control over a vacuum deposition setup.

Intravenous haloperidol and cocaine alter the distribution of T CD3+ CD4+ , non-T/NK and NKT cells in rats.

The modulation of dopamine transmission evokes strong behavioural effects that can be achieved by commonly used psychoactive drugs such as haloperidol or cocaine. Cocaine non-specifically increases dopamine transmission by blocking dopamine active transporter (DAT) and evokes behavioural arousal, whereas haloperidol is a non-specific D2-like dopamine receptor antagonist with sedative effects. Interestingly, dopamine has been found to affect immune cells in addition to its action in the central nervous system. Here, we address the possible interactions between haloperidol and cocaine and their effects on both immune cells and behaviour in freely moving rats. We use an intravenous model of haloperidol and binge cocaine administration to evaluate the drugs' impact on the distribution of lymphocyte subsets in both the peripheral blood and the spleen. We assess the drugs' behavioural effects by measuring locomotor activity. Cocaine evoked a pronounced locomotor response and stereotypic behaviours, both of which were completely blocked after pretreatment with haloperidol. The results suggest that blood lymphopenia, which was induced by haloperidol and cocaine (except for natural killer T cells), is independent of D2-like dopaminergic activity and most likely results from the massive secretion of corticosterone. Haloperidol pretreatment prevented the cocaine-induced decrease in NKT cell numbers. Moreover, the increased systemic D2-like dopaminergic activity after cocaine administration is a significant factor in retaining T CD3+ CD4+ lymphocytes and non-T/NK CD45RA+ cells in the spleen.

Nanoparticle Tracking Analysis of Urinary Extracellular Vesicle Proteins as a New Challenge in Laboratory Medicine.

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.

Long-Term Effects of Suramin on Renal Function in Streptozotocin-Induced Diabetes in Rats.

In short-term diabetes (3 weeks), suramin, a drug used clinically, affects renal function and the expression of vascular endothelial growth factor A (VEGF-A), which may be involved in the pathogenesis of diabetic nephropathy, the main cause of end-stage renal disease. In the present study, we evaluated the long-term (11 weeks) effects of suramin (10 mg/kg, i.p., once-weekly) in diabetic rats. Concentrations of VEGF-A, albumin, soluble adhesive molecules (sICAM-1, sVCAM-1), nucleosomes, and thrombin-antithrombin complex (TAT) were measured by ELISA, total protein was measured using a biuret reagent. Glomerular expression of VEGF-A was evaluated by Western blot, mRNA for VEGF-A receptors in the renal cortex by RT-PCR. The vasoreactivity of the interlobar arteries to acetylcholine was assessed by wire myography. Long-term diabetes led to an increased concentration of VEGF-A, TAT, and urinary excretion of total protein and albumin, and a decrease in the concentration of sVCAM-1. We have shown that suramin in diabetes reduces total urinary protein excretion and restores the relaxing properties of acetylcholine relaxation properties to non-diabetic levels. Suramin had no effect on glomerular expression VEGF-A expression and specific receptors, and on sICAM-1 and nucleosomes concentrations in diabetic rats. In conclusion, the long-term effect of suramin on the kidneys in diabetes, expressed in the reduction of proteinuria and the restoration of endothelium-dependent relaxation of the renal arteries, can be considered as potentially contributing to the reduction/slowing down of the development of diabetic nephropathy.

Oral Coenzyme Q10 supplementation leads to better preservation of kidney function in steroid-resistant nephrotic syndrome due to primary Coenzyme Q10 deficiency.

Primary Coenzyme Q10 (CoQ10) deficiency is an ultra-rare disorder caused by defects in genes involved in CoQ10 biosynthesis leading to multidrug-resistant nephrotic syndrome as the hallmark kidney manifestation. Promising early results have been reported anecdotally with oral CoQ10 supplementation. However, the long-term efficacy and optimal prescription remain to be established. In a global effort, we collected and analyzed information from 116 patients who received CoQ10 supplements for primary CoQ10 deficiency due to biallelic pathogenic variants in either the COQ2, COQ6 or COQ8B genes. Median duration of follow up on treatment was two years. The effect of treatment on proteinuria was assessed, and kidney survival was analyzed in 41 patients younger than 18 years with chronic kidney disease stage 1-4 at the start of treatment compared with that of an untreated cohort matched by genotype, age, kidney function, and proteinuria. CoQ10 supplementation was associated with a substantial and significant sustained reduction of proteinuria by 88% at 12 months. Complete remission of proteinuria was more frequently observed in COQ6 disease. CoQ10 supplementation led to significantly better preservation of kidney function (5-year kidney failure-free survival 62% vs. 19%) with an improvement in general condition and neurological manifestations. Side effects of treatment were uncommon and mild. Thus, our findings indicate that all patients diagnosed with primary CoQ10 deficiency should receive early and life-long CoQ10 supplementation to decelerate the progression of kidney disease and prevent further damage to other organs.

Variation of the clinical spectrum and genotype-phenotype associations in Coenzyme Q10 deficiency associated glomerulopathy.

Primary Coenzyme Q10 deficiency is a rare mitochondriopathy with a wide spectrum of organ involvement, including steroid-resistant nephrotic syndrome mainly associated with disease-causing variants in the genes COQ2, COQ6 or COQ8B. We performed a systematic literature review, PodoNet, mitoNET, and CCGKDD registries queries and an online survey, collecting comprehensive clinical and genetic data of 251 patients spanning 173 published (47 updated) and 78 new cases. Kidney disease was first diagnosed at median age 1.0, 1.2 and 9.8 years in individuals with disease-causing variants in COQ2, COQ6 and COQ8B, respectively. Isolated kidney involvement at diagnosis occurred in 34% of COQ2, 10.8% of COQ6 and 70.7% of COQ8B variant individuals. Classic infantile multiorgan involvement comprised 22% of the COQ2 variant cohort while 47% of them developed neurological symptoms at median age 2.7 years. The association of steroid-resistant nephrotic syndrome and sensorineural hearing loss was confirmed as the distinctive phenotype of COQ6 variants, with hearing impairment manifesting at average age three years. None of the patients with COQ8B variants, but 50% of patients with COQ2 and COQ6 variants progressed to kidney failure by age five. At adult age, kidney survival was equally poor (20-25%) across all disorders. A number of sequence variants, including putative local founder mutations, had divergent clinical presentations, in terms of onset age, kidney and non-kidney manifestations and kidney survival. Milder kidney phenotype was present in those with biallelic truncating variants within the COQ8B variant cohort. Thus, significant intra- and inter-familial phenotype variability was observed, suggesting both genetic and non-genetic modifiers of disease severity.

X-ray reflectivity from curved surfaces as illustrated by a graphene layer on molten copper.

The X-ray reflectivity technique can provide out-of-plane electron-density profiles of surfaces, interfaces, and thin films, with atomic resolution accuracy. While current methodologies require high surface flatness, this becomes challenging for naturally curved surfaces, particularly for liquid metals, due to the very high surface tension. Here, the development of X-ray reflectivity measurements with beam sizes of a few tens of micrometres on highly curved liquid surfaces using a synchrotron diffractometer equipped with a double crystal beam deflector is presented. The proposed and developed method, which uses a standard reflectivity θ-2θ scan, is successfully applied to study in situ the bare surface of molten copper and molten copper covered by a graphene layer grown in situ by chemical vapor deposition. It was found that the roughness of the bare liquid surface of copper at 1400 K is 1.25 ± 0.10 Å, while the graphene layer is separated from the liquid surface by a distance of 1.55 ± 0.08 Šand has a roughness of 1.26 ± 0.09 Å.

Hypertriglyceridemia, a causal risk factor for atherosclerosis, and its laboratory assessment.

Epidemiological and clinical studies show a causal association between serum triglyceride (TG) level, the number of triglyceride-rich lipoproteins (TRLs) and their remnants, and the increased risk of atherosclerosis and cardiovascular disease (CVD) development. In light of current guidelines for dyslipidemia management, the laboratory parameters reflecting TRL content are recommended as part of the routine lipid analysis process and used for CVD risk assessment, especially in people with hypertriglyceridemia (HTG), diabetes mellitus, obesity and low levels of low-density lipoprotein cholesterol (LDL-C), in which high residual CVD risk is observed. The basic routinely available laboratory parameters related with TRL are serum TG and non-high-density lipoprotein cholesterol (non-HDL-C) levels, but there are also other biomarkers related to TRL metabolism, the determination of which can be helpful in identifying the basis of HTG development or assessing CVD risk or can be the target of pharmacological intervention. In this review, we present the currently available laboratory parameters related to HTG. We summarise their link with TRL metabolism and HTG development, the determination methods as well as their clinical significance, the target values and interpretation of the results in relation to the current dyslipidemia guidelines.

Mild X-linked Alport syndrome due to the COL4A5 G624D variant originating in the Middle Ages is predominant in Central/East Europe and causes kidney failure in midlife.

A study of 269 children enrolled into a National Registry for children with persistent glomerular hematuria identified 131 individuals with genetically confirmed X-linked Alport Syndrome. A single variant c.1871G>A p.Gly624Asp (G624D) in COL4A5 was predominant and accounted for 39% of X-linked Alport Syndrome in unrelated Polish families (44 of 113). To evaluate its origins, the genetic variation in a 2.79 Mb segment encompassing the COL4A5 locus on chromosome X was assessed. All G624D alleles were found on the same rare haplotype background, indicating a founder effect dating back to the 12-13th century. The phenotypic data of 131 children with X-linked Alport Syndrome and their 195 affected adult relatives revealed that the G624D variant was associated with a significantly milder clinical course in comparison to other pathogenic COL4A5 variants. Furthermore the clinical course of this genetically uniform cohort was milder than that observed in individuals with other COL4A5 missense mutations. In spite of the benign clinical manifestation throughout childhood and early adulthood, the G624D variant confers significant risk for both kidney failure and deafness in males, albeit 20-30 years later than that observed in individuals with other COL4A5 pathogenic variants (50% cumulative risk of starting dialysis at 54 years (95% confidence interval: 50-62) v. 26 years (95% confidence interval: 22-30)). Thus, males with G624D are candidates for existing and emerging therapies for Alport Syndrome.

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.

Non-HDL-C/TG ratio indicates significant underestimation of calculated low-density lipoprotein cholesterol (LDL-C) better than TG level: a study on the reliability of mathematical formulas used for LDL-C estimation.

OBJECTIVES: Low-density lipoprotein cholesterol (LDL-C) is the main laboratory parameter used for the management of cardiovascular disease. The aim of this study was to compare measured LDL-C with LDL-C as calculated by the Friedewald, Martin/Hopkins, Vujovic, and Sampson formulas with regard to triglyceride (TG), LDL-C and non-high-density lipoprotein cholesterol (non-HDL-C)/TG ratio. METHODS: The 1,209 calculated LDL-C results were compared with LDL-C measured using ultracentrifugation-precipitation (first study) and direct (second study) methods. The Passing-Bablok regression was applied to compare the methods. The percentage difference between calculated and measured LDL-C (total error) and the number of results exceeding the total error goal of 12% were established. RESULTS: There was good correlation between the measurement and calculation methods (r 0.962-0.985). The median total error ranged from -2.7%/+1.4% (first/second study) for Vujovic formula to -6.7%/-4.3% for Friedewald formula. The numbers of underestimated results exceeding the total error goal of 12% were 67 (Vujovic), 134 (Martin/Hopkins), 157 (Samspon), and 239 (Friedewald). Less than 7% of those results were obtained for samples with TG >4.5 mmol/L. From 57% (Martin/Hopkins) to 81% (Vujovic) of underestimated results were obtained for samples with a non-HDL-C/TG ratio of <2.4. CONCLUSIONS: The Martin/Hopkins, Vujovic and Sampson formulas appear to be more accurate than the Friedewald formula. To minimize the number of significantly underestimated LDL-C results, we propose the implementation of risk categories according to non-HDL-C/TG ratio and suggest that for samples with a non-HDL-C/TG ratio of <1.2, the LDL-C level should not be calculated but measured independently from TG level.

Synaptic Recruitment Enhances Gap Termination Responses in Auditory Cortex.

The ability to detect short gaps in noise is an important tool for assessing the temporal resolution in the auditory cortex. However, the mere existence of responses to temporal gaps bounded by two short broadband markers is surprising, because of the expected short-term suppression that is prevalent in auditory cortex. Here, we used in-vivo intracellular recordings in anesthetized rats to dissect the synaptic mechanisms that underlie gap-related responses. When a gap is bounded by two short markers, a gap termination response was evoked by the onset of the second marker with minimal contribution from the offset of the first marker. Importantly, we show that the gap termination response was driven by a different (potentially partially overlapping) synaptic population than that underlying the onset response to the first marker. This recruitment of additional synaptic resources is a novel mechanism contributing to the important perceptual task of gap detection.

Organothiol Monolayer Formation Directly on Muscovite Mica.

Organothiol monolayers on metal substrates (Au, Ag, Cu) and their use in a wide variety of applications have been extensively studied. Here, the growth of layers of organothiols directly onto muscovite mica is demonstrated using a simple procedure. Atomic force microscopy, surface X-ray diffraction, and vibrational sum-frequency generation IR spectroscopy studies revealed that organothiols with various functional endgroups could be self-assembled into (water) stable and adaptable ultra-flat organothiol monolayers over homogenous areas as large as 1 cm2 . The strength of the mica-organothiol interactions could be tuned by exchanging the potassium surface ions for copper ions. Several of these organothiol monolayers were subsequently used as a template for calcite growth.

Apolipoprotein E gene polymorphism and renal function are associated with apolipoprotein E concentration in patients with chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) associates with complex lipoprotein disturbances resulting in high cardiovascular risk. Apolipoprotein E (APOE) is a polymorphic protein with three common isoforms (E2; E3; E4) that plays a crucial role in lipoprotein metabolism, including hepatic clearance of chylomicrons and very low-density lipoprotein (VLDL) remnants, and reverse cholesterol transport. It demonstrates anti-atherogenic properties but data concerning the link between polymorphism and level of APOE in CKD patients are inconclusive. The aim of our research was to assess the relationship between APOE gene polymorphism and APOE concentration and its redistribution among lipoproteins along with CKD progression. METHODS: 90 non-dialysed CKD patients were included into the study. Real time PCR was used for APOE genotyping. APOE level was measured in serum and in isolated lipoprotein fractions (VLDL; IDL + HDL; HDL). Kidney function was assessed using eGFR CKD-EPI formula. RESULTS: The population was divided into three APOE genotype subgroups: E2(ε2ε3), E3(ε3ε3) and E4(ε3ε4). The highest APOE level was observed for the E2 subgroup (p < 0.001). APOE concentration positively correlated with eGFR value in the E2 subgroup (r = 0.7, p < 0.001) but inversely in the E3 subgroup (r = - 0.29, p = 0.02).). A lower concentration of APOE in the E2 subgroup was associated with its diminished contents in HDL and IDL + LDL particles. In the E3 subgroup, the higher concentration of APOE was related to the increased number of non-HDL lipoproteins. CONCLUSION: In patients with CKD, APOE genotype as well as renal function are associated with the concentration of APOE and its redistribution among lipoprotein classes.

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.

Insulin increases filtration barrier permeability via TRPC6-dependent activation of PKGIα signaling pathways.

Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner. TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes. Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca2+ overload.