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.

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.

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.

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.

Flaxseed (Linum Usitatissimum L.) Supplementation in Patients Undergoing Lipoprotein Apheresis for Severe Hyperlipidemia-A Pilot Study.

Being rich in polyunsaturated fatty acids, flaxseed (Linum usitatissimum L.) is thought to be able to decrease lipid levels and dampen inflammation. In this pilot study, we aimed to determine whether flaxseed supplementation could improve the profiles of lipids and inflammatory mediators in patients with severe hyperlipidemia resistant to conventional lipid-lowering pharmacotherapy and requiring lipoprotein apheresis. To this end, six patients received, blindly-in addition to their normal lipoprotein apheresis regimen-a 10-week dietary supplementation with flaxseed (28 g/d) administered in biscuits. This was followed by a 10-week washed out-period and a 10-week supplementation phase with whole wheat placebo. Blood samples were collected at the end of each phase, before the lipoprotein apheresis session. The primary endpoint was the lipid profile and the secondary endpoints were the concentrations of inflammatory mediators and tolerability. Flaxseed supplementation was well-tolerated and resulted in a consistent and significant decrease in total cholesterol and low-density lipoprotein (LDL) levels. The median (and range) percentage decrease was 11.5% (0-18.8) and 7.3% (4.4-26.6), for cholesterol (p = 0.015) and LDL-C (p = 0.003), respectively. On the other hand, there was no significant effect of flaxseed on lipoprotein(a) (Lp(a)), C-reactive protein (CRP), and interleukin 6 (IL-6) concentrations. These observations indicate that flaxseed can produce a cholesterol- and LDL-lowering effect in patients treated with lipoprotein apheresis. Thus, flaxseed supplementation may help to control cholesterol in this patient population. The flaxseed supplementation protocol applied may be of use for further adequately-powered studies to validate and extend our findings.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.

Renal vasculature reactivity to agonist of P2X7 receptor is increased in streptozotocin-induced diabetes.

BACKGROUND: Diabetic nephropathy is characterized by the dysfunction of renal microvasculature. The involvement of the P2X7 receptor, being a part of the purinergic system, is presumable in this process. The aim of our study was to investigate the P2X7 receptor-mediated renal microvasculature response and renal metabolism of extracellular adenine nucleotides in diabetic rats. METHODS: Study was performed on streptozotocin-induced diabetic Wistar rats. The vascular response to BzATP, an agonist of the P2X7 receptor, was monitored based on the changes of cortical blood flow (CBF), glomerular filtration rate (GFR) and glomerular inulin space (GIS). The renal interstitial fluid (RIF) was probed by microdialysis technique and concentrations of ATP and adenosine were measured. Activity on NTDPase and 5'-nucleotidases was measured on renal membranes. RESULTS: Diabetic kidneys were characterized by decreased ATP RIF and increased adenosine RIF concentrations with accompanied enhancement of NTDPase and 5'-nucleotidase activities. BzATP induced a more pronounced increase of CBF and decrease of GFR and GIS in diabetes rats. These effects were abolished by A438079, an antagonist of the P2X7 receptor. CONCLUSIONS: It is possible that increased P2X7 receptor reactivity may be involved in the pathogenesis of diabetic nephropathy.

Chronic renal denervation increases renal tubular response to P2X receptor agonists in rats: implication for renal sympathetic nerve ablation.

BACKGROUND: Kidney noradrenergic innervation regulates tubular function. Adenosine triphosphate (ATP)-a co-transmitter of norepinephrine-acts on purinoceptors, including ion channel receptor, P2X. P2X receptor agonists, α,ß-methylene ATP (α,ß-meATP) and ß,γ-methylene ATP (ß,γ-meATP), induce natriuresis. Regarding the functional co-localization of adrenoceptors and P2X receptors, we evaluated rat renal tubular system sensitivity to natriuretic action of P2X receptor agonists in chronically denervated kidney. METHODS: Clearance studies with α,ß-meATP and ß,γ-meATP (intravenous infusion rate, 2 µmol/kg + 20 nmol/kg/min) were performed after bilateral surgical kidney denervation (DNx) and sham-operation (Sham). Na/K-ATPase activity was measured in isolated rat renal proximal tubules. RESULTS: In DNx compared with Sham, saline infusion significantly increased renal sodium and urine excretion and P2X receptor agonist infusion was significantly more natriuretic and diuretic. In DNx and Sham, respectively, α,ß-meATP increased fractional excretion of sodium (FE(Na)) by 2 ± 0.3 and 0.6 ± 0.1% and urine (FE(V)) by 1.6 ± 0.3 and 0.9 ± 0.2%; ß,γ-meATP had similar effects. In both groups of rats, natriuretic and diuretic actions were abolished by P2 receptor blocker (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate, PPADS), mean arterial blood pressure and glomerular filtration rate remained unchanged during infusion of P2X receptor agonists and antagonist and basal Na/K-ATPase activities in isolated proximal tubules were similar. Both α,ß-meATP and ß,γ-me-ATP decreased the Na/K-ATPase activity, with 20% inhibition (P < 0.05) in denervated and innervated rats; these inhibitory effects were abolished in the presence of PPADS. CONCLUSIONS: Decreased renal sympathetic activity enhances the natriuretic effect of P2X receptor stimulation. This effect is probably not related to altered Na/K-ATPase activity in renal proximal tubules.