Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells.

Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E). Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry and confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells. CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardiolipin content. Also CsA released cytochrome c into cytosol and provoked cellular apoptosis. Vit E pretreatment inhibited the effects that CsA induced on mitochondrial structure and function in LLC-PK1 cells and avoided apoptosis. CsA modifies mitochondrial LLC-PK1 cell physiology with loss of negative electrochemical gradient across the inner mitochondrial membrane and increased lipid peroxidation. These features are related to apoptosis and can explain the cellular damage that CsA induces. As Vit E inhibited these effects, our results suggest that they were mediated by an increase in ROS production by mitochondria.

[Cyclosporin A causes oxidative stress and mitochondrial dysfunction in renal tubular cells]. / La ciclosporina A origina estrés oxidativo y disfunción mitocondrial en células tubulares renales.

Reactive oxygen species (ROS) have been implicated in cyclosporin A (CsA) nephrotoxicity. As mitochondria are one of the main sources of ROS in cells, we evaluated the role of CsA in mitochondrial structure and function in LLC-PK1 cells. We incubated cells with CsA 1 microM for 24 hours and studies were performed with flow citometry and confocal microscopy. We studied mitochondrial NAD(P)H content, superoxide anion (O2.-) production (MitoSOX Red), oxidation of cardiolipin of inner mitochondrial membrane (NAO) and mitochondrial membrane potential (DIOC2(3)). Also we analyzed the intracellular ROS synthesis (H2DCF-DA) and reduced glutation (GSH) of cells. Our results showed that CsA decreased NAD(P)H and membrane potential, and increased O2.- in mitochondria. CsA also provoked oxidation of cardiolipin. Furthermore, CsA increased intracellular ROS production and decreased GSH content. These results suggest that CsA has crucial effects in mitochondria. CsA modified mitochondrial physiology through the decrease of antioxidant mitochondrial compounds as NAD(P)H and the dissipation of mitochondrial membrane potential and increase of oxidants as O2.-. Also, CsA alters lipidic structure of inner mitochondrial membrane through the oxidation of cardiolipin. These effects trigger a chain of events that favour intracellular synthesis of ROS and depletion of GSH that can compromise cellular viability. Nephrotoxic cellular effects of CsA can be explained, at least in part, through its influence on mitochondrial functionalism.

Cyclosporine increases local glomerular synthesis of reactive oxygen species in rats: effect of vitamin E on cyclosporine nephrotoxicity.

BACKGROUND: We report an investigation of the effects of cyclosporine (CsA) on kidney function, the glomerular synthesis of reactive oxygen species, the peroxidation of lipids, and the levels of thromboxane B2 (TXB2). The effect of the simultaneous administration of the antioxidant vitamin E (Vit E) and CsA in rats was also evaluated. METHODS: Adult male Wistar rats were treated for 30 days with CsA (30 mg/kg/day), with Vit E (0.05 mg/ml), with CsA plus Vit E, or with the vehicle used for administration of CsA, namely 12.6% ethanol. RESULTS: CsA induced kidney failure and increased the glomerular synthesis of superoxide anion, H2O2, malonyldialdehyde, and TXB2. Vit E minimized the adverse effects of CsA on kidney function and the glomerular synthesis of these compounds. CONCLUSIONS: Our results suggest that the acute decrease in glomerular filtration rate induced by CsA might be mediated by the synthesis of reactive oxygen species and subsequent peroxidation of lipids, which increases the levels of TXB2. Treatment with Vit E prevented these effects, suggesting a possible role for antioxidants in the prevention of CsA nephrotoxicity.

Adenosine induces a calcium-dependent glomerular contraction.

Glomeruli isolated from rat kidney cortex were incubated with adenosine in the presence or absence of verapamil and calcium and their change in cross-sectional area was recorded. Adenosine induced a 10% decrease in glomerular cross-sectional area. This decrease was blocked by verapamil or a calcium-free medium. The results suggest that the effect of adenosine in the kidney could be due to glomerular constriction, and that this constriction depends on the entry of calcium into glomerular cells.

Atrial natriuretic peptide inhibits glomerular contraction induced by angiotensin II and platelet activating factor.

Isolated rat glomeruli were incubated with angiotensin II, PAF and atrial natriuretic peptide (ANP). Angiotensin II and PAF produced a significant glomerular contraction as evidenced by the decrease in glomerular cross-sectional area. The addition of ANP abolished this effect. These results suggest that ANP could modulate glomerular filtration rate, not only by inducing changes in intrarenal hemodynamics, but also by preventing the effect of some hormones on the filtration surface and subsequently on the ultrafiltration coefficient.

Antioxidant nutrients protect against cyclosporine A nephrotoxicity.

The immunosuppressive drug cyclosporine A (CsA) has been successfully used in several diseases with immunological basis and in transplant patients. Nephrotoxicity is the main secondary effect of CsA treatment. Although the mechanisms of nephrotoxitity are not completely defined, there is evidence that suggests the role of reactive oxygen species (ROS) in its pathogenesis. It has been demonstrated in numerous in vivo and in vitro experiments that CsA induced renal failure and increased the synthesis of ROS, thromboxane (TX) and lipid peroxidation products in the kidney. Furthermore, CsA modified the expression and activity of several renal enzymes (ciclooxygenase, superoxide-dismutase, catalase and glutathione-peroxidase). Antioxidant nutrients (e.g. Vitamins E and C) can neutralize some of the effects that CsA produced in the kidney. Thus, Vit E inhibited the synthesis of ROS and TX and the lipid peroxidation process induced by CsA in kidney structures. Antioxidants can also improve renal function and histological damage produced by CsA administration. Although there are few data in humans treated with CsA, the possibility exists that antioxidants can also neutralize CsA nephrotoxicity and LDL oxidation. Thus, antioxidant nutrients could have a therapeutic role in transplant patients treated with CsA.

Cyclosporin A-induced hydrogen peroxide synthesis by cultured human mesangial cells is blocked by exogenous antioxidants.

Cyclosporin A (CsA) is the immunosupressor most widely used in transplanted patients for preventing organ rejection, but it has some toxic side effects in vascular beds and kidney. The purpose of this work was to study if H2O2, a reactive oxygen species, is involved in the CsA-induced toxic effects on kidney in vitro. Human mesangial cells (HMC) in culture were incubated in presence of CsA (10[-5]-10[-8]M) and H2O2 was measured by flow cytometry. The specificity of the probe used in this method was demonstrated as fluorescence was not detected when superoxide anion generated through a Xanthine-Xanthine oxidase system was present, but fluorescence was noted when H2O2 was present in the incubation medium, both directly and after addition of superoxide dismutase to the medium thus promoting H2O2 synthesis. CsA induced a significant dose and time-response increased H2O2 synthesis by cultured HMC. This increase appeared 5 min after CsA addition, being maximal between 15-45 min at CsA concentration of 10(-7)M. When HMC were preincubated with antioxidants as vitamin E or selenium, the CsA-induced H2O2 production was partially blocked. In addition, selenium also induced an increased activity of glutathion peroxidase in HMC after 24 hours of incubation, suggesting that it exerted its H2O2 scavenging action through the modulation of the activity of this enzyme.

[Minimal changes nephrotic syndrome associated to penicillamine treatment]. / Síndrome nefrótico por cambios mínimos asociado a tratamiento con penicilamina.

We describe three patients with minimal change nephrotic syndrome associated with penicillamine treatment. Two patients had systemic sclerosis and one had rheumatoid arthritis. Cumulative dose of D-penicillamine was similar in all cases, and nephrotic syndrome appeared after 15-33 months of treatment. The drug was stopped and nephrotic syndrome disappeared in 2-4 months, suggesting a possible causal relationship between penicillamine and minimal change disease.

C3 glomerulopathy: A new complement-based entity.

C3 glomerulopathy is a new, recently described entity that has changed the perspective, treatment and classification of a number of glomerular diseases. It encompasses 2 similar but clearly differentiated pathologies -the dense-deposit disease and C3 glomerulonephritis itself. The alternative complement pathway plays a fundamental role in its pathogenesis and, specifically, the mutations and defects in its regulatory factors (mainly factor H and factor I), as well as the presence of acquired autoantibodies (C3 nephritic factor), which generates an unbridled activation of the system, and ultimately, a deposit of its products at the glomerular level. Its poor prognosis and onset in young populations makes the detailed study of new therapeutic alternatives for this disease essential. Recently eculizumab, an anti-C5 antibody, has demonstrated effectiveness in the treatment of these patients.

The nephrologist's role in metformin-induced lactic acidosis.

Metformin is an antihyperglycemic agent commonly used in diabetic patients. It is very effective and is able to reduce the plasma glucose and HbA1C. However, in some patients, specially those with comorbidities, metformin can provoke severe lactic acidosis with high morbimortality. Treatment of the lactic acidosis induced by metformin is based on the use of supportive general measures; in severe cases, procedures of extrarrenal purification like hemodialysis or continuous hemodiafiltration have been successfully used.

Atrial natriuretic peptide inhibits angiotensin II-induced contraction of isolated glomeruli and cultured glomerular mesangial cells of rats: the role of calcium.

We examined the contractile effect of angiotensin II on isolated rat glomeruli and cultured glomerular mesangial cells, as well as the possible involvement of calcium in this contraction. In addition, the effect of rat atrial natriuretic peptide (ANP) on angiotensin II-induced contraction was studied. Computer-assisted image analysis was used to measure glomerular cross-sectional area and planar mesangial cell surface area in several experimental conditions. Angiotensin II induced a decrease in glomerular cross-sectional area and planar mesangial cell surface area that was both time and dose dependent. This effect was partially blocked by preincubation with verapamil or TMB-8, the latter compound being more effective. Incubation with ANP prevented angiotensin II-induced reduction of glomerular cross-sectional area and planar mesangial cell surface area and reversed contraction in mesangial cells. Incubation with TMB-8 impaired the effect of ANP. These data suggest that angiotensin II could exert part of its physiologic effects in the kidney through calcium-dependent mesangial and glomerular contraction. ANP inhibited this contraction, possibly explaining some of its renal effects.

Changes in glomerular cross-sectional area induced by platelet activating factor.

Isolated glomeruli from normal rats were incubated with platelet-activating factor 10(-6) M at variable incubation times (8, 15, 30 and 45 min) and with different concentrations (0 to 10(-6) M) for 20 min. In addition, the platelet-activating factor effect (10(-6) or 10(-8) M, 20 min) was tested in the presence of BN-52021 (10 or 50 micrograms/ml), verapamil (10(-5) M), acetylsalicylate of lysine (10(-3) M), and in a free-calcium media with EGTA 2 mM. Glomerular microphotographs were taken before and 20 min after adding the substances, and glomerular cross-sectional area was measured using a computerised technique. Platelet-activating factor induced a significant time-dependent reduction in glomerular cross-sectional area, from a concentration of 10(-8) M. BN-52021, verapamil, and the free-calcium media inhibited platelet-activating factor-induced reduction of glomerular cross-sectional area, but acetylsalicylate of lysine did not. Platelet-activating factor-induced reduction in glomerular cross-sectional area seems to be dependent on the interaction of platelet-activating factor with a specific glomerular receptor, with a subsequent modification of the intracellular concentration of calcium. Arachidonic acid metabolites from the cyclo-oxygenase pathway do not seem to be involved in these phenomena. Results suggest that platelet-activating factor could modulate glomerular filtration rate not only by inducing changes in systemic or intrarenal haemodynamics, but also by modifying the filtration surface, thus reducing the Kf.

Acute myelogenous leukemia treated with daunomycin associated with nephrotic syndrome.

We report a 33-year-old patient with a diagnosis of acute myelogenous leukemia that developed a nephrotic syndrome 9 days after starting treatment with daunomycin. Pathological studies of the kidneys revealed minimal change disease with IgM deposits. Possible pathogenetic mechanisms are discussed.