Resistance exercise training ameliorates chronic kidney disease outcomes in a 5/6 nephrectomy model.

Chronic kidney disease (CKD) is defined by decreased glomerular filtration rate (GFR) or increased albumin excretion leading to renal injury. However, exercise training is an important non-pharmacological intervention that ameliorates and protects against Diabetes Mellitus, cardiovascular disease, and CKD. AIM: Our aim was to evaluate the capability of resistance exercise training (RET) to improve CKD outcomes and the contribution of the renal and muscular Akt/mTOR signaling pathway for RET beneficial effects on a CKD model. MAIN METHODS: Male Wistar rats were subjected to RET, followed for 10 weeks, and randomly divided into 5 groups: Sham: Sham-operated; sedentary and nephrectomy (5/6Nx) (SNS); exercising post-5/6Nx (SNE); exercising pre-5/6Nx (ENS); exercising pre- and post-5/6Nx (ENE). The systolic blood pressure (BP) was measured. Creatinine, proteinuria, and blood urea nitrogen (BUN) were evaluated. After euthanasia Renal and muscular Akt/mTOR signaling pathways were analyzed. KEY FINDING: Our study showed that the SNS presented renal injury, hypertension, weight and muscular mass loss and a higher mortality rate. SNS group also decreased renal IL-10 and increased TNF-alfa and TGF-Beta. Renal AKT, mTOR, and rpS6 pathway were increased, PTEN was decreased on SNS. And muscular Akt and mTOR were decreased on SNS. SIGNIFICANCE: The RET before and after the 5/6Nx ameliorates all these parameters mentioned above, suggesting that RET is a good non-pharmacological approach to diminish complications frequently found in CKD. We also suggest that the AKT-m-TOR pathway can play an important role in these beneficial outcomes of RET on the CKD animal model.

P2X7 siRNA targeted to the kidneys increases klotho and delays the progression of experimental diabetic nephropathy.

Previous studies in our laboratory have suggested that P2X7 could contribute to the progression of diabetic nephropathy and modulated klotho expression. The aim of this study was to investigate if P2X7 receptor is related to the expression of klotho in the onset of diabetic nephropathy in rats. Seven-week-old male Wistar rats weighing 210 g were all uninephrectomized; two-third of the animals were induced to diabetes with 60 mg/kg streptozotocin i.v., and one-third received its vehicle (control rats). At 4th day of the fifth week of the protocol, half of the diabetic rats received a small interfering RNA targeting for P2X7 mRNA, and the other half received its vehicle. Euthanasia was made at the eighth week. Diabetic animals reproduced all classic symptoms of the disease; besides, they showed reduced renal function and low NO bioavailability; also, SOD1, SOD2, and catalase were increased, probably due to the oxidative stress which was elevated in this situation. Metabolic data of diabetic rats did not change by silencing P2X7 receptor. For the other hand, silencing P2X7 was able to contribute to balance oxidative and nitrosative profile, ultimately improving the renal function and increasing plasma and membrane forms of klotho. These findings suggest that the management of P2X7 receptor can benefit the kidneys with diabetic nephropathy. Further studies are needed to show the therapeutic potential of this receptor inhibition to provide a better quality of life for the diabetic patient.

Esculin reduces P2X7 and reverses mitochondrial dysfunction in the renal cortex of diabetic rats.

AIMS: To evaluate the effects of esculin treatment on P2X7 receptor and mitochondrial dysfunction in the renal cortex of diabetic rats. MAIN METHODS: Male Wistar rats, 7 weeks old, were unilaterally nephrectomized. Part of these animals were induced to diabetes using streptozotocin (60 mg/kg). Diabetes was confirmed 48 h after induction, with blood glucose levels ≥200 mg/dL. Part of control and diabetic animals were selected to receive daily doses of esculin (50 mg/kg), during 8 weeks. The animals were placed in metabolic cages at the eighth week of protocol for 24 h urine collection and a small aliquot of blood was collected for biochemical analysis. After this procedure, the animals were euthanized and the remaining kidney was stored for histopathological analysis, Western blotting and mitochondrial high-resolution respirometry. KEY FINDINGS: Although esculin did not change metabolic parameters, renal biochemical function, neither TBARS in DM rats, esculin reduced P2X7 levels in these animals and restored mitochondrial function via glycolysis substrates and ß-oxidation. Besides, at the histological analysis, we observed that esculin reduced inflammatory infiltrates and collagen IV deposits as compared to diabetic group. SIGNIFICANCE: Esculin attenuated the development of renal injuries caused by hyperglycemia, proinflammatory and oxidative mechanisms mediated by P2X7 receptor, as seen by histological findings and improved mitochondrial function in diabetic animals. This suggests that esculin could be used as an adjuvant therapy to prevent the diabetic nephropathy.

In vitro cleavage by asbestos fibers of the fifth component of human complement through free-radical generation and kallikrein activation.

Chrysotile and crocidolite fibers incubated in normal human plasma (NHP) generated from the C5 component of complement C5a-type fragments that stimulated polymorphonuclear leukocyte (PMN) chemotaxis. Absorption of NHP with antiserum against C5a totally abolished neutrophil chemotactic activity. Asbestos fibers also produced C5a small peptides in the presence of ethylene glycol bis(beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA) but not ethylene diamine tetraacetic acid (EDTA). Activation of C5 was significantly inhibited when asbestos fibers were pretreated with iron chelators such as sodium dithionite (DTN), deferoxamine (DFX), or ascorbate (AA). Concentration-related inhibition of C5 activation was also observed when asbestos fibers were added concurrently to plasma in the presence of DFX, 1,3-dimethyl-2-thiourea (DMTU), a strong hydroxyl scavenger, or aprotinin (APR), a specific protease inhibitor. Further, chrysotile and crocidolite significantly increased plasma kallikrein activity. Data demonstrate that asbestos-induced C5 activation plays a role in inflammatory reactions characteristic of asbestosis through mechanisms involving iron ions, hydroxyl radicals, and oxidized C5-ike fragments. The ferrous ions present at the asbestos fiber surface trigger this activation and catalyze, via Fenton reaction, the production of hydroxyl radicals, which in turn convert native C5 to an oxidized C5-like form. This product is then cleaved by kallikrein, activated by the same asbestos fibers, yielding an oxidized C5a with the same functional properties as C5a.

Wollastonite fibers in vitro generate reactive oxygen species able to lyse erythrocytes and activate the complement alternate pathway.

Wollastonite fibers were tested in vitro for their ability to produce reactive oxygen species (ROS) with two different systems: a cell-free reactive mixture containing deoxyribose and a polymorphonuclear leukocyte suspension. After adding the fibers, we measured the thiobarbituric acid-reactive substances produced by deoxyribose degradation and luminol-enhanced chemiluminescence, respectively. Compared with asbestos, wollastonite fibers produced higher ROS levels both in the PMN suspensions and in the cell-free reactive mixtures. A large amount of these ROS were not hydroxyl radicals. Indeed we obtained remarkable differences in ROS generation between unground and ground wollastonite fibers and negative results with fibers modified with ferric chloride and dithionite. In addition, ROS generation was partially inhibited (by 46-54%) in the reactions performed in the presence of 1,3-dimethyl-2-thiourea (DMTU), a strong hydroxyl radical scavenger. Wollastonite fibers were also analyzed for their ability to lyse erythrocytes and activate complement. Hemolytic potency was about twice that of chrysotile and half that of crocidolite. The levels of complement activation (via the alternate pathway) were about four-fifths of those measured in zymosan-activated plasma (a typical stimulus used to activate the alternate pathway), equal to those obtained with crocidolite, and two-thirds of those found with chrysotile. The addition of DMTU markedly reduced both these activities. Since asbestos fiber toxicity is mainly due to hydroxyl radical generation, our results indicate that wollastonite fibers are probably less toxic than asbestos fibers.

[Reactive oxygen species produced by the addition of sepiolite and vermiculite (expanded or not) to suspensions of human polymorphonuclear phagocytes and bovine alveolar macrophages]. / Specie reattive dell'ossigeno indotte dall'addizione di sepiolite e vermiculite (espansa e non) a sospensioni di fagociti, polimorfonucleati umani e macrofagi alveolari bovini.

We have studied a sample of commercial sepiolite and two samples of commercial vermiculite, which are clay minerals advised to replace asbestos. We have in vitro tested their abilities to produce reacting oxygen species (ROS) after they have been added to suspension of human polymorphonuclear leukocytes and bovine alveolar macrophages. The behaviour of sepiolite and vermiculite have been compared with those of asbestos fibres given by Unione Internationale contre le Cancer (UICC) and with kaolin and illite. Sepiolite was not able to induce ROS production, while vermiculite was able to induce a relevant ROS generation, even if the values were always lower than that obtained from chrysotile. Kaolin was able to generate a high ROS production.

Biological effects of contaminated silicon carbide particles from a workstation in a plant producing abrasives.

A sample of silicon carbide dust taken in the field from a plant producing abrasives was studied in vitro. The SiC particles (part unmilled and part milled) were able to disturb the structure of erythrocyte membranes and to lead to blood red-cell lysis; they also either interfered with complement and activated the alternate pathway, or interacted with biological media and polymorphonuclear leucocyte membranes, thus eliciting reactive oxygen species production. These in vitro properties were detected both in original large particles and unmilled particles, over 40% of which were of respirable size. The ability of these SiC particles to produce complement activation in vitro lends support to the previous hypothesis, that the radiographic opacities found in two workers employed in the same area of the plant from which the dust tested was taken are due to a reaction by pulmonary interstitial structures to SiC particle inhalation. It is speculated that SiC particles could act like asbestos, the ability of which to activate complement through the alternate pathway is considered to be one of the mechanisms by which the initial asbestotic lesions and subsequent fibrotic inflammatory infiltrates are generated in the lung.

In vitro biological effects of clay minerals advised as substitutes for asbestos.

We studied one sample of commercial sepiolite and two samples of commercial vermiculite--clay minerals proposed as replacements for asbestos--and tested in vitro their abilities to activate complement, to lyse erythrocytes, and to elicit the production of reactive oxygen species (ROS) with human polymorphonuclear leukocytes (PMN) or bovine alveolar macrophages (AM); their behavior was compared with that of asbestos fibers obtained from the Union International Contra Cancer (UICC) as reference standards, as well as with kaolinite and illite, main members of the clay mineral family. Since in short-term in vitro tests the biological activity of mineral particles seems especially related to the active sites on their surface, we first measured the specific surface area of each mineral. Sepiolite was unreactive in two of the three tests we used (complement activation and ROS production) and able to lyse a minimal percentage of red blood cells. Vermiculite was shown to be incapable of activating complement, to have a moderate hemolytic activity and a high ability to elicite ROS production, although lower than that of chrysotile. Sepiolite, therefore, might be of more interest than vermiculite, given the low level of biological effects detected during the tests used to compare both clay minerals with asbestos fibres. The ROS production does not seem to require phagocytosis. A high ROS production was observed with kaolinite: this result casts doubt on the ability of pathogenic mineral dusts in vitro to induce a greater release of ROS than nonpathogenic mineral dusts.

[Importance of the specific surface area in the study of the biological effects of dusts of industrial use]. / Importanza della superficie specifica nello studio degli effetti biologici di polveri ad uso industriale.

We have studied a sample of commercial sepiolite and two samples of commercial vermiculite which are advised to replace asbestos, and we have in vitro tested their abilities to activate the complement and to lyse erythrocytes. Their behaviours has been compared with those of asbestos fibres given by Union Internationale Contre le Cancer (UICC) as reference standards. We have first measured the specific surface area and the weight of each mineral. Sepiolite has been inactive in complement activation and little able to lysis red blood cells on the base of surface area; on the base of the weight it has been unreactive in complement activations but very reactive in red blood cells lysis. We believe that in the in vitro assays to evaluate the biological effects of the substitutes of asbestos it is very important to considerate as the weight and as the specific surface are of each clay mineral.

[Acrylonitrile and chemotaxis: in vitro experiments]. / Acrilonitrile e chemiotassi: esperimenti in vitro.

We have verified that chemotaxis of isolated human polymorphonuclear leukocytes (PMN) was a target of in vitro toxicant effect of acrylonitrile (ACN). This toxicant induced a significant dose dependent decreasing of chemotaxis with 50% inhibition (IC50) occurring at 15 mM. We assume that PMN from workers exposed to ACN reacts vivo in a similar way to PMN exposed in vitro to ACN. We propose therefore to use chemotaxis assay as a biomarker of early biological effect of ACN in workers, since for their monitoring there are so far only internal dose indicators but no suitable effect indicators.

In vitro effect of acrylonitrile on specific activities of human polymorphonuclear leucocytes.

N-Formyl-methionyl-leucyl-phenylalanine (F-MLP)-induced chemotaxis and luminolamplified chemiluminescence were studied in isolated human polymorphonuclear leucocytes (PMN) and exposed to acrylonitrile (ACN) in vitro. Chemotaxis was reduced at ACN concentrations of 3 mm and above, while respiratory burst was affected at ACN concentrations of 18mm and above. The ACN concentrations were highly correlated both with decreased chemotactic activity (r = 0.75; P < 0.05) and with decreased respiratory burst (r = 0.93; P < 0.01).

Human polymorphonuclear leukocyte chemotaxis as a tool in detecting biological early effects in workers occupationally exposed to low levels of n-hexane.

Human polymorphonuclear leukocytes (PMN) were chosen to measure two cellular end points--chemotaxis and respiratory burst--and to verify whether they could function as biomarkers of early effect in detecting occupational exposure to n-hexane of apparently healthy shoe workers, without any electroneuromyographic (ENMG) abnormality. Chemotaxis, but not respiratory burst, was found to be impaired. A negative linear correlation between chemotaxis of PMN of those workers that had been exposed to n-hexane versus 2,5-hexanedione (2,5-HD) urinary concentrations were found. This negative trend is consistent with our previous in vitro experimental findings: it was observed that the progressive addition of 2,5-HD to PMN suspensions inhibited chemotaxis in a dose-dependent mode, while chemiluminescence was not modified. Now we have confirmed in vivo that chemotaxis is more sensitive than the respiratory burst response to 2,5-HD. Such results justify the interest in the behaviour of PMN harvested from workers exposed to n-hexane. Since significant inhibition of chemotactic activity was observed in some workers whose urinary 2,5-HD levels were lower than 5 mg l-1, which is the biological exposure index suggested by ACGIH, this study suggests that PMN chemotaxis may be proposed as a useful biomarker in detecting occupational exposure to low level of n-hexane.

Chemotactic activity of human polymorphonuclear leukocytes and industrial xenobiotics: a brief review.

This review deals with some of our contributions to the use of chemotaxis, as a tool in evaluating effects of industrial xenobiotics on PMN, either in vitro or ex vivo. In vitro experiments have shown that lead, arsenic, styrene and 2,5-hexanedione, a major neurotoxicant metabolite of n-hexane, reduce chemotaxis. The most important results of ex vivo experiments have confirmed those obtained in vitro with styrene and 2,5-hexanedione: a significant reduction of chemotaxis was indeed observed in PMN harvested from workers exposed to low levels of n-hexane or styrene who did not show any sign of biochemical or clinical alteration. After 3 weeks under non-exposed conditions, the chemotactic indexes were markedly increased in most of the workers which were exposed to styrene and in all the workers exposed to n-hexane, all of whom have shown a reduced chemotaxis at the first blood sampling. Moreover chemotaxis was found to be significantly reduced at relative low levels of lead: results of the in vitro and ex vivo experiments show a comparable ranking of midpoint inhibition concentrations. We are only at the dawn of the understanding of the relation between occupational xenobiotics and PMN chemotaxis. This research area is still promising for the future, since PMN chemotaxis seems to be adequate and it must therefore enter in well-defined study protocols for investigating the potential immunotoxicity of occupational chemicals to which humans are exposed at low levels.

Effects of lead on polymorphonuclear leukocyte (PMN) functions in occupationally exposed workers.

Previous in vitro experiments have shown that lead can inhibit PMN chemotaxis, phagocytosis and superoxide formation. Moreover, we have observed an inhibition of PMN chemotaxis in workers occupationally exposed to lead with a mean blood lead concentration of 3.06 mumol/l. The present study was carried out to evaluate locomotion and luminol assisted chemiluminescence (CL) of polymorphonuclear leukocytes (PMN) harvested from ten lead occupationally exposed workers with blood lead concentrations of 1.59 mumol/l (SD 0.27 mumol/l). Since lipids affect PMN activity and lipid composition is modified in erythrocytes of lead workers, PMN lipids were also studied. Ten healthy male subjects of the same age were taken as controls. Chemotaxis, i.e. locomotion stimulated through a specific membrane receptor, was impaired in the PMN of lead workers, but random migration, i.e. unstimulated cell locomotion, and respiratory burst were both unmodified. Cholesterol and phospholipids were not changed, but the percentage of arachidonic acid was significantly increased. The release of LTB4, generated by the oxidative metabolism of arachidonic acid, was increased. CL, which detects reactive oxygen species (ROS), was unmodified, but this lack of change could be the result of an increase in ROS, due to the augmentated percentage of arachidonic acid, and of a decrease in ROS, due to a direct inhibitory effect of lead on ROS generation. On the basis of the results from these ex vivo experiments, the conclusion that chemotaxis is the PMN function primarily affected by lead was confirmed. PMN are considered to be one of the first cellular targets for the action of lead; low exposure to lead modifies their activity and mainly modifies chemotaxis and LTB4 production.

Impairment of chemotaxis of polymorphonuclear leukocytes from lead acid battery workers.

Since lead impairs in vitro the functions of macrophagic cells, we have studied the chemotactic activity of polymorphonuclear leukocytes (PMNs) obtained from lead acid battery workers who were removed from exposure one month before, because they had an abnormal lead absorption. Controls were 18 age matched subjects without any history of occupational lead exposure. Both lead acid battery workers and controls had no alterations of the blood haematological and metabolic parameters. Chemotaxis was carried on in Boyden chambers using zymosan activated serum as chemotactic stimulus. The chemotactic indexes are 56.4 +/- 8.7 in acid battery workers and 75.6 +/- in controls. The difference, which is statistically significant, shows that lead workers have an impairment of PMNs chemotactic activity.

Activation of the alternative complement pathway and generation of stimulating factors for granulocytes by glass fibers.

Continuous-filament glass fibers coated with organic agents, candidate asbestos substitutes, were assessed for their ability to elicit from normal human serum complement-derived cleavage products which are able to stimulate the chemotaxis and the respiratory burst of polymorphonuclear leukocytes. Glass fibers generated chemoattracting and respiratory stimulating factors for polymorphonuclears from human serum. The effect was dose related for chemotaxis from the serum fiber concentration of 75 micrograms/ml to 1,250 micrograms/ml. The serum chemoattracting activity, as well the respiratory stimulation, were dramatically impaired when serum had been preliminarily absorbed with antiC5 antiserum. Since the impairment of chemotactic activity occurred also in the presence of EDTA, but not in the presence of EGTA, we assumed an activation of the alternative complement pathway. Glass fibers were studied in comparison to a UICC sample of Canadian chrysotile asbestos, which is able to activate in vitro the alternative complement pathway. Glass fibers exhibited less ability than asbestos fibers to generate complement cleavage products with chemotactic activity for polymorphonuclears; however, they produced an activity about equal to 80% of a chemotactic standard stimulus such as zymosan-activated plasma.

In vitro impairment of human granulocyte functions by lead.

Chemotaxis and receptor independent phagocytosis of human polymorphonuclear leukocytes (PMNs) exposed to lead in vitro (concentrations between 1.2 microM and 115 microM) were studied. Chemotaxis was measured in Boyden chambers and phagocytosis was investigated using latex beads. Additional methods were also applied. Superoxide anion formation from PMNs activated with preopsonized zymosan was quantified as superoxide dismutase-inhibitable reduction of ferricytochrome c. Steady state fluorescence polarization was performed using trimethylammonium diphenylexatriene (TMA-DPH). Lead concentrations were highly correlated both with decreased chemotactic activity (r = 0.70 p less than 0.01) and with decreased phagocytosis (r = 0.68 p less than 0.01). Ferricytochrome c reduction was not significantly affected. An increase in fluorescence polarization was recorded at the highest concentration of lead used, i.e. 57.6 microM and 115 microM, both in unstimulated PMNs and in PMNs activated with N-formyl-methionyl-leucyl-phenylalanine chemotactic peptide (n-FMLP). Moreover, an increase in the fluorescence polarization was observed in PMNs pretreated with a microtubule disrupting drug, exposed to lead concentrations of 14.4 microM and 57.6 microM and then activated with n-FMLP; no increase was recorded at the lowest lead concentrations used, i.e. 1.2 microM and 3.6 microM. The possible interaction of lead with the membrane-cytoskeleton apparatus is discussed.

Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione.

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and gamma-valerolactone were used; 2,5-hexanedione at 75 micrograms/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of fluorescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.

[Modification of granulocyte chemotaxis during anesthesia for surgery]. / Modificazione della chemiotassi di granulociti durante anestesia per intervento chirurgico.

The PMNs of fourteen patients undergoing general anesthesia for surgical operations were examined to determine whether abnormal chemotaxis occurs during these procedures. Neutrophil chemotaxis was determined immediately before anesthesia and after the anesthetic somministration. Anesthetic agents included 0.2-1% enflurane, 0.3-1% halothane or N20-02 (60:40). Neutrophil chemotaxis was reduced an average of 10.9% by fluorinated anesthetics, no significant modification (+8.1 +/- 13.0%) was detected by N20-02.