Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes.

Articular cartilage is an avascular and aneural tissue with limited capacity for regeneration. On large articular lesions, it is recommended to use regenerative medicine strategies, like autologous chondrocyte implantation. There is a concern about morphological changes that chondrocytes suffer once they have been isolated and cultured. Due to the fact that there is little evidence that compares articular cartilage chondrocytes with cultured chondrocytes, in this research we proposed to obtain chondrocytes from human articular cartilage, compare them with themselves once they have been cultured and characterize them through genetic, phenotypic and morphological analysis. Knee articular cartilage samples of 10 mm were obtained, and each sample was divided into two fragments; a portion was used to determine gene expression, and from the other portion, chondrocytes were obtained by enzymatic disaggregation, in order to be cultured and expanded in vitro. Subsequently, morphological, genetic and phenotypic characteristics were compared between in situ (articular cartilage) and cultured chondrocytes. Obtained cultured chondrocytes were rounded in shape, possessing a large nucleus with condensed chromatin and a clear cytoplasm; histological appearance was quite similar to typical chondrocyte. The expression levels of COL2A1 and COL10A1 genes were higher in cultured chondrocytes than in situ chondrocytes; moreover, the expression of COL1A1 was almost undetectable on cultured chondrocytes; likewise, COL2 and SOX9 proteins were detected by immunofluorescence. We concluded that chondrocytes derived from adult human cartilage cultured for 21 days do not tend to dedifferentiate, maintaining their capacity to produce matrix and also retaining their synthesis capacity and morphology.

Characterization of N-diethylnitrosamine-initiated and ferric nitrilotriacetate-promoted renal cell carcinoma experimental model and effect of a tamarind seed extract against acute nephrotoxicity and carcinogenesis.

Renal cell carcinoma (RCC), the commonest malignancy in adult kidney, lacks of early signs, resulting often in metastasis at first diagnosis. N-Diethylnitrosamine (DEN)-initiated and ferric nitrilotriacetate (FeNTA)-promoted RCC may be a useful experimental model, but it is not well characterized. In this study, histological alterations and oxidative stress markers were analyzed at different times throughout RCC development, histological subtype was re-evaluated in the light of current classification, and a tamarind seed extract (TSE) effect was examined. Male Wistar rats experimental groups were control, TSE, DEN, DEN+FeNTA, and TSE+DEN+FeNTA. TSE was given 2 weeks before DEN administration (200 mg/kg) and throughout the experiment. Fourteen days after DEN treatment, two FeNTA doses (9 mg Fe/kg) for acute nephrotoxicity study, and increasing FeNTA doses (3-9 mg Fe/kg) twice a week for 16 weeks for carcinogenesis protocol, were administered. In acute study, necrosis and renal failure were observed and TSE ameliorated them. Throughout carcinogenesis protocol, preneoplastic lesions were observed since 1 month of FeNTA treatment, which were more evident at 2 months, when also renal cysts and RCC were already detected. RCC tumors were obtained without changes in renal function, and clear cell histological subtype was identified in all cases. 4-Hydroxy-2-nonenal and 3-nitro-L: -tyrosine levels increased progressively throughout protocol. TSE decreased both oxidative stress markers and, although there was no statistical difference, it delayed RCC progress and decreased its incidence (21 %). This study brings an insight of the time course events in this carcinogenesis model, identifies clear cell subtype and establishes TSE renoprotective effects.

Hepatoprotective effect of acetonic and methanolic extracts of Heterotheca inuloides against CCl(4)-induced toxicity in rats.

A model of hepatotoxicity by carbon tetrachloride (CCl(4)) in rats was used in order to evaluate the protective potential of the acetonic and methanolic extracts of Heterotheca inuloides. Pretreatment with the two H. inuloides extracts attenuated the increase in the activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) observed in CCl(4)-induced liver injury. The protective effect was confirmed by the analysis of tissue slides stained with hematoxylin-eosin and periodic acid/Schiff's reagent. Additionally, the two extracts are scavengers to the superoxide radical as was observed by electron paramagnetic resonance. Due to the fact that the methanolic extract resulted in a better protective effect in the previous experiments, it was used to investigate in more detail the mechanism of hepatoprotection. Quercetin, one of the main components of the extract, with known hepatoprotective and antioxidant activity was used as a positive control. Pretreatment of animals with the methanolic extract or quercetin, was associated with the prevention of 4-hydroxynonenal and 3-nitrotyrosine increase in the liver, two markers of oxidative stress. Furthermore, the decrease in the activity of several antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase in CCl(4)-induced liver injury was alleviated by the pretreatment with H. inuloides methanolic extract or quercetin. These results suggest that the hepatoprotective capacity of H. inuloides methanolic extract is associated with its antioxidant properties, which would also explain the biomedical properties attributed to this plant.

Sulforaphane protects against cisplatin-induced nephrotoxicity.

Cisplatin (cis-diamminedichloroplatinum II, CDDP) is a chemotherapeutic agent that induces nephrotoxicity associated with oxidative/nitrosative stress. Sulforaphane (SFN) is an isothiocyanate produced by the enzymatic action of myrosinase on glucorophanin, a glucosinolate contained in cruciferous vegetables. SFN is able to induce cytoprotective enzymes through the transcription factor Nrf2. The purpose of this study was to evaluate whether SFN induces a cytoprotective effect on the CDDP-induced nephrotoxicity. Preincubation of LLC-PK1 cells with 0.5-5 microM SFN by 24 h was able to prevent, in a concentration-dependent way, CDDP-induced cell death. Immunofluorescent staining confirmed the nuclear translocation of Nrf2 after treatment with SFN. In the in vivo studies, CDDP was given to Wistar rats as a sole i.p. injection at a dose of 7.5 mg/kg. SFN (500 microg/kg i.v.) was given two times (24 h before and 24 after CDDP-injection). Animals were killed three days after CDDP-injection. SFN attenuated CDDP-induced renal dysfunction, structural damage, oxidative/nitrosative stress, glutathione depletion, enhanced urinary hydrogen peroxide excretion and the decrease in antioxidant enzymes (catalase, glutathione peroxidase and glutathione-S-transferase). The renoprotective effect of SFN on CDDP-induced nephrotoxicity was associated with the attenuation in oxidative/nitrosative stress and the preservation of antioxidant enzymes.

Garlic powder ameliorates cisplatin-induced nephrotoxicity and oxidative stress.

cis-Diamminedichloroplatinum (II) (cisplatin) is an effective chemotherapeutic agent successfully used in the treatment of a wide range of tumors. Nevertheless, nephrotoxicity has restricted its clinical use. The use of more than a few antioxidants has shown that reactive oxygen species are involved in cisplatin-induced nephrotoxicity. In the present work the effect of garlic powder, a recognized antioxidant, on cisplatin-induced nephrotoxicity and oxidative and nitrosative stress was studied. Rats were fed with a 2% garlic powder diet for 4 weeks. A single injection of cisplatin (7.5 mg/kg) induced tubular damage and an increase in the following markers of renal injury 3 days later: blood urea nitrogen, serum creatinine, and urinary excretion of N-acetyl-beta-D-glucosaminidase. The cisplatin injection also increased 3-nitrotyrosine and 4-hydroxy-2-nonenal immunostaining in renal cortex and medulla. It was found that the garlic powder feeding was able to prevent by 40-59% the alterations in the markers of renal injury studied, by 33% the histological damage, and by 38-75% the increase in markers of oxidative and nitrosative stress. It is concluded that the ability of garlic powder to ameliorate cisplatin-induced renal injury is associated with its antioxidant properties. Our data support the use of garlic powder as a renoprotective agent.

S-allylcysteine ameliorates ischemia and reperfusion induced renal damage.

The effect of the garlic-derived antioxidant S-allylcysteine (SAC) on renal injury and oxidative stress induced by ischemia and reperfusion (IR) was studied in this work. Rats were anesthetized and subjected to right nephrectomy; 15 min later ischemia was induced for a period of 40 min and then the rats were subjected to a reperfusion period of 6 h after which they were killed to obtain blood and the left kidney. SAC was given at a dose of 100 mg/kg 30 min before nephrectomy, 15 min before ischemia, immediately before reperfusion and 2 h after reperfusion. IR-induced renal injury was evident by the increase in blood urea nitrogen (BUN) and serum creatinine as well as by the renal structural damage which was assessed by histological analysis. IR-induced oxidative stress was evident by the increase in immunostaining with 4-hydroxy-2-nonenal (4-HNE). SAC treatment was able to ameliorate the increase in BUN and serum creatinine and to decrease the structural damage. This protective effect was associated with a decrease in the immunostaining for 4-HNE. It is concluded that the antioxidant properties of SAC are involved in its protective effect on renal ischemia and reperfusion injury.

PJ34, a poly adenosine diphosphate-ribose polymerase inhibitor, attenuates chromate-induced nephrotoxicity.

Potassium dichromate (K(2)Cr(2)O(7))-induced nephrotoxicity is associated with oxidative stress. In addition, the activation of the polyadenosine diphosphate-ribose [poly(ADP-ribose)] polymerase-1 (PARP-1) plays a role in the pathophysiology of some diseases associated with oxidative stress. To clarify the potential role of PARP-1 in this experimental model, N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyacetamide HCl (PJ34), a highly specific inhibitor of this enzyme, was used. Nephrotoxicity was induced in rats by a single sc injection of K(2)Cr(2)O(7); studies were performed 2 days later. PJ34 was given intraperitoneally (15 mg/kg) 1 hr before and 1, 5, 24, 26, 31 and 46 hr after K(2)Cr(2)O(7) injection. Nephrotoxicity was evaluated by histological analysis and by measuring blood urea nitrogen, serum creatinine, serum glutathione peroxidase activity and urinary excretion of N-acetyl-beta-D-glucosaminidase. PARP-1 activation was evaluated by the immunostaining of poly(ADP-ribose). In addition, the following markers of oxidative stress were evaluated: 3-nitrotyrosine, 4-hydroxy-2-nonenal, malondialdehyde and protein carbonyl content. K(2)Cr(2)O(7) increased poly(ADP-ribose) content suggesting the PARP-1 activation in this model. PJ34 significantly ameliorated the K(2)Cr(2)O(7)-induced: (i) nephrotoxicity, (ii) poly(ADP-ribose) accumulation and (iii) oxidative stress. It is concluded that PARP-1 is activated and involved, at least in part, in K(2)Cr(2)O(7)-induced nephrotoxicity in rats.

Protective effects of apocynin against cisplatin-induced oxidative stress and nephrotoxicity.

Cis-diamminedichloroplatinum (II) (cisplatin) is an effective chemotherapeutic agent successfully used in the treatment of a wide range of tumors; however, nephrotoxicity has restricted its clinical use. Several studies have shown that reactive oxygen species are involved in cisplatin-induced nephrotoxicity, including hydrogen peroxide, hydroxyl radical and superoxide anion (O(2)(-)). The source of O(2)(-) in cisplatin-induced renal damage has not been established. The aim of this study was to investigate if NADPH oxidase is involved in cisplatin-induced nephrotoxicity using apocynin, a widely used NADPH oxidase inhibitor. Rats were studied 3 days after a single injection of cisplatin (7.5mg/kg, i.p.). Apocynin was given in the drinking water (2g/L) 7 days before and 3 days after cisplatin injection. Apocynin treatment was able to ameliorate the renal histological damage and the increase in blood urea nitrogen, serum creatinine, and urinary excretion of total protein, N-acetyl-beta-d-glucosaminidase and glutathione-S-transferase induced by cisplatin. In addition, the protective effect of apocynin was associated with the amelioration of cisplatin-induced oxidative and nitrosative stress. Our data suggest that O(2)(-) derived from NADPH oxidase triggers some of the side effects due to cisplatin administration.

Nordihydroguaiaretic acid attenuates potassium dichromate-induced oxidative stress and nephrotoxicity.

Larrea tridentata also known as Creosote bush, Larrea, chaparral, greasewood or gobernadora has been used in the folk medicine for the treatment of several illnesses. The primary product that is present at high concentrations in the leaves from this plant is nordihydroguaiaretic acid (NDGA) which is a powerful antioxidant. On the other hand, potassium dichromate (K(2)Cr(2)O(7))-induced nephrotoxicity is associated with oxidative stress. The aim of this work was to study the effect of NDGA on K(2)Cr(2)O(7)-induced nephrotoxicity and oxidative stress. Nephrotoxicity was induced by a single injection of K(2)Cr(2)O(7) (15 mg/Kg). A group of K(2)Cr(2)O(7)-treated rats was administered NDGA by mini osmotic pumps (17 mg/Kg/day). The results show that NDGA was able to ameliorate the structural and functional renal damage evaluated by histopathological analysis and by measuring proteinuria, urinary excretion of N-acetyl-beta-d-glucosaminidase, serum creatinine, and serum glutathione peroxidase activity. In addition, immunostaining of 4-hydroxy-2-nonenal and 3-nitrotyrosine, markers of oxidative and nitrosative stress, respectively, was ameliorated by the NDGA treatment. These data strongly suggest that the antioxidant properties of NDGA are involved in its renoprotective effect in K(2)Cr(2)O(7)-treated rats.

Selective iNOS inhibition reduces renal damage induced by cisplatin.

Cisplatin is a chemotherapeutic agent used in the treatment of several cancer tumors; however, nephrotoxicity has restricted its use. Reactive oxygen species and peroxynitrite, which is formed by the reaction between superoxide anion and nitric oxide (NO*), are implicated in cisplatin-induced nephrotoxicity. In contrast, both toxic and beneficial effects of NO* have been suggested in cisplatin-induced nephrotoxicity. Therefore, nowadays the role of NO* in this experimental model remains controversial. The aim of the present work was to elucidate the role of NO* in cisplatin-induced renal damage using N-[3-(aminomethyl)benzyl]acetamidine (1400W), a selective and irreversible inhibitor of iNOS. The mRNA levels of iNOS were increased in cisplatin-treated rats. The administration of 1400W reduced the cisplatin induced histological damage, renal dysfunction (increase in proteinuria and kidney injury molecule expression and decrease in creatinine clearance), tubulointerstitial infiltration, oxidative stress (increase in renal malondialdehyde and inmmunostaining for 4-hydroxy-2-nonenal) and nitrosative stress (immunostaining for 3-nitrotyrosine). In addition, the administration of 1400W was unable to modify systolic blood pressure in control rats. Our data demonstrate that selective iNOS inhibition reduces the cisplatin-induced nephrotoxicity and nitrosative stress which strongly suggest that in this experimental model (1) the NO* production is toxic and (2) iNOS is the main source of NO*.

Protective effects of garlic powder against potassium dichromate-induced oxidative stress and nephrotoxicity.

Potassium dichromate (K(2)Cr(2)O(7))-induced nephrotoxicity is associated with oxidative stress. In the present work the effect of garlic powder, a recognized antioxidant, on K(2)Cr(2)O(7)-induced nephrotoxicity and oxidative stress was studied. Rats were fed a 2% garlic powder diet for 1 month. A single injection of K(2)Cr(2)O(7) (15 mg/kg) to rats induced tubule interstitial damage and an increase in the following markers of renal injury 2 days later: blood urea nitrogen (4.6-fold), serum creatinine (9.7-fold), proteinuria (35.9-fold), urinary excretion of N-acetyl-beta-d-glucosaminidase (12.9-fold) and glutathione-S-transferase (2.3-fold) and a decrease of 65% in serum glutathione peroxidase activity. In addition, K(2)Cr(2)O(7) injection increased the following nitrosative and oxidative stress markers in kidney: 3-nitrotyrosine (1.9-fold), 4-hydroxy-2-nonenal (2.1-fold), malondialdehyde (1.8-fold) and protein carbonyl content (1.7-fold). It was found that garlic powder feeding was able to prevent by 44-71% the alterations in the markers of renal injury studied, by 55% the histological damage, and by 47-100% the increase in markers of oxidative and nitrosative stress. It is concluded that the ability of garlic powder to ameliorate K(2)Cr(2)O(7)-induced renal injury is associated with its antioxidant properties. Our data support the use of garlic powder as a renoprotective agent.

Renoprotective and antihypertensive effects of S-allylcysteine in 5/6 nephrectomized rats.

Progressive renal damage and hypertension are associated with oxidative and nitrosative stress. On the other hand, S-allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract (AG), has antioxidant properties. The effects of SAC and AG on blood pressure, renal damage, and oxidative and nitrosative stress were studied in five-sixths nephrectomized rats treated with SAC (200 mg/kg ip) and AG (1.2 ml/kg ip) every other day for 30 days. Proteinuria and serum creatinine and blood urea nitrogen concentrations were measured on days 0, 5, 10, 15, and 30, and systolic blood pressure was recorded on days 0, 15, and 30. The degree of glomerulosclerosis and tubulointerstitial damage, the immunostaining for inducible nitric oxide synthase, 3-nitrotyrosine, poly(ADP-ribose), and the subunits of NADPH oxidase p22phox and gp91phox, and the activity of SOD were determined on day 30. SAC and AG reduced hypertension, renal damage, and the abundance of inducible nitric oxide synthase, 3-nitrotyrosine, poly(ADP-ribose), p22phox, and gp91phox and increased SOD activity. Our data suggest that the antihypertensive and renoprotective effects of SAC and AG are associated with their antioxidant properties and that they may be used to ameliorate hypertension and delay the progression of renal damage.

Evaluation of oxidative stress in D-serine induced nephrotoxicity.

It has been suggested that oxidative stress is involved in d-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24h) after a single i.p. injection of d-serine (400mg/kg). Control rats were injected with l-serine (400mg/kg) and sacrificed 24h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-beta-d-glucosaminidase (NAG) were measured 24h after d-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before d-serine injection: (a) alpha-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl(2)), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24h, respectively, and KIM-1 mRNA levels increased significantly on 6-24h. Histological analyses revealed tubular necrosis at 12h. The activity of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase remained unchanged at all times studied. Protein carbonyl content, MDA, 4-HNE, and ROS remained unchanged at all time-points studied. GSH content decreased transiently on 9 and 12h. Interestingly, fluorescent products of lipid peroxidation decreased significantly on 3-24h. HO-1 expression was undetectable by Western blot and the immunohistochemistry studies revealed that the intensity of HO-1 staining was weak. The administration of PBN, FeTPPS, ATZ, SnCl(2), and SnMP did not prevent or enhance renal damage induced by d-serine. Our data taken as a whole suggest that oxidative stress is not involved in the early phase of the nephrotoxicity induced by d-serine.

Las quimiocinas, una nueva familia de citocinas en el reclutamiento de células inflamatorias / Chemokines, a new family of citokine for inflammatory cell recruitment

El reclutamiento de células es un aspecto crucial en el desarrollo de la respuesta inflamatoria aguda y crónica, incluidas las reacciones de hipersensibilidad aguda y tardía. De la acción combinada de moléculas de adhesión, quimiocinas y sus receptores, entre otros factores, los leucocitos obtienen la información que les indica la dirección de migración a los diferentes sitios en donde son requeridos. Las quimiocinas son un grupo numeroso de citocinas de naturaleza peptídica que se caracterizan por tener una región conservada de 4 cisteínas en grupos de dos, lo que permite clasificarlas en subfamilia alfa y beta. En general las quimiocinas de la subfamilia beta atraen sobre linfocitos y macrófagos. Además de su actividad quimiotáctica, las quimiocinas están implicadas en procesos biológicos fundamentales como la hematopoyesis, angiogénesis y actividad antitumoral, entre otras. Las quimiocinas no son secretadas únicamente por células inmunes, sino también por otros tipos celulares como fibroblastos, queratinocitos, células de músculo liso y de endotelio vascular. En algunas condiciones patológicas, las quimiocinas son también importantes, tal es el caso de las enfermedades alérgicas en las que estos factores son patogénicamente esenciales. En enfermedades autoinmunes, como la artritis reumatoide, existen receptores para quimiocinas en la sinovia inflamada; y en enfermedades infecciosas, como la infección por virus de la inmunodeficiencia humana, los receptores para ciertas quimiocinas participan también como correceptores para el VIH, participando así en la resitencia natural a la infección por este agente infeccioso. La participación de las quimiocinas en el reclutamiento preferencial de leucocitos podría ser modulada farmacológicamente, lo cual puede conducir al control terapéutico de enfermedades inflamatorias de causas diversas