RESUMEN
Zona occludens-2 (ZO-2) is a protein present at the tight junction and nucleus of epithelial cells. ZO-2 represses the transcription of genes regulated by the Wnt/ß-catenin pathway. This pathway plays a critical role in podocyte injury and proteinuria. Here, we analyze whether the overexpression of ZO-2 in the glomerulus, by hydrodynamics transfection, prevents podocyte injury mediated by the Wnt/ß-catenin pathway in the mouse model of adriamycin (ADR) nephrosis. By immunofluorescence and immunogold electron microscopy, we show that ZO-2 is present in mice glomerulus, not at the slit diaphragms where nephrin concentrates, but in the cytoplasm and at processes of podocytes. Our results indicate that in the glomeruli of mice treated with ADR, ZO-2 overexpression increases the amount of phosphorylated ß-catenin, inhibits the expression of the transcription factor snail, prevents nephrin and podocalyxin loss, reduces podocyte effacement and massive fusions, restrains proteinuria, and supports urea and creatinine clearance. These results suggest that ZO-2 could be a new target for the regulation of hyperactive Wnt/ß-catenin signaling in proteinuric kidney diseases.
Asunto(s)
Glomérulos Renales/metabolismo , Podocitos/efectos de los fármacos , Podocitos/fisiología , Proteinuria/prevención & control , Proteína de la Zonula Occludens-2/fisiología , Animales , Doxorrubicina , Masculino , Ratones , Ratones Endogámicos BALB C , Nefrosis/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/biosíntesis , Vía de Señalización Wnt/efectos de los fármacos , Proteína de la Zonula Occludens-2/biosíntesis , beta Catenina/metabolismoRESUMEN
Renal tight junctions (TJ) play a central role in modulating the paracellular pathway. We examined the function, quantity and distribution of TJ proteins: occludin and claudin-2 (cln-2), on proximal tubule in a model of acute renal failure (ARF) associated with oxidative damage. Since ERK1/2-p modulates TJ integrity, we studied their participation in dichromate (Cr(6+)) toxicity. We evaluated whether co-administration of the antioxidant alpha-tocopherol (alpha-TOC) prevents Cr(6+) toxicity in TJ. Female Wistar rats received potassium dichromate 15 mg/kg, s.c. (5.3 mg/kg of Cr(6+)) single dose, with or without alpha-TOC (125 mg/kg, p.o., daily). Two and 7 days after Cr(6+) treatment, oxidative damage was assessed by renal lipid peroxidation (LPO), proximal function was estimated by sodium and glucose fractional excretions. Occludin, cln-2, and ERK1/2-p were detected by immunofluorescence and Western blot. ARF induced by Cr(6+) provoked augment in the sodium and glucose urinary looses, increases in occludin quantity (6.6- and 15-fold on days 2 and 7, respectively) and the mislocation of cln-2. Electrophoresis migration showed a higher molecular weight band only in the Cr(6+)-administered groups, suggesting occludin hyperphosphorylation. Alpha-TOC treatment diminished the LPO, improved tubular function, and preserved TJ location and expression. In summary, we show disruption of occludin and cln-2 in ARF induced by Cr(6+)-intoxication. This study provides evidence of the beneficial effect of alpha-TOC on TJ structure and function undergoing oxidative damage, and we suggest the participation of ERK1/2 in the mechanisms leading to protection by the antioxidant.
Asunto(s)
Antioxidantes/farmacología , Cromatos/antagonistas & inhibidores , Cromatos/toxicidad , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/enzimología , alfa-Tocoferol/farmacología , Animales , Western Blotting , Centrifugación por Gradiente de Densidad , Cromo/toxicidad , Claudinas , Creatinina/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Glucosa/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Ocludina , Ratas , Ratas Wistar , Uniones Estrechas/patologíaRESUMEN
Exposure to hexavalent chromium (Cr(6+)) causes mutagenic, carcinogenic, and toxic effects, some of which have been associated with its oxidative capacity. In the kidney, Cr(6+) has been claimed to provoke necrosis of the proximal tubular cells. Our aim was to assess the functional involvement of the different segments that form the nephron in a model of acute renal failure caused by potassium dichromate and the participation of oxidative damage in this process. We also studied the possible protective effect of alpha-tocopherol (alpha-TOC) against renal damage. Wistar female rats 200g body weight (bw) received potassium dichromate (15mg/kg, sc, single dose). Lipid peroxidation and renal function were evaluated on days 0, 1, 2, 3, 7, 10, and 14. A second group received alpha-TOC (125mg/kg, by gavage) 5 days before and during dichromate exposure (same dose as for the first group), and was monitored at 0, 2, and 7 days of exposure. Creatinine clearance, glucose and sodium fractional excretions, p-aminohippurate uptake, free-water and osmolal clearances were also measured. Thiobarbituric acid-reactive substances were quantified in renal cortex. The results revealed altered proximal tubule function, decreased glomerular filtration, and distal segment dysfunction, accompanied by oxidative damage 48h after exposure to dichromate. In the alpha-TOC-treated group proximal reabsorptive and secretory functions were preserved, suggesting that oxidative damage is a participating mechanism in dichromate toxicity on these functions. In contrast alpha-TOC did not prevent glomerular or distal dysfunction, indicating selectivity of the protection afforded by this compound on the toxicity of dichromate, at the several components of the nephron.
Asunto(s)
Enfermedades Renales/prevención & control , Túbulos Renales Proximales/efectos de los fármacos , Dicromato de Potasio/toxicidad , Sustancias Protectoras/farmacología , alfa-Tocoferol/farmacología , Animales , Creatinina/sangre , Creatinina/orina , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Enfermedades Renales/inducido químicamente , Enfermedades Renales/metabolismo , Túbulos Renales Distales/efectos de los fármacos , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Proteinuria/inducido químicamente , Proteinuria/prevención & control , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
In mammals, neonatal positive calcium balance is required for adequate growth. Parathyroid hormone (PTH) plays a central role in this process mainly through its action on the distal nephron. We studied the effect of PTH on cytosolic calcium in distal segments from neonatal rat kidney. PTH elicited a concentration-dependent increase in cytosolic calcium in neonatal distal nephron (EC(50)=0.5 nM) but not in proximal tubules. At similar PTH concentrations the response was higher in the neonatal than in the adult tubules. The response was associated with protein kinase C (PKC), since phorbol myristate acetate (100 nM) increased [Ca(2+)]i, and staurosporin, an inhibitor of PKC, decreased (10 nM) or suppressed (100 nM) the PTH effect. cAMP analogues did not change [Ca(2+)]i. The response was diminished in low external calcium (0.1 mM) and absent at zero calcium, indicating dependency on external calcium. Resting calcium decreased from 80+/-10.8 to 28.6+/-2.6 nM at zero [Ca(2+)]e. PTH and nifedipine increased cytosolic calcium in an additive fashion. We show for the first time that PTH increased cytosolic calcium in the distal nephron of neonatal kidney, in a concentration-dependent pattern and in association with PKC activation. Higher sensitivity of the neonatal tubule might facilitate absorption of this cation during the neonatal period, when growth requires a positive balance of calcium.
Asunto(s)
Calcio/metabolismo , Túbulos Renales Distales/metabolismo , Hormona Paratiroidea/fisiología , Proteína Quinasa C/fisiología , Animales , Calcio/análisis , Citosol/metabolismo , Femenino , Masculino , Modelos Animales , Nefronas/metabolismo , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
BACKGROUND: Retinoic acid (RA) exerts beneficial effects on vascular remodelling and experimental nephritis, and plays a role in kidney development. Pathological changes caused by acute renal failure (ARF) result in high mortality. We determined whether RA ameliorates ARF-induced pathology caused by potassium dichromate (PD). METHODS: Adult Wistar female rats (210-250 g) were randomly allocated to four groups: (i) an ARF group that received PD [15 mg/kg body weight (bw), single dose subcutaneously]; (ii) a group that received PD plus RA (1 mg/kg bw) beginning at 5 days before PD and that continued for 14 additional days; (iii) a group that received PD plus thyroxine (T(4); 8 micro g/100 g bw) with RA; and (iv) a group that received only the vehicle for PD (saline solution). We evaluated functional, biochemical and morphological characteristics of the kidneys. RESULTS: PD-induced alterations in serum creatinine, creatinine clearance (C(cr)) and fractional excretion of sodium (FeNa) were less severe when rats received RA. PD increased lipoperoxidation and this alteration was partially blocked by RA. Animals undergoing ARF showed severe histological injury (brush border loss, acidophilia, oedema, pyknosis, karyorhexis, cell detachment and disruption of the basement membrane). These alterations were less severe in RA-treated rats, indicating a protective effect on functional and morphological alterations. Alterations in urinary sediment were reduced by RA. The simultaneous administration of T(4) with RA did not produce additional protection. CONCLUSION: RA exerted beneficial effects on the duration and severity of renal damage induced by PD in a model of renal failure resembling ARF in humans. The protective effect of RA may be mediated by diminished lipoperoxidative damage.