RESUMO
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.
Assuntos
Antioxidantes/farmacologia , Cromatos/antagonistas & inibidores , Cromatos/toxicidade , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/enzimologia , alfa-Tocoferol/farmacologia , Animais , Western Blotting , Centrifugação com Gradiente de Concentração , Cromo/toxicidade , Claudinas , Creatinina/metabolismo , Feminino , Imunofluorescência , Glucose/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Confocal , Ocludina , Ratos , Ratos Wistar , Junções Íntimas/patologiaRESUMO
BACKGROUND: The tight junction (TJ) regulates the passage of ions and molecules through the paracellular pathway. In multicellular organisms, epithelial sheets function as a barrier between a variety of environments and the internal media. Therefore, TJs are required to control the passage of diverse molecules in different epithelia. The mammalian nephron constitutes a particularly relevant model of this diversity, since the paracellular transport in this organ is significantly different along the various tubular segments. Here, we have analysed the distribution of claudins-7 and -8 in Henle's loops and collecting ducts isolated from rabbit kidneys. METHODS: Renal segments were manually isolated from newborn and adult rabbit kidneys and processed for immunofluorescence. The distribution of claudins-7 and -8 was studied by confocal microscopy. RESULTS: The localization of claudins-7 and -8 along Henle's loops and collecting ducts is remarkably different. While claudin-8 displays a clear cell border distribution in Henle's segment, claudin-7 shows a non-specific cytosolic staining. Moreover, in the collecting ducts, claudin-8 localizes at the TJ region, while claudin-7 shows a basolateral staining. This pattern is present from the newborn stage. The distribution of claudins along the mammalian kidney has been found to vary in different mammalian species. Accordingly, in the rabbit, we have found the expression of claudin-8 at the descending and ascending thin limbs of Henle, a distribution that differs from that found in the mouse by others. CONCLUSION: In the rabbit Henle's loop, claudin-8 is present at the cellular borders of the descending and ascending thin limbs, while claudin-7 displays no specific labelling. Instead, at the collecting duct, both claudins are present but exhibit a different subcellular distribution.
Assuntos
Túbulos Renais Coletores/metabolismo , Alça do Néfron/metabolismo , Proteínas de Membrana/metabolismo , Animais , Claudinas , Imunofluorescência , Técnicas In Vitro , Túbulos Renais Coletores/citologia , Alça do Néfron/citologia , Masculino , Microscopia Confocal , Coelhos , Frações Subcelulares/metabolismoRESUMO
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.
Assuntos
Nefropatias/prevenção & controle , Túbulos Renais Proximais/efeitos dos fármacos , Dicromato de Potássio/toxicidade , Substâncias Protetoras/farmacologia , alfa-Tocoferol/farmacologia , Animais , Creatinina/sangue , Creatinina/urina , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Túbulos Renais Distais/efeitos dos fármacos , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Proteinúria/induzido quimicamente , Proteinúria/prevenção & controle , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
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.
Assuntos
Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Tretinoína/farmacologia , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/urina , Animais , Creatinina/metabolismo , Diurese/efeitos dos fármacos , Combinação de Medicamentos , Sinergismo Farmacológico , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Natriurese/efeitos dos fármacos , Dicromato de Potássio , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Tiroxina/farmacologia , Redução de Peso/efeitos dos fármacosRESUMO
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.
Assuntos
Cálcio/metabolismo , Túbulos Renais Distais/metabolismo , Hormônio Paratireóideo/fisiologia , Proteína Quinase C/fisiologia , Animais , Cálcio/análise , Citosol/metabolismo , Feminino , Masculino , Modelos Animais , Néfrons/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
BACKGROUND: Permeability properties of mammalian nephron are tuned during postnatal maturation. The transepithelial electrical resistance (TER) and complexity of tight junctions (TJs) vary along the different tubular segments, suggesting that the molecules constituting this structure change. We studied the differential expression of occludin and several claudins in isolated renal tubules from newborn and adult rabbits. METHODS: Isolated renal tubules from newborn and adult rabbits were processed for occludin, claudin-1 and claudin-2 immunofluorescence, and Western blot detection of claudin-1 and -2. Claudin-5 was detected in whole kidney frozen sections. RT-PCR from isolated tubules was performed for claudins-1 to -8. RESULTS: Immunofluorescence revealed that occludin, claudin-1 and -2 were present at the cell boundaries at the neonatal stage of development. Claudin-1 was detected in the tighter segments of the nephron (distal and collecting duct), while claudin-2 was found in the leaky portions (proximal). Claudin 5 was found in the kidney vasculature. PCR amplification revealed the presence of claudins-1 to -4 in tubules of newborns. In adults, claudins-1, -2 and -4 were present in proximal, Henle's loop and collecting segments; claudin-3 was in proximal and collecting tubules, while claudins-5 and -6 were absent from all tubular portions. Claudin-7 was restricted to proximal tubules, while claudin-8 was present in proximal and Henle's segments. CONCLUSIONS: The pattern of occludin distribution is present from the neonatal age. Claudins-7 and -8 are up-regulated after birth. Each tubular segment expresses a peculiar set of claudins that might be responsible for the permeability properties of their TJs.