The concentrations in the renal homogenates were determined from standard curves produced with GSH or GSSG standard dilutions. The final results are presented as nanomoles of GSH or GSSG/milligram of protein and the GSH/GSSG ratio (Rahman et al., 2006). Nitric oxide production analyses were done indirectly by the Griess reaction method, which detects nitrite, the NO degradation product (Green et al., 1982). Briefly, the homogenate samples mentioned above were reacted with 1% sulfanilamide for 10 min following reaction with nafitiletilenodiamine. selleck chemicals llc The resultant product was determined by absorbance at 540 nm in a spectrophotometer. The left kidneys of both groups were used for the histological

analyses performed by fixation in 10% formaldehyde and dehydrated in increasing aqueous solutions of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for 30 min each. After dehydration, the NVP-BGJ398 nmr samples

were embedded in paraffin and sectioned (4 and 7 μm) in a microtome (model MRP-03, Lupe Indústria e Comércio Ltda.) These sections were fixed on slides and all tissues were stained with hematoxylin and eosin (H/E) for structural assessment of the tissue, Picrosirius Red to measure the surface density of collagen and periodic acid–Schiff (PAS) to allow visualization of the basement membrane. The sections were mounted on coverslips using Entellan® mounting medium. The fields were photographed randomly. Quantification was obtained using Image-Pro Plus (Media Cybernetics). The percentage of intense red color with the picrosirius red staining was given by comparing it to total renal tissue in the photograph (100%). The percentage of interstitial space was obtained by measuring the spaces in relation to the total area photographed. Right kidneys

were used to obtain membrane-enriched fractions to measure the activities of sodium pumps and protein kinases, and for protein identification. Membrane fractions of kidneys from control rats and rats exposed to MCYST-LR were prepared from the outer cortex (cortex corticis), a region of the kidney in which more than 90% of the cell population corresponds to proximal tubules (Whittembury and Proverbio, 1970; Proverbio and Aspartate Del Castillo, 1981). The external portion of the cortex was carefully removed with a Stadie Riggs microtome and carefully dissected with small scissors to eliminate contamination. The fragments were homogenized in a Teflon and glass homogenizer using 4 ml of solution (250 mM sucrose, 10 mM Hepes–Tris (pH 7.6), 2 μM EDTA, 1 mM phenylmethylsulfonyl fluoride and 0.15 mg/ml of soybean trypsin inhibitor) per gram of kidney slices in an ice bath. The homogenate was centrifuged at 755×g for 10 min at 4 °C. The supernatant was collected and stored at −20 °C or in liquid nitrogen. Protein content was determined by the Folin phenol method ( Lowry et al., 1951).