The suppression of MAPK/NOX/MMP signaling prompts renoprotection conferred by prenatal naproxen in weaning preeclamptic rats
All raw data of the study entitled "The suppression of MAPK/NOX/MMP signaling prompts renoprotection conferred by prenatal naproxen in weaning preeclamptic rats". we tested the hypothesis that renal programming incited by preeclampsia is modulated by gestationally administration of nonsteroidal antiinflammatory drugs (NSAIDs). The data showed that naproxen was more advantageous than celecoxib or diclofenac in relieving functional and morphological manifestations of the erupted renal injury. Molecularly, the suppression of the upregulated offending signals of inflammatory (arachidonate-derived prostanoids, p-ERK2), oxidative (NOX2/4), fibrotic (MMP9), and autophagic (LC3) cascades provides a mechanistic basis for the privileged action of naproxen.
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Studies were undertaken in the N(gamma)-nitro-L-arginine methyl ester (L-NAME) model of PE to assess the influence of gestational exposure to NSAIDs (celecoxib, diclofenac or naproxen) on PE-evoked renal damage in weaning dams. Gene and protein expression studies were also undertaken to determine the roles of arachidonate end products, MAPK signaling, and downstream oxidative, fibrotic, and autophagic effectors in the interaction. Pregnant female rats were randomly assigned into 5 different groups; n = 5-6 rats each: (i) pregnant non-PE control rats, (ii) PE rats, (iii) PE/celecoxib (10 mg/kg/day) (iv) PE/diclofenac (0.5 mg/kg/day), and (v) PE/naproxen (1 mg/kg/day). NSAIDs were co-administered with L-NAME starting at GD14 till GD20. At weaning (3 weeks post-labor), dams were euthanized by i.p. injection of an overdose of thiopental (100 mg/kg) and blood was collected via cardiac puncture, spun (800 × g, 4 °C, 20 min), and serum was stored at -80 °C until used for the measurement of creatinine levels. Portions of the left kidneys were dissected, immediately frozen in liquid nitrogen, and stored at −80 °C for protein and gene expression studies. Right kidneys were fixed in 10% formaldehyde and embedded in paraffin blocks for histopathological examination and morphometric analysis. The levels of serum and urine creatinine were assessed using Jaffe' reaction, then creatinine clearance (CrCl) was calculated. Urine protein level was measured using pyrogallol red method. Total protein levels in kidney tissues were measured using the Lowry method. The left kidney portions were homogenized in phosphate buffered saline in ratio of 1:9 and used to determine prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2α) (Enzo Life Sciences, NY, USA) and thromboxane A2 (TXA2) (LifeSpan BioSciences, Inc, Seattle, USA) using commercially available ELISA kits. Real time reverse transcriptase-polymerase chain reaction (qRT-PCR). Total RNA was isolated from kidney tissue using the miRNeasy kit according to the manufacturer's instructions. The isolated RNA was reverse transcribed into complementary DNA (cDNA) using reverse transcriptase, amplified, and detected by qRT-PCR using specific primers. The primer sequences of the genes examined; beclin-1, microtubule-associated protein 1A/1B-light chain 3 (LC3), MMP2, MMP9, NADPH oxidase 2 (NOX2), NADPH oxidase 4 (NOX4) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blotting. The ReadyPrepTM protein extraction kit (total protein) provided by Bio-Rad Inc (Catalog #163-2086) was employed according to manufacturer instructions was added to each sample of the homogenized tissues of all different groups. Primary antibodies of phospho-p38 mitogen-activated protein kinases (p-p38 MAPK), phospho-c-Jun N-terminal kinase (p-JNK), extracellular signal-regulated protein kinase (ERK) p-Erk1, and p-ERK2 were diluted in TBST according to manufactured instructions.