| Part I. Expression of MAPEG superfamily and generation of related inflammatory mediators in AAI induced renal injury in caninesObjective:To establish an canine model of AAI induced renal injury, and determine the changes in the generation of MAPEG related inflammatory mediators-LTC4 and PGE2, as well as the expressions of MAPEG superfamily in this process, so as to explore the relationship between MAPEG superfamily and pathological changes caused by AAI in canine models.Methods:Canines were treated with AAI (3 mg/kg/day) orally, and the canines in control group were given equal amount of empty capsules (n=4), all animals were subjected to continuous administration for 10 days. The general state and body weight of animals were observed daily. Blood and urine samples were collected at certain time points and analyzed to evaluate the extent of renal impairment. Animals were euthanized after drug withdrawal and sarificed to collect organ samples. Hematoxylin-eosin (H&E) and Periodic Acid-Schiff (PAS) staining were applied on paraffin embedded kidney sections to evaluate the pathological status of kidneys. Microstructure of renal proximal tubule was observed by transmission electron microscopy (TEM). The changes of cysLTs, PGE2 in renal cortex and medulla were detected by EIA. Western blot and immunohistochemistry (IHC) were applied for determining the alterations in expression and distribution of MAPEG family associated with generation of inflammatory mediators (FLAP, mGST2, mGST3, LTC4S and mPGES-1) in kidneys. HPLC was established to evaluate the activity of LTC4 synthesis enzymes in renal microsomal in vitro and the contribution of mGST2 in cysLTs synthesis.Results:1. Cannies with AAI (3 mg/kg/day) treatment for 10 days were associated with body weight loss, increased blood urea nitrogen (BUN), creatinine (crea) in serum and appearance of urine proteins, suggesting that AAI induced renal injury model was established successfully. Increased Bax/Bcl-2 ratio and caspase 3 activation were observed in AAI induced renal injury in canines. Pathological examination showed the renal cortex had more severe impairment compared to medulla in AAI treated canines, and that renal proximal tubule is the main toxic target of AAI. Epithelial cell swelling, tubule brush border loss, mitochondrial damage were occurred in renal proximal tubular, and part of the proximal tubule basement membrane were exposed. In addition, male dogs were more susceptible to AAI than females.2. The levels of cysLTs, PGE2 were affected by AAI in the canine model. Renal cortical cysLTs in AAI treated canines was elevated and PGE2 was decreased, whereas both of them were found with no significant changes in the renal medulla. Moreover, MAPEG family related to generations of cysLTs and PGE2 were widely expressed in renal cortex, which presented as an increased expression of FLAP and mGST3, as well as decreased expression of LTC4S and mGST2 in AAI treated canines. Furthermore, the upregulation of FLAP and mGST3 mainly occurred in renal proximal tubules that damaged by AAI. In addition, according to LTC4 synthase catalytic activity test, mGST2 was not involved in the generation of LTC4 in renal cortex, suggesting that elevated level of renal cysLTs induced by AAI was mainly due to the increased expression of FLAP and mGST3. On the other hand, the renal expression of mPGES-1 was down-regulated by AAI, which sugguested to consistent with PGE2 generation.Conclusions:1. Renal injury in canines was induced by continuous high-dose oral administration of AAI, renal cortex was found to be with more severe impairment than medulla, and renal proximal tubular was considered to be the main target of AAI. MAPEG superfamily was considered to be involved in this process.2. Renal injury in canines induced by AAI was associated with cysLTs synthesis that trigged by FLAP and mGST3, as well as the decreased PGE2 mediated by mPGES-1. PartⅡ. MAPEG activated inflammatory signaling pathway in AAI induced renal tubular epithelial cell apoptosisObjective:Present study was attempted to eluciate the role of MAPEG family in cysLTs, PGE2 generation in AAI induced renal proximal tubular epithelial cell (LLC-PK1) apoptosis, as well as to explore the regulatory mechanisms by MAPK activation involved in this process, so as to clarify the pathogenesis of aristolochic acid nephropathy from a new perspective.Methods:Cell viability and morphological changes observed as indexes of AAI caused toxicity on LLC-PK1 cells. JC-1 staining was performed to determine mitochondrial membrane potential (△Ψm), AO/EB and DAPI staining were used to evaluate nuclear damage. Proteins expression associated with mitochondrial dependent apoptosis were detected by western blot analysis. Temporl and dose dependent expression of MAPEG family was also studied. CysLTs generation was detected by EIA. HPLC was established to evaluate the effect of AAI on the catalytic activity of renal microsomal LTC4 synthesis enzymes, as well as the role of mGST2 in cysLTs synthesis. After adding the FLAP inhibitor (MK886), cell viability and morphology changes were determined; activation of caspase 3 was applied as an indicator to evaluate the reversiblity effect on AAI induced cell injury. The effect of exogenous PGE2 on cell viability in AAI stimulated apoptosis was also studied. Moreover, temporal changes and dose response on MAPK activation in AAI treated cell were evaluated by western blot analysis. Cell viability, mitochondrial membrane potential and caspase 3 activation were applied to evaluate the effect of inhibition ERK activation (by U0126) in AAI induced cells injury. Western blot method was used to evaluate the effect of U0126 on regulation of MAPEG expression associated with cysLTs and PGE2 generation in cells exposed to AAI.Results:1. AAI triggered the mitochondrial/caspase apoptotic pathway in LLC-PK1 cells, indicated by enhanced Bax/Bcl-2 ratio, loss of△Ψm, cytochrome c release, and caspase 3 activation.2. The generation of cysLTs was raised by AAI in a concentration-dependent manner, along with increased expression of FLAP and mGST3. Furhtermoer, inhibition of FLAP by MK886 was confirmed to alleviate AAI induced apoptosis.3. Expression of mPGES-1 was reduced in an early phase of AAI induced apoptosis, and exogenous PGE2 can improve cell viability reduced by AAI.4. ERK phosphorylation was triggered by AAIalong with inhibition of P38 phosphorylation. AAI induced apoptosis in renal proximal tubule cells was reversed by U0126, as well as the expression of MAPEG family in the model.Conclusions:1. AAI upregulated cysLTs synthesis in LLC-PK1 cells, which was sugguested to be related to the increased expression of FLAP and mGST3, but mGST2 and LTC4S may not participate in the process. Inhibition of FLAP can enhance the survival of the renal proximal tubular cells exposed to AAI, which was further confirmed the role of cysLTs synthesis in AAI induced renal proximal tubular cells apoptosis.2. AAI reduced mPGES-1 expression in LLC-PKl cells, suggesting that generation of PGE2 might play an important role in AAI induced renal proximal tubule injury.3. AAI induces LLC-PKl cell apoptosis via mitochondria dependent pathway, inhibition of ERK activation protects LLC-PKl cells from AAI induced apoptosis may due to its effect on the process of cysLTs generation by blocking the increased expression of FLAP and mGST3, as well as mPGES-1 to alter PGE2 generation, which in turn initiated the caspase cascade and eventual apoptosis. |
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