The Protective Effects And The Mechanism Of The Nitro-oleic Acid On Acute And Chronic Renal Injury In Mice

Endotoxemia is a systemic inflammatory response to a blood-borne infection that is associated with an extremely high rate of morbidity and mortality. Endotoxemia often leads to sepsis, DIC and the failure of multiple organs, which can cause the mortality rate as high as 60%-70%.Endotoxemia often cause multiple organ damage, especially the heart, liver and kidney. ARF is considered a critical prognostic factor in endotoxic shock, with the 50% coincidence, the mortality rate for septic patients with acute renal failure (ARF) is approximately doubled compared with patients with sepsis alone. Although there is great progress in the pathology and physiology of the ARF, but the management of sepsis and sepsis-induced ARF is largely supportive. Therefore, novel therapies to prevent or treat this devastating disease are urgently required.Recently, nitrated free fatty acid (NO2-FA), notably nitroalkene derivatives of linoleic acid (nitrolinoleic acid; L NO2) and nitro-oleic acid (OA-NO2), are identified as endogenous molecules with several attractive signaling properties. These derivatives are formed via NO-dependent oxidative reactions. They were detected in healthy human blood, indicating their capability to act in physiological concentration ranges. To date, there are three major mechanisms by which nitroalkenes appear to mediate cell signaling. LNO2 and OA-NO2, at physiological concentrations, serve as potent ligands for proxisome proliferator-activated receptor subtype gamma (PPARy). Moreover, nitroalkenes are electrophiles and thus can nitroalkylate proteins and small peptides such as glutathione through reaction with cysteine thiols and histidine., which is independent on proxisome proliferator-activated receptor pathway. Increasing in vitro evidence demonstrates that nitroalkenes exert potent anti-inflammatory actions.Therefore, the nitroalkenes has multiple advantage, for example, endogenous molecules without side effects, and notably several attractive signaling properties. Nitrooleate arouses the widespread interest because of its content rich, the structure simple, and it will benefit the patients if it is applied in clinic. The present study seeks to examine the potential therapeutic effects of OA-NO2 in LPS-induced inflammation and renal injury.Objectives1. TO produce LPS-induced endotoxemia model in mice.2. To illustrate the potential therapeutic effects of OA-NO2 in LPS-induced inflammation and renal injury.3. To illustrate the potential therapeutic effects of OA-NO2 in LPS-induced heart and liver injury. Methods1. Animal protocolMale C57BL/6 mice (8-week-old) were maintained on a standard rodent chow and had free access to water. OA-NO2 was dissolved in 100% DMSO at 1 mg/ml. Mice were pretreated for 48 h with DMSO (LPS vehicle) or OA-NO2 (LPS OA-NO2) at 0.2 mg/kg/d via a micro-osmotic pump, and then both groups were treated with a single intraperitoneal injection of LPS at 10 mg/kg. The third group received an i.p. injection of saline only and served as controls. Functional studies were done at 18 h after LPS injection.A separate experiment was performed to compare the anti-inflammatory effects of OA-NO2 versus OA; OA was delivered at the same dose (0.2 mg/kg/d) via the same route as OA-NO2. 2. Echocardiography.In vivo cardiac function was assessed using echocardiography.28 male C57BL/6 mice (8-week-old) divided into cont, LPS and LPS+OA-NO2 groups, and the OA-NO2 pretreatment, LPS injection were the same as the previous.3. Blood pressure measurements.MAP was determined by telemetry. The radiotelemetric device was implanted into mice through catheterization of the carotid artery.Animals were allowed to recover from surgery for 1 week. MAP was recorded 2 days prior to and 18 h after LPS injection.4. Measurement of body temperature and hematocritRectal temperature was measured before and 18 h after LPS injection using a digital thermometer. At 18 h after LPS injection, hematocrit was determined.Briefly, blood was collected from tail cut using a capillary glass. The tube was centrifuged in a microcentrifuge machine. The total height of sample and height of the red blood cell column were measured.5. Measurement of biochemical parameters.Plasma BUN,Cr,AST,ALT and LDH were measured.6. Real-time RT-PCR.Total RNA was isolated from fresh tissue, and the mRNA expression ofβ-actin, TNF-α, iNOS, MCP-1, ICAM-1, VCAM-1, MCP-1 were determined.7. Westen BlotThe protein was isolated from fresh tissue, and protein expression of COX-2, iNOS in kidney were determined.8. ELISA measurementThe plasma, heart, liver and kidney TNF-acontent, also the kidney PGE2 content were measured using EIA kits, respectively.9. Statistical analysisValues shown represent means±SE. Data were analyzed using unpaired t test or ANOVA followed by a Bonferroni posttest. A P value<0.05 was considered significant. Results1. Body temperature and hematocritThe body temperature decreased significantly at 18 h after LPS injection (P< 0.01). Pretreatment of OA-NO2 for two 48h significantly improved LPS-induced hypothermia (P< 0.05). Hematocrit was significantly decreased that was attenuated by pretreatment with OA-NO2 (P< 0.01).2. Renal functionLPS injection elevated plasma BUN and Cr 4-,1.5-fold (P< 0.05).Pretreatment with OA-NO2 attenuated the rise of plasma BUN (P< 0.05) and almost completely normalized plasma creatinine levels (P< 0.05). While, renal histological changes in response to LPS were not evident and thus were not used as a parameter for evaluating the effect of OA-NO2 (data not shown).3. Proinflammatory cytokines, chemokines, and adhesion moleculesLPS induced renal TNF-a mRNA and protein 14-fold,4-fold increase. Pretreatment with OA-NO2 attenuated the rise of TNF-a(P< 0.01). Similar results were obtained concerning circulating TNF-a (P< 0.05).The fold inductions between LPS vehicle and LPS OA-NO2 groups were 27.1-vs.9.7-fold for MCP-1,12.0-vs. 4.8-fold for ICAM-1, and 4.2-vs.1.8-fold for VCAM-1.4. Renal iNOS expression.At 18 h post LPS, renal iNOS mRNA exhibited a more than 1000-fold increase that was reduced by 80% in LPS OA-NO2 mice. Similarly, LPS injection induced a 70-fold increase in renal iNOS protein expression that was reduced by 90% with OA-NO2. Renal cGMP content exhibited a similar pattern of changes as renal iNOS expression.5. Renal COX-2 expressionRenal COX-2 mRNA and protein increase 8.3-and 17.1-fold, respectively, by LPS injection; they were attenuated to 2.1-and 8.9-fold by OA-NO2.while renal content of PGE2 exhibited a 1.5-fold increase that was almost normalized by pretreatment with OA-NO2 (P< 0.05). 6. Hepatic injuryLPS injection elevated plasma AST, ALT strikingy (P< 0.05). Pretreatment with OA-NO2 lowered plasma AST and ALT. By ELISA, the increase in hepatic TNF-a content was less in LPS OA-NO2 vs. LPS vehicle mice (Fig.7C). LPS injection induced parallel increases in mRNA expression of hepatic TNF-a, IL-1, ICAM-1, MCP1, iNOS and COX-2; these increases were all attenuated by OA-NO2 (P<0.05).7. Cardiac injury and hypotentionEchocardiography revealed that LPS injection significantly reduced the ejection fraction (EF) (P< 0.01). Plasma LDH was elevated by LPS injection (P< 0.05) that was attenuated by OA-NO2 (P< 0.05). LPS-induced hypotension was evident in both groups. MAP in LPS OA-NO2 mice tended to be consistently-10 mmHg higher than in LPS vehicle animals.8. Effectiveness of OASeparate experiments determined whether the protective effect of OA-NO2 in endotoxin-induced endotoxemia was specific to the nitrated form. The indices of systemic inflammation (body temperature and hematocrit) and renal dysfunction (plasma BUN and creatinine) in the endotoxemic mice were significantly attenuated by OA-NO2 but was unaffected by OA, documenting the lack of anti-inflammatory effect of OA. This finding suggests that the protective effect of OA-NO2 in endotoxic shock is attributable to the nitration of the fatty acid.Conclusion1. Pretreatment with OA-NO2 for 48 h attenuated the systemic inflammation and improved the multiple organ dysfunction in the LPS induce endotoxemia in mice.2. The data showed the mechanism about the anti-inflammation of the OA-NO2, which was via suppress the production and release of the inflammation markers, such as TNF-α,MCP1,ICAM-1,VCAM-1,iNOS and COX-2. Protective Effects of Nitro-Oleic Acid Against Cisplatin-induced Nephrotoxicity in MiceBackgroundCisplatin(cis-diamminedichloroplatinum, CDDP) is widely used in the treatment of a variety of malignancies. Howerer, the full clinical utility of the drug is limited by its adverse effects. One of the most common side effects is cisplatin nephrotoxicity. Cisplatin is a potent toxin to renal tubules and is associated with a cumulative decline in renal function. Approximately 25-35% of patients administered with cisplatin develop acute renal failure (ARF). But the management of cisplatin nephrotoxicity is largely supportive. Therefore, novel therapies to prevent or treat this devastating disease are urgently required.Our previous study indicated that nitro-oleic acid (OA-NO2) protected against LPS induced endotoxic ARF via suppress the production and release of inflammation marker and mediators. As the OA-NO2 is characterized with the endogenous molecules, high capability to act in low concentration ranges, and without any side effects so far. So it may provide the novel therapeutic drug for the clinic. But we ask ourselves several questions:Are the protective effects of the OA-NO2 specific to the LPS induced ARF? Does the drug protect against other model of renal injury? To answer these questions, we design the present study to explore more details of the drug. We investigated whether OA-NO2 ameliorates cisplatin-induced renal dysfunction in mice. To explore whether the protective effect was PPAR gamma dependent, we repeated the model in inducible PPAR gamma knockout mice. Objectives1. To produce cisplatin-induced renal dysfunction in mice.2. To illustrate the potential therapeutic effects of OA-NO2 in cisplatin-induced renal dysfunction.3. To illustrate the whether the protective effect was on PPAR gamma dependent.Methods1. Animal protocolMale C57BL/6 mice (8-week-old) were maintained on a standard rodent chow and had free access to water. OA-NO2 was dissolved in 100% DMSO at 100 mg/ml. Mice were pretreated for 48 h with DMSO (DDP vehicle) or OA-NO2 (DDP+OA-NO2) at20 mg/kg/d via a micro-osmotic pump, and then both groups were treated with a single intraperitoneal injection of DDP at 20 mg/kg. The third group received an i.p. injection of saline only and served as controls. Functional studies were done at 18 h after DDP injection.A separate experiment was performed to compare the anti-inflammatory effects of OA-NO2 versus OA; OA was delivered at the same dose (20mg/kg/d) via the same route as OA-NO2.A separate experiment was performed in wild type and inducible PPAR gamma knockout mice. The pretreatment of the OA-NO2 and the DDP was the same as the described above.2. General condition and body weightAt 3days after DDP injection, evaluate the general condition and weighted the mice.3. Measurement of biochemical parameters.Plasma BUN and Cr were measured.4. Real-time RT-PCRTotal RNA was isolated from fresh tissue, and the mRNA expression of GAPDH, TNF-α,IL-β,MCP-1,ICAM-1,VCAM-1,MCP-1,COX-2,mPGES-1,P47phox and gp91phox mRNA were determined. 5. Westen BlotThe protein was isolated from fresh tissue, and protein expression of TNF-α,COX-2,P47phox and gp91phox in kidney were determined.6. Histological examinationKidney tissue was fixed, embedded in paraffin and cut into sections. The kidney damage was examined in haematoxylin-eosin(HE) stained sections.7. ELISA measurementThe plasma TNF-a content, and the kidney PGE2, TBARS content were measured using EIA kits, respectively.8. Statistical analysisValues shown represent means±SE. Data were analyzed using unpaired t test or ANOVA followed by a Bonferroni posttest. A P value<0.05 was considered significant.Results1. General condition and body weightAfter DDP injection, the mice were sick with less food and water intake less and their activity decrease obviously. The body weight decrease were no statistical difference between the DDP and OA-NO2 treatment groups.2. Kidney indexDDP induced the kidney hypertrophy and swelling, with increased kidney index (P<0.05). Pretreatment with OA-NO2 attenuated the rise of kidney index. 3. Renal function and histologyLPS injection elevated plasma BUN and Cr 4.5-,4-fold (P< 0.05).Pretreatment with OA-NO2 improved the renal dysfunction (P< 0.05). Histological examination revealed necrosis, protein cast, vacuolation and desquamation of epithelial cells in renal tubules after cisplatin injection. However, pretreatment with OA-NO2 dramatically improved the histological damage.4. kidney oxidative stress conditionCisplatin elevated the kidney TBARS and induced the mRNA and protein expression of the p47 phox and gp91 phox subunits. Pretreatment with OA-NO2 attenuated the rise of kidney TBARS, p47phox and gp91phox subunits expression. 5. Proinflammatory cytokines, chemokines, and adhesion moleculesCisplatin induced renal TNF-a mRNA and protein expression increase, also the circulating TNF-a. Pretreatment with OA-NO2 attenuated the rise of TNF-a(P< 0.01). The fold inductions between DDP vehicle and DDP+ OA-NO2 groups were 3.8-vs. 1.8-fold for ICAM-1, and 5.1-vs.1.7-fold for VCAM-1. Pretreatment with OA-NO2 almost normalized DDP induced the increase of MCP-1 and IL-1β.6. Renal COX-2 expression.Renal COX-2 mRNA and protein increase 6- and 4- fold, respectively, by cisplatin injection; they were attenuated to 1.6- and 2-fold by OA-NO2.while renal content of PGE2 exhibited a strikingly increase that was almost normalized by pretreatment with OA-NO2 (P< 0.05).7. Effectiveness of OASeparate experiments determined whether the protective effect of OA-NO2 in cisplatin nephrotoxicity was specific to the nitrated form. The indices of renal dysfunction (plasma BUN and creatinine, histological changes) in the mice were significantly attenuated by OA-NO2 but was unaffected by OA, documenting the lack of anti-inflammatory effect of OA. This finding suggests that the protective effect of OA-NO2 in cisplatin nephrotoxicity is attributable to the nitration of the fatty acid.8. Effectiveness of OA-NO2 in whole body inducible PPAR y knockout mice Similarly, pretreatment with OA-NO2 for 48 h attenuates the renal dysfunction in both the wild type and knockout mice, indicating the protective effects of the OA-NO2 was independent on the PPARy pathway.Conclusion1. Pretreatment with OA-NO2 for 48h attenuated the cisplatin nephrotoxicity in mice.2. The mechanism of the protection of the OA-NO2 was via anti-inflammation and anti-oxidative stress. 3. The protective effects of the OA-NO2 was independent on the PPARy pathway. Renoprotective effect of Nitro-Oleic Acid in DOCA-salt hypertensive MiceBackgroundThe role of aldosterone in hypertension has received increasing attention during recent years. High serum levels of the hormone preceded the development of hypertension in a population-based, long-term study. The use of a synthetic mineralocorticoid, DOCA, which is an aldosterone analog, together with a high-salt diet (DOCA-salt), is a well-established means of inducing hypertension. This hypertension model is relevant to human primary aldosteronism The increasingly frequent diagnosis of primary hyperaldosteronism, the recognition of non-genomic effects of the hormone and the availability of the more specific receptor blocker eplerenone have also drawn attention to the role of aldosteron mineralcorticoid hypertension with renal dysfunction. Aldosterone induces inflammation, oxidative stress and fibrosis in the kidney dependent or independent on blood pressure, and blockade of the hormone reduces nephrosclerosis in several models of hypertensive renal damage.Our previous study demonstrated that nitro-oleic acid protect against LPS-induced and cisplatin induced kidney injury via anti-inflammation and anti-oxidative stress. So this current study was undertaken to investigate whether the nitro-oleic acid ameliorates DOCA-salt induced kidney injury in mice.Objectives1. TO produce DOCA-salt hypertensive model in mice.2. To illustrate the potential therapeutic effects of OA-NO2 in DOCA-salt induced renal injury. 3. To illustrate the potential mechanism of the protective effects.Methods1. Animal protocolMale C57BL/6 mice (8-week-old) were maintained on a standard rodent chow and had free access to water. OA-NO2 was dissolved in 100% DMSO at 10mg/ml. Mice were pretreated for 48 h with DMSO (DOCA vehicle) or OA-NO2 (DOCA+ OA-NO2) at 2 mg/kg/d via a micro-osmotic pump, and then both groups were implanted with a DOCA pellet together with a high-salt diet for two weeks. The third group received an surgery only and served as controls. Functional studies were done at 2 weeks after DOCA-salt treatment.2. Blood pressure measurementSystolic blood pressure was measured by a tail-cuff method. All animals were habituated to the blood pressure measurement device for 7 days. At 11 day post DOCA treatment, they all underwent 2 cycles of 20 measurements recorded per day for 4 days.3. Food and water intake, urine collectionThe mice were put in the metabolic cages to collect urine and measure food and water intake at 14 days post DOCA-salt treatment.4. ELISA measurementThe plasma and kidney TNF-αcontent, also the urine albumin and 8-isoprostane were measured using EIA kits, respectively.5. Real-time RT-PCR.Total RNA was isolated from fresh tissue, and the mRNA expression of GAPDH,TNF-α,IL-β,MCP-1,ICAM-1,VCAM-1,MCP-1,COX-2,mPGES-1,P47phox,gp91phox,podocin and nephrin were determined.6. Westen BlotThe protein was isolated from fresh tissue, and protein expression of TNF-α,COX-2,nephrin,P47phox and gp91phox in kidney were determined. 7. EM examinationEM was performed to examined the injury of the podocyte.8. Statistical analysisValues shown represent means±SE. Data were analyzed using unpaired t test or ANOVA followed by a Bonferroni posttest. A P value<0.05 was considered significant.Results1. Measurements of blood pressureAfter DOCA-salt treatment for 14 days, the systolic blood pressure increased strikingly. Pretreatment with OA-NO2 decrease a small degree but significant systolic blood pressure.2. Food and water intake, body weightThere were no differences of the food intake in all mice. DOCA-salt increased the water intake strikingly. Pretreatment with OA-NO2 decreased the water intake but not significant. DOCA-salt decreased the body weight significantly, while pretreatment with OA-NO2 attenuated the decrease of body weight.3. Urine volume and urine albuminDOCA-salt increased the urine volume and urine albumin strikingly. Pretreatment with OA-NO2 attenuated the rise of the urine albumin.4. Kidney histologyDOCA-salt induced the kidney hypertrophy with pale outlook. Pretreatment with OA-NO2 improved the kidney outlook and hypertrophy. According to PAS staining, DOCA-salt induced kidney cortical and outer medulla tubules dilation and hypertrophy, the basement membrane atrophy and brush border discontinuity. Pretreatment with OA-NO2 attenuated the kidney injury.5. Podocyte injuryDOCA-salt induced the podocytic process widely fuge and disappear, while the podocyte markers nephrin and podocin decrease. Pretreatment with OA-NO2 improved podocyte injury. 6. Kidney oxidative stress conditionDOCA-salt elevated the kidney TBARS and urine 8-isoprostane, together with high expression of the p47 phox and gp91 phox subunits. Pretreatment with OA-NO2 attenuated the rise of kidney TBARS, urine 8-isoprostane, p47phox and gp91phox subunits expression.7. Kidney inflammationDOCA-salt induced renal TNF-a mRNA and protein expression increase. DOCA-salt also increased the mRNA expression of IL-1β,MCP-1,ICAM-1,VCAM-1,MCP-1,COX-2. Pretreatment with OA-NO2 improved the inflammation induced by DOCA-salt.Conclusion1. Pretreatment with OA-NO2 for 48 h decreased the systolic blood pressure induced by DOCA-salt.2. Pretreatment with OA-NO2 attenuated the urine albumin and podocyte damage induced by DOCA-salt.3. The data showed the effects of the OA-NO2 via anti-inflammatory and anti-oxidative stress...