Effects And Mechanisms Of SEH Gene Disruption On Streptozotocin-induced Diabetic Nephropathy In Mice

Research Backgrounds and AimsDiabetic nephropathy is the leading cause of chronic renal failure and one of the mostserious long term complications for diabetic patients. DN is initiated by hyperglycemia andis characterized by mesangial expansion and thickening of the tubular and glomerularbasement membranes due to excessive matrix accumulation, associated with progressiverenal deterioration. Central to DN is the apoptosis of renal proximal tubular epithelial cells,which has been observed in diabetic rodents and humans. Numerous transgenic mousestudies have also shown that the disruption of insulin signaling might contribute to defectsin both peripheral insulin action and β-cell function. The exact molecular mechanismsresponsible for these defects have yet to be clearly identifed; however, the insulin signalingcascade and downstream signaling components are attractive therapeutic targets for thetreatment of diabetes and diabetes-related complications.Cytochrome P-450(CYP) epoxygenases metabolize arachidonic acid (AA) to fourregioisomeric epoxyeicosatrienoic acids (5,6-,8,9-,11,12-, and14,15-EET), which playcrucial and diverse roles in cardiovascular homeostasis. Epoxide hydrolases hydrolyzeEETs into biologically less active dihydroxyeicosatrienoic acids (DHETs). Previous studiesindicate that soluble epoxide hydrolase (sEH, Ephx2) is the primary enzyme involved inthe in vivo hydrolysis of EETs. The inhibition of sEH results in the accumulation of EETs due to the decreased rate of conversion to DHETs. Pharmacological inhibitors of sEH havebeen shown to be protective in animal models of renal injury. The sEH inhibitor CDU(1-cyclohexyl-3-dodecylurea) has been shown to protect kidney vasculature and glomerulifrom injury in angiotensin-induced hypertension. AUDA [(3-adamantan-1-yl-ureido)-dodecanoic acid], a similar sEH inhibitor, lowered blood pressure, increased the urinaryEET/DHET ratio, and decreased macrophage infltration in the kidneys of rats withsalt-sensitive hypertension. AUDA also lowered blood pressure and increased urinary saltand water excretion in angiotensin-induced hypertensive mice. Moreover, inhibition of sEHwith AUDA attenuated the progression of the renal damage associated with hypertensionand type II diabetes.Similar to sEH inhibition studies, research utilizing sEH-defcient mice have revealedthe benefcial role of EETs in renal and cardiovascular systems. In a deoxycorticosteroneacetate plus high salt (DOCA-salt) hypertensive model, sEH-defcient mice had decreasedblood pressure, attenuated renal infammation, and decreased glomerular injury. Numerousnonvascular animal models have also shown a benefcial role for EETs in attenuating theprogression of diabetic symptoms. Genetic disruption or inhibition of sEH preventshyperglycemia, promotes insulin secretion, and reduces islet apoptosis in streptozotocin(STZ)-induced diabetic mice (28). In our previous gene therapy study with CYP2J3, anEET-producing epoxygenase, insulin resistance and diabetic manifestations were alleviatedin db/db diabetic mice and in rats with fructose-induced insulin resistance. Numerousstudies have linked the benefcial functions of EETs to the activation of prosurvival andantiapoptotic pathways. EETs stimulate endothelial cell growth and angiogenesis viamitogen-activated protein kinase (Erk1/2) and phosphatidylinositol3-kinase (PI3K)/Aktsignaling pathways. CYP epoxygenase overexpression signifcantly protects endothelialcells from TNF-α-induced apoptosis, at least in part, through the activation of the PI3K/Aktsignaling cascade and the expression of the antiapoptotic protein Bcl-2.In this study, we hypothesize that sEH gene disruption and the resultant increase in EET production reduced the detrimental effects associated with diabetic nephropathy,attenuated renal tubular apoptosis, and delaying the progression of diabetic nephropathy.Therefore, we investigated the effects and possible molecular mechanisms of sEH genedisruption on diabetic nephropathy in STZ-induced mice. We also probed the anti-injuryeffects and possible molecular mechanisms of sEH siRNA or synthetic EETs treatment onHK-2cells. The aim of the study is to explore whether CYP epoxygenase-EETs-sEHsystem could protect against diabetic nephropathy in the whole animal and cell level, andfurther defined CYP epoxygenase-EETs-sEH system could delay the progress of diabeticnephropathy.In Vivo Study sEH Gene Disruption Attenuates Nephropathyin Streptozotocin-induced Diabetic MiceResearch aims: The effects and possible molecular mechanisms of sEH genedisruption on streptozotocin-induced diabetic mice were investigated.Research methods:1. Wild type C57BL/6mice and the sEH-defcient (Ephx2-/-) mice on a pure C57BL/6genetic background were housed (4per cage) in a temperature (25±3°C) and humidity (50±20%)-controlled room with a12:12-h light-dark cycle and access to normal chow adlibitum plus water. At8wk of age, wild-type (WT) and sEH-defcient mice were injectedwith STZ (50mg/kg ip, made freshly in50mM sodium citrate buffer, pH4.5) for5consecutive days or with50mM sodium citrate solution only as control. Tail vein bloodglucose levels were evaluated1wk after the last STZ injection and glucose levels>250mg/dl were considered diabetic mice. Then, sEH-defcient and WT mice were divided intocontrol and diabetic groups (4groups,10mice each). All animals were sacrificed24wklater with pentobarbital sodium (50mg/kg body wt).24-h urine samples were collected.Blood samples, hearts and kidneys were taken, frozen with liquid nitrogen, and stored at-80°C refrigerator; 2. Serum concentrations of glucose, HbA1c, blood urea nitrogen (BUN) and creatininewere measured by the related method;3. ELISA was carried out to measure14,15-DHET levels in urine;4.4μm thick kidney sections were stained with periodic acid Schiff (PAS) andMasson’s trichrome staining for morphological changes and evaluation of the extent ofrenal injury.6. Apoptotic cells were detected by use of a terminal dUTP nick end-labeling(TUNEL) assay on paraffn-embedded kidney sections with a commercially available kit todetect end-labeled DNA, and in situ apoptosis analysis was carried out according to themanufacturer’s instructions.7. Western blot was carried out to detect sEH, cleaved caspase-3, Bcl-2，Bcl-xl，Bax,PI3K, p-AKT, p-NOS3Ser1177, p-eNOS Thr495and p-AMPK Thr172expression kidney ofmice.Research results:1. Compared with control mice, administration of STZ to mice signifcantly increasedthe ratio of kidney weight to body weight, blood glucose, Hb A1c, BUN, plasma creatinine,and urinary albumin (P<0.05), suggesting establishment of the early phase of diabeticnephropathy and diabetic renal dysfunction. Interestingly, the increases in blood glucoselevels, HbA1c, the ratio of kidney weight to body weight, plasma creatinine, BUN, andurinary microablumin were reversed in sEH-defcient diabetic mice (P<0.05).2. PAS and Masson’s trichrome staining of WT diabetic mice showed that increasedcollagen deposition and severe glomerular injury in the WT diabetic mice (P<0.05),whereas sEH gene disruption signifcantly suppressed these pathological changes insEH-defcient diabetic mice (P<0.05). Quantifcation of the degree of glomerular injuryshowed that sEH-defcient diabetic mice had signifcantly reduced mesangial injury scorecompared to STZ-treated WT mice (P<0.05).3. STZ-administration increased the number of apoptotic cells as evaluated by TUNEL staining in kidneys of WT mice. sEH gene disruption signifcantly mitigated thiseffect (P<0.05). Kidneys from WT diabetic mice had downregulated levels of theantiapoptotic proteins Bcl-2and Bcl-xl, and upregulated levels of the proapoptotic proteinBax. However, decrease in antiapoptotic Bcl-2and Bcl-xl and the increase in proapoptoticBax were partially reversed in sEH-defcient diabetic mice (P<0.05). In the kidneys ofSTZ-treated WT mice, there was a markly increase in both caspase-3cleavage by Westernblot and caspase-3activity (P<0.05). All these pathological changes in diabetes weremitigated in sEH-defcient diabetic mice (P<0.05).4. PI3K and AKT phosphorylation levels were significantly reduced in the kidneys ofSTZ-treated WT mice, whereas sEH gene disruption partially reversed these effects(P<0.05). NOS3has two well-studied phosphorylation sites, the activating Ser1177site andthe inhibitory Thr495site. Akt has been proven to activate NOS3by phosphorylating Ser1177.NOS3phosphorylation levels have significant difference between the nondiabetic WT andsEH-defcient mice. Following STZ-administration, WT mice had downregulatedphosphorylation of the NOS3-activating Ser1177residue and upregulated phosphorylation ofthe NOS3-inhibitory Thr495residue (P<0.05). Interestingly, NOS3phosphorylation levelsin STZ-treated sEH-defcient mice were partially restored to nondiabetic levels (P<0.05). InWT mice, AMPK phosphorylation was downregulated following STZ treatment, an effectthat was partially mitigated in sEH-defcient diabetic mice (P<0.05). In conclusion, thesefindings suggested that sEH gene disruption mitigated diabetic nephropathy progression byactivating the PI3K/AKT/NOS3and AMPK signaling pathways.In Vitro Study Effects and Mechanisms of sEH Gene Inhibition onTNF-α-induced Apoptosis in HK-2CellsResearch aims: The effects and mechanisms of sEH gene inhibition onTNF-α-Induced Apoptosis in HK-2Cells 1. HK-2cells were cultured with DMEM/F12medium with10%FBS at5%CO2incubator with37℃. When HK-2cells grew to90%confluence in75cm2flasks,0.25%trypsin was used to passage. Then, when cells grew to70%-80%confluence, HK-2cellswere cultured with DMEM/F12medium with0.1%fetal bovine serum for starvation of12hfor synchronization.2. HK-2cells were transfected with siRNA control and siRNA targeting human sEHby use of Lipofectamine2000according to the manufacturer’s protocol. Apoptosis wasinduced by incubating HK-2with TNF-α plus actinomycin D. HK-2cells were gathered24h later, flow cytometry assays were implemented to evaluate the effects of sEH geneinhibition on TNF-α induced-apoptosis in HK-2cells.3. HK-2cells were transfected with siRNA control and siRNA targeting human sEHby use of Lipofectamine2000according to the manufacturer’s protocol. Apoptosis wasinduced by incubating HK-2with TNF-α plus actinomycin D. HK-2cells were gathered24h later, and caspase-3activity was measured.4. HK-2cells were transfected with siRNA control and siRNA targeting human sEHby use of Lipofectamine2000according to the manufacturer’s protocol. Apoptosis wasinduced by incubating HK-2with TNF-α plus actinomycin D. HK-2cells were gathered24h later, and cell proteins were extracted. The protein expression levels of Bcl-2, Bcl-xland Bax in HK-2cells were detected by Western blot.Research results:1. sEH gene inhibition significantly attenuated TNF-α-induced apoptosis in HK-2cells. The results displayed that sEH gene inhibition significantly decreased apoptotic cellsratio. The apoptotic cells ratios in Control group were (4.6±0.6)%; the apoptotic cellsratios in TNF-α-treated group were (33.5±2.7)%; while the apoptotic cells ratios in TNF-α+siRNA-control group and TNF-α+siRNA-sEH group were (34.6±2.6)%and (12.5±1.2)%respectively. The differences of the apoptotic cells ratios between TNF-α-treatment groupand TNF-α+siRNA-sEH group had statistically significance (P<0.05). 2. sEH gene inhibition significantly attenuated TNF-α-induced caspase-3activityincrease in HK-2cells. The results displayed that caspase-3activity was signifcantlyincreased by TNF-α treatment, which was attenuated by sEH gene inhibition (P<0.05).3. TNF-α significantly downregulated Bcl-2and Bcl-xl expression levels (P<0.05),and significantly upregulated Bax expression levels in HK-2cells (P<0.05). sEH geneinhibition significantly inhibited the downregulation of Bcl-2and Bcl-xl expression levels(P<0.05), and the upregulation of Bax expression levels TNF-α-induced apoptosis in HK-2cells (P<0.05).In Vitro Study Effects and Mechanisms of Exogenous EETs onTNF-α-induced Apoptosis in HK-2CellsResearch aims: The effects and mechanisms of exogenous EETs on TNF-α-inducedapoptosis in HK-2cells.Research methods:1. HK-2cells were cultured with DMEM/F12medium with10%FBS at5%CO2incubator with37℃. When HK-2cells grew to90%confluence in75cm2flasks,0.25%trypsin was used to passage. Then, when cells grew to70%-80%confluence, HK-2cellswere cultured with DMEM/F12medium with0.1%fetal bovine serum for starvation of12h for synchronization.2. Flow cytometry assays were implemented to evaluate the effects of exogenous EETson TNF-α induced-apoptosis in HK-2cells.3. AKT, PI3K,MAPK, and MEK inhibitors AKTI, LY294002, Apigenin and PD98059and HK-2cells, as well as14,15-EET were added together for24h, and then flowcytometry assays were implemented to evaluate the effects of exogenous EETs on TNF-αinduced-apoptosis in HK-2cells.4. Apoptosis was induced by incubating HK-2with TNF-α plus actinomycin D. EETsand HK-2cells were incubated together for24h, and then cells were gathered, and caspase-3activity were measured.5. AKT, PI3K,MAPK, and MEK inhibitors AKTI, LY294002, Apigenin and PD98059and HK-2cells, as well as14,15-EET were added together for24h, and then cells weregathered, and caspase-3activity were measured.6. Apoptosis was induced by incubating HK-2with TNF-α plus actinomycin D. EETsand HK-2cells were incubated together for24h, and then cells were gathered, and cellproteins were extracted. The protein expression levels of Bcl-2, PI3K, P-AKT andP-Erk1/2in HK-2cells were detected by Western blot.7. AKT, PI3K,MAPK, and MEK inhibitors AKTI, LY294002, Apigenin and PD98059and HK-2cells, as well as14,15-EET were added together for24h, and then cells weregathered, and cell proteins were extracted. The protein expression levels of Bcl-2in HK-2cells were detected by Western blot.Research results:1.8,9-EET,11,12-EET and14,15-EET significantly attenuated TNF-α-inducedapoptosis in HK-2cells. The results displayed that EETs significantly decreased apoptoticcells ratio. The apoptotic cells ratios in Control group were (4.5±0.6)%; the apoptotic cellsratios in TNF-α-treated group were (31.1±1.9)%; while the apoptotic cells ratios in TNF-α+8,9-EET group, TNF-α+11,12-EET group, TNF-α+14,15-EET group were(14.1±1.2)%，(12.1±1.1)%and (8.8±1.0)%respectively. The differences of the apoptoticcells ratios between TNF-α-treatment group and TNF-α+8,9-EET group, TNF-α+11,12-EET group, TNF-α+14,15-EET group had statistically significance (P<0.05).2. AKT, PI3K, MAPK, and MEK inhibitors AKTI, LY294002, Apigenin and PD98059significantly reversed the decrease in TNF-α-induced apoptosis after14,15-EET treatment.The apoptotic cells ratios in Control group were (4.6±0.5)%; the apoptotic cells ratios inTNF-α-treated group were (26.5±2.1)%; the apoptotic cells ratios inTNF-α+14,15-EET+AKTI group， TNF-α+14,15-EET+LY294002group， TNF-α+14,15-EET+Apigenin group and TNF-α+14,15-EET+PD98059group were (19.5±1.2)%， (19.9±1.3)%，(21.0±1.4)%and (22.6±1.5)%respectively. The differences of the apoptoticcells ratios between TNF-α+14,15-EET+AKTI group, TNF-α+14,15-EET+LY294002group, TNF-α+14,15-EET+Apigenin group, TNF-α+14,15-EET+PD98059group andTNF-α+14,15-EET group had statistically significance (P<0.05).3.8,9-EET,11,12-EET and14,15-EET significantly attenuated TNF-α-inducedcaspase-3activity increase in HK-2cells. The results displayed that caspase-3activity wassignifcantly increased by TNF-α treatment, which was attenuated by synthetic EETstreatment (P<0.05).4. AKT, PI3K, MAPK, and MEK inhibitors AKTI, LY294002, Apigenin andPD98059significantly reversed the reduction in TNF-α-induced caspase-3activity increaseafter14,15-EET treatment. Pretreatment of HK-2cells with AKTI, LY294002, Apigeninand PD98059increased TNF-α-induced caspase-3activity and attenuated the protectiveeffects of14,15-EET (P<0.05).5. The Bcl-2, P-MAPK, PI3K and P-AKT expression levels in HK-2cells werereduced after TNF-α treatment (P<0.05), which were reversed by EETs (P<0.05).6. AKT, PI3K, MAPK, and MEK inhibitors AKTI, LY294002, Apigenin and PD98059significantly reversed the increase in TNF-α-induced Bcl-2reduction after14,15-EETtreatment. Pretreatment of HK-2cells with AKTI, LY294002, Apigenin and PD98059decreased TNF-α-induced Bcl-2reduction and attenuated the protective effects of14,15-EET (P<0.05).Statistical AnalysisResults are expressed as mean±SEM. Multiple comparisons between two groups wereperformed with unpaired t-test; among three or more groups they were carried out withANOVA and Newman-Keuls tests for post hoc analyses. P<0.05was consideredsignifcantly different.Conclusions 1. sEH gene disruption signifcantly reduced the detrimental effects associated withdiabetic nephropathy, improved renal function, and attenuated renal tubular apoptosis.2. sEH gene disruption signifcantly increased antiapoptotic Bcl-2and Bcl-xl anddecreased proapoptotic Bax. Further more, sEH gene disruption signifcantly attenuateddiabetic nephropathy, at least in part, through PI3K/AKT/NOS3and AMPK signalingpathways in the kidney.3. sEH gene inhibition attenuated the increase of caspase-3activity and decreasedapoptosis after TNF-αaddition, which were associated with increase Bcl-2and Bcl-xlexpression levels, as well as and decrease Bax expression levels.4. Exogenous EETs activated phosphorylation of MAPK and PI3K/AKT signalingmolecules, which might contribute to the observed antiapoptotic effects of EETs in HK-2cells.5. These fndings highlight the benefcial role of the CYP epoxygenase-EETs-sEHsystem in the pathogenesis of diabetic nephropathy and suggest that the sEH inhibitorsavailable may be potential therapeutic agents for this condition.