| (1)in vivo studyObjective:We established the kidney injury model of rats induced by aldosterone to investigate the role of oxidative stress,ERS and Cx43 in podocyte injury in vivo.Methods:24 male Sprague-Dawley（SD）rats were randomly divided into 4groups:Control group（n=6,received Sham operation,1%NaCl water）;Aldo-infused group（n=6,received a right uninephrectomy,1%NaCl water,aldosterone were pumped at0.75μg/h concentration continuously by an subcutaneous embedded osmotic minipump two weeks after the surgery）;Aldo+Cx43 SCR group（n=6,received a right uninephrectomy,1%NaCl water,two weeks after the surgery,aldosterone were pumped at0.75μg/h concentration by subcutaneous embedding an osmotic minipump while Cx43SCR were pumped at 50u M concentration by subcutaneous embedding an osmotic minipump respectively）;Aldo+Cx43 AS group（n=6,received a right uninephrectomy,1%NaCl water,aldosterone were pumped at 0.75μg/h concentration by an osmotic minipump embedded subcutaneously while Cx43 AS were pumped at 50u M concentration by another osmotic minipump two weeks after the surgery）.At the beginning of the experiment and at week 4 during the treatment period,we measured the systolic blood pressure（SBP）of the rat tail arteries.When one day before the terminal of experiment,we collected twenty-four-hour urine samples in metabolic cages.Enzyme-linked immunosorbent assay（ELISA）kits were used to determine urinary protein excretion.Renal tissues were fixed and sliced.TUNEL staining was used to detect apoptosis situation in glomerular podocyte.Immunohistochemistry or immunofluorescence were used to detect the expression of Cx43、Casepase12、WT1and NOX4 respectively in glomeruli.Western blot was used to detect the expression of NOX4,Cx43,GRP79,Bax,Bcl-2 and Caspase12.DHE staining was applied to ROS production in kidney tissues.Results:Compared with the control group,the SBP,urine volume in 24 hours,urinary protein/creatinine ratio,and kidney/body weight ratio in Aldo-infused group were significantly increased.Aldo treatment induced higher oxidative stress（detected by DHE staining and NOX4 expression）,increased ERS markers（including GRP78 and Caspase12）,and increased Cx43 expression.Meanwhile,increased number of podocyte apoptosis assessed by TUNEL,and decreased number of WT1 positive cells suggested more podocyte injury.Compared with the Aldo+Cx43 SCR group,treatment with Cx43 AS declined the Cx43 expression and attenuated Aldo-induced oxidative stress,ERS,as well as podocyte injury in the glomeruli.（2）in vitro studyObjective:Rat podocyte was cultured and treated with aldosterone to observe the role of oxidative stress,ERS,and Cx43 in podocyte injury in vitro.Methods:The MPC5 conditionally immortalized ratpodocyte cell line was cultured and the cellular differentiation was induced without interferon-γat 37°C for 10～14 days.Podocytes were then exposed to different concentrations of Aldo(10-9M,10-8M,and 10-7M)or 10-7M Aldo treatment for different time（12,24,and 36h）respectively.Western blot was applied to detect the protein expression of Cx43,GRP78,and Caspase12.After establishing the time point for Aldo-induced oxidative stress,ERS,and apoptosis in podocytes,N-acetyl-L-cysteine（NAC,an antioxidants agent）,tauroursodeoxycholic acid（TUDCA,an ERS antagonist）,and Cx43 siRNA transfection oligo were applied to intervene podocytes with or without Aldo.A cell death detection ELISA was appliedto detect the apoptosis of podocyte.Western blot was used to estimate the expression of Cx43,GRP78,Caspase12,Bax,and Bcl-2.DCFDA probes were applied to detect the active oxygen content in podocytes.Results:The Cx43 expression levels after stimulation with Aldo were up-regulated in a dose dependant manner(10-9M,10-8M,and 10-7M)and time-dependent manner（12,24,and 36 h）.Compared with the control cells,Aldo increased podocyte injury（as indicated by the increased number of apoptotic podocytes and increased expression of Bax/Bcl-2）,enhanced oxidative stress（as evidenced by the increased ROS content）,and increased ERS（increased expression of GRP78 and Caspase12）.However,pretreatment with NAC,or TUDCA,or Cx43 siRNA attenuated Aldo-induced oxidative stress,ERS and podocyte injury.Summary:（1）Excessive ERS is involved in Aldo-induced podocyte injury.（2）Increased oxidative stress is involved in Aldo-induced podocyte injury.（3）There is cross-talk between ERS and oxidativestress in Aldo-induced podocyte injury.（4）The up-regualtion of Cx43 is involved in Aldo-induced podocyte injury.（5）Cx43 regulated podoycte injury induced by aldosterone through oxidative stress and ERS. |
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