| Part one Short-term effects of unilateral ureteral obstruction on the pyroptosis in the contralateral kidneyObjective:To observe the pyroptosis of the contralateral kidney with short-term UUO rat model as well as the signal regulation.Methods:Wistar rats were divided into four groups:Sham group,UUO group,?UUO+EPL?group,and?UUO+TCM?group.Except for dissection the ureter with the Sham group,other groups were subjected left ureteral obstruction.After UUO,eplerenone(100mg·kg-1·d-1)and TCM(1.92g·kg-1·d-1)were given the corresponding groups for10-day.The contralateral kidneys?right kidney?of the obstruction were taken for studying.Pathological changes and DNA damage of the contralateral kidneys were observed by HE and TUNEL staining;the expression of NLRP3,caspase-1,IL-1?,SGK-1 and NF-?B?p65?were observed by immunohistochemistry and Western blotting;transcription of NLRP3,caspase-1,and IL-1?genes were detected by RT-PCR;immunohistochemistry was used to detect the expression of macrophage marker F4/80;the co-expression of F4/80 and NLRP3 was observed by immunofluorescence.The above data were expressed as mean±standard deviation,analyzed by one-way ANOVA and SNK-q test,the test level was P<0.05.Results:1.Pathological changes and DNA damage of the contralateral kidneyThe morphology of tubule epithelial cells was almost normal and the number of TUNEL positive cells was small in Sham group;in UUO group,inflammatory cells infiltrated,some tubule epithelial cells were incomplete,chromatin aggregation,cell lysis,and compared with the Sham group,the number of TUNEL positive cells was significantly higher?P<0.001?;only a few inflammatory cells were found around the blood vessels in?UUO+EPL?group,and the number of TUNEL positive cells was significantly lower than the UUO group?P<0.001?.2.The proteins and genes expression of NLRP3,caspase-1,IL-1?were detected by immunohistochemistry,Western blot and RT-PCRThe three proteins were mainly expressed in distal tubular cytoplasm,and NLRP3 was also expressed in renal interstitial.The expression of NLRP3 and caspase-1 in the UUO group was significantly higher than that in the Sham group?P<0.01?,while the expression of which decreased obviously after treated by eplerenone?P<0.01?.The expression of IL-1?in the UUO group was also increased than that in the Sham group?P<0.05?,and its expression was down-regulated after intervented with eplerenone?P<0.05?.3.The expression of SGK-1 and NF-kB were tested by Western blotCompared with Sham group,the expressions of SGK-1 and NF-kB?p65?in UUO group were remarkable rised?P<0.001?,while the expressions of those in?UUO+EPL?group were reduced?P<0.01,P<0.05?.4.The observation of macrophage by immunohistochemistryF4/80 positive cells were mainly distributed in renal interstitial.The number of positive cells in Sham group was very small,and increased significantly in UUO group?P<0.001?;compared with UUO group,the number was decreased striking in?UUO+EPL?group?P<0.01?.5.Co-expression of NLRP3 and F4/80 was observed with immuno-fluorescenceThe co-expression positive area in UUO group was mainly in the renal interstitial,and the number was more than that in Sham group?P<0.001?.Compared with the UUO group,the number was reduced in?UUO+EPL?group?P<0.01?.Conclusion:Short-term UUO can induce the contralateral renal injury,which is related to serum aldosterone.Aldosterone activates the NLRP3/caspase-1/IL-1?pathway to induce pyroptosis through the SKG-1/NF-kB signaling pathway in the contralateral kidney.Part two The role of cell pyroptosis in the progression of chronic renal failure induced by ureteral obstruction and the interventionmechanism of Huayujiedu herbsObjective:The 180-day UUO rats were used to simulate the pathological process of renal failure induced by long-term ureteral obstruction renal so as to analyze the mechanism and verify the pathogenesis of"turbid toxin injuring kidney"and the protective effect of Huayujiedu herbs.Methods:Wistar rats were divided into five groups:Sham group,Cut group,UUO group,Spironolactone group?Spi group?,and TCM group.The UUO surgery was the same as that in part one.Spi group(20mg·kg-1·d-1)and TCM group(1.92g·kg-1·d-1)rats were intervened for 180 days continuously.Blood,urine and the contralateral kidneys were taken for examination after UUO 180-day.Kidney index,blood pressure,and renal function?Cr,BUN?were measured.The pathological changes of kidney were observed by HE and Masson staining.Serum aldosterone and MR were detected respectively by ELISA and immunohistochemistry.The expression of SGK-1 and NF-kB?p65?were detected by Western blot.DNA damage was observed by TUNEL staining.The expression of NLRP3,caspase-1,and IL-1?were tested by immunofluorescence or Western blot.The statistics were the same as that in the part one.Results:1.General state of ratsThe kidney index in all obstruction groups and Cut group was significantly increased than that in the Sham group?P<0.001?.The systolic blood pressure?SBP?in UUO group rats was higher than that in Sham group on 5 and 6months after UUO,but which was declined obviously after intervention.There was no significant difference between the Cut group rats and the Sham group rats.2.Renal function and pathology examinationCompared with Sham group,serum Cr and BUN levels were significantly increased in UUO group rats?P<0.001?.Compared with UUO group,the Cr and BUN levels reduced in Spi group and TCM group rats?P<0.01,P<0.001?.There was no significant difference in renal function between Cut group and Sham group rats.HE staining showed that the glomeruli and renal tubules were normal in the kidneys of Sham group rats,with a small amount of inflammatory cells in the kidney interstitial.A large number of inflammatory cells were observed in the kidneys of UUO group rats,and some tubule cells were atrophic,exfoliated,vacuolated,cytolytic;tubular morphology was incomplete and cavity enlarged.Inflammatory cell still existed in the kidneys of Spi group and TCM group rats,but the number was small.In the Cut group rats,the glomeruli and tubule were almost normal,but the tubular epithelial cells were slightly swollen and some tubular cross-sectional area increased.Masson staining showed that collagen deposition in UUO group kidneys was significantly increased than that in Sham group,which was cord-like or flaky.Collagen in Spi group and TCM group kidneys was less than that in UUO group.There was no significant difference between the Cut group and Sham group kidneys.3.Detected Serum aldosterone,renal tissue MR,and aldosterone effector factorsSerum aldosterone in UUO group rats elevated remarkably than in Sham group rats?P<0.05?.There was no significant difference between the intervention groups and UUO group rats.The immunohistochemistry showed that the number of MR located in nucleus in UUO group kidneys was significantly increased than that in Sham group kidneys,which mainly distributed in distal tubules.Compared with the UUO group kidneys,the number of activated MR was reduced in the intervention groups kidneys.Western blot showed that compared to the Sham group rats,the expression of SGK-1 and NF-kB?p65?were significantly elevated in UUO group rats?P<0.001,P<0.01?,while they were all down-regulated obviously?P<0.001,P<0.05?.4.Detected the characteristics and signal pathway of pyroptosisTUNEL positive cells which implied DNA damage were mainly located in the distal tubules in UUO group kidneys,which was more than that in Sham group kidneys?P<0.001?;The number of positive cells decreased in Spi and TCM groups kidneys?P<0.001?.NLRP3,caspase-1,and IL-1?were detected by immunofluorescence and Western blot.The three proteins were mainly expressed in the cytoplasm of tubule cells.Compared with the Sham group kidneys,the expression of three proteins was significantly up-regulated in UUO group kidneys?P<0.001,P<0.01,P<0.05?,however,which became weaker in the drug intervented groups kidneys?P<0.001,P<0.01,P<0.05?.Conclusion:The pathological and functional changes of the contralateral kidney caused by long-term ureteral obstruction are related to the cell pyroptosis which induced by the actived MR;Huayujiedu herbs inhibit the pyroptosis of renal cells by NLRP3/caspase-1/IL-1?pathway to reduce inflammation,so as to delay the progress of CKD.Part three The study on pyroptosis of HK2 cell induces by aldosterone and treated by serum containing Huayujiedu herbsObjective:To observe the effect of aldosterone on the pyroptosis of human proximal tubule epithelial cells as well as the signal regulation,and verify the intervention effect of Huayujiedu herbsMethods:Preparation of serum containing Huayujiedu herbs;HK2 was divided into 4 groups:control group?Cont?,aldosterone group?Ald,1?mol/L aldosterone?,eplerenone group?EPL,1?mol/L aldosterone and 10?mol/L eplerenone?,TCM group?TCM,10%TCM serum and 1?mol/L aldosterone?.DNA damage was detected by TUNEL;LDH activity of cell supernatant was detected by biochemical instrument;expression of MR,SGK-1,NF-?B?p65?,NLRP3,caspase-1,and IL-1?was detected by immunofluorescence and/or Western blot.The statistics were the same as the first part.Results:1.Detected MR activation and effector molecular of aldosteroneAfter stimulated by aldosterone,MR entered the nucleus from the cytoplasm and was activated.The number of activated MR cells in the Cont group was small.The number of MR-activated cells in Ald group increased significantly;compared with Ald group,the number of activated MR decreased obviously in EPL group and TCM group.The expression of SGK-1 and NF-?B?p65?in Ald group was markedly higher than that in Cont group?P<0.001,P<0.05?;compared with Ald group,the expression levels of above protein were lower obviously in EPL group and TCM group?P<0.001,P<0.05?.2.DNA and cell membrane damage were detected by TUNEL and LDH respectivelyImmunofluorescence showed TUNEL positive cells were green fluorescence.The number of TUNEL positive cells was very small in the Cont group;compared with the Cont group,the number of DNA-damaged cells in the Ald group elevated significantly;positive cells in EPL and TCM groups were reduced remarkably.The LDH activity of cell culture supernatant in Ald group was dramatically higher than that in Cont group?P<0.001?.3.Detected the signal molecules of pyroptosis by immunofluorescence and Western blot.The expression of NLRP3 protein was mainly in the cytoplasm.Compared with Cont group,the relative expression of NLRP3 of was significantly enhanced in Ald group?P<0.01?;compared with Ald group,the relative expression in the intervention groups was decreased markedly?P<0.01?.Caspase-1 and IL-1?showed green fluorescence in cytoplasm.The expressions of caspase-1 and IL-1?in Ald group were significantly stronger than those in Cont group?P<0.01,P<0.05?;after treated by eplerenone and serum containing Huayujiedu herbs,their expression became weaker.?P<0.01,P<0.05?.Conclusion:Aldosterone activates MR to trigger HK2 cell pyroptosis through NLRP3/caspase-1;eplerenone and serum containing Huayujiedu herbs can reduce HK2 cell pyroptosis which is involved in MR activation.General conclusion:1.Unilateral ureteral obstruction can induce cell pyroptosis in the contralateral kidney;2.Unilateral renal obstruction can lead to structural and functional changes in the contralateral kidney with time,and progress to renal failure;3.The mechanism of chronic renal failure induced by unilateral renal obstruction is related to aldosterone,and the activation of mineralocorticoid receptor plays a key role in this process,and MR blockers can alleviate renal injury;4.Aldosterone activates the NLRP3/caspase-1/IL-1?pathway to regulate the cell pyroptosis by SGK-1/NF-?B signal;5.Huayujiedu herbs can down-regulate the expression of NLRP3/caspase-1/IL-1?to reduce cell pyroptosis,and delay the progression of CKD by SGK-1/NF-?B signaling pathway. |
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