| Part I The dynamic change of autophagy in renal ischemia/reperfusion and relationship with acute kidney injuryObjective: To investigate the expression of autophagy and apoptosis in a time-dependent manner and to explore the relationship between them in renal ischemia/repefusion injury both in vivo and in vitro.Methods: Eighty male BALB/c mice were randomly divided into sham operati on group(Sha m, n=40) and ischemia/reperfusion group(I/R, n=40). The bilateral renal arteries of the mice were clamped and then opened to establish the renal I/R model. The blood and kidneys were collected at 0h, 2h, 5h, 12 h, 1day, 2day, 3day and 7day after reperfusion. The renal tubular cells cultured in vitro were randomly divided into Normoxic group(Nor), Hypoxia group(H6h,H12 h,H24h) and Hypoxia/Reoxygenation group(H/R2 h,H/R4 h,H/R8 h,H/R24h) according to the different designated time of incubation. Renal function was evaluated. Renal tubule injuries were observed by hematoxylin-eosin(HE) staining. Cell apoptosis was evaluated after Annexin-V/PI staining and cell viability was detected by CCK-8 assay. The expression of LC3, p62, Bcl-2 and Bax were detected with Western blotti ng assay. The morphol ogy of autophagy in kidney and cells were observed under an electron microscope.Results: 1.In vivo experiment: Western blotting analysis showed that the autophagy of renal tissue began to increase at 2h after ischemia/reperfusion(p<0.05), showed by the inc reased ratio of LC3-II/LC3-I and decreased expression of p62 protein. The level of autophagy reached the peak until 2 days of reperfusion(p<0.001), then declined at 3-day but was still higher than Sham group(p<0.05), and finally returned to normal level at 7 days. Electron microscopy observed that autophagosomes engulfed dysfunctional mitoc hondria and other cytoplasmic components when the mice were subjected to 24-h reperfusion.The apoptosis of renal tissue occurred later than autophagy, the ratio of Bcl-2/Bax started to decrease at 12 h of reperfusion, which was obviously lower than Sham group(p<0.01).The apoptosis in renal tissue reached the peak after 1 day reperfusion(p<0.001), then reduced gradually along with the prol ongation of reperfusion, and the ratio of Bcl-2/Bax had been restored to normal until 3 days after I/R. It was also found that Scr and BUN concentrations began increasing 2 and 5-h after reperfusion respectively, reached the maximum at 24-h after reperfusion(Scr: 130.00±30.07 umol/L vs. 14.00±6.60 umol/L; BUN: 45.57±8.67 mmol/L vs. 8.18±2.17 mmol/L, p<0.001), began decreasing 2 days after reperfusion, and returned to the normal level 7 and 2 days after reperfus ion respectively. HE staining showed the renal tissue was damaged obviously 24-h after reperfusion. After 7 days reperfusion, the renal tubular da mage was reduced significantly, although vacuolar degenerati on was still observed. 2. In vitro experiment: Western bl otting analysis showed autophagy was increased significantly at 12-h(p<0.05) and further increased at 24-h after hypoxia incubation(p<0.001). However, after 4 and 8-h reoxygenation, autophagy began decreasing although was still higher than that in normoxic group(p<0.05). Electron microscopy also showed that autophagosomes engulfed the cytoplasmic components when cells were exposed to 24-h hypoxia and subsequent 4-h reoxygenation. Finally, autophagy declined to normal after 24-h reoxygenation. The cellular apoptosis began to increase after 24 h of hypoxia incubation. The ratio of Bcl-2/Bax in H/R4 h and H/R8 h groups decreased significantly(p<0.05), and did not recover to normal until 24 h of reoxygenation(p<0.001). Flow cytometry analysis also found that apoptosis began to increase after 24 h of hypoxia and further increased after 4 and 8h of reoxygenation incubation. Cell viability began to decrease after 24 h of hypoxia(p<0.05) and further decreased at 4h and 8h of reoxygenation treatment(p<0.01,p<0.001). Of note, the cell apoptosis and viability did not return to normal until 24 h of reoxygenation treatment.Conclusions: In the early stage of renal I/R, autophagy increased earlier than apoptosis, whereas in the recovery stage of I/R injury, autophagy de clined later than apoptosis. Autophagy might actas a stress response to postpone the occurrenceof apoptosis in early stage of AKI, and contribute to the kidney repair in recovery stage of AKI.Part II The effects and mechanism of regulation autophagy in re nal ischemia/reperfusion injuryObjective: To investigate the effect of autophagy inhibitor(3-MA) or autophagy activator(Rapamycin) pretreatment on renal ischemia/repefusion injury and to elucidate the role of autophagy in acute kidney injury.Methods: 1. In vivo interventi on of autophagy experiment: Forty male BALB/c mice were randomly divided into SD1+vehgroup(n=5), SD1+3-MAgroup(n=5), ID1+veh group(n=5), ID1+3-MA group(n=5); SD1+vehgroup(n=5), SD1+Rap group(n=5), ID1+veh group(n=5), ID1+Rap group(n=5); 3-MA or Rapamycin was administered with intraperitoneal injection 2-h before 30-min ischemia for autophagy suppression or induction, respectively. The blood and kidneys were collected after 24 h reperfusion. Renal function was evaluated by monitoring blood urea nitrogen(BUN) and serum creatinine(Sc r) with an automatic biochemical analyzer. The kidney tissue slices were stained with hematoxylin-eosin(HE) to observe the renal tubule injury. The expression of LC3, p62, Bcl-2, Bax, p-m TOR, p-P70S6 K and p-4E-BP1 were detected with Western blotti ng assay. The morphology of apoptosis in kidney tissue was observed with TUNEL staining. 2. In vitro intervention of autophagy experiment: The renal tubular cells were randomly divided into Nor+vehgroup, Nor +3-MA group, H/R+veh group, H/R+3-MA group; Nor+vehgroup, Nor+Rap group, H/R+veh group, H/R+ Rap group. 3-MA or Rapamycin was administered 1h or 4h before normoxic and hypoxia incubation, respectively. The expression of LC3, p62, Bcl-2, Bax, p-m TOR, p-P70S6 K and p-4E-BP1 in renal tubular cells were detected with Western blotting assay. The morphology of cell apoptosis was observed with TUNEL staining.Results: 1. In vivo intervention experiment: Added with 3-MA, the autophagy of renal kidney in mice subjected to I/R 24 h was inhibited significantly, decreased the convertion of LC3-I to LC3-II as well as degradation of p62. 3-MA pretreatment contributed to the increase of apoptosis in mice subjected to renal I/R 24 h injury. Compared with ID1+veh group, the ratio of Bcl-2/Bax decreased obviously in ID1+3-MA group(p<0.05). Morphologically, the number of TUNEL positive cells in ID1+3-MA group also increased significantly(ID1+3-MA vs ID1+veh: 62.67±10.97 vs 38.34±7.37,p<0.05).The BUN and Scr concentra tions in ID1+3-MA group increased to 1.47 and 1.49 fold respectively, as compared with ID1+veh group(BUN: 66.78±11.13 mmol/L vs. 45.57±10.39 mmol/L, p<0.05; Scr: 186.26±33.75 umol/L vs. 125.21±27.82 umol/L, p<0.05). HE staining also showed that 3-MA treatment aggravated renal ischemia/reperfusion injury. On the contrary, in the presence of Rapamycin autophagy further increased when mice were subjected to I/R injury. As shown that the ratio of LC3-II/LC3-I increased and the expression of p62 dec reased significantly in ID1+Rap group than ID1+veh group(p<0.05).The ratio of Bcl-2/Bax in mice increased after renal I/R injury in the presence of Rapamycin. TUNEL assay also showed the number of apoptotic cell reduced obviously when Rapamycin was administrated(ID1+Rap vs ID1+veh: 19.43±4.16 vs 38.34±7.37,p<0.05). The loss of renal function after renal I/R injury were partly but significantly alleviated in the presence of rapamycin( I D1+Rap vs ID1+veh: Scr : 75.36±24.21 umol/L vs 125.21±27.83 umol/L,BUN:27.3±7.99 mmol/L vs 45.58±10.39 mmol/L,p<0.05). HE-staining also showed renal tubular cell necrosis, degeneration, tubule casts and renal interstitial infiltration of inflammatory cells were alleviated significantly with addition of Rapamycin. Western blotting analysis showed that the phosphorylations of m TOR, P70S6 K and 4E-BP1 in mice subjected to renal I/R injury were obviously lower tha n SD1+veh group(p<0.001). Added with Rapamycin, m TOR signal pathway was inhibited, resulting in further inhibition of phosphorylations of m TOR, P70S6 K and 4E-BP1 in kidney after I/R injury. 2. In vitro intervention experi ment: Western blotting analysis showed that 3-MA inhibited the expression of autophagy induced by renal tubular cells H/R injury, and decreased the ratio of Bcl-2/Bax, aggravated the cellular apoptosis significantly. Confocal microscope also observed the number of TUNEL positive cells increased after 3-MA treatment when renal tubular cell subjected to H/R injury. Rapamycin pretreatment activated autophagy induced by H/R stimulation in renal tubular cell, and attenuated the apoptosis obviously which also was shown by reduced number of TUNEL positive cells. m TOR activation was not inhibited during the incubation of H/R. There was no significantly difference of phosphorylations of m TOR, P70S6 K and 4E-BP1 between H/R+veh group and Nor+veh group. In the presence of Rapamycin, m TOR activation was inhibited, showing that the expression of phosphorylations of m TOR, P70S6 K and 4E-BP1 were lower than H/R+veh group.Conclusions: Autophgy induced in renal I/R injury might play a role of renoprotecti on. The upregulation of autophagy by Rapamycin during renal I/R injury may be associated with its inhibition of expression ofphosphorylations of m TOR, P70S6 K and 4E-BP1.Part III The effects of Klotho on autophagy induced in renal ischemia/reperfusion injuryObjective: Observed the effects of Klotho on Autophagy induced in renal ischemia/reperfusion to explore the renoprotective mechanism of Klotho in AKI.Methods: 1. In vivo experiment: Twenty five male BALB/c mice were randomly divided into SD1+veh group, ID1+veh group, ID1+kl group, ID1+3-MA group, ID1+3-MA+kl group(n=5 per group). 3-MA was administered with intraperitoneal injection 2-h before 30-min ischemia.Recombinant Klotho protein was injected intraperitoneally30 min and 2 h after reperfusion, respectively. Blood and kidneys were collected after 24 h reperfusion for ananlysis.The expression of LC3 and p62 in renal tissue were detected with Western blotti ng assays and autophagosomes were observed and counted under electron microscope.Renal function was evaluated by monitoring blood urea nitrogen(BUN) and serum creatinine(Scr) with an automatic biochemical analyzer. The kidney tissue slices were stained with hematoxylin-eosin(HE) to observe the renal tubule injurywhich was scored accordingto the percentage of damaged tubules.The apoptosis in kidney was stained with TUNEL assay. 2. In vitro experiment: TCMK-1 cells randomly divided into Nor group,Nor+kl group, H/Rgroup, H/R+kl group. Recombinant Klotho protein was addedin the culture medium 4h before hypoxia incubation and at the beginni ng of reoxygenation treatment, respectively.The cells were collected 4h after reoxygenation for analysis. The expression of LC3, p62, Bcl-2 and Bax in renal tubular cells were detected with Western blotti ng assay as well as autophagosomes were observed and counted under electron microscope.T he number of Annexin-V+/PI- cells were detected with flow cytometry.Results:1. In vivo experiment: It was shown by Western blotting assay that the ratio of LC3-II/LC3-I in kidney tissue at 24 h after reperfusion increased significantly in the presence of Klotho protein as well as the expression of p62 decreased. The number of autophagosomes in kidney tissue observed under electron microscope also increased with Klotho protein pretreatment. However, added with 3-MA blocked the up-regulation of autophagy of Klotho protein. The levels of blood BUN and Scr at 24 h of reperfusion declined obviously in the presence of Klotho protein. HE-staining and tubular damage scores also indicated that renal tubular epithelial cells degeneration, necrosis and casts were reduced by Klotho protein as well as the numbr of TUNEL positive cells.But added with 3-MA bl ocked the anti-apoptosis of Klotho protein. 2. In vitro experiment: Both Western blotting assay and electron microscope observati on indicated that autophagy in TCMK-1 exposed to H/R injury increased significantly in the presence ofrecombinant Klotho proteinc omparing with H/R group.The ratio of Bcl-2/Bax in TCMK-1 also elevated drastically.The number of Annexin-V+/PI- cells after hypoxia/reoxygenation injury decreased obviously with Klotho protein incubation.Conclusion: Klotho protein might attenuate apoptosis in renal ischemia/reperfusion injury by up-regulating autophagy to alleviate the renal tissue damage.Part IV The mechanism of Klotho on autophagy in renal tubular cells induced by hypoxia/reoxygenation injuryObjective: Mornitored the regulation of recombinant or overexpression Klotho protein on IGF-1/Akt/FOXO1 signal pathway in renal tubular cells exposed to hypoxia/reoxygenation injury, to explore the mechamism of Klotho on Autophagy in AKI.Methods: TCMK-1 cells were randomly divided into Nor group, Nor+Kl group, H/R group, H/R+Kl group. IGF-1 pretreatment and Ad-Klotho infection experi ment groups: Nor+Lac Z group, H/R+Lac Z, H/R+Ad-Kl, H/R+Lac Z+IGF-1, H/R+Ad-Kl+IGF-1. Western blotting detected the phosphorylation of IGF-1/Akt/FOXO1 signal pathway and expression of LC3 as well as p62. The cell apoptosis was detected by flow cytometry assay. The nucleus translocation of FOXO1 was observed under confocal microscope.Results: The phosphorylation of IGF-1/Akt/FOXO1 signal pathway increased significantly after renal tubular cells exposed to H/R injury. However, the phosphorylations of IGF-1R, Akt and FOXO1 were inhi bited obviously with addition of recombinant Klotho protein which also contributed to the nucleus translocation of FOXO1 in renal tubular cells subjected to H/R injury. Added with IGF-1 further activated the IGF-1/Akt/FOXO1 signal pathway drastically when tubular cells exposed to H/R injury, whereas overexpression of Klotho protein could inhi bit the activation of IGF-1/Akt/FOXO1 pathway by IGF-1. The apoptosis was further increased significantly in the presence of IGF-1 comparing with H/R+Lac Z group. But overexpression of Klotho protein could decrease the cell apoptosis significantly after H/R incubation, and alleviate the aggravation of cell apoptosis induced by IGF-1.Conclusions: Klotho might up-regulate autophagy in renal tubular cells after H/R injury via inhibiting phosphorylation of IGF-1/Akt/FOXO1 signal pathway and contributing to the nucleus translocation of FOXO1. |
PDF Full Text Request