| ObjectiveSepsis is a critical clinical syndrome with high morbidity and mortality. The key to cure sepsis is to control the sepsis-induced organ dysfunction. Kidney is the most vulnerable organ during sepsis and sepsis-induced acute kidney injury (SAKI) is closely related to the prognosis. Malfunction of tubular epithelial cells (TECs) is the leading cause of AKI. Several reports suggest that TECs can actively secrete cytokines and participate in the processes of several diseases. High mobility group box-1 (HMGB1) is a highly conserved pro-inflammation mediator in mammals. Once being released into blood, HMGB1 acts as a danger associated molecular pattern (DAMP) which can promote inflammation via its receptors such as Toll like receptor 4 (TLR4). Up-regulation of soluble mediators acts as the key that is involved in the pathogenesis of sepsis-induced organ dysfunction and almost all of the serum-derived molecules smaller than the molecular weight cut off of the filtration barrier can enter the tubular system and interact with TECs. Because the molecular weight of HMGB1 is approximately 25 kDa and the vascular permeability of the kidneys is enhanced during sepsis, we hypothesize that serum derived HMGB1 can enter the renal tissue and urine, stimulate the TECs to mediate inflammation, and promote the occurrence of SAKI during sepsis. In this study, we examined our hypothesis using in vivo and in vitro systems.MethodsPart 1:85 adult male SD rats were randomly assigned to:control group (n=5); cecal ligation and puncture (CLP) group (n=20); CLP+HMGB1 injection (CLP+H) group (n=20); sham operation+HMGB1 injection (sham OP+H) group (n=20) and CLP+splenectomy (CLP+S) group (n=20). Rats were sacrificed at different time points after the treatments. Serum and renal TECs were separated while urine was concentrated by ultrafiltration. Serum and urine HMGB1 levels were assessed by Western-blotting, intrarenal distribution of HMGB1 was investigated by immunofluorescence, His-tagged rHMGB1 was used as a marker to determine the distribution of serum derived HMGB1 and IL-1/IL-6 mRNA transcription levels in TECs were measured with Real-time PCR.Part 2:Western-blotting and immunohistochemistry were used to evaluate the expression of TLR4 on TECs of normal rats. Expression and localization of TLR4 on NRK52E cells were assessed with Western-blotting and immunofluorescence,Part 3:Rat tubular epithelial cell (NRK52E) were cultured in vitro and divided into:control group; HMGB1 group and HMGB1+LPS RS group. TECs were serum free before stimulation. Apoptosis rates were measured by flow cytometry, phosphorylation of proteins associated to MAPK/NF-kB pathway were investigated by Western-blotting, IL-1/IL-6 mRNA transcription levels were measured with Real-time PCR and secretions of IL-1/IL-6 were measured by protein chip assay.Part 4:1) 45 adult male SD rats were randomly assigned to:control group (n=5); cecal ligation and puncture (CLP) group (n=20); CLP+splenectomy (CLP+S) group (n=20). TECs were separated and TIMP2 mRNA transcription level was assessed by Real-time PCR, Urine was concentrated and TIMP2 level was investigated by Western-blotting.2) NRK52E was cultured and divided into:control group; HMGB1 group and HMGB1+LPS RS group. TECs were serum free before stimulation. Cell cycle was analyced by flow cytometry, TIMP2 mRNA level was measured with Real-time PCR and secretion of TIMP2 protein was measured by protein chip assay.ResultsPart 1:1) Serum and urine HMGB1 levels were increased significantly in CLP group compared to both control group and CLP+S group (P<0.05); 2) Expression of HMGB1 was distributed widely in the renal tissue of CLP group but was not co-located with the nuclei; 3) Urine His-tagged rHMGB1 was higher in CLP+H group, and His-tagged rHMGB1 was distributed widely except the nucleus region in the renal tissue of CLP+H group; 4) IL-1 and IL-6 mRNA expressions were increased significantly since 6h after the treatment in CLP group (P<0.05).Part 2:1) TLR4 was expressed normally by TECs; 2) TLR4 was localized on the membrance of TECs.Part 3:1) Apoptosis of TECs stimulated with HMGB1 did not rise significantly (P>0.05); 2) Phosphorylation levels of proteins associated to MAPK/NF-kB pathway were increased significantly in HMGB1 group compared to that of both control group and HMGBl+LPS RS group (P<0.05); 3) IL-1 and IL-6 mRNA and proteins were increased significantly in HMGB1 group (P<0.05).Part 4:1) The proportion of TECs in the cell cycle G1 phase was increased significantly in HMGB1 group (P<0.05) but not in HMGB1+LPS RS group (P>0.05); 2) TIMP2 mRNA and protein were increased significantly in HMGB1 group (P<0.05), but not in HMGB1+LPS RS group (P>0.05); 3) In vivo, TIMP2 mRNA expressions in both CLP and CLP+S groups were increased significantly (P<0.05), but TIMP2 mRNA expression in CLP group was significantly higher than that of CLP+S group (P<0.05). TIMP2 protein was increased in the urine of the the CLP animal group.Conclusions(1) HMGBlwas significantly increased in blood and renal tissue during sepsis. Serum derived HMGB1 can distribute widely in renal tissue.(2) TLR4 was normally expressed and localized on the menbrance by TECs.(3) Interaction between HMGB1 and TLR4 mediated MAPK/NF-kB signal pathway of TECs activation, furthermore, leading to TECs to participate in inflammatory processes through active production of IL-1 and IL-6.(4) Interaction between HMGB1 and TLR4 indcued TECs cell cycle Gl arrest and TIMP2 secretion. These results suggest that this interaction has a close relationship with SAKI. |
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