Role Of Tec Non-receptor Kinase On Acute Kidney Injury In Sepsis Induced By LPS And Its Mechanism

1. Introduction Sepsis is a very heterogeneous clinical syndrome broadly defined as the systemic host response to an infection, characterized by excessive release of various inflammatory mediators. It will cause a wide range of cell and tissue damage, may result in further septic shock, severe sepsis or multiple organ dysfunction syndrome.Acute kidney injury(AKI) is the most common complication and an independent risk factor for mortality. Recent studies have indicated that the pathogenetic mechanism in septic AKI is totally different from that in non-septic AKI. The acute inflammatory response to sepsis plays an important role in AKI. Tec(kinase expressed in hepatocelluar carcinoma Tyrosine) is a nonreceptor tyrosine kinase which was found in the liver cancer. The Tec family kinase Src, Syk were confirmed to be related to the inflammatory response. Tec kinase family Src, Tec were confirmed to be phosphorylated in the early stages of the inflammatory response. The mechanism of Tec kinase in inflammatory response to sepsis is seldom studied at present. Whether Tec kinase is involved in sepsis induced AKI, it is rarely documented. The purpose of this study is to create a model of acute kidney injury in sepsis, and to investigate the expression of Tec kinase in kidney, and to explore the possible mechanism of Tec kinase in acute kidney injury induced by sepsis.2. Objectives 1) In vivo, the expression of Tec kinase and the effect of Tec kinase inhibitor in the kidney of AKI induced by Sepsis was studied. And we explored the possible mechanism of Tec kinase in AKI via TLR4, MYD88 expression, NF?B-p65 pathway activation and renal interstitial macrophage infiltration. 2) In vitro, the effect of Tec kinase on the release of inflammatory cytokines induced by LPS in RAW264.7 macrophages was observed. Through Tec kinase inhibitors and si RNA to understand its effect on the secretion of inflammatory mediators and activation of MAPK and NF?B-p65. We expect these studies to further understanding of Tec kinase possible mechanism in the development of sepsis induced AKI, and providing a new target molecule for the treatment of AKI.3. Materials and methods Part? Tec kinase expression in AKI induced by sepsis in mice. 32 healthy male spf C57BL/6 mice were divided into 2 groups with 16 mice in each group. LPS group received intraperitoneal injection of LPS 20mg/kg, the control group was given the same amount of solvent. At 1h, 6h, 24 h, 48 h, 4 mice from each group were sacrificed, checked serum IL-1?, TNF-?, BUN, serum creatinine, Cystatin C, isolated kidneys, the histological pathology was detected by HE staining, the expression of Tec kinase was observed by immunohistochemistry, the expression of Tec kinase, TLR4, NF?B-p65, p-NF?B-p65 was detected by Western blot. Part II Protective effects of Tec kinase inhibitor LFM-A13 on septic acute kidney injury in mice 36 healthy male spf C57BL/6 mice were divided into 6 groups with 6 mice in each group. LPS group received intraperitoneal injection of LPS 20mg/kg, the control group was given the same amount of solvent. LFM-A13(40mg/kg, 60mg/kg, 80mg/kg) were administered i.p. immediately after i.p. injection of LPS20mg/kg. The volume of peritoneal perfusion fluid of mice was 1ml/20 g. Mice were sacrificed at 24 h. checked serum IL-1?, TNF-?, BUN, serum creatinine, Cystatin C, isolated kidneys, the histological pathology was detected by HE staining, the expression of Tec kinase and macrophage infiltration was observed by immunohistochemistry, the expression of Tec kinase, TLR4, myd88, NF?B-p65, p-NF?B-p65 was detected by Western blot. Cultured rat renal tubular epithelial cell NRK-52 E, with different concentrations of LPS(0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml, 10ug/ml) stimulation, at different times(LPS 0.1ug/ml 0min, 15 m, 30 min, 60 min, 120min), to detect the expression of Tec kinase by Western blot. Pretreated the cells 1h with different concentrations of LFM-A13(50u M, 75 u M, 100 u M), then stimulated with LPS(0.1ug/ml), to detect the expression of Tec kinase by Western blot. Part III Effect and mechanism of inhibiting the expression of Tec kinase on LPS induced RAW264.7 macrophage proinflammatory cytokine production Cultured macrophages RAW264.7, Pretreated RAW264.7 cells 1h(25u M, 75 u M) with inhibitor LFM-A13, and stimulated 1h with 0.1ug/ml LPS, The expression of ICAM-1 in RAW264.7 cells were detected by flow cytometry and q RT-PCR. Western blot detected the expression and phosphorylation of I?B?, NF?B-p65 and TAK-1. Laser confocal detect nuclear transfer of NF?B-p65 in RAW264.7 cells. Tec kinase si RNA interfered RAW264.7 cell, stimulated with LPS 0.1ug/ml 1h after transfection. The expression of TAK1, p-p38/p38 and p-NF-?B-p65 were detected by Western blot.4. Results Part ? In vivo, acute kidney injury model in mice was successfully established by LPS intraperitoneally. LPS at dosage of 20mg/kg induced slight injury to renal tissue with elevated Cys-C level at 1h, high level of BUN in blood at 6h and obvious renal dysfunction at 24 h. The renal function and tissue injury tend to recover at 48 h. The inflammatory factors such as TNF-??and IL-1? increased at 1h after LPS injection i.p. The level of IL-1? and TNF-? reached the peak at 6h and 1h respectively. Compared with control, the level of tec and TLR4 protein expression was significantly higher in LPS exposed group at 1h, and began to decline at 6h, and continuously elevated at 48 h. The expression of NF?B-p65 and its phosphorylation was evidently increased at 6h after LPS exposure. IHC staining confirmed that tec protein expressed high in renal tubular epithelial cells in septic mice at 1h. Part II In vivo, the level of Cr, BUN and Cys-C in blood obviously increased at 24 h after LPS injection intraperitoneally. The renal tissue sustained severe injury significantly measured by pathological scores. Pretreatment with LFM-A13 improved the function of kidney in mice at each dosage, and decreased the score of renal injury. ELISA has showed that LFM-A13 significantly reduced the release of IL-1? and TNF-? in mice exposed LPS. LFM-A13 can evidently abrogate the expression of tec protein, My D88, TLR4, NF?B-p65 and its phosphorylated protein measured by western blot. IHC analysis presented that LFM-A13 markedly down-regulated expression of tec kinase in renal tubular epithelial cells and macrophage infiltration in renal tissue of septic mice. Different concentration of LPS was used to stimulate NRK-52 E cells in vitro. Tec kinase protein expressed highly after LPS exposure, even at concentration of 0.1ug/ml. Expression of tec protein increased in NRK-52 E cells after 0.1ug/ml LPS stimulus at 30 min and continuously elevated at 120 min. LPS-induced change was prevented by LFM-A13 at 50 u M, 75 u M and 100 u M. Part III The FCM, q RT-PCR showed that LFM-A13 alleviated on the product of ICAM-1 and decreased the m RNA expression. LFM-A13 inhibited the phosphorylation of NF-k B and TAK1 in RAW264.7 cells induced by LPS. There is no statistical difference of p65 between groups after LPS stimulation. The p65 protein in RAW264.7 cell cytoplasm stimulated by LPS was significantly lower than the control group, while p65 protein expression in the nucleus was significantly higher than the cytoplasm. I?B? expression after LPS stimulation was significantly down-regulated with higher level of phosphorylation protein in cytoplasm. LFM-A13, the inhibitor of tec kinase, obviously suppressed the activation of NF-k B signal. Laser confocal detected nuclear transfer of NF?B-p65 was also suppressed by LFM-A13. Consistent with LFM-A13, silencing tec kinase m RNA by tec si RNA(Tec mus-790 RNAi) significantly abrogated the activation of p38 MAPK and NF-k B, and also reduced phosphatase TAK1.5. Conclusion 1) In vivo, acute kidney injury model in mice was successfully established by LPS(20mg/kg) intraperitoneally. The inflammatory factors such as TNF-? and IL-1? were measured highly at 1h in blood. The injury of renal function was presented at 6h and reached the peak at 24 h, then begin to decline. The tec kinase was up-regulated early in renal tissue induced by LPS, accompanied with increased expression of TLR4, activation of NF-?B pathway and release of inflammatory factors. The results show that tec kinase is involved in the early initiation of acute kidney injury in sepsis and may contribute to the development of renal injury induced by sepsis. 2) Tec kinase was positively regulated in renal tissue in mice model of sepsis and also expressed in renal tubular epithelial cells assessed by immunohistochemical staining. LFM-A13 can inhibit the expression of tec protein in renal tubular epithelial cells both in vivo and in vitro; reduce macrophage infiltration; and improve renal dysfunction and pathological injury through TLR/My D88 inactivated NF-?B pathway and dependent on cytokines 3) Pretreatment with LFM-A13 markedly attenuated the expression of tec kinase in RAW264.7 cells after LPS stimulus. The mechanism includes the down-regulated phosphorylation of TAK1, inhibition of NF-?B pathway and decrease of inflammatory cytokines.