1.23KD RhALR Expression, Purification, Monoclonal Antibody Preparation And Function Of A Preliminary Exploration 2.rhALR On Human Renal Tubular Epithelial Cells Hypoxia / Reoxygenation Inflammation Intervention And Mechanism

Background and objectiveAugmenter of liver regeneration (ALR) is a growthfactor which widely exists in eukaryocyte. It not only expresses in liver, but also in kidney. Human ALR has two subtypes whose molecular is 15KD and 23KD.15KD ALR is located in cytoplasm and 23 KD ALR is in mitochondria. They own the same termination code and different initiator code. However, there are lots of studies about 15KD ALR, there is little known about 23 KD ALR.In our previous study, we found in the rats models of acute kidney injury(AKI),15KD ALR was increased in 6h after AKI, and recovered to basic line in 72h.15KD ALR can inhibit tubular cell apoptosis and promote its generation. We conclude that 15KD ALR take apart in the progress of AKI. In the early phase of AKI, it is common to see the dysfunction of mitochondria. Moreover, the two subtypes of ALR have a highly comparability, we think that 23KD ALR may have the same biological function as 15KD ALR.In our work here, we construct the plasmid pET28a-hALR. Recombinant human 23KD ALR was expressed and purified. The monoclonal antibody against 23KD rhALR was prepared for the detection of its function. The difference biological function between 15KD rhALR and 23KD rhALR was tested.Methods1. Construction of plasmid pET28a-hALRThe plasmid pET28a with the tag of 6×His was used. Total RNA was extracted from HepG2 cells and transferred to cDNA according to manufacturer’s instruction. The main fragment of hALR cDNA contained BamH Ⅰ/Xho Ⅰ sites was amplified by PCR. The fragment and plasmid were cut by BamH Ⅰ/Xho Ⅰ restriction endonuclease at the same time, and connected by T4 ligase. The constructed plasmid was identified by restriction endonuclease and PCR, and analyzed by gene sequencing.2. The expression of recombinant human ALRThe plasmid pET28a-hALR was transferred to BL21. The protein was producted under the right time points and the concentration of IPTG. The protein was purified by affinity chromatogaphy and tested by Western blot. 23KD rhALR was collected and diayzed under 4℃.3. Preparation of monoclonal antibody against 23KD rhALRBalb/c mouses were immuned by 23KD rhALR. Then mouses were all sacrificed. Mouse myeloma cell line Sp2/0 was used to fuse the splenocytes of immunized mouses. Limited dilution method was used to clone the bybridoma cells. ELISA and Western blot was used to exam the titer of monoclonal antibody. The immnoglobulin subtype was test by IsoQuick strips.4. The comparision of 15KD rhALR and 23KD rhALR on the promotion of generation on HepG2 cells and cisplatin induced HepG2 apoptosis.5. The concentration of 23KD rhALR was tested in normal people and AKI patients blood and urine by indirect ELISA.Results1. Plasmid pET28a-hALR was successfully constructed. pET28a-hALR and pET28a were digested by restriction endonuclease and analyzed by PCR. The fragment of 23KD hALR was found in pET28a-hALR and not in pET28a. The sequence of 23KD hALR was determined and analyzed. Gene sequencing result was correct after blasting in Gene bank.2. BL21 containing pET28a-hALR was inducted by different concentration of IPTG and time points, and then was analyzed on SDS-PAGE. The 23KD recombinant human ALR was expressed most high under the condition of 0.6mM IPTG and 5h.3. BL21 was collected after inducing, resuspended in Binding buffer which contained lysozyme. BL21 was broken by ultrasound and supernatant was collected and purified by Ni2+-NTA resin. In the progress of purify, the purified product was identified by SDS-PAGE. The concentration of imidazole was chosen in 100mM in Binding buffer and 500mM in Elution buffer.4. One of hybridomas was screened out, named 2C11. Cells were cultured and injected into mice abdominal. After 10-15 days, ascites was get. Each of mouses can produce 2ml ascites. The title of monoclonal antibody was 10-5, tested by ELISA. Its subtype of Ig was IgG1, and the light chain was kappa. Indirect ELISA and Western blot suggested that antibody specifically reacted with 23KD rhALR.5.15KD rhALR can promote HepG2 cells generation and recede HepG2 apoptosis, however,23 KD rhALR can not.6. The concentration of 23KD ALR was tested negatively in normal people and AKI patients blood and urine by indirect ELISA.Conclusion1. The recombinant plasmid of pET28a-hALR was constructed successfully.2. Recombinant human 23KD ALR was expressed and purified. It will lay the foundation for monoclonal antibody production.3. The high titer, specific monoclonal antibody had been successful acquired.4. Exogenous 23KD rhALR can not promote HepG2 cells generation and inhibit HepG2 apoptosis induced by cisplatin. There is different biological function between 15KD ALR and 23KD ALR.5. The concentration of 23KD ALR was tested negatively in normal people and AKI patients blood and urine by indirect ELISA. We suggested that the amount of ALR in samples was low, and indirect ELISA could not serve as a sensitive detection measure for clinic. In the other hand, the way of collection and preserve should be improved.Background and objectiveAcute kidney injury (AKI) is a frequent and serious complication in hospitalized patients that occurs when nephrotoxicity, infection, or operation damages the kidneys. Although we can avoid it happens by getting away from pathogenesis, there is still lack of treatment for AKI.Ischemia-reperfusion injury (IRI) is one of the major causes of AKI. Studies have demonstrated that tubular cells were turn to apoptosis or necrosis. Renal tissues were infiltrated by inflammatory cells. In animal models, rats have a strong inflammatory reaction after IRI. So, inflammatory reaction plays a central role in the progress of IRI. The mitogen-activated protein kinase (MAPKs) signaling pathway regulates diverse cellular programs. In mammals, there are more than a dozen MAPK genes; the best known are the extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinase, and p38. MAPKs have been implicated in post-I/R cell survival, necrosis, and apoptosis. MAPKs and NF-κB play a central role in the balance of inflammation.Augmenter of liver regeneration(ALR) was found as a growthfactor. In the previous study, we found that it can protect renal function in the rats with ischemia reperfusion injury. ALR can inhibit inflammation through down-regulation of NF-κB in NRK-52E cells underwent hypoxia reoxygenation.In our research, we used hypoxia reoxygenation to mimic ischemia reperfusion injury and human tubular cells (HK-2) as the research object. HK-2 cells were treated with 15KD rhALR or ALR antibody. We transferred shRNA to HK-2 cells to interfere endogenous ALR expression. After hypoxia reoxygenation, cytokines and chemokines were examined. The phosphorylation of ERK, p38 and JNK and the expression of NF-κB in nuclear were detected. This study is tended to explain the mechanism of ALR on inflammatory response.Methods1. We used HK-2 as the research object. shRNA was transferred into HK-2 cells to interfere endogenous ALR expression. Cells were randomly divided two groups. In group A, there are four groups:normal group, hypoxia reoxygenation group,15KD rhALR group and 15KD rhALR+ALR Ab group. In group B, there are four groups:normal group, hypoxia reoxygenation group, shRNA/control group, shRNA/ALR group. All cells were pre-starved 24h by using free culture and then changed with complete culture before hypoxia reoxygenation. Based on the previous study, besides normal group, cells were hypoxia for 6h and reoxygenation for 12h.2. To the study the relationship between exogenous ALR and endogenous ALR, HK-2 cells were divided into two groups:normal group and 15KD rhALR group (25μg/ml,50μg/ml). After treated with 15KD rhALR for 24h, endogenous ALR expression was tested by Real-time PCR and Western blot.3. The viability of HK-2 cells which were transferred shRNA was measured by MTS.4. The level of TNF-α、IL-6、MCP-1 was tested by ELISA and Real-time PCR.5. The expression of pERK/ERK, pp38/p38, pJNK/JNK and NF-κB p65 was detected by Western blot.6. The NF-κB p65 nuclear translocation was observed by laser confocal microscopy.Results1. In order to knockdown ALR gene, we used shRNA to infect HK-2 cells. We observed that the efficiency of infection was above 80%. The expression of mRNA and protein of ALR was examined by qPCR and Western blot analysis, respectively. shRNA/control-infected cells had no significantly impact on ALR expression levels compared to normal cells (P>0.05). However, ALR expression in knockdown cells was markedly lower than that in normal or shRNA/control cells (P<0.05).2. To observe the effects of ALR on the viability of HK-2 cells, we examined cell growth using the MTS assay at three different time points (24h,48h,72h). Knockdown of ALR expression did not affect cell proliferation at any time point.3. Compared with hypoxia reoxygenation group, the expression of TNF-a, IL-6, MCP-1 was remarkably decreased in 15KD rhALR group (P<0.05). Compared with 15KD rhALR group, the expression of TNF-a, IL-6, MCP-1 in 15KD rhALR+15KD ALR Ab group was increased (P<0.05).4. Compared with hypoxia reoxygenation group, in 15KD rhALR group, the phosphorylation of ERK, p38, JNK level was lower (P<0.05). To further verity the p65 nuclear translocation data, we analyzed the cells by immunofluorescence. NF-κB translocated to the nucleus after H/R. In contrast, NF-κB in the 15KD rhALR group remained in the cytoplasm. In 15KD rhALR+15KD ALR Ab group, NF-κB was mainly expressed in nucleus.5. Compared with shRNA/control group, the expression of TNF-a, IL-6, MCP-1 in shRNA/ALR group was remarkably decreased (P<0.05).6. Compared with shRNA/control group, in shRNA/ALR group, the phosphorylation of ERK, p38, JNK level was lower (P<0.05). ALR knockdown reduced the expression of NF-κB in the nucleus (P<0.05).7. We examined the expression of endogenous ALR in HK-2 cells, and observed low ALR expression in the control group. However, when cells were subject to hypoxia for 6h and reoxygenation for 12h, endogenous ALR expression was increased, as determined by Western blotting and Real-time PCR. Administration of ALR to cells significantly inhibited endogenous ALR protein and mRNA expression. Moreover, cells cultured with 50 μg/ml 15KD rhALR had lower endogenous ALR expression than cells cultured with 25 μg/ml hALR (P<0.05).Conclusion1. Exogenous 15KD rhALR can decrease endogenous ALR expression in HK-2 cell.2. Exogenous 15KD rhALR can attenuate inflammatory response after hypoxia reoxygenation in HK-2 cells, while ALR antibody can partly get rid of this protection.3. Exogenous 15KD rhALR can inhibit the phosphorylation of ERK, p38, JNK and decrease the expression of NF-κB in the nucleus.4. shRNA/ALR can markedly decrease ALR expression in HK-2 cell. Knockdown of endogenous ALR can inhibit the inflammatory response in HK-2 cell after hypoxia reoxygenation.5. shRNA/ALR can alleviate the phosphorylation of ERK, p38, JNK, regulate NF-κB p65 expressed in cell nucleus.