Protective Effect Of KIM-1 On Renal Tubular Epithelial Cell Hypoxic Injury And HIF-1 Signal Pathway Regulation

KIM-1 (kidney injury molecule-1) is a newly discovered type I transmembraneous protein, which is characteristically expressed on the top membrane of epithelial cells of kidney proximal tubules following hypoxic injury. Hence, KIM-1 is an ideal premonition molecule for renal ischemic/hypoxic injury. Recent studies have shown KIM-1 to be a functional molecule, which contributes to renal injury and repair. KIM-1 is specifically expressed by dedifferentiated renal tubular epithelial cells, suggesting that it may contribute to dedifferentiation, proliferation, migration, and continuous reconstruction of renal tubular epithelial cells. In vitro studies have demonstrated that KIM-1 is a unique phosphatidylserine receptor, which endues renal tubular epithelial cells with phagocytotic function and, thus, helps scavenge apoptotic and necrotic cells and relieve tubular obstruction. Nevertheless, the precise function of KIM-1 is not clear yet, and there is no direct evidence that supports the protective function of KIM-1. KIM-1 is a transmembraneous protein, and its extracellular structure includes an immunoglobin-like functional domain which comprises 6 cysteine residuals and a threonine /serine-rich mucoprotein domain. The short intracytoplasmic domain includes a conserved tyrosine phosphorylation site, which is phosphorylated under the action of phosphatase inhibitors. KIM-1 may exert adhesion function and activate intracellular signaling pathways. The ectodomain of KIM-1 can be shed into the tubular lumen and this ectodomain shedding is mediated by matrix metalloproteinases (MMPs) possibly involving MAP kinase activation. MAPK plays an important role in signal transduction pathways activated by multiple stressors or inflammatory factors, and mediates physiological and pathophysiological changes in cell function. However, the role of MAPK in KIM-1 cleavage function is still unclear. In light of the molecular structure of KIM-1, we postulate that KIM-1 may serve as an important signal molecule in tubular hypoxic injury, and regulate apoptosis/survival of renal tubular epithelial cells, thus protecting kidneys against hypoxic injury.After KIM-1 has been functionally characterized, it is critical to explore its expression regulating mechanism. Essentially, the response of renal tubular epithelial cells to hypoxia is an oxidative stress reaction. KIM-1 is a specific hypoxia induced functional gene of renal tubular epithelial cells, and its expression is necessarily regulated by multiple stress associated transcription factors. Hypoxia induction factor-1(HIF-1) is a classic oxygen dependent protective nuclear transcription factor, which plays an important role in ischemic/hypoxic responses. HIF-1 target genes already identified do not include genes specifically expressed by kidneys. In pilot studies, we found that HIF-1αand KIM-1 are closely associated with hypoxia /reoxygenation of HK2 cells. With the use of TFsitescan software, we identified HIF-1 binding sites in KIM-1 gene promoter region. Hence, we postulate that KIM-1 is regulated by HIF-1 signal pathway in hypoxia induced injury to renal tubular epithelial cells and that KIM-1 contributes to renal injury and repair as a target gene of HIF-1.In the present study, the protective effect and mechanism of KIM-1 in hypoxic injury to renal tubular epithelial cells were investigated in human proximal tubular epithelial cells (HK2), and the relationship of KIM-1 expression and HIF-1 was analyzed to elucidate the mechanism by which HIF-1 regulates KIM-1. The purpose of the study was to provide experimental basis for KIM-1 targeted renal failure therapy.Part 1 Protective effect of KIM-1 on hypoxic injury to HK2 cells Objective: To construct pcDNA-hKIM-1 -HK2 cell strain that stably expresses KIM-1 and observe the effect of KIM-1 on the proliferation and apoptosis of HK2 cells following hypoxic injury. Methods:1. Construction of pcDNA-hKIM-1 -HK2 cell strain: pcDNA- hKIM-1 plasmids were PCR identified, amplified and transfected into HK2 cells. HK2 cells were then screened repeatedly with G418 medium to obtain pcDNA-hKIM-1 -HK2 cell strain that stably expressed KIM-1. Then HK2 cells were characterized by immunocytochemistry, PCR and Western Blot to confirm high KIM-1 expression by pcDNA-hKIM-1 -HK2 cells.2. Protective effect of KIM-1 on hypoxia induced HK2 cells apoptosis: pcDNA- hKIM-1 -HK2 cells were observed after pretreatment with pcDNA-hKIM-1 -HK2, HK2 and KIM-1 antibodies and hypoxic injury for cell morphology, cell viability and apoptosis, so as to determine the protective effect of KIM-1 expression on hypoxic injury to HK2 cells.Results1. The results showed a bright band at approximately 100bp, which was consistent with the theoretical prediction, suggesting correct plasmid construction. Immunocyto- chemical study suggested that KIM-1 expression was located to cytomembrane, and PCR and Western-Blot suggested that the transfected HK2 cells expressed KIM-1 at high levels.2. pcDNA-hKIM-1 -HK2 cells showed obvious action against hypoxic injury, and their proliferation ability was higher than the control group. The number of apoptotic cells and the apoptosis index as determined by flow cytometry at early, middle and late hypoxic stages were obviously lower in the study group than the control group (p<0.01).Part 2 Mechanism of soluble KIM-1 cleavage for relieving tubular hypoxic injuryObjective: To explore the mechanism by which KIM-1 extracellular domain cleavage relieves hypoxic injury and the effect of the cleavage on apoptosis related signal molecules.Methods:1. The content of soluble cleaved KIM-1 in media of pcDNA-hKIM-1 -HK2 cells, hypoxic and normal HK2 cells and the effect of KIM-1 antibody on soluble KIM-1 expression were analyzed by ELISA to observe KIM-1 extracellular domain cleavage.2. KIM-1 cleavage was blocked by SB203580, a p38MAPK inhibitor to observe the morphology, viability and apoptosis of pcDNA-hKIM-1 -HK2 cells after hypoxic injury, so as to determine whether KIM-1 extracellular domain cleavage mediates KIM-1's protection of cells from hypoxic injury.3. ERK, p38MAPK, JNK, and AKT were detected by Western-blot in pcDNA-hKIM-1 -HK2 cells after pretreatment with the p38MAPK inhibitor and hypoxia, so as to investigate the anti-apoptotic signal mechanism of KIM-1 extracellular domain cleavage against cell injury.Results1. KIM-1 was expressed at high levels on cytomembrane of pcDNA-hKIM-1 -HK2 cells and hypoxic HK2 cells, and soluble KIM-1 was detected in cell culture media, suggesting that soluble cleaved KIM-1 can be released into media from membranous KIM-1 through extracellular domain cleavage.2. SB203580 suppressed KIM-1 cleavage and KIM-1 mediated action against hypoxic injury. HK2 cells pretreated with SB203580 showed decrease in proliferative activity when compared to the control group, and the number of apoptotic cells and the apoptosis index as determined by flow cytometry at early, middle and late hypoxic stages was obviously higher in the study group than the control group (p<0.01). Moreover, the suppressive action was dose dependent.3. After pretreatment of HK2 cells with p38MAPK inhibitor, ERK and AKT expression did not change obviously during hypoxic injury; however, phosphorylated ERK and AKT decreased significantly, and phosphorylated P38 and JNK decreased to certain degrees. Soluble KIM-1 may suppress apoptosis through the PI3K-AKt/PKB and ERK pathways.Part 3 KIM-1 in HIF-1 signal pathway regulation of hypoxic injury to HK2 cells Objective: To observe the correlation between KIM-1 and HIF-1αexpression in HK2 cells during hypoxic injury and the effect of HIF-1αexpression on KIM-1 expression. Then ChIP and EMSA were performed to verify the binding of HIF-1 gene and KIM-1 gene promoter region to test the regulation of KIM-1 expression by the HIF-1 signal pathway during hypoxic injury to renal tubular epithelial cells.Methods:1. KIM-1 and HIF-1αmRNA and protein expression levels were measured by Real Time RT-PCR and Western-blot in HK2 cells at various hypoxia /reoxygenation stages. Double labeling immunofluorescence staining and laser confocal microscopy revealed the location of KIM-1 and HIF-1αexpression in HK2 cells, so as to determine the correlation between KIM-1 and HIF-1αexpression.2. HK2 cells were pretreated by the HIF-1αagonist CoCl2 and the HIF-1αinhibitor Rapamycin, and then the effect of upregulating or downregulating HIF-1αexpression on KIM-1 mRNA and protein expression was observed to determine the regulatory effect of HIF-1αon KIM-1.3. Detection of KIM-1 promoter region and HIF-1αbinding site: Co- immunopre- cipitation of chromatin-HIFαprotein of hypoxia HK2 cells was performed using ChIP kit. Primers were designed according to the predicted sequences of KIM-1 promoter region and HIF-1αbinding site, and the presence of binding site was detected by Real Time RT-PCR.4. Detection of KIM-1 promoter and HIF-1αbinding capability: Using HRE site of KIM-1 promoter region as template, double-strand oligonucleotide probes with biotin labeled 3'-end were designed, and dynamic changes in probe and HIF-1αbinding were determined by EMSA in HK2 cells at various time points of hypoxic injury. Meanwhile, the effect of upregulating or downregulating HIF-1αexpression with CoCl2 and Rapamycin on the binding dynamics was observed.Results1. In HK2 cells, KIM-1 and HIF-1αprotein and gene expression increased simultane- ously during hypoxia, and decreased simultaneously during reoxygenation, suggesting the close correlation of KIM-1 and HIF-1αexpression. Immunocytochemical study suggested that KIM-1 and HIF-1αwere co-expressed in hypoxic HK2 cells, and that KIM-1 was expressed on cytomembrane, while HIF-1αwas expressed in cell nuclei.2. CoCl2 or Rapamycin upregulated or downregulated HIF-1αexpression and, thus, promoted or suppressed KIM-1 expression, respectively (p<0.01). Moreover, this regulatory effect was dose dependent.3. After hypoxia of HK2 cells, primers were designed for KIM-1 promoter HIF-1αbinding site and the amplified fragment was detected at 227bp, which was consistent with the predicted size. In non-hypoxic HK2 cells, no KIM-1 promoter and HIF-1αbinding site were detected. The results suggested that KIM-1 promoter region contained HIF-1αbinding site, and that this binding must depend on hypoxia.4. Using KIM-1 promoter region HRE site as template, double strand oligonucleotide probes were designed and the probes bound HIF-1αduring hypoxic injury in HK2 cells. Moreover, upregulating or downregulating HIF-1αexpression can promote or suppress this binding capability.Conclusions:In the study, in vitro results confirmed the protective effect of KIM-1 on hypoxia induced injury to HK2 cells, and KIM-1 extracellular domain cleavage through p38-MAPK pathway activated the downstream PI3K-AKt/PKB and ERK pathways, promoted AKT and ERK phosphorylation, thus promoting cell proliferation and antagonizing apoptosis. Moreover, KIM-1 expression in HK2 cells during hypoxic injury was regulated by HIF-1α. As a molecule downstream of HIF-1α, KIM-1 contributed to tubular repair after hypoxia.