The Regulation For Expression Of C3aR And The Role Of Activation Of It In Renal Tubular Epithelial Cells

BACKGROUNDThe complement system is a cascade of biological systems, with complex and sophisticated regulatory mechanisms, containing more than 30 plasma and cell-associated proteins. Complement cascade is a set of soluble and cell-bound components, regulators and receptors, which can not only be activated in response to pathogens, but also due to the presence of autoantibodies, apoptotic, necrotic or ischemic cells and tissues. Complement activation is known to occur through three different pathway:classical pathway, alternate pathway and mannose-binding lectin. All the three pathways lead to activation of C3 component by C3 convertases, release of C3b opsonin, C5 conversion, and eventually membrane attack complex(C5b-9) formation. C5b-9 disrupts the phospholipid bilayer of target cells causing cell injury and necrosis. The complex is also capable of activating neutrophils, endothelium, and epithelium. Complement system also is an important part of the innate immunity, they can recognize conserved antigens derived from common pathogens, and make rapid and strong response. Except as participation in the body’s host defense, the normal or abnormal activation of complement system are also involved in the occurrence and progression of a variety of diseases including kidney disease. Previously, people pay more attention on the effect of MAC, the production of activation of complement system, in kidney disease, but in recent years, the role of C3a which is a small fragment derived from C3 complement cleaved by C3 convertase and its specific receptor C3aR has received extensive attention gradually by scholars in the pathogenesis of kidney disease.Complement activation via three main pathways generates C3 convertase and a set of effector molecules, including the large fragment C3b and its metabolites(iC3b, C3dg); the small fragments(C3a). C3b then through a cascade of enzymatic reactions generates the terminal complex C5b-9. C3a is a peptide containing approximately 77 amino acid residues. As a potent inflammatory cytokine, C3a not only participate to the innate immune response of the body, but also show a various of biological effects via acting on extensive immune or non-immune cells. For example, anaphylatoxin C3a regulate vasodilation, increase the permeability of small blood vessels, mediate contraction of smooth muscles. C3a has a strong chemotaxis for phagocytes(neutrophils, macrophages) and make them migrate to local injury or inflammatory tissues. C3a also promote release of histamine by basophils and mast cells, and induce oxidative burst in macrophages, neutrophils and eosinophils. It also regulate tissue regeneration and fibrosis, such as contribute to regeneration of liver.C3aR is the receptor which can specifically combine with anaphylatoxin C3a, it’s a member of G protein-coupled receptors(GPCR) superfamily and has a seven helical transmembrane domains connected by intracellular and extracellular loops. In the past decade, expression of C3aR has been reported in many cell types including bone marrow-derived cells and non-bone marrow-derived cells. It is well-know that expression of C3aR on monocytes, neutrophils, macrophages, T cells and mast cells, of course the non-bone marrow-derived cells such as activated astrocytes, endothelial cells, epithelial cells and smooth muscle cells could be detected C3aR mRNA and protein. Recent studies showed that in the kidney tissues, tubular epithelial cells and podocytes also could express C3aR. In variety of kidney disease model mice renal tissues, C3aR expression was up-regulated significantly at both the mRNA and protein levels. This up-regulated expression started before the onset of kidney disease, indicating that abnormal activation of C3a/C3aR axis may be involved in the development of disease, rather than simply a consequence.The relationship between pathogenesis of kidney disease and activation of complement system is complicated. Many researches have shown that the complement cascade were involved in development of kidney diseases including variety of autoantibodies mediated glomerulonephritis, C3 glomerulopathy, renal ischemia-reperfusion(I/R) injury and antibody-mediated renal allograft rejection. Moreover, each complement component or regulatory factors plays different role under several pathophysiological conditions. As a part of complement system, the role of C3a/C3aR axis in kidney disease is gradually given more and more attention by most scholars, in addition, some literatures support that the normal or abnormal activation of C3a/C3aR axis plays a more direct and significant role in kidney disease, indicating that C3a/C3aR axis may be a new therapeutic target in kidney disease.Diabetic nephropathy(DN), one of the most common complication of diabetes, is a progressive fibrotic kidney disease. Furthermore, it is one of the main causes of death in patients with diabetes. In previous work, we observed that the increased expression of C3aR in renal tissue from patients with diabetic nephropathy, then we hypothesized that the activation of C3a/C3aR axis may play an important role in DN, but the mechanism by which factor induces C3aR expression increased is not fully understood. Here, in order to better understand the regulatory factors for expression of C3aR in renal tissues, we studied the regulation of expression for C3a receptors in human renal tubular epithelial cells (HK2 cells) by multiple inflammatory cytokines(IFN-γ、C3a、IL-1β、TNF-α、TGF-β). In addition, tubulointerstitial fibrosis and glomerular sclerosis are both major characteristic of kidney damage in DN patients. Some studies have shown that epithelial-to-mesenchymal transition played an important role in renal fibrosis. We speculated that activation of C3a/C3aR axis might induce epithelial-to-mesenchymal transition in kidney thereby accelerating the progression of renal fibrosis. Therefore, We established a method for primary culture and identification of mouse renal tubular epithelial cells and explored whether the activation of C3aR could induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells.Part I Inflammatory cytokines regulate the expression of receptors for complement C3a on human renal tubular epithelial cellsObjectionIn previous work, we found that the increased expression of C3aR in renal tissue from patients with diabetic nephropathy, but the mechanism by which factor induces C3aR expression increased is not fully understood. Here, in order to better understand the regulatory factors for expression of C3aR in renal tissues, we studied the regulation of expression for C3a receptors in human renal tubular epithelial cells (HK2 cells) by multiple inflammatory cytokines.MethodHK2 cells were suspended into the appropriate amount of culture medium:1:1 DMEM/F12 and supplemented with fetal bovine serum 10%. HK2 cells were seeded in 12-well plates at density of 1 × 105/well. Then the plates were left at 37℃ and 95% air-5% CO2 in a standard humidified incubator. Replaced with 0.1% fetal bovine serum DMEF/F12 medium for synchronization 12-18 hours when the cell density approximately reach 80% confluence. Multiple inflammatory cytokines(IFN-γ、C3a、 IL-1β、TNF-α、TGF-β) were used to be co-cultivation with synchronized HK2 cells. According to the literature, the stimulation groups of each inflammatory cytokines were divided into the concentration and time gradient group, then the levels of C3aR mRNA in HK2 cells were tested by Real-time PCR. Based on the results of mRNA expression, We used IFN-y to stimulate HK2 cells and detected the levels of C3aR protein by immunocytochemistry staining and Western blotting.ResultWe discovered that inflammatory cytokine interferon-gamma (IFN-γ) can significantly promote the expression of C3aR in HK2 cells (Compared with control group, C3aR mRNA expression levels increase about 5 times, P= 0.001<0.05, protein levels were up-regulated approximately 1.6-fold, P=0.046<0.05) and the up-regulation of C3aR on mRNA levels was dose- and time-dependent for it. However, other cytokines such as:TNF-α、TGF-β、IL-1β and even biologically active polypeptide C3a were not observed visible impact on the expression of C3aR in HK2 cells.ConclusionThis present study not only proved tubular epithelial cell surface expressed C3a receptor (C3aR), but also proved that inflammatory cytokine interferon-y could significantly enhance the expression of C3aR in HK2 cells, indicating that the C3a/C3aR axis may be involved in the regulation of renal local inflammatory reaction. It also suggested that C3a/C3aR axis was a certain degree of correlation with the occurrence and development of kidney diseases.Part Ⅱ C3aR agonists transforming the phenotype of mouse primary renal tubular epithelial cellObjectiveTo establish a method for primary culture and identification of mouse renal tubular epithelial cells and explore whether the activation of C3aR can induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells.MethodFirst, the tubular segments isolated from mouse kidneys were used for primary culture, steps were summarized below. After sacrificing and disinfecting mice, the kidneys were removed into ice-cold PBS solution at sterile environment. Then the renal cortices were dissected visually in ice-cold PBS solution and sliced into pieces of about 1mm wide. The fragments were transferred to 5ml collagenase solution at 37℃ and digested for 1 hour. After digestion, the supernatant was sieved trough a nylon sieves(pore size 80 um), the fragments remaining in the sieve could be gently rolling with a sterile mortar. After gently washing by warm PBS solution, the longer tubular fragments remain in the sieve were resuspended by flushing the sieve in the reverse direction with warm(37℃) PBS containing BSA 1%. The tubular fragments present in BSA solution were centrifuged for 5min at 170g, washed, and then resuspended into the appropriate amount of culture medium. After that, the tubular fragments were seed onto collagen-coated 12-well plates and left unstirred for 48 hours at 37℃ and 95% air-5% CO2 in a standard humidified incubator. The medium was then replaced every 2 days. After 7 days, cell cultures were organized as a confluent monolayer. Used tripsin digestion in the subculture of renal tubular epithelial cells. The identification of the renal tubular epithelial cells was performed by immunocytochemistry and immunofluorescence staining. After stimulating by C3aR agonists, the expression of E-cadherin、α-SMA and collagen I at mRNA levels in renal tubular epithelial cells were detected by Real-time PCR. On the other way, We observed the cytoskeleton of epithelial cells by phalloidin staining.ResultAfter 48 hours, most of renal tubular segments were adherence and a small amount of epithelial cells in the segments climbed out. Subsequently, cells gradually increased and grown as an island. The cells connected tightly and most of them were short spindle or polygonal. After 7 days, cell cultures were organized as a confluent monolayer. Cells showed a typical cobblestone paving kind and had a strong transparency and refraction. The results of immunocytochemistry and immunofluorescence staining showed E-cadherin staining were positive around the nucleus and in the cytoplasm while control group without special staining. Treating with C3aR agonists, expression of E-cadherin mRNA deceased and expression of both a-smooth muscle actin and collagen I mRNA increased. In addition, the expression trend of three molecules in does-and time-dependent manner respectively. The cytoskeleton staining showed that treatment of renal tubular epithelial cells with C3aR agonists induced the formation of actin stress fibers in a time-dependent manner. when the stimulation time over 12 hours the formation of actin stress fibers increased significantly.ConclusionWe successfully established the method for primary culture and identification of mouse renal tubular epithelial cells and provided experimental basis for the study of mouse renal tubular epithelial cells. We found that C3aR agonists could promote expression of α-SMA and collagen I while suppress E-cadherin in mouse renal tubular epithelial cells. Furthermore, the formation of actin stress fibers also increased obviously by stimulation with C3aR agonists. In summary, it suggested that the activation of C3aR could induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells and play an essential role in the development of renal fibrosis. Moreover, it indicated C3aR may become a new therapeutic target in kidney diseases.