The Experimental Research On Expression Of NGAL-NGALR In Glomerulonephritis And Implicated Mechanisms

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25KDa protein which belongs to the lipocalin superfamily. NGAL was initially detected in activated neutrophils, in accordance with its role as an innate antibacterial factor. NGAL receptor (NGALR) is one of the cellular receptors of NGAL identified recently, which was first isolated from murine lymphocyte FL5.12 cells. This specific receptor probably has a fundamental role in NGAL endocytosis and cellular trafficking.NGAL protein accumulation in the blood and urine can only be detected within a few hours in acute kidney injury (AKI). A large number of clinical trials demonstrate that the sharp increase of NGAL accumatation in the blood and urine promise a bad prognosis of AKI.However, recent reports show that NGAL might associate with some chronic kidney diseases. Some scientists have performed unbiased profiling of gene expression in remnant kidneys of two mouse strains (FVB/N and B6D2F1)that reacted differently to nephron reduction and found 70 genes which expression levels differed significantly 2 months after nephron reduction. Among these transcripts, NGAL was the maximal transcriptional induction in damaged FVB/N kidneys as compared with well-preserved kidneys from B6D2F1 mice. They have revealed NGAL correlated with lesion progression in mouse and human with CKD(such as human autosomal dominant polycystic kidney disease) and identified NGAL was one of the key effectors of renal damage and cystogenesis. Another group have found NGAL-mediated tubule epithelium cell proliferation effects might be regulated by the inflamatary cytokine through its action on NGALR.Furthermore, recent evidence also suggests that Patients with systemic lupus erythematosus, IgA nephropathy, membranous (MGN) or membranoproliferative glomerulonephritis (MPGN) have higher urinary and serum NGAL levels compared to normal controls. It is generally known that HMC play a crucial role in glomeruli. Aberrant proliferation of mesangial cells and extracellular matrix deposition is a common finding in several types of glomerulonephritis. But several studies have investigated the expression and significance of NGAL-NGALR in glomerulonephritis and human mesangial cell.In our experiments we used molecular biologic technologies and cell culture to examine the expression of NGALR in glomerulonephritis and human mesangial cell,to explore the effect of inflammatory cytokines on NGALR expression and to identify the significance of NGAL in HMC and the implicated mechanisms. Our research should provide a useful data for investigation of the possible roles of NGAL-NGALR in the pathophysiological processes of chronic kidney disease.Part I Increased expression of neutrophil gelatinase-associated lipocalin receptor by interleukin-1?in human mesangial cells via MAPK/ERK activationPurpose Neutrophil gelatinase-associated lipocalin (NGAL), one of the most promising next-generation biomarkers in clinical nephrology, has received extensive attention. However, the biological function of its receptor (NGALR) remains unclear. Here, we have assessed the expression pattern of NGALR in injured glomeruli and explored the possible mechanism of the NGALR involvement in inflammation in HMC.Methods and Results The expression pattern of NGALR was detected by immunohistochemistry in biopsy samples of 93 glomerulonephritis patients and healthy controls, and the regulation of NGALR by the proinflammatory cytokines, TGF-?1, TNF-?and IL-1?in HMC was analyzed by real-time PCR and Western blotting. NGALR was found to be predominately expressed in glomeruli. The expression of NGALR was significantly higher in acute proliferative glomerulo-nephritis and lupus nephritis than that in other types of glomerulonephritis or healthy kidney tissues. In in vitro experiments, both mRNA and protein levels of NGALR were dramatically induced by treatment of IL-1?, whereas TGF-?1 or TNF-?did not have the same effect. Further investigation showed that the IL-1?-induced NGALR expression is mediated via the MAPK/ERK signaling pathway by using pharmacological inhibitors. Interestingly, the basal mRNA levels of NGAL detected in HMC, could be induced by IL-1?. However, NGAL protein could not be detected, even with IL-1?treatment. The ability of HMC to express NGAL protein was ascertained by exogenous administration of NGAL.Conclusions The data shows that NGALR is differentially expressed in human glomerular disease and is significantly up-regulated by Il-1?in HMC via MAPK/ERK activation. Furthermore, exogenous NGAL can be uptaken into HMC.Part II NGAL regulates CTGF, fibronectin,collagen type IV and cell cycle distribution in mesangial cells:possible roles in the progression of renal fibrosisPurpose To identify the effect of NGAL on secretion and cell cycle of HMC and the implicated mechanisms; to investigate the possible roles of NGAL in the progression of renal fibrosis.Methods and Results We studied the effect of NGAL on secretion and cell cycle of HMC and the implicated mechanisms by immunoblotting, real-time PCR,siRNA, immunofluorescence,CCK8 assay and flow cytometry. The results displayed that NGAL induced the mRNA and protein expression of fibronectin,collagen type IV and CTGF, when HMC exposured to NGAL (50 ng/ml) for the indicated times.The activation of Smad2/3 was up-regulated and the MAPK signal pathway had no effect. When NGALR was knocking-down by siRNA in HMC, the increased protein expression of collagen type IV and CTGF induced by NGAL was significantly attenuated. Meanwhile,the cell cycle distribution in HMC treated with NGAL was changesd:incubation of cells with NGAL caused a G0/G1 stagnation in cell cycle progression and no increased apoptosis. Cyclin proteins that cyclin D1, p21 and p27 were increased, whereas CDK2 was decreased in HMCs treated with 50 ng/ml NGAL, and CDK4 had no change, the Akt activation was attenuated by treating with NGAL.Conclusions NGAL induced the mRNA and protein expression of fibronectin,collagen type IV and CTGF by activated Smad2/3; NGAL caused a G0/G1 stagnation in cell cycle progression of HMC by increased cyclin D1, p21 and p27 and decreased CDK2.