Li Chien Chang

Urokinase plasminogen activator receptor and its soluble form in common biopsy-proven kidney diseases and in staging of diabetic nephropathy

OBJECTIVES Soluble urokinase plasminogen activator receptor (suPAR), derived from membrane bound uPAR, is associated with inflammatory diseases. In the present study, we explored the expression of uPAR/suPAR in common biopsy-proven kidney diseases and the relationship between suPAR and staging of type 2 diabetic nephropathy (DN). DESIGN AND METHODS Serum samples for suPAR and renal tissues for uPAR staining were investigated in various common kidney diseases. The levels of serum suPAR were measured and adequate cut-off values of different stage of DN were calculated by receiver operating characteristic (ROC) curve. RESULTS In our results, the expression of uPAR on renal tissues was pronounced in the majority of kidney diseases. Comparing of expression of uPAR among different kidney diseases, it was strongest in minimal change disease (MCD) and weakest in chronic interstitial nephritis. Serum suPAR in most kidney diseases, except of MCD, was significantly elevated and was highest in DN. As for DN and suPAR, we found that suPAR progressively increased with staging of DN. Moreover, suPAR was linearly and negatively related to estimated glomerular filtration rate and positively related to the amount of proteinuria. By ROC curve, the cut-off values of suPAR in DN for assessing development increased with the progression of the disease. CONCLUSIONS We concluded that uPAR/suPAR is elevated in most kidney diseases and that suPAR is a useful biomarker for assessing stages of DN.

Involvement of F-Actin in Chaperonin-Containing t-Complex 1 Beta Regulating Mouse Mesangial Cell Functions in a Glucose-Induction Cell Model

The aim of this study is to investigate the role of chaperonin-containing t-complex polypeptide 1 beta (CCT2) in the regulation of mouse mesangial cell (mMC) contraction, proliferation, and migration with filamentous/globular-(F/G-) actin ratio under high glucose induction. A low CCT2 mMC model induced by treatment of small interference RNA was established. Groups with and without low CCT2 induction examined in normal and high (H) glucose conditions revealed the following major results: (1) low CCT2 or H glucose showed the ability to attenuate F/G-actin ratio; (2) groups with low F/G-actin ratio all showed less cell contraction; (3) suppression of CCT2 may reduce the proliferation and migration which were originally induced by H glucose. In conclusion, CCT2 can be used as a specific regulator for mMC contraction, proliferation, and migration affected by glucose, which mechanism may involve the alteration of F-actin, particularly for cell contraction.

Effects of fungal statins on high-glucose-induced mouse mesangial cell hypocontractility may involve filamentous actin, t-complex polypeptide 1 subunit beta, and glucose regulated protein 78.

Glomerular hyperfiltration is associated with mesangial cell hypocontractility. How 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) influence mesangial cell contraction is unclear. We investigated the effect of statins on mesangial cell hypocontractility and identified candidate proteins and filamentous/globular (F/G)-actin involved in this process. A high-glucose-induced mouse mesangial cell hypocontractility model was treated with fungal statins, simvastatin (Sim), lovastatin (Lov), and pravastatin (Pra). The optimum statin dose was determined by an 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and then applied to a cell model. A 2-dimensional gel/matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis was used to evaluate protein expression cells incubated in the presence of a normal level of glucose (N), a high level of glucose (H), and a high level of glucose plus Sim (H + S). Candidate proteins were analyzed. Finally, the ratio of F/G actin in groups N, H, and H+S was evaluated. The MTT assay showed that Sim and Lov exerted dose- and time-related inhibition of proliferation of mesangial cells at N, but Pra had no effect. The optimum doses selected for Sim was 1 microM and for Lov was 3 microM, which were 1 increment before significant proliferation inhibition. Both doses reversed cell hypocontractility significantly, but Sim was chosen for further proteomic and F/G actin analyses. Proteomic analysis of groups N, H, and H + S showed that 18 proteins were involved in hypocontractility. These proteins were grouped and analyzed based on their known functions. Two selected proteins, TCP-1beta and GRP78, that were upregulated and downregulated, respectively, were confirmed by Western blot and immunohistochemistry. In regard to the F/G actin, group H had a significantly lower ratio than that of group N, and group H + S returned to a level similar to that of group N. In conclusion, Sim and Lov both seem to reverse mesangial cell hypocontractility. The process of Sim reversal of mesangial cell hypocontractility may involve F-actin, TCP-1beta, and GRP78.

Phosphotriesterase-related protein sensed albuminuria and conferred renal tubular cell activation in membranous nephropathy

BackgroundMembranous nephropathy (MN) is a common cause of nephrotic syndrome that may progress to end-stage renal disease (ESRD). The formation of MN involves the in situ formation of subepithelial immune deposits and leads to albuminuria; however, the underlying mechanism of how MN leads to ESRD remains unclear. The aim of this study was to investigate the expression and biological functions of phosphotriesterase-related protein (PTER) in MN.ResultsIn the progression of MN, the expression of PTER increased significantly and was mainly expressed in the renal tubular cells. Both mRNA and protein expression levels of PTER were increased in a concentration- and time-dependent manner in the in vitro albuminuria tubular cell model. Silencing the expression of PTER by RNA interference diminished albuminuria-induced inflammatory and pro-fibrotic cytokines production.ConclusionsOur findings reveal that PTER may sense albuminuria in the progression of MN, induce tubular cell activation and lead to ESRD.

Association among Fibrinolytic Proteins, Metabolic Syndrome Components, Insulin Secretion, and Resistance in Schoolchildren

We investigated the role of urokinase plasminogen activator (uPA) and its soluble receptors (suPAR) and plasminogen activator inhibitor-1 (PAI-1) in metabolic syndrome (MetS) components, insulin secretion, and resistance in schoolchildren. We enrolled 387 children, aged 10.3 ± 1.5 years, in Taipei. Anthropometry, fibrinolytic proteins, MetS components, insulin secretion, and resistance were measured. Subjects were divided into normal, overweight, and obese groups. Finally, the relationship between fibrinolytic proteins and metabolic syndrome in boys and girls was analyzed. In boys, PAI-1 was positively associated with body mass index (BMI) percentile, hypertriglyceride, insulin secretion, and resistance. In girls, PAI-1 was positively associated with obesity, hypertriglyceridemia, and insulin secretion. In girls, uPA was positively associated with insulin secretion. suPAR was positively associated with high-sensitivity C-reactive protein in both boys and girls, and with BMI percentile and body fat in girls. The obese boys had higher suPAR and PAI-1 levels than the normal group. The obese girls had higher uPA, suPAR, and PAI-1 than the normal group. Boys and girls with MetS had higher PAI-1. Fibrinolytic proteins, especially PAI-1, are associated with MetS components and insulin secretion in children. Fibrinolytic proteins changes were more likely to occur in girls than in boys.

Significance of the urokinase-type plasminogen activator and its receptor in the progression of focal segmental glomerulosclerosis in clinical and mouse models

BackgroundsuPAR biomarker generally considered a pathogenic factor in FSGS. However, studies have been published that dispute this conclusion. The current study was designed to investigate the roles of uPA and suPAR in FSGS in clinical and mouse models.MethodsClinical subjects including those with biopsy-proven FSGS and MCD were enrolled. To verify the role of uPA in FSGS, Adriamycin was used to induce FSGS in uPA knockout (uPA−/−) and BALB/c (WT) mice. Proteinuria and suPAR, the cleaved/intact forms of the circulating suPAR, and possible proteases involving cleavage of the suPAR were also studied.ResultsFSGS clinical cases presented significantly higher serum levels of suPAR and Cr and lower serum levels of uPA. In the mice model, the uPA−/− group exhibited faster disease progression and worsening proteinuria than the WT group. In addition, the uPA−/− group had higher plasma suPAR levels, glomerular cell apoptosis, and dysregulation of the Th1/Th2 balance. In an analysis of suPAR variants in FSGS, both the intact and cleaved forms of the suPAR were higher in clinical subjects and the mouse model. However, the process of suPAR cleavage was not mediated by enzymatic activities of the uPA, elastase, or cathepsin G.ConclusionsA deficiency of uPA accelerated the progression of Adriamycin-induced mouse FSGS model. Decrease of serum uPA levels may be an indicator of the progression of FSGS in clinical subjects and animal models.

Chaperonin-Containing t-Complex Protein-1 Subunit β as a Possible Biomarker for the Phase of Glomerular Hyperfiltration of Diabetic Nephropathy

In cell model, we discovered the association between chaperonin-containing t-complex polypeptide 1 subunit β (TCP-1β) and early diabetic nephropathy (DN). In this study, we further explored the relationships between TCP-1β and type 2 diabetic mellitus (DM). To mimic the clinical hyperfiltration state, a type 2 DM mice model was established by feeding a high-fat diet in combination with treatment of streptozotocin and nicotinamide. Blood and urine were collected to determine creatinine clearance (C cr), and kidney tissues were harvested for evaluation of TCP-1β expression by immunohistochemistry and Western blot. Meanwhile, clinical subjects of healthy controls and type 2 DM were recruited to strengthen the evidence with urine TCP-1β. Results showed that C cr and the expression of TCP-1β in kidney were significantly higher one week after hyperglycemia development, suggesting that the hyperfiltration state was successfully established in the mice model. TCP-1β was expressed predominantly on renal tubules. By using the estimated glomerular filtration rate to index progression in clinical investigation, urine TCP-1β level was associated with the hyperfiltration phase in type 2 DM patients. Conclusively, we confirmed that TCP-1β is a possible biomarker for early nephropathy of type 2 DM, but further mechanistic study to elucidate its cause and pathway is needed.

Attributes of antiangiogenic factor plasminogen kringle 5 in glomerulonephritis

CONTEXT Plasminogen kringle domain (K) 5 is known to inhibit endothelial cell growth, but limited data are available investigating the relationship between K5 and glomerulonephritis (GN). OBJECTIVE To understand the relationships among K5, GN, and glomerular endothelial cells in GN mice models and human subjects. DESIGN Two mice models of GN and 2 categories of human GN biopsy samples were collected to gain insight into the disease mechanism from the laboratory to bedside. In the mechanistic animal study, membranous nephropathy (MN) and focal segmental glomerulosclerosis mice models were used. Kringle domain 5 in the diseased kidney was located by immunofluorescence and quantified by Western blotting. In the kinetic animal study, different MN time points were stained with K5, immunoglobulin G, and C3 by immunofluorescence. CD31 and proliferating cell nuclear antigen were evaluated by immunohistochemical double staining for alterations in the glomerular endothelial cells. Biopsy samples from patients diagnosed with antibody (Ab)-mediated and non-Ab-mediated GN were collected for K5 analysis. RESULTS The expression level of K5 was found to be significant in MN, but not in focal segmental glomerulosclerosis, and was markedly elevated in the diseased glomeruli along the capillary walls. Kringle domain 5 levels increased steadily with the evolution of MN, appearing after the deposition of Abs. In altered glomerular endothelial cells, CD31 decreased with the evolution of MN. In human subjects, K5 occurred only in patients with Ab GN. CONCLUSIONS Kringle domain 5 might be involved in the progression of Ab-mediated GN and associated with the alteration of MN glomerular endothelial cell growth.

Atorvastatin from target screening attenuates endothelial cell tube formation and migration by regulating urokinase receptor-related signaling pathway and F/G actin

Background Angiogenesis and cytoskeletal transformation are common denominators of many pathological developments. The relationship between angiogenesis, urokinase plasminogen activator receptor (uPAR) signaling pathway, and cytoskeletal transformation is still unknown. In this study, a pGL3‐uPAR promoter reporter system combined with Bio‐Plex mRNA analysis was established for discovering uPAR modulators to analyze this interconnection. Methods After screening a set of clinically used drugs, atorvastatin (Ator) was found to significantly affect uPAR expression and its ideal dose, 1 &mgr;M, was determined for cell study. Mouse endothelial cell (mEC) models, including tube formation for angiogenesis and wound healing assay for migration, were employed to test the effects on angiogenesis and cytoskeletal transformation with (Group Ator) and without (Group C) the treatment of Ator. Results The mEC tube formation and migration was significantly decreased in Group Ator. Regarding cytoskeleton changes, the ratio of F/G actin by Western blotting and the assembly of F‐actin (lamellipodia) by immunofluorescence were attenuated. Furthermore, uPAR and all uPAR‐related factors, including integrin &agr;5&bgr;3, phosphorylated‐focal adhesion kinase, and Rac, revealed a significant reduction when compared with Group C. Conclusion We conclude that close regulatory machinery spans angiogenesis, uPAR signaling, and cytoskeletal transformation, and that uPAR modulator Ator can decrease the reorganization of actin cytoskeleton, which may lead to a new approach in angiogenesis.