The Study Of The Therapeutic Effect Of PLNPK On Rat Anti-GBM Glomerulonephritis And Its Therapeutic Mechanisms

Objective:To observe the therapeutic effect of pentapeptide compound PLNPK on anti-glomerular basement memberane (GBM) glomerularnephritis in rats, and investigate its possible therapeutic mechanism.Methods:1. The GBM antigen was extracted from the renal cortex of Wistar rats. The rabbits were immunized by rat GBM antigen to prepare the rabbit anti rat GBM serum. Glomerulonephritis was induced in male Wistar rats by preimmunization via an i.p. injection of rabbit IgG and followed by an intravenous injection of anti-GBM serum.2. The rats with nephritis were divided into 3 treatment groups, which received i.p. injections of PLNPK (400μg/kg/d), PLNPK (200μg/kg/d) and normal saline (N.S.) respectively, another group of healthy rats served as a healthy control group. The therapeutic effect of PLNPK was evaluated by urinary protein detection, serumal biochemical indexes detection, renal index, renal histology, immunofluorescence, and ultrastructure examination by transmission electron microscopy.3. The macrophages (Mφ) infiltrated into glomeruli were detected by CD68 immunohistological stain 7 days after the nephritis had been induced, and a quantitive analysis was performed to invest whether or not PLNPK could inhibite Mcp infiltration into glomeruli.4. Total RNA and protein were extracted from renal cortex, monocyte chemoattractant protein (MCP)-1 mRNA and protein expression were detected by Real-Time PCR and Western Blot in order to observe whether PLNPK has any influence on MCP-1 expression in kidney.5. Rat glomerular mesangial cells (MC) and rat glomerular mesangial cell strain, RMC, were cultured, and after stimulated by recombined rat IL-1β, Real-Time PCR and Western Blot were performed to detecte whether PLNPK could down regulate the expression of MCP-1 in above cells in vitro.6. The effect of PLNPK on p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation in MC and RMC was detected by Western Blot.Results:1. The 24hrs urinary protein of glomerular nephritis (GN) control group was much higher than that of healthy control from the second day after heterologous antibody had been injected (P<0.01), crescents could be observed in glomeruli, and heterologous antibody and auto-antibody deposited along GBM, which indicated that rat anti-GBM nephritis had been successfully induced.2. After treated with PLNPK (200,400μg/kg/d) for 2 weeks, the urinary protein level of rats with nephritis was markedly reduced.6 weeks after the nephritis was induced, PLNPK could raise the level of serumal total protein (TP) and albumin (ALB), reduce serumal total cholesterol (TC) and serumal urea nitrogen (BUN), but the effect on serumal triglyceride (TG) and creatinine (Cr) was not significant. PLNPK could degrade the kidney index of the GN rats, reduce the formation of cerescents in glomeruli, lessened the deposition of auto-antibodies on the GBM. Ultrastructural analysis showed that PLNPK could ameliorate the injury of GBM, reduce the level of GBM thickening and foot processes fusion.3. Considerable Mφinfiltrated into glomeruli one week after anti-GBM serum injection in GN control group, but no Mφwere seen in glomeruli of healthy rats. PLNPK (200 and 400μg/kg/d) could reduce the number of Mφin glomeruli (P<0.01 vs nephritis-induced group).4. The expression of MCP-1 mRNA and protein was up regulated significantely in GN control group one week after nephritis was induced, P<0.01 vs healthy control. After treated with PLNPK (200,400μg/kg/d), both mRNA and protein expression of MCP-1 was reduced, P<0.01 vs GN control group.5. The MCP-1 mRNA and protein expression was enhanced after stimulated by recombined rat IL-1βfor 5 and 12 hours respectively in MC and RMC, PLNPK (1.6,3.2,6.4mg/ml) reduced the IL-1βstimulated MCP-1 mRNA and protein expression in above cells, P<0.01 compared with IL-1βstimulated control. 6. The phosphorylation of p38 MAPK was inhanced after stimulated by rat IL-1βfor 30 minutes in MC and RMC, PLNPK (1.6,3.2,6.4mg/ml) pretreatment could reduce the phosphorylation level of p38 MAPK in those cells, P<0.01 vs IL-1βstimulated control.Conclusion:PLNPK can ameliorate rat anti-GBM glomerular nephritis, the possible mechanism (at least partly) is though inhibiting the phosphorylation of p38 MAPK, blocking the activation of MC, to reduce the expression of MCP-1 in MC, and decrease the Mφinfiltration into glomeruli, then reduced the injury of kidney.