Experimental Study Of Effect Of Rapamycin On Anti-GBM Glomerulonephritis In Rat

BackgroundChronic kidney diseases are emerging as a worldwide public health problem. The number of patients who have ESRD is ever increasing. Glomerulonephritis is one of the most common causes of established renal failure worldwide. Therefore, the development of novel therapeutic agents that interrupt the uncontrollable response to renal injury has been eagerly anticipated.Anti-glomerular basement membrane (GBM) disease or Goodpasture's disease, is a rare renal±pulmonary syndrome responsible for cases of acute renal failure presenting due to rapidly progressive glomerulonephritis with a poor prognosis.For investigating mechanisms of glomerulonephritis and to test approaches to specific immune intervention, animal model of Goodpasture's disease known as the nephrotoxic serum (NTS) nephritis, has been induced by immunization with administration of a heterologous antibody against glomerular basement membrane. Severe proliferative and necrotizing glomerulonephritis with crescent formation that leads to glomerulosclerosis in rats, resembling human anti-GBM glomerulonephritis. At present, it is thought that the pathogenesis of anti-GBM nephritis is affected by multiple factors. The development of anti-GBM disease is associated with both cell-mediated and humoral immunity. But the precise pathogenesis is unknown. Treatment of anti-GBM nephritis by immunosuppressants has emerged as a new challenge. Takeda et al. reported the beneficial effect of the immunosuppressant mycophenolate mofetil (MMF) in preventing the progression of anti-GBM nephritis.Rapamycin (RAPA) was derived from a macrocyclic lactone produced by Streptomyces hygroscopicus. Rapamycin binds to a specific cytosolic protein, FK binding protein-12, and this complex inhibits the activation of the mammalian target of rapamycin (mTOR), a kinase required for cell cycle progression. mTOR is an evolutionarily conserved serine/threonine kinase, acting as a rheostat to regulate the rate of cell growth and cell proliferation by controlling the mammalian translational machinery via regulating its downstream targets, the ribosomal protein S6 kinase (S6Ks) and the eukaryotic initiation factor 4E-binding proteins. So rapamycin acts by①inhibiting the phosphorylation of 4E-BP1 and preventing the release and transcription of eIF-4E;②inhibiting the activation of cdk2- cyclin E and the synthesis of DNA mediated by P27③inhibiting the activation of P70S6 kinase and the phosphorylation of ribosomal protein S6, and reducing the synthesis of ribosome/ transcription factor, and inhibiting the transition of T cells from G1 phase to S phase.Rapamycin is a Food and Drug Administration-approved immunosuppressive drug to inhibit organ transplant rejection. In addition to its immunosuppressive actions, rapamycin inhibits growth factor-mediated proliferation and survival of many nonimmune cells, including endothelial cells, renal tubular cells, and fibroblasts. Recently, it has been reported that serum-stimulated mesangial cell proliferation is inhibited by very low level of rapamycin. Rapamycin reduce expression of the proinflammatory and profibrotic genes and markedly attenuate both the inflammatory and fibrotic responses associated with renal disease. Rapamycin can ameliorate proteinuria-associated tubulointerstitial inflammation and fibrosis in experimental membranous nephropathy. Rangan et al. confirmed that rapamycin has renoprotective effects when administered during the early stages of an focal segmental glomerulosclerosis pattern of chronic renal injury. Others have reported that the same effect of rapamycin is beneficial in polycystic kidney disease, in which excessive tubular cell proliferation seems to play a pathogenic role. Rapamycin also inhibit compensatory renal hypertrophy after unilateral nephrectomy and significantly attenuate tubulointerstitial damage in a UUO-induced rat model of renal fibrosis. The renoprotective effects of rapamycin could be attributed to its anti-proliferative,anti-inflammatory and anti-fibrotic properties.On the basis of above theories, we postulate that rapamycin may be useful in preventing and treating anti-GBM nephritis. If the hypothesis was confirmed, we could find the valuable theory evidence of rapamycin treatment in preventing and treating anti-GBM nephritis. Part I Set up of anti-GBM glomerulonephritis model in ratObjectiveIn the study, we set up anti-GBM glomerulonephritis model by injection of rabbit anti-rat GBM nephrotoxic serum. Based on the animal model, we discuss the mechanism of anti-GBM glomerulonephritis.MethodsRabbit anti-rat GBM nephrotoxic serum was raised by repeated immunization of New Zealand White rabbits with particulate rat GBM. Accelerated anti-GBM nephritis was induced in male Sprague-Dawley rats. Rats were divided into control group (n=8) and model group (n=8). Rats were placed in metabolic cages for collection of 24-hour urine specimens on day 4, day 7 and on day 14. Blood samples were obtained at the end of experimental period on day 15. The levels of 24-urinary protein, serum urea nitrogen, serum creatinine, serum cholesterol and serum albumin were determined by standard methods using an autoanalyzer. Direct immunofluorescence microscopic studies to assess deposition of IgG in glomeruli were carried out on frozen sections. Sections were cut and stained with PAS,Masson and HE. Paraffin sections were used for immunohistochemistry for macrophages (monoclonal antibody ED1). The levels of transforming growth factor beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were determined by ELISA. Results1. The urinary protein increased significantly from the 4th to the 14th day in the model group, compared with the control group (P<0.01).2. The levels of serum urea nitrogen and serum cholesterol markedly increased in the model group, compared with the control group (P<0.01). The level of serum creatinine markedly increased in the model group, compared with the control group (P<0.05).The level of serum albumin markedly decreased in model group(P<0.01).3. The kidney weight in the model group was heavier than that in the control group (P<0.05).4. IgG deposited along with GBM were observed in the model group by immunoflurescence statining and were not observed in the control group.5. Crescent and intratubular protein cast formation were observed in the model group (P<0.01).6. Meanwhile, significant up-expression of ED-1+ on the glomeruli were observed in the model group (P<0.01).7. The levels of TGF-beta1, MCP-1, VEGF in the model group were higher than that in the control group (P<0.05).ConclusionsIn the model used in this study, Sprague-Dawley rats that were given a single injection of rabbit anti-rat GBM serum develop sustained anti-GBM antibody synthesis, linear deposition of IgG on the GBM, albuminuria, focal necrotizing glomerulonephritis with crescent formation. This model will be of value for investigating mechanisms of immunity in glomerulonephritis, and for attempting novel forms of immunotherapy prior to trials in man. Part II Experimental study of effect of Rapamycin on anti-GBM-induced glomerulonephritis in ratObjectiveIn the study, we set up anti-GBM glomerulonephritis model in rat. Based on the animal model, we want to study the therapeutic effects and mechanism of rapamycin on anti-GBM glomerulonephritis in rats.MethodsAnti-GBM glomerulonephritis was induced in male Sprague-Dawley rats. Thirty-four rats were divided into five groups, as follows:group M (n=6) model rats;group R7 (n=7)in which RAPA treatment by daily gavage(1 mg·kg^-1·d^-1) was started 7 days after the injection of serum; group R4 (n=7)in which RAPA treatment by daily gavage (1 mg·kg^-1·d^-1) was started 4 days after the injection of serum; group Rl(n=7) in which RAPA treatment by daily gavage(1 mg·kg^-1·d^-1) was started 1 day after the injection of serum; group C (n=7) control rats; Rats were placed in metabolic cages for collection of 24-hour urine specimens. Blood samples were obtained at the end of experimental period. Levels of 24h-urinary protein, serum urea nitrogen, serum creatinine, serum cholesterol and serum albumin were determined by standard methods using an autoanalyzer. Direct immunofluorescence microscopic studies to assess deposition of IgG in glomeruli were carried out on frozen sections. Sections were cut and stained with PAS. Paraffin sections were used for immunohistochemistry for ED-1+, proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (alpha-SMA). The total RNA was extracted from the renal tissues. The expression of PCNA, MCP-1 and TGF-beta1 mRNA were detected by real-time PCR method. The total protein was extracted from the renal tissues. The protein contents were detected by Western blot.Results1. The urinary protein increased significantly in the model group, compared with the control group(P<0.01).Compared to the model group, the levels of 24h-urinary protein in the R1 and R4 group were lower (P<0.05).2. The serum creatinine increased significantly in the model group, compared with the control group (P<0.05). Compared to the model group, the level of serum creatinine in the R1 group was lower (P<0.05).3. RAPA treatment reduced glomerular crescent formation in a model of anti-GBM nephritis in the R4 group and R1 group (P<0.01). There are no differences of intratubular protein cast formation in the R7 group, R4 group, R1 group and model group (P>0.05).4. Immunohistochemical method showed that the expression of ED-1 in the model group was higher than that in the control group (P<0.01). Compared to the model group, the expression of ED-1 in the R1 and R4 group were lower (P <0.05).5. Immunohistochemical method showed that the expression of PCNA in the model group was higher than that in the control group (P<0.01). Compared to the model group, the expression of PCNA in the R4 group and R1 group were lower (P<0.05).6. Immunohistochemical method showed that the expression of alpha-SMA in the model group was higher than that in the control group (P<0.01). Compared to the model group, the expression of alpha-SMA of R1 group was lower (P<0.05). 7. Real time-PCR method showed that the expression of MCP-1 mRNA in the model group was higher than that in the control group (P<0.01). Compared to the model group, the expression of MCP-1 mRNA in the R4 group and R1 group were lower (P<0.05).8. Real time-PCR method showed that expression of PCNA mRNA in the model group was higher than that in the control group (P<0.05).9. Real time-PCR method showed that the expression of TGF-beta1 mRNA in the model group was higher than that in the control group (P<0.01). Compared to the model group, the expression of TGF-beta1 mRNA in the R1 group was lower (P<0.05).10. Western blot method showed that the expression of PCNA protein in the model group was higher than that in the control group (P<0.01). Compared to the model group, the expression of PCNA protein in the R1 and R4 group were lower (P<0.05).Conclusions1. RAPA inhibited development of proteinuria and glomerular crescent formation in anti-GBM nephritis.2. RAPA suppressed renal inflammation as evidenced by decreased monocyte/ macrophage infiltration and downregulated expression of the MCP-1 mRNA and TGF-beta1 mRNA. Rapamycin blocked PCNA protein expression and inhibited the expression of alpha-SMA. Moreover, rapamycin directly inhibited cell proliferation.