The Suppressive Effect Of Rapamycin On Renal Inflammation In Diabetic Rats

ObjectiveDiabetic nephropathy is one of the major chronic complications of diabetes, and it is also a major etiology of end stage renal disease. The precise pathogenesis of diabetic nephropathy is unknown. At present, it is thought that the pathogenesis of diabetic nephropathy is affected by multiple factors, including metabolic disorders, hemodynamic abnormalities, oxidative stress, activation of polyol pathway and genetic predisposition. Recently the role of inflammation in the pathogenesis of diabetic nephropathy has been paid more and more attention to.The kidney resident cells can produce inflammatory cytokines ,such as TNF-α, IL-1, IL-6, NO in pathologic conditions, and these cytokines amplify the inflammation by autocrine and paracrine and lead to an inflammatory cascade. Recently it is proposed that the diabetic nephropathy be considered as an inflammatory disease that link metabolism and immunology.Macrophage infiltration in glomeruli mediated by MCP-1 is the early event in diabetic nephropathy. It has been proved that high glucose concentration and mesangial expansion can stimulate the expression of MCP-1 mRNA and protein in human mesangial cells, which is mediated by IkappaB-NF-kappaB pathway. It is showed that the excretion of MCP-1 in urine increase with the extent of the renal injury. Osteopontin (OPN) is an extracellular matrix protein secreted by multiple cells, such as osteoblasts, lymphocytes,macrophages, epithelial cells and vascular smooth muscle cells. As a growth factor , OPN can stimulate the proliferation and the migration of vascular smooth muscle cells and the glomerular mesangial cells;OPN can also recruit the macrophage as a macrophage chemokine. IL-1 > TNF-a and PDGF can activate PKC ,which can activate the transcriptionof OPN and recruit the macrophages.At present, though it is not practical to treat the diabetes with immunosuppressive drugs in clinical setting , the results of animal experiments are promising. MMF (Mycophenolate mofetil) is a common immunosuppressive drug after renal transplantation. It is shown, in vitro, MMF can suppress the proliferation of glomerular mesangial cells;MMF can also ameliorate the pathological changes and reduce the excretion of proteinuria in mesangial proliferative glomerulonephritis in animal model . MMF can suppress the expression of the adhesion molecule and reduce the macrophage infiltration. Brazilian Utimura R reported in 1999: MMF treatment largely prevented the macrophage infiltration and reduced the glomerulosclerosis in STZ-induced diabetic rats. This protection could not be attributed to a hemodynamic or metabolic effect, and likely resulted from its anti-proliferative and anti-inflammatory properties. Mizoribine is a common anti-rejection drug after renal transplantation in Japan. Kikuchi Y reported in 2005:Administration of Mizoribine for 8 weeks inhibited renal macrophage accumulation, the expression of MCP-1,OPN and TGF-p\ prevented the progression of glomerulosclerosis and interstitial fibrosis in non-insulin-dependent diabetic kidneys.Rapamycin was extracted from the culture fluid of Streptomyces hygroscopicus by Ayerst Institute in the early 1970s. Rapamycin binds to a member of the FK binding protein (FKBP) family, FKBP12. The complex binds to the protein kinase, that is , mammalian target of rapamycin (mTOR). Rapamycin acts by ?inhibiting the phosphorylation of 4E2BP1 and preventing the release and transcription of eIF24E;?inhibiting the activation of cdk2- cyclin E and the synthesis of DNA mediated by P27(Dinhibiting 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 Gl phase to S phase. Rapamycin is known to inhibit responses induced by a number of cytokines IL-2, IL-4, IL-7,and IL-15 and exert its anti-inflammatory property via inhibition of the activation of NF-kappaB, which canreduce the expression of some cytokines and chemokines and inhibit the proliferation of lymphocytes and monocytes. At present, Rapamycin is mainly used to prevent and treat the acute or chronic rejection after renal transplantation, but there are few literatures about the effect of rapamycin on the renal inflammation in diabetic rats.The aim of present study was to investigate the effect of rapamycin on renal inflammation in diabetic rats, observe the biochemical changes in blood, urine and renal pathological changes after administration of rapamycin by gavage for 8 weeks in streptozocin-induced diabetic rats. We researched on the effect of rapamycin on the blood glucose, blood pressure, proteinuria, renal function ,the expression of MCP-1,OPN and the macrophage infiltration in the kidney.Methods32 male SD rats were randomly divided into normal control group (NC) and diabetic mellitus group(DM). Diabetes was induced by a single intraperitoneal injection of streptozocin (60mg/kg). The diabetic rats were redivided into diabetic control group (DC)and rapamycin group (DM+RAPA). Rapamycin group were administrated by gavage(6mg/kg, TIW) for 8 weeks. 8 weeks later, 24h urine was collected for urine creatinine and urine albumin. Blood was collected for blood glucose, total protein, albumin, serum creatinine, high density lipoprotein, low density lipoprotein, cholesterol ,et al. Then the urine albumin excretion rate and the creatinine clearance rate were calculated according to related formula. The morphological changes were observed in HE-stained, PAS-stained sections by light microscope and the changes of glomerular basement membrane and visceral epithelial foot process were observed by electron microscope. Serial sections were used to detect MCP-1,OPN, EDI-positive cells by immunohistochemistry. The results of immunohistochemistry were analysed by OPTIMAS image analysis system and the results were semi-quantified by positive index: positive area*mean gray value /total area.ResultsAfter induction of diabetes, the rats showed obvious polydispia, diuresis, and polyphagia. Compared to the normal control group, the body weights of diabetic rats were smaller (PO.05) , the urine volumes were more (P<0.05) , the level of urine albumin excretion rate was higher (PO.05) . The mesangial expansion, mesangial matrixproliferation and vacuolus degeneration of some tubular epithelial cells were observed in diabetes rats by light microscope. Basement membrane diffuse thickening and the extensive effacement of visceral epithelial foot process were observed in diabetes rats by electron microscope. Compared to NC rats, DM+RAPA rats had less body weights (P<0.05 ) , more urine volumes (P<0.05 ) , higher level of urine albumin excretion rate (PO.05), but there were no statistical differences compared with DC rats. The mesangial expansion, mesangial matrix proliferation and vacuolus degeneration in some tubular epithelial cells were observed in DM+RAPA rats by light microscope. Segmental basement membrane thickening and effacement of visceral epithelial foot process were observed in DM+RAPA rats by electron microscope, and we also observed the increase of lysosome and vacuolus degeneration of mitochondria in some renal tubular epithelial cells.We observed the strong expression of MCP-1 in glomerular capillary tuft area and mesangial area, while there was no or rare expression of MCP-1 in normal rats. By immunohistochemistry, rapamycin suppressed the expression of MCP-1 in glomerular capillary tuft area and mesangial area (PO.05) in diabetic rats. We observed the strong expression of OPN in renal cortex tubules, tuft of Helen and conducting tubules in diabetic rats, while there was no or rare expression of OPN in normal rats (PO.05) . Rapamycin suppressed the expression of OPN in renal cortex tubules ,tuft of Helen and conducting tubules in diabetic rats (PO.05) in diabetic rats. There was no or rare macrophage infiltration in normal rats glomeruli or tubules, while ED-1-positive cells significantly increased in glomeruli and tubules in diabetic rats.Rapamycin significantly reduced the number of ED-1-positive cells infiltrated in glomeruli (PO.05) , but didn't affect the number of ED-1-positive cells infiltrated in tubules (P>0.05) .We also observed that the creatinine clearance rate is higher (PO.01) in diabetic rats than normal control rats. Rapamycin intervention significantly reduced creatinine clearance rate of diabetic rats (PO.01) . The glomerular volumes are larger in diabetic rats than in normal rats (PO.05) ,while they are smaller in DM+RAPA rats than in diabetic rats (PO.05) . Conclusion1 Administration of rapamycin can suppress the expression of MCP-1 protein in glomeruli and OPN protein in renal tubules in STZ-induced diabetic rats.2 Administration of rapamycin can reduce the number of the macrophage infiltrated in glomeruli in STZ-induced diabetic rats.3 Administration of rapamycin can ameliorate the renal hyperfiltration , reduce the volume of glomeruli and inhibit the hypertrophy in diabetic rats.