Regulating Effect Of Calcineurin Inhibitor On Diabetic Kidney Inflammation And Its Potential Mechanism

BackgroundDiabetic nephropathy is a serious microvascular complication of diabetes mellitus andthe major cause of chronic renal failure. DN is the first cause of ESRD （30%⁴0%） inEurope and the United States and other developed countries, second in China, and thereis a potential. The pathogenesis of DN is very complicated and has not been fullyelucidated at present. The theory of inflammation is paid more attention in recent yearsand inflammatory mechanism mainly mediated by macrophages is considered the keyfactor of sustainable development. DN in renal tissue inflammation formation is closelyrelated to the glomerular filtration barrier permeability changes and proteinuria. Theouter layer of podocytes in the filtration barrier, the injury is involved in the formationof protein in the urine DN early pathological process. The typical feature of DN is theaccumulation of matrix protein，which ultimately lead to glomerular sclerosis andinterstitial fibrosis. In the past few years, glomerular disease was focused on. But now，renal tubular lesions and renal tubulointerstitium fibrosis which playing a important roleof DN is gradually known by people. The pathological change of tubulointerstitium isthe main basis of pathology progress in DN. At the same time, it is one of the mostimportant influential factors in the prognosis of DN. Renal tubular epithelialmyofibroblast transdifferentiation （TEMT） maybe the key link in the tubulointerstitiumfibrosis of DN. Non-activated macrophages have no injury effect, only activatingmacrophages can be cells which have active biological function. Toll-like receptors isone of the key factor involved in the immune inflammation reaction, and thought to bethe principal part which mediated immune and inflammatory reaction. Tacrolimus （FK506） is a new type of immunosuppressant, which is mainly used for organ transplant,rheumatism immunological diseases and refractory nephrosis. There are reported thatFK506play suppress the immune, anti-inflammatory activity may be related to theinhibition of calcineurin （CaN）, osteopontin （OPN）, NF-kappaB and a variety ofinflammatory cytokine expression.Objective（1） The purpose of the study was to investigate inhibiting effect of the inhibitor FK506for calcineurin on renal hypertrophy and its mechanism in early diabetic rats.（2） Thestudy aimed to investigate the protective effete of FK506on the podoeytes in diabeticrat model，and evaluates its machanism.（3） The study aimed to investigate theprotective effect of FK506on renal tubule-interstitiurn in diabetic rat mode，andevaluate its machanism.（4） The study aimed to investigate the protective effect ofFK506on macrophage accumulation，proliferation and activition in the kidney of earlydiabetic rats．MethodsForty adult male Sprague-Dawley rats were separated into four groups at random.Control group （n=10）, model group （n=10）, model group treated with tacrolimus0.5mg·kg^-1d^-1（n=10）, model group treated with tacrolimus^-1.0mg·kg^-1d^-1（n=10）. Diabeteswas induced with streptozotocin (65mg·kg^-1) in rats, and tacrolimus (0.5or^-1.0mg·kg^-1d^-1) was orally administered once a day for4weeks. After4weeks, thefollowing determinations were done in samples: plasma glucose （BG）、liver function、kidney function and plasma lipid were determined according to standard methods.24hours urinary albumin excretion rate （AER） was determined by Enzyme Immunoassay（EIA）. Relative kidney weight （RKW） and Creatinine clearance rate （Ccr） were measured. Kidney pathologic injury was observed by light microscopy and an electronmicroscope．Immunofluorescence to detect the renal tissue of Nephrin and Podocinexpression situation. The expression of Ostepontin（OPN）、NF-κB-p65、transforminggrowth factor β1（TGFβ1）、E-cadherin and alpha-smooth muscle actin （α-SMA）、Vimentin and ED-1positive cells,NF-κB-p-p65positive cells, were detected byimmunohistochemistry. TLR2and ED-1double positive cells, PCNA and ED-1doublepositive cells, TLR4and ED-1double positive cells, iNOS and ED-1double positivepositive macrophages were measured with double immunohistochemistryimmunostaining in the kidney. The expression of calcineurin （CaN）,1α type Ⅳcollagen, alpha-smooth muscle actin （alpha-SMA）, Nephrin, Podocin, NF-κB p65andNF-κB p-p65were detected by Western blot.Results1. Clinical and metabolic parameters: There was a significant increase in fasting BGand plasma lipid （P<0.01）,but body weight （BW） was significantly decrease （P<0.01）in model group and FK506group compared with control group.The BG、plasma lipidand liver function of FK506group were not differnt with model group. Increased RKWwas significantly reduced by FK506treatment with1.0mg/kg （P<0.05）, elevated AERwas markedly attenuated by FK506treatment with0.5and1.0mg/kg （P<0.05,0.01）.Ccr was not changed by FK506treatment with0.5or1.0mg/kg.2. Renal pathologicmorphology: Comparaed with control group, indices for tubulointerstitial injury （TII）and glomerular volume （VG） were significantly increased in model group （P<0.05,0.01）. Elevated glomerular volume was significantly attenuated by FK506treatmentwith0.5and1.0mg/kg （P<0.05）, and increased indices for tubulointerstitial injury wereonly ameliorated by FK506treatment with1.0mg/kg （P<0.01）. Under the electronmicroscope, group DM showed a significantly widened glomerular basement membrane, disordered, wide and fused podocytic-process. The above lesions in groups FK506treatment with0.5or1.0mg/kg were less compared with group DM（P＜0.05）.3. The protective effect of the FK506on the podocytes in kidney of diabetic rats:Western blot analysis noted that the expression of CaN protein was increased2.4fold inthe kidney in DM group, FK506treatment with0.5and1.0mg/kg could reduceexpression of CaN protein by38.0%and73.2%. There was a finely dotted linearepithelial staining of Nephrin and Podocin in control group glomeruli. In contrast, thestaining of glomeruli from untreated diabetic rats was attenuated, more dispersed andclustered, this diabetic-induced loss of glomerular Nephrin and Podocin expression waslargly prevented in FK506-treated diabetic rats. Western blot analysis showed that theexpression of Nephrin and Podocin protein was reduced in the kidney of diabetic rats,and FK506treatment significantly increased the expression of Nephrin and Podocinprotein （P<0.01）.4. The protective effect of the FK506on therenaltubule-interstitium in kidney of diabetic rats: TGFβ1immunostaining wasfound in grestest aboundance in the glomerular and tubulointerstitial of model groupcomparaed with control group（P<0.01），TGFβ1expression in FK506treatment with0.5and1.0mg/kg were significantly lower than that in model group（P<0.05,0.01）.Compared with control group, E-cadherin in renal tubular epithelial cells wasmarkedly decreased （P<0.01）, conversely, α-SMA and Vimentin significantly increasedin diabetic rats （P<0.01）.FK506treatment with0.5and1.0mg/kg could reduce theincreased expression of α-SMA and Vimentin （P<0.01）, at the same time, increased theexpression of E-cadherin （P<0.01）. Western blot analysis noted that the expression ofα-SMA protein was increased3.5fold in the kidney in model group, FK506treatmentwith0.5and1.0mg/kg could reduced increased expression of α-SMA protein by40.7%and69.1%.The expression of1α type Ⅳcollagen in kidney were significantly increasedin model group and reduced by FK506treatment （P<0.05,0.01）. 5. The effect of the FK506on macrophage accumulation, proliferation andactivition in the kidney of diabetic rats: There were marked accumulation of ED-1+cells in diabetic kidney, which were not inhibited by treatment with FK506treatment（P<0.01）. ED-1and TLR2double positive cells,ED-1and TLR4double positive cellsED-1+PCNA+cells, ED-1+iNOS+cells were significantly increased in kidneys frommodel group（P<0.01）, while they were significantly inhibited by FK506treatment（P<0.01）. Immunohistochemistry showed that elevated NF-κB p-p65expression indiabetic rates was significantly attenuated by FK506treatment with0.5and1.0mg/kg d（P<0.01）. Western blot analysis demonstrated that the expression of NF-κB p65andNF-κB p-p65in the kidney of diabetic rats were increased by5.35and7.57foldsrespectively as compared to control. FK506treatment with0.5and1.0mg/kg d couldreduce the increased expression of NF-κB p65by26.32%and47.37%, and couldreduce the increased expression of NF-κB p-p65by56.67%and70.00%.ConclusionFK506could ameliorate renal structure and function injury in early experimentaldiabetic rats, which mechanism may be partly correlated with suppression of NF-κBp-p65、OPN、CaN、α-SMA、Vimentin、TGFβ1and1α（IV） collagen in renal tissue.FK506can up-regulate the expression of E-cadherin, Nephrin and Podocin. IncreasedTLR2, TLR4, PCNA and iNOS expression on macrophage in the kidney of earlydiabetic rats may be correlated with inflammatory response caused by macrophageactivation. FK506may directly or indirectly downregulate TLR2, TLR4, PCNA andiNOS expression on macrophage and inhibit inflammatory response related toTLR-NF-κB signal transduction in the kidney.