The Mechanisms Of Calcitriol Inhabiting The Renal Interstitial Fibrosis Of UUO Rats And Endothelial Cells Involved In The Regulation Of Fibrosis

Objective:By observing calcitriol inhibition of interstitial fibrosis inUUO rats and endothelial cells of TGF-β1,ICAM-1,HIF-1α,VEGF,VEGFR-2gene and protein levels expressed affect. Explore the mechanism of theinhibitory effect of calcitriol on renal interstitial fibrosis from endothelial cellpoint.Methods:1In vivo experimental observations calcitriol inhibition of interstitial fibrosisin UUO rats.1.1One hundred and eighty six weeks old male Sprague-Dawley rats wererandomLy assigned to five groups, each group had36animals:①UUOoperation group,②s ham operated group③U UO+high dosecalcitriol④U UO+low dosecalcitriol⑤U UO+Benazopril group.Calcitriol wasadministered daily at a dose6ng/100g、3ng/100g dissolved in olive oil1mLin high calcitriol and low treatment groups, benazopril was administered dailyat a dose0.17mg/100g dissolved in NS1mL day.The UUO operation grouponly left ureteral ligation.The UUO operation group and sham operated groupwas administered daily at a dose olive oil1mL after UUO and daily for2weeks.1,3,5,7,10,14days each group were sacrificed six rats.Specimens weretaken.1.2Take the obstructed kidney by HE staining observed10and14days groupin renal tissue pathological changes. Sirius scarlet staining observed14daysgroup kidney fibrosis.1.3The7、10、14days group expression of TNF-α levels by ELISA.1.4The7、10、14days group expression of TGF-β1,ICAM-1by RT-PCR andwestern blot. 1.5The1,3,5days group expression of VEGF,VEGFR-2,HIF-1α by westernblot.2Cytology test of calcitriol on endothelial cells involved in the mechanism offibrosis.2.1Endothelial cells culture： Eahy926，Cultured under high glucose DMEMmedium containing10%fetal bovine serum at37°C,5%of CO2, saturatedhumidity，to Eahy926cells growth monolayer spare. Eahy926cell weredivided into the following groups;①Cocl2control group(cobaltchloride),②B lank control group(Equal volume ofD-Hank’s solution),③Highdose group of calcitriol(final concentration10-8mol/L),④Medium dosegroup of calcitriol(final concentration10-9mol/L),⑤L ow dose groupofcalcitriol(final concentration10-10mol/L).2.2Proliferation effect of calcitriol in The cobalt chloride stimulation Eahy926cells measured by MTT.2.3The expression of VEGF levels in each group by ELISA.2.4The effect of calcitriol in Eahy926cell determined by RT-PCR andwestern blot.Detecting the relative content of Eahy926cells mRNA onVEGF,VEGFR-2,HIF-1α,ICAM-1and protein on VEGFR-2,HIF-1α,ICAM-1.2.5Endothelial cell apoptosis rate detected by flow cytometry in eachexperimental group.3The enumeration data was recorded as±s.Results:1Effect of calcitriol on pathological changes in rat renal interstitial fibrosis.After UUO, the tumafaction and atrophy in renal parenchyma ofobstructed,kidneys were measured obviously.Sham operated group did notchange significantly.HE staining observed UUO group found tubulointerstitialinflammatory cells increased，Varying degrees of tubular cell degeneration,atrophy and necrosis.The High dose calcitriol, low dose calcitriol,thebenazepril group Kidney fibrosis milder UUO group at the same point in time.Sirius scarlet staining observed UUO group product collagen increase,decrease in each drug treatment group collagen, No significant changes in sham operated group.2Calcitriol changes on7,10,14days serum TNF-α concentrations.TNF-α concentrations of each drug treatment groups and UUO groupthan the sham operated group increased(P<0.05). The longer obstruction,theincreasing expression of TNF-α. High and low dose of calcitriol andbenazepril can reduce the UUO rat serum concentration of TNF-α(P<0.05).3Effect of calcitriol on the mRNA and protein expression of TGF-β1,ICAM-1in different groups rats.The protein and mRNA expression of TGF-β1,ICAM-1in UUO groupand each drug treatment groups significantly increased than the sham operatedgroup (P<0.05), The longer obstruction,the increasing protein and mRNAexpression of TGF-β1,ICAM-1in UUO group. High and low dose of calcitrioland benazepril can significantly reduce the same point in time UUO rat TGF-β1,ICAM-1protein and mRNAexpression(P<0.05).4Effect of calcitriol on the protein expression of HIF-1α,VEGFR-2,VEGF indifferent groups rats.4.1The protein expression of HIF-1α in1,3,5days UUO group significantlyincreased what compared with sham operated group (P<0.05),but significantlyinhibited what compared with Benazopril/Calcitriol treatment group(P<0.05).4.2The protein expression of VEGF in1,3,5days UUO group compared withsham operated group (P>0.05) but significantly inhibited what compared withBenazopril/Calcitriol treatment group (P<0.05).4.3The protein expression of VEGFR-2in1,3,5days UUO group comparedwith sham operated group (P>0.05), UUO group significantly higher thansham operated group (P<0.05), in5days UUO group increased compared withsham operated group (P>0.05) and significantly inhibited compared withBenazopril/high dose Calcitriol treatment group (P<0.05).5Proliferation effect of calcitriol in the cobalt chloride stimulation Eahy926cells measured by MTT.The Eahy926cell growth was inhibited since the final concentration of Calcitriol increased to10-9mol/L what compared with control group (P<0.05).6Change of calcitriol on the concentration of VEGF induced by cobaltchloride Eahy926cell supernatant.Each drug treatment groups and cobalt chloride concentration of VEGFstimulation group than the control group increased(P<0.05), The eachcalcitriol dose group VEGF were significantly increased compared with cobaltchloride stimulation group(P<0.05), The most obvious VEGF expressionwhen calcitriol a final concentration of10-9mol/L.7Calcitriol induced by cobalt chloride Eahy926cell of VEGF, VEGFR-2,HIF-1α, ICAM-1mRNA expression changes.Cobalt chloride stimulation group HIF-1α, ICAM-1mRNA expressioncompared with the control group increased(P<0.05).After24h stimulated bydifferent dose of calcitriol.The expression of HIF-1α、ICAM-1mRNA wasinhibited what compared with Cocl2control group (P<0.05), VEGF,VEGFR-2expression than cobalt chloride stimulation group washigher(P<0.05).8Calcitriol induced by cobalt chloride Eahy926cells VEGFR-2, HIF-1α,ICAM-1protein changes.Cobalt chloride stimulation group HIF-1α, ICAM-1protein expressionwas significantly higher than blank group(P<0.05). VEGFR-2compared withthe blank control group increase was not obvious（P>0.05）. After24hstimulated by different dose of calcitriol, each groups HIF-1α, ICAM-1proteinexpression compared with cobalt chloride stimulation group wasinhibited(P<0.05). VEGFR-2protein expression compared with cobaltchloride stimulated group increased(P<0.05).9The result of Flow cytometry.After24h stimulated by Cobalt chloride， the apoptosis rate significantlyincreased what compared with control group (P<0.05),After24h stimulated bydifferent dose of calcitriol, The apoptosis rate was inhibited what comparedwith Cocl2control group (P<0.05). Conclusions:1Calcitriol could inhibits renal interstitial fibrosis in UUO rats.2Calcitriol may be related to the inhibition of in UUO rat renalinterstitial fibrosis raised the VEGF,VEGFR-2protein expression, reduceHIF-1α, ICAM-1,TGF-β1protein expression. Through affect the fibrosiscytokine expression, reduce the destruction of the peritubular capillaries,reduce the transdifferentiation of renal tubular epithelial cells and fibroblastssecrete extracellular matrix, thereby reducing renal interstitial fibrosis.3Calcitriol have a role in the Eahy926cells in the treatment of cobaltchloride, VEGF,VEGFR-2expression increased, HIF-1α,ICAM-1expressionwas decreased(the same as uuo rats), shown to reduce the rate of endothelialcell apoptosis. Calcitriol may affect the endothelial cells of fibrosis-relatedcytokines, receptors and adhesion molecule expression, Suppress localinflammatory reaction, protection the kidney capillary network and tubularepithelial cells and indirectly reduce local TGF-β1levels. Play the role ofagainst renal interstitial fibrosis. The results reveal calcitriol part of themechanism of Eahy926cell pathway to play the role of renal interstitialfibrosis.