| BackgroundGlobally,the incidence of chronic kidney disease(CKD)is increasing,threatening the health of approximately 10%to 13%of the world’s adults and becoming an important public health problem in the world.The occurrence and development of chronic kidney disease(CKD)has brought a great burden to the individual,family,socioeconomic and public health.Therefore,the effective prevention and treatment of CKD has become a difficult and important point in the current kidney disease research.CKD condition is persistent and gradually progresses to end-stage renal disease(ESRD),and the life-long renal replacement therapy or kidney transplantation ultimately is needed,in which abnormal activation of RAS in chronic progression of CKD play an important role in the process,RAS activation can cause water,electrolyte and acid-base balance disorders,leading to high blood pressure and renal fibrosis,and promote the development of CKD and cardiovascular disease.RAS includes four components,namely:angiotensinogen(AGT),renin(renin),angiotensin converting enzyme(ACE)and angiotensin Ⅱ receptor(AT1 and AT2).Under normal physiological conditions,RAS widely exists in the blood circulation system,its components from the liver,kidneys and lungs and other different organs of the body synthesis of secretion,by a series of complex and strict regulation and control mechanisms.However,a large number of studies have shown that after kidney damage,the lesion of the kidney can activate all the components of RAS,and abnormal activation of RAS and promote hypertension,CKD and cardiovascular disease occurrence and development.At present,the clinical treatment of RAS drugs are mainly angiotensin Ⅱ receptor blockers(ARB)and angiotensin converting enzyme inhibitors(ACEI)and the two renin-angiotensin system(RAS)block ARB and ACEI can reduce the blood pressure and urinary protein to a certain extent,delay the progress of CKD,but these drugs in blocking RAS when the effect is limited,with "angiotensin Ⅱ escape" phenomenon,and can not fully inhibit RAS,Blocking the progress of CKD to ESRD,which means that there may be other CKD pathogenesis related to the continued activation of RAS,leading to the progress of CKD to ESRD.Therefore,in addition to ACEI,ARB drugs,the urgent need for further study of kidney injury-related pathological and physiological mechanisms to find new treatment strategies and drugs to block or prevent the occurrence of hypertension and CKD occurrence and development.Hedgehog signaling pathway is a conserved,regulated signal cascade that regulates multiple biological processes,such as embryonic development,repair and tumorigenesis,and plays an important role in the stability of tissues..Sonic hedgehog(Shh)is one of the most abundant ligands of the hedgehog signaling pathway.It is a kind of secreted extracellular signal protein and plays an important role in regulating the development of mammalian organogenesis.It is a kind of morphogenesis of many tissues and organs Occurrence factor.Therefore,abnormalities of various components in the Shh signaling pathway can lead to severe developmental abnormalities.Under normal circumstances,when the body after the completion of development,Shh signal gets into a resting state.Abnormal activation of Shh signaling can cause a variety of tissues and organs Of the abnormal disease.The mechanism of action of Shh is mainly two kinds of classical and non-classical pathways,the classic pathway is "shh-Ptchl-Smo-Gli-1"that shh through this cascade reaction finally cause the activation of Gli-1 transcription factor into the nuclear regulatory target genes.Non-classical pathway can be divided into Gli-1 activation but not dependent on Ptchl and Smo activation and Ptchl or Smo activation but does not dependent on Gli-1 activation.In addition,cyclopamine(CPN),as a specific inhibitor of Smo,can effectively inhibit the activity of Smo,thus effectively blocking the shh signaling pathway.Pre-study showed that after renal injury,shh are synthesized and secreted in renal tubular epithelial cells specifically and then transmitted to renal interstitial fibroblasts in paracrine pathway to promote differentiation and fibrosis of fibroblasts,leading to the occurrence and development of renal fibrosis.In the course of the development of CKD,the activation of RAS and the abnormal activation of Shh signaling pathway all promote the fibrosis of kidney through a certain mechanism.At present,there is no research about the correlation between the Sonic hedgehog signal pathway and RAS.Therefore,it is presumed that there may be some direct or indirect association between the Sonic hedgehog signal pathway and the RAS,and the Sonic hedgehog signal promotes the occurrence and development of CKD by regulating RAS,ultimately leading to a rapid progress of CKD toward ESRD.This study focused on the interaction between shh and RAS in CKD and its specific mechanism.In this study,we worked in the following two aspects:1.In vitro experiments,HKC-8 cells were incubated with the recombinant shh protein to simulate the biological effects of shh on HKC-8 and explore whethere the activation of shin signaling pathway on RAS;In vivo experiment:the model of CD1 mouse 5/6 nephrectomy(5/6NX)were used to simulate CKD of human,overexpressing shh and inhibition of Smo with CPN to further explore and confirm the activation of RAS of shh and related mechanisms.This study provides a novel and effective strategy and method to prevent CKD and CKD-related hypertension,delay or even effectively block the development of CKD to ESRD.ObjectiveTo investigate the effect of shh signaling pathway on the regulation of RAS in CKD,and evaluate the effectiveness and feasibility of inhibiting shh signaling with CPN in prevention and treatment of CKD.Methods1.Cell experiments1)The expression of RAS(ACE,AT1,AGT,Renin)in HKC-8 cells transfected with shh plasmid(pFlag-shh):plasmid pcDNA3 and shh were transfected respectively into HKC-8 cells,the transfection time was 72hours,the expression of RAS(ACE,AT1,AGT,Renin)protein were detected by Western blot.2)The expression of nucleic acid of Ptchl,Ptch2,Smo,Gli-1,Gli-2 and Gli-3 in HKC-8 cells transfected with pFlag-shh:HKC-8 cells were divided into three groups,one group was transfected with plasmid pcDNA3,the transfection time was 6 hours,the other two groups transfected pFlag-shh,the transfection time was 6hours and 12hours.The transfection quality was 4ug,.The expression of nucleic acid of Ptch1,Ptch2,Smo,Gli-1,Gli-2 andGli-3 was detected by Real-time PCR.3)The effects of CPN on the expression of RAS(ACE,AT1,AGT,Renin)after HKC-8 transfection with pFlag-shh:HKC-8 cells were divided into three groups,in which two groups were transfected with pcDNA3 and pFlag-shh respectively.Another group was incubated with CPN at a concentration of lOumol/L,1 hour before shh transfection.The transfected plasmid was 4ug.The protein expression of RAS(ACE,AT1,AGT,Renin)was detected by Western blot.4)The expression of MAPK after HKC-8 was stimulated by recombinant shh protein:HKC-8 cells were divided into six groups with a concentration of 50ng/ml shh to stimulate HKC-8,stimulation time was 0,0.25hour,0.5 hour,1 hour,3 hours,6 hours,respectively,and then Western blot was used to detect p-P38,p-ERK,ERK,p-JNK and JNK.5)The effect of CPN on the expression of MAPK after HKC-8 stimulated byrecombinant shh protein:HKC-8 cells were divided into three groups,in which a group was a control group,a group with shh stimulation,the last group with shh stimulation with CPN 1hour before incubation.The concentration of CPN was lOumol/L,the stimulation concentration of shh was 50ng/ml,and the stimulation time was 15min.The expression of p-p38,p38,p-ERK,ERK,p-JNK and JNK were detected by Western blot.6)Effects of MAPK inhibitor(U0126,SP,SB)on RAS expression after HKC-8 stimulated by Shh:HKC-8 cells were divided into three groups,one group as control group,one group with shh stimulation,and the last group(U0126,SP,SB)concentration was 10umol/L,shh stimulation concentration was 50ng/ml,stimulation time was 72hours.MAPK inhibitor(U0126,SP,SB)was pre-incubated for 1hour.The expression of RAS protein was detected by Western blot.7)The expression of p-PLC、p-PKC(p-PKC α/βⅡ,p-PKCζ)and PKC(PKC α/βⅡ,PKCζ)in HKC-8 cells after stimulation with recombinant shh protein:The stimulation time was 0 min,l min,5 min,10 min,15 min and 30 min,respectively,when stimulated with shh at a concentration of 50 ng/ml.Cells were harvested after stimulation.The protein expression of p-PLC、PKC(p-PKC α/βⅡ)and PKC(PKC α/βⅡ)was detected by Western blot.2.Animal experiments1)Effects of overexpressing shh and the inhibition of Smo with CPN on RAS:Male CD1 mice were randomly divided into sham operation group,5/6NX empty plasmid group(5/6NX+pcDNA3),5/6NX overexpressing Shh plasmid(5/6NX+pFlag-shh),5/6NX overexpressing shh plasmid with CPN treatment group(5/6NX+pFlag-shh+CPN).The mice were sacrificed 2/3 on the side of the mouse,and the other side of the kidney was cut after one week.The late three group was injected with pcDNA3 or pFlag-shh by tail vein at the 2nd,3rd,4th and 5th week after 5/6 NX.CPN was injected intraperitoneally from the 2nd week to the day before sacrifice.In addition,CPN was injected intraperitoneally from the 2nd week to the end of the experiment.In addition,the blood pressure was measured at the 2nd,3rd4th,5th and 6th week after 5/6 NX.The blood,urine and kidneys were collected at the 6th week after 5/6NX.The rats were sacrificed for renal function test,urinary protein,urine creatinine,immunohistochemistry,immunofluorescence and Western blot respectively.2)Serum and urine creatinine as well as blood urea nitrogen levels were measured by using automatic biochemical analyzer.3)Urinary albumin level was determined using rat Albuminuria ELISA Quantization kit.4)The protein expression of RAS(ACE、AT1、AGT、Renin)、Fn、α-SMA、p-PKC(p-PKC α/βⅡ)、PKC(PKC α/βⅡ)、MAPK(p-p38、p38、p-ERK、ERK、p-JNK、JNK)were examined by western blot.5)Renal morphological changes were measured by PAS staining.Collagen deposition was examined by Masson-Trichromatic staining.6)The expression of Fibronectin were examined by immunofluorescence.The location and expression of RAS(ACE、AT1、AGT、Renin)、Fn and α-SMA were determined by immunohistochemistry.ResultsCell experiments1)The expression of RAS(ACE,AT1,AGT,renin)in HKC-8 cells transfected with pFlag-shh:the results of Western blot showed that the expression of ACE,AT1 and rennin protein were significantly increased;in additional,the expression of mRNA of ACE、AT1、renin and AT1 was significantly increased by Real-time PCR.2)The expression of mRNA of Ptch1,Ptch2,Smo,Gli-1,Gli-2 and Gli-3 in HKC-8 cells transfected with pFlag-shh:real-time PCR showed that compared with pcDNA3 group,there was no significant change in the expression of Smo mRNA in group transfected pFlag-shh for 6 hours.However,the expression of Smo mRNA in group transfected shh plasmid for 12 hours was significantly higher than the group transfected pFlag-shh for 6 hours.3)The effect of CPN on the expression of RAS(ACE,AT1,AGT,Renin)after HKC-8 transfection with pFlag-shh:western blot analysis showed that the expression of ACE,AT1 and Renin protein in group transfected pFlag-shh was significantly increased compared with pcDNA3 group,and the expression of ACE,AT1 and Renin protein in CPN group was significantly decreased compared with group transfected pFlag-shh.4)The expression of MAPK after HKC-8 was stimulated by recombinant shh protein:western blot showed that the expression of p-P38,p-ERK and p-JNK protein was significantly increased and reached the peak at 0.25hour.Then,at 0.5hour,lhour,3hours and 6hours,the expression of p-P38,p-ERK and p-JNK protein decreased gradually compared with the 0.25 hour protein;however,the expression of P38,ERK and JNK did not change at all time points.5)The effect of CPN on the expression of MAPK after HKC-8 stimulated by recombinant shh protein:western blot analysis showed that CPN could effectively inhibit the expression of p-P38,p-ERK and p-JNK protein induced by shh on HKC-8.However,P38,ERK,JNK protein expression was no significant change.6)The effect of MAPK inhibitor(U0126,SP,SB)on the expression of RAS after HKC-8 stimulated by recombinant shh protein:Western blot analysis showed that MAPK inhibitor could effectively inhibit the protein expression of AT1,renin and ACE induced by shh on HKC-8.7)The expression of p-PLC、p-PKC(p-PKC α/βⅡ,p-PKCζ)and PKC(PKCα/βⅡ,PKCζ)in HKC-8 cells after stimulation with recombinant shh protein:western blot analysis showed that the expression of p-PLC、p-PKCα/βⅡ and pKC αprotein increased with the extension of time;However,the protein expression of PKC α/βⅡ and PKCζ did not change significantly.Animal experiment1)The blood pressure was measured by ail-cuff technique at the 4th、5th and 6th week postoperatively indicated that:SBP and MBP levels in the 5/6NX+pcDNA3 group were significantly higher than those in the sham-operated group(p<0.05);SBP and MBP levels in the 5/6NX+pFlag-shh group were significantly higher than those in the 5/6NX+pcDNA3 group(p<0.05);however,SBP and MBP levels in CPN-treated group were significantly lower than those in 5/6NX+pFlag-shh group(p<0.05).2)PAS staining showed that glomerular sclerosis,mesangial stromal hyperplasia,tubular basement membrane thickening and tubular dilatation occurred in 5/6NX+pcDNA3 mice compared with ctr group.Compared with 5/6NX+pcDNA3,the glomerular sclerosis,mesangial stromal hyperplasia,renal tubular basement membrane thickening and tubule dilatation were further aggravated in the 5/6NX+pFlag-shh group,and inflammatory mediators were found in the renal interstitial.CPN treatment group can significantly relieve glomerular,renal tubular and renal interstitial lesions of the above in the 5/6NX+pFlag-shh mice.3)MSSON staining showed that compared with ctr group,5/6NX+pcDNA3 group had a significant increase in renal interstitial cells.Compared with 5/6NX+pcDNA3,the amount of renal collagen fibers in the pFlag-shh group was significantly increased in the renal interstitial tissue,while the CPN treatment group significantly inhibited the deposition of collagen fibers in the 5/6NX+pFlag-shh group.4)The effects of overexpressing shh and inhibiting shh signaling pathway with CPN on RAS:western blot results showed that the protein expression of ACE,ATI and Renin in 5/6NX+pFlag-shh group was significantly increased compared with 5/6NX+pcDNA3 group;however,CPN could effectively inhibit the expression of ACE,ATI and Renin induced by overexpression of shh;IH results showed that the expression of Renin,ATI and ACE protein in 5/6NX+pcDNA3 group was significantly higher than that in ctr group,and the expression of 5/6NX+pFlag-shh group was significantly higher than that of 5/6NX+pcDNA3 group,the protein of Renin,ATI and ACE was distributed in renal tubular epithelial cells.And the treatment of CPN significantly inhibited the expression of Renin,AT1 and ACE in renal tubular epithelial cells in 5/6NX+pFlag-shh group.5)The effects of overexpressing shh and inhibiting shh signaling pathway with CPN on p-PKC α/βⅡ and PKC α/βⅡ:western blot results showed that the protein expression of p-PKC α/βⅡ and PKC α/βⅡ in 5/6NX+pFlag-shh group was significantly increased compared with 5/6NX+pcDNA3 group;however,CPN could effectively inhibit the expression of p-PKC α/βⅡ and PKC α/βⅡ induced by overexpression of shh.6)The effects of overexpressing shh and inhibiting shh signaling pathway with CPN on p-ERK and ERK:western blot results showed that the protein expression of p-ERK in 5/6NX+pFlag-shh group was significantly increased compared with 5/6NX+pcDNA3 group,ERK protein expression did not change significantly;however,CPN could effectively inhibit the expression of p-ERK induced by overexpression of shh,ERK protein expression did not change significantly.7)he effects of overexpressing shh and inhibiting shh signaling pathway with CPN on Fn and a-SMA:western blot results showed that the protein expression of Fn and a-SMA in 5/6NX+pFlag-shh group was significantly increased compared with 5/6NX+pcDNA3 group;however,CPN could effectively inhibit the expression of Fn and α-SMA induced by overexpression of shh;IH results showed that the expression of Fn and a-SMA in 5/6NX+pcDNA3 group was significantly higher than that in ctr group(P<0.05),Compared with 5/6NX+pcDNA3,the expression of Fn,α-SMA was significantly increased in 5/6NX+pFlag-shh,and mainly distributed in renal interstitial.The treatment of CPN can significantly inhibit the expression of 5/6NX+pFlag-shh group of Fibronectin and α-SMA in renal interstitial.Conclusions1.Shh secreted by renal tubular epithelial cells can act on itself by autocrine pathway.Then,Shh activates MAPK through its nonclassical signaling pathway,and then regulates the transcription of target gene RAS.,and finally results in high blood pressure and promotes fibrosis;2.Targeting inhibition of Smo protein of shh signaling pathway by CPN can effectively inhibit the activation of RAS expression,low blood pressure and reduce renal fibrosis,thereby delaying the slow progress of CKD. |
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