Study On The Intervention Of ?-Klotho Protein To Delay Renal Interstitial Fibrosis By Xiaozheng Sanjie Method

Chronic kidney disease(CKD),one of the major threat to human health problems,is increasingly at an alarming rate of growth in the global range,but still lacing any effective treatment,caused heavy economic burden to the country and the prevalence of family.Renal interstitial fibrosis(RIF)is a common pathway of CKD development into ESRD.However,there is no effective way to control RIF.The discovery of?-Klotho protein has an important influence on the research of RIF.It is found that?-Klotho's role is not limited to regulate calcium and phosphorus metabolism.It can not only reflect the state of renal function,but also inhibit TGF-?1 pathway,Wnt pathway,EMT process and other ways to reduce the RIF,might be a key factor controlling the development of CKD,and has potential value in the treatment of CKD.The theory of "miniature mass of renal collateral"(Shen Luo Wei Xing Zheng Jia)is the classic interpretation of the common pathogenesis of CKD,the treating method " Xiaozheng Sanjie" and "Shenyanfangshuai solution" which is based on the theory,are effective in clinical practices and fundamental experiments.This study will use Klotho protein as the breakthrough point,through the clinical observation to explore the pathogenesis of "miniature mass of renal collateral" theory,and through the whole animal experiment to explore the effect of target and effect mechanism of Shenyanfangshuai solution".Objective:1.Through the observation of TCM syndrome distribution characteristics of different observation CKD and laboratory index of correlation,correlation of focus on the distribution of TCM syndromes and serum?-Klotho level and IL-6,IL-18,TNF-?,evolution and characteristics of the different stages of disease of TCM key pathogenesis,to explore the theory of "miniature mass of renal collateral".2.To study on UUO rat model of bilateral renal tissue injury and Klotho,TGF-?1 expression,the focus on the "Shenyanfangshuai solution" on the intervention effect of bilateral kidneys,provide the basis for the formation of specific mechanisms for "Xiaozheng Sanjie" regulation of kidney by micro Zhengjia interpretation,which provides the basis for further study on pharmacodynamics.Methods:1.To collect the clinical data,TCM syndrome data and serum of CKD2-4 patients,analyze the serum soluble?-Klotho,IL-6,IL-18,TNF-? and clinical indicators,and the correlation between different TCM syndromes.2.SD rats were used as the research object,and the obstructive renal tubulointerstitial fibrosis model was established by unilateral ureteral ligation.The experiment was divided into 4 groups:sham operation group,model group,Lotensin group and Shenyanfangshuai solution group,respectively after 7 days,14 days,21 days to collect serum and plasma and urine samples,and sacrifice the animal,the kidneys.Test the weight of rats,serum creatinine,urea nitrogen,urinary Kim-1,NGAL,pathological sections HE,PAS,Masson and Tunel staining,observe pathological changes and apoptosis of cells;immunohistochemistry double kidney?-Klotho,?-SMA and Col I expression;expression in renal tissue was detected by real-time fluorescence quantitative PCR Klotho,TGF-?1 mRNA.Results:1.The serum soluble?-Klotho protein in patients with CKD stage 2 was significantly lower than that in healthy subjects(P=0.003),but not continue to fall(P>0.05).After the age of?-Klotho was corrected,the above characteristics still exist.The levels of IL-6,IL=18 and TNF-?were increased in different degrees(P<0.05)at CKD3 and 4 stages,which was lower than that of Klotho.2.The level of serum logKlotho was positively correlated with eGFR(y=0.297),and negatively correlated with age(y=-3.43),negatively correlated with Logscr(?=-0.348),and negatively correlated with serum IL-18(y=-0.234).In TCM syndrome,logKlotho level was negatively correlated with Yin and yang deficiency syndrome(?=-0.383),which was negatively correlated with blood stasis syndrome(y=-0.296).3.After UUO,the contralateral kidney appeared a series of early tissue injury,including urinary Kim-1 increased in 14 days and 21 days(P<0.05),urinary NGAL increased significantly in 14 days,21 days(P<0.01),renal tubular injury score increased in 14 days,21 days(P=0.05),renal tubular epithelial cells apoptosis in a the number in the 21 days have increased trend(P=0.077)changes.4.Shenyanfangshuai solution can maintain the body weight of UUO rats in the normal level,at the same time,the blood creatinine,urea nitrogen in different degrees of decline,so that urinary Kim-1,NGAL maintained at a relatively low level.5.In the ligated kidney,after using Shenyanfangshuai solution,each point of the renal epithelial cell apoptosis,tubular damage and interstitial fibrosis in different degree is reduced,and the overall effect is better than Lotensin;the mechanism may be related to Shenyanfangshuai solution reduced the expression of ?-SMA and Col I,upregulate the expression of?-Klotho protein,inhibition of TGF-?1 pathway.6.Shenyanfangshuai solution prevented renal tubular epithelial cell injury,apoptosis in the non ligated kidney,and the effect of lotensin.The mechanism may be related to the upregulation of?-Klotho expression and the inhibition of TGF-?1 pathway in the rat.Conclusion:1.The level of serum soluble ?-Klotho in the CKD2 phase was significantly lower than that in the healthy population,which may be a predictor of CKD progression.With Yin and yang deficiency,blood stasis has certain relevance,suggesting decreased level of ?-Klotho may be the key factor leading to the formation of miniature mass of renal collateral.2.In the UUO model,Shenyanfangshuai solution can up regulate the expression of Klotho and inhibition of TGF-?1 pathway,reduce the pathological products such as to reduce cell apoptosis,tissue injury,delayed the progression of fibrosis in the ligated kidney;while in the collateral kidney,Shenyanfangshuai solution can up regulate the expression of Klotho and inhibition of TGF-?1 signaling way to prevent tissue damage,cell apoptosis.