Analysis Of Risk Factors Of Chronic Kidney Disease And The Effect Of Traditional Chinese Medicine Tangshen Recipe On Renal Interstitial Fibrosis

Background:Chronic kidney disease(CKD)is a global public health problem.As estimated by epidemiological studies,the global prevalence of CKD is about 8%?16%.Approximately 10.8%of adults in China suffer from CKD,and some of CKD patients fail to receive effective treatment and are progressed into end-stage renal disease(ESRD),posing a heavy economic burden on society and families.As an important potential risk factor for cardiovascular events and cerebrovascular diseases,CKD has received extensive attention from medical workers.Research on the risk factors that affect the occurrence and development of CKD has also drawn special attention from nephrology community.Some factors including hypertension,hyperglycemia,and hyperlipidemia have been well established as promising risk factors for the progression of CKD,whereas there is currently a paucity of data on the impact of thyroid hormone levels on its progression.Renal interstitial fibrosis is an important pathological process for the progression of CKD to ESRD.Currently,effective treatment drugs for CKD are still lacking in the clinical practice.Chinese medicine has a significant advantage in preventing and treating renal interstitial fibrosis.Tangshen Formula is a compound preparation of Chinese medicine for the treatment of DKD developed on the basis of inheriting clinical experience of Chinese Medicine doctors.Clinical trials have shown that Tangshen Formula can improve renal function and increase glomerular filtration rate,yet evidence from animal studies is lacking to confirm the anti-fibrotic effect of Tangshen Formula.Objective:(1)Utilizing a retrospective study design,we decide to identify potential risk factors for the progression of CKD.(2)As a risk factor for the progression of CKD,we construct a predictive model to judge the occurrence and development of thyroid hormone and CKD.(3)We aimed to evaluate the impact of Tangshen Formula on the alleviation of renal interstitial fibrosis,and further to detemine the best therapeutic dose,and meanwhile to explore the mechanism of Tangshen Formula on renal interstitial fibrosis.Methods:(1)Study 1:A retrospective study was conducted to recruit CKD patients aged between 18 and 80 who were hospitalized in our hospital for at least two times during the period between January 2010 and December 2018.The baseline characteristics,primary diseases,and clinical biochemical indexes of CKD patients at the initial hospitalization were extracted and analyzed.Spearman correlation analysis,univariate and multivariable Logistic regression analyses were employed to explore potential risk factors responsible for the progression of renal function.(2)Study 2:On the basis of CKD patients from Study 1,patients with complete thyroid hormone levels were selected as the case group,and meanwhile subjects without clinically-confirmed CKD from the same hospital formed the control group.By means of ?2 test,Wilcoxon rank-sum test,Spearman correlation analysis,and Logistic regression analysis,a risk prediction model for the development of CKD was constructed.Additionally,a combination of multiple statistics,such as net reclassification improvement and integrated discrimination improvement,were adopted to assess prediction performance from both calibration and discrimination aspects.Furthermore,a generalized regression analysis was used to assess the impact of thyroid hormone changes on eGFR changes by using data from the first and last admissions after adjusting for other confounding factors.(3)Study 3:Use UUO model mice to evaluate the curative effect of Tangshen Formula on renal interstitial fibrosis,and to select the best therapeutic dose;and initially explore the mechanism of Tangshen Formula in treating renal interstitial fibrosis with optimal dosage.90 male C57BL/6 mice aged 6-8 weeks were selected,and after 3 days of adaptive feeding,they were randomly divided into 6 groups according to body weight:sham operation group,UUO model group,UUO model with Tangshen Formula low-dose group,UUO model with Tangshen Formula medium-dose group,UUO model with Tangshen Formula high-dose group,UUO model with fosinopril group,and each group has 15 animals.The sham operation group was used as the normal control group.Unilateral ureteral ligation surgery was performed 7 days after gavage with medicine or equal volume of distilled water,and last 7 days after surgery.The sham operation group and UUO model group were given equal volume of distilled water.After the mice sacrificed,HE staining and Masson trichrome staining were used to observe the pathological damage of the kidney tissues of the mice in each group,to evaluate the degree of renal interstitial fibrosis,and to determine the optimal dosage of Tangshen Formula in the treatment of UUO model mice.Immunohistochemical staining and Western Blot were used to analyze the molecular changes of renal interstitial fibrosis related factors TGF-?1,E-cadherin and Vimentin,and the expression levels of Col I and Col III in the kidney of each group.Result:(1)Study 1:A total of 5603 CKD patients were eligible for inclusion in this study,and 64.9%of them were males and 65.9%were aged 61?80 years.1372 patients(24.5%)had a diagnosis of primary diseases,with the top 3 high incidences being secondary glomerular disease(828 cases,60.3%),primary glomerular disease(291 cases,21.2%),and renal vascular disease(96 cases,7.0%).By means of Spearman correlation analysis and Logistic regression analysis,the significant risk factors responsible for the progression of CKD included the decrease of free thyroxine,free triiodothyronine,thyroxine,triiodothyronine,blood calcium,hemoglobin,and high density lipoprotein cholesterol,and the increase of thyroid stimulating hormone,high-sensitivity C-reactive protein,uric acid,homocysteine,blood ?2-microglobulin,24-hour urine protein,inorganic phosphorus,serum potassium,and triglycerides.(2)Study 2:A total of 3274 CKD patients and 289 healthy controls were eligible for analysis.On the basis of the risk prediction model for the impact of thyroid hormone on the development of CKD,our results showed that per 0.2 pg/mL increment in FT3 can reduce the risk of CKD stage 1-4 by 35%?38%,and per 0.3 ng/dL increment in FT4 can reduce the risk of developing CKD stage 5 by 21%(odd ratio[OR],0.79,95%confidence interval(CI)0.69?0.89).Per 0.5 ?IU/mL increment of TSH can increase the risk of developing CKD stage 5 by 8%(OR,1.08,95%CI:1.02?1.14).The interaction between FT3 and FT4 can be used to predict the progression of CKD stage 5(high FT3-low FT4:OR,1.81 95%CI;1.35-2.55;low FT3-high FT4:OR,17.72;95%CI,7.18?43.74;low FT3-Low FT4:OR,22.28;95%CI,9.68?51.30).Subgroup analysis showed that TSH had no predictive effect.Further investigation on the correlation between the first and last thyroid hormone changes and eGFR changes was conducted in 620 patients with regular admission and routine treatment of CKD The changes of FT3,FT4 and TSH between the first and last admissions were significantly correlated with eGFR changes(P<0.001).After adjusting for the interval between the first and last admissions,age,gender,diabetes,and hypertension at the time of first admission,the changes in FT3 and FT4 were significantly related to eGFR changes(P<0.05),and with the rapid decrease of FT3 and FT4,eGFR declined rapidly,and FT3 and FT4 can play a synergistic role in predicting the progress of CKD.(3)Study 3:It was found that the medium dose of Tangshen Formula is the best dose for the treatment of renal interstitial fibrosis in UUO model mice.Differentiation is related to excessive deposition of extracellular matrix.Compared with the sham operation group,the UUO model group had severe renal interstitial damage and significantly increased fibrosis area.The Tangshen Formula medium and high-dose group and Fosinopril group can significantly improve renal interstitial damage and kidney collagen deposition;Moreover,the middle-dose and high-dose therapeutic effects of Tangshen Formula are equivalent,so we chose the middle-dose of Tangshen Formula with lower drug concentration to explore its mechanism of action to improve renal interstitial fibrosis.Immunohistochemistry and Western blot results showed that compared with the sham operation group,UUO model group kidney tissue TGF-?1 and extracellular matrix-related proteins Col ?,Col ? expression levels were significantly increased;renal tubular epithelial cell interstitial.As the markers of epithelial-mesenchymal transition of renal tubular epithelial cells,the expression levels of ?-SMA and vimentin protein were significantly increased,and the expression level of E-cadherin protein was significantly reduced,while Tangshen Formula could significantly reverse the expression of the above proteins in UUO model mouse kidneyConclusion:(1)The risk factors for the progression of CKD disease are related to anemia,electrolyte disturbance,and dyslipidemia,and among them the changes in various thyroid hormones represent a special concern(2)Changes in serum free triiodothyronine and free thyroxine levels can predict the risk of CKD progression,and eGFR declines faster when free triiodothyronine and free thyroxine levels decrease heavily(3)Chinese medicine Tangshen Formula improves renal interstitial fibrosis in the treatment of CKD,of which the middle dose is the best therapeutic dose,and its mechanism may be related to the suppression of TGF-?1 expression in renal tissue and the reduction of renal tubular epithelial-mesenchymal transition.