動靜脈廔管及腎絲球腎炎之治療策略探討

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(1)國立台灣師範大學生命科學系（所）生理組博士論文論文題目動靜脈廔管及腎絲球腎炎之治療策略探討. The Therapeutic Strategy Exploration of Arteriovenous Fistula Stenosis and Glomerulonephritis. 博士研究生：張皓翔 (Hao-Hsiang Chang). 指導教授：鄭劍廷博士 Chiang-Ting Chien, Ph.D.. 中華民國一 0 六年一月.

(2) Content Chapter 1 Introduction and Literature Review ......................................12 1.1 Arteriovenous Fistula Stenosis ........................................................12 1.2 Glomerulonephritis and Oxidative Stress ........................................13 Chapter 2. Material and Method ............................................................16 2.1 A population-based Method for Evaluation of Statin on AVF Stenosis Prevention................................................................................16 2.2 Animal model to Exploration of the Anti-oxidative Mechanisms in Therapeutic Application of Mesenchymal Stem Cell for Glomerulonephritis ................................................................................20 Chapter 3. Results .................................................................................27. 3.1 Therapeutic Effect of Statin on AVF Stenosis ................................27 3.2 Theapeutic Effects of Mesenchymal Stem Cell on GN ..................30 Chapter 4 Discussion and Conclusion ...................................................35 4.1 Statins Possess a Dose-responsive Effect of Protecting AVF from Stenosis ..................................................................................................35 4.2 Hypoxic Preconditioning Promote the Ability of Stem Cell to Activate Intrinsic Anti-oxidative Defense Systems ..............................40 Reference ..................................................................................................65. 1.

(3) Tables Table 1 Demographic characteristics, comorbid diseases and medications exposure between statin users and nonusers ... 46 Table 2 Crude and adjusted hazard ratio of vascular access recreation for statin users and nonusers .............................. 48 Table 3 Risk factors of vascular access recreation by multivariate Cox proportional hazards model, by different types of vascular access ................................................................... 49 Table 4 Crude and adjusted hazard ratio of vascular access recreation for different statins ............................................ 51 Table 5 Demographic characteristics, comorbid diseases and medications exposure between statin users and nonusers in propensity-score matched cohort........................................ 52 Table 6 Crude and adjusted hazard ratio of vascular access recreation for statin users and nonusers in propensity-score matched cohort .................................................................. 54 Table 7 Hazard ratio of permanent hemodialysis access recreation for statin users in propensity-score matched cohort ........................................................................................... 55 Table 8 Crude and adjusted hazard ratio of vascular access recreation for statin users by time-dependent Cox regression models ............................................................................... 56 Table 9 Crude and competing-risk adjusted hazard ratio of vascular access recreation for statin users .......................... 56. 2.

(4) Figures Figure 1 The experimental grouping and design were displayed in the eight groups. ...............................................................57 Figure 2 Cumulative incidence of vascular access recreation for statin users and nonusers by the multivariate Nelson-Aalen method...........................................................58 Figure 3 The recruitment of MSCs and HMSCs in injured kidneys. ..............................................................................59 Figure 4 Mesenchymal stem cell transplantation ameliorated disease activity in a rat anti-Thy1.1-induced GN model. ..60 Figure 5 Immunohistochemical stains for ED1, GRP78, LC3-II, Caspase1, TUNEL and Collagen IV in the study groups. . ..............................................................................................61 Figure 6 The index of ROS, ROS enzymatic scavenger expression, Nrf2 expression, and anti-oxidative response protein expressions among the experimental groups. ..........63. 3.

(5) Preface The growing end stage renal disease (ESRD) population and expanding cost of renal replacement therapy (RRT) have great burden on both public health and economics. Glomerulonephritis (GN) is still one of the leading causes of ESRD, and has limited immunosuppressive treatment options with non-satisfactory outcomes. Pathophysiologic feature of GN is immune mediated inflammation related mesangial proliferation and damage. Stem cells had been proved the potential of self-renewal and the ability of immune modulation and provide one potential therapeutic option to the unmet need of GN. Autologous arteriovenous fistula (AVF) is widely recommended as the first choice of long term vascular access for long term hemodialysis. However, high stenosis rates of AVF remained a medical challenge today. The pathophysiology of AVF stenosis is characterized by endothelial dysfunction and vascular smooth muscle proliferation and transmigration. Statins are well known to reduce inflammation and improve endothelial function, thus provide a potential drug of choice for prevention of AVF stenosis. In this thesis, we explored the two clinical problems into pathophysiologic understandings, and tested the novel treatments for them focusing on the anti-inflammatory and anti-oxidative mechanisms.. 4.

(6) Abstract The growing end stage renal disease (ESRD) population and expanding cost of renal replacement therapy (RRT) have great burden on both public health and economics. Autologous arteriovenous fistula (AVF) is widely recommended as the first choice of long term vascular access for long term hemodialysis. However, high stenosis rates of AVF remained a medical challenge today. Glomerulonephritis (GN) is still one of the leading causes of ESRD, and has limited immunosuppressive treatment options with non-satisfactory outcomes. This thesis is to explore the potential beneficial effect of statin on AVF and the anti-oxidative therapeutic mechanism of stem cell on GN. The pathophysiology of AVF stenosis is characterized by endothelial dysfunction and vascular smooth muscle proliferation and transmigration. Statins are well known to reduce inflammation and improve endothelial function, thus provide a potential drug of choice for prevention of AVF stenosis. The protective effects of statins against stenosis for permanent hemodialysis access have been repeatedly demonstrated in animal studies, but remain controversial in human studies. This thesis evaluates the association between statin use and permanent hemodialysis access patency using a nationwide hemodialysis cohort. A total of 9862 pairs of statin users and non-users, matched by age and gender, were selected for investigation from 75404 new hemodialysis patients during 2000-2008. The effect of statins on permanent hemodialysis access patency was evaluated using Cox proportional hazards models. Compared with non-users, statin users had an overall 18% risk reduction in the composite 5.

(7) endpoint in which angioplasty and recreation were combined (adjusted hazard ratio=0.82 [95%CI, 0.78-0.87]) and 21% in recreation of permanent hemodialysis access (adjusted hazard ratio=0.79 [95%CI, 0.69-0.80]).. Specifically,. the. protective. effect. was. found. for. arteriovenous fistula (adjusted hazard ratio= 0.78[95% CI, 0.73-0.82] for composite endpoint and 0.74 [95% CI, 0.69-0.80] for vascular recreation), but not for arteriovenous grafts (adjusted hazard ratio= 1.10 [95% CI, 0.98-1.24] and 0.94 [95% CI, 0.83-1.07]). These findings suggest that statins use after permanent HD access creation possess a dose-responsive effect of protecting AVF from stenosis for patients undertaking hemodialysis. The beneficial effect on permanent HD access outcomes is a universal class effect and the effect size is associated with the statins’ potency. The use of statin may reduce failure of AVF, therefore, promoting patient outcomes and reducing health-care costs. These results have important therapeutic implications for future prospective randomized control studies. Pathophysiologic feature of GN is immune mediated inflammation related mesangial proliferation and damage. Stem cells had been proved the potential of self-renewal and the ability of immune modulation and provide one potential therapeutic option to the unmet need of GN. The anti-oxidative ability of stem cells is one potential mechanism in the therapeutic application, but scarcely studied. This study aimed to investigate the mechanisms of the anti-oxidative effects involved in the use of normoxic and hypoxic conditions treated mesenchymal stem cells to treat anti-Thy1.1 induced rat glomerulonephritis. Proteinuria, 6.

(8) histochemical staining and western blotting were used to explore the therapeutic effects and anti-oxidative mechanisms. Mesenchymal stem cell transplantation ameliorated proteinuria and glomerulosclerosis glomerulonephritis rats. Hypoxic conditioning significantly enhanced these therapeutic effects. Normoxic and hypoxic mesenchymal stem cells demonstrated a consistent reduction in macrophage/monocyte infiltration, levels of stress index proteins, autophagy and apoptosis. In addition, they both promoted intranuclear nuclear factor (erythroid-derived 2)-like expression and ameliorated elevated nuclear factor kappa B expression in diseased kidneys. Hypoxic preconditioning treated mesenchymal stem cells significantly enhanced the expression of anti-oxidative response elements. including. glutamate-cysteine. ligase. catalytic. subunit,. glutamate-cysteine ligase modifier subunit, glutathione peroxidase, catalase,. Mn. and. Cu/Zn. superoxide. dismutase.. Anti-oxidative. mechanisms play a role in the therapeutic effect of mesenchymal stem cells in glomerulonephritis. Hypoxic preconditioning is one effective strategy to activate intrinsic anti-oxidative defense systems by promoting the Nrf2 pathway signaling, rescuing ROS scavengers and increasing anti-oxidative responsive element proteins. The results of this thesis provide evidence supporting therapeutic options to permanent hemodialysis vascular access failure and glomerulonephritis. Key. words:. arteriovenous. fistula,. mesenchymal. glomerulonephritis, oxidative stress, statin. 7. stem. cell,.

(9) 中文摘要日益增加的末期腎病人數顯著地增加腎臟取代治療的費用與公共衛生的負擔。如何減少末期腎病的發生，及提升腎臟取代病患的照護品質是面對這一問題的關鍵。本論文針對血液透析病患動靜脈廔管阻塞問題及腎絲球腎炎治療等臨床困境進行研究。自體動靜脈廔管因為感染率較低、生活品質較佳與整體醫療花費較少等因素，是長期血液透析病患建立血管通路的第一選擇，也是使用最廣泛的。然而，自體動靜脈廔管的阻塞率高，且目前仍缺乏有效的藥物或方法來預防阻塞改善通暢率，因此，血液透析病患自體動靜脈廔管功能喪失仍是臨床上未解決的困境。腎絲球腎炎則是導致末期腎病的重要病因之一，腎絲球腎炎的治療目前僅有一些效果有限的免疫調節治療，許多慢性腎絲球腎炎病患最終仍發展至末期腎病卻苦無有效治療，在本論文中透過臨床大資料分析探討藥物史塔汀對自體動靜脈廔管阻塞的影響，以及以間質幹細胞治療腎絲球腎炎的大鼠模式，評估其療效與治療機轉，並針對抗氧化機轉深入研究，期許成果能對此醫學難題提供進一步的治療基礎。動靜脈廔管阻塞的疾病生理特徵是內皮細胞功能失調、血管平滑肌增生與移轉，而史塔汀藥物具有抗發炎、改善內皮細胞功能的特性，具有潛在改善動靜脈廔管阻塞的病理機轉，是預防靜脈廔管阻塞的選 8.

(10) 擇藥物之一。史塔汀藥物對動靜脈廔管的保護作用已在動物實驗中多次被證實，然而，人類的臨床研究卻無一致性的結論。本論文利用全國性的血液透析世代研究資料庫來分析處方史塔汀類藥物與血液透析病患血管通路通暢度的關係。總共 9862 對依年齡、性別配對的史塔汀使用者與非使用者，自 2000-2008 年間 75404 位新增加的血液透析病患中選出。史塔汀對於自體動靜脈廔管阻塞的影響利用 Cox proportional hazards 模式分析。與非使用者相比，史塔汀使用降低 18%動靜脈廔管需要進行血管整型術或重建的風險(adjusted hazard ratio=0.82 [95%CI, 0.78-0.87]) ，減少 21%重建風險 ( 風險比 =0.79 [95%CI, 0.69-0.80])，這種保護的效果只在自體動靜脈廔管有顯著意義(風險比=0.78[95% CI, 0.73-0.82])，而對使用人工血管的動靜脈廔管無保護效果(風險比=1.10 [95% CI, 0.98-1.24])。本研究的結果顯示在血液透析病患建立動靜脈廔管後使用史塔汀具有保護血管通路通暢的效果。這種效果在不同史塔汀中都出現，而其保護效力與史塔汀的效力強弱有關。使用史塔汀能降低血液透析病患血管通路重建風險，進而降低醫療花費與提昇病患生活品質，這分析的結果可提供將來臨床治療動脈廔管的重要參考依據。腎絲球腎炎的疾病生理特徵是免疫相關的發炎現象，導致腎小球内繫膜细胞增生與受損，而幹細胞證實具備自我修復與免疫調節 9.

(11) 的特性提供的腎絲球腎炎符合病理機轉的治療選擇。相較於幹細胞的抗發炎與抗纖維化作用被廣泛探討，氧化壓力的傷害也是腎絲球腎炎重要損傷來源，抗氧化作用雖是幹細胞重要潛在治療機轉，卻是很少研究詳細地探索其作用，本論文透過使用一般濃度氧氣及缺氧狀態培養之間質幹細胞治療 anti-Thy1.1 引發大鼠腎炎的模式，以尿蛋白、組織化學染色與西方墨點法等方法來確認幹細胞的治療效果與分析其抗氧化的機制。結果發現經腎動脈注射間質幹細胞能緩解腎炎大鼠的蛋白尿、降低腎絲球硬化程度，低氧環境培養可以強化此療效。無論一般濃度氧氣或缺氧狀態培養之間質幹細胞均顯示一致性的減少巨噬細胞/單核球浸潤、壓力指標蛋白表現、自噬作用與凋亡指標蛋白表現，在抗氧化壓力的部分，則能提昇核內的 Nrf2 表現，減低 NFkB 的表現。經低氧前處理的幹細胞能強化細胞抗氧化壓力反應組成 (ARE anti-oxidative response elements)包括 GCLC (glutamate-cysteine ligase catalytic subunit)、GCLM (glutamate-cysteine ligase modifier subunit)、麩胱甘肽過氧化酵素(glutathione peroxidase)、錳超氧化物歧化酶、銅/鋅超氧化物歧化酶的表現。由此結果可知，低氧前處理可藉由活化 Nrf2 的訊息、加強抗氧化壓力反應組成，提升內在抗氧化能力達到減低傷害的目標，而抗氧化機轉在間質幹細胞治療的腎絲球腎炎上扮演重要角色。 10.

(12) 本論文利用臨床大資料分析及動物實驗等方法，針對醫學上仍難以克服兩個的困境，提供史塔汀藥物降低動靜脈廔管阻塞與幹細胞治療腎絲球腎炎的實證基礎。關鍵字: 動靜脈廔管、間質幹細胞、腎絲球腎炎、氧化壓力、史塔汀. 11.

(13) Chapter 1. Introduction and Literature Review. 1.1 Arteriovenous Fistula Stenosis Autologuos arteriovenous fistula (AVF) is universally recommended to be the first choice of long term vascular access for patients receiving hemodialysis 1-3. Many national guidelines suggest AVF for hemodialysis in terms of infection, patient quality of life and medical costes. The maintenance of AVF patency remains a challenge to current medicine in terms of stenosis. Several advances had been made to facilitate the maintenance of AVF patency. These included ultrasound assessment for operation, proper timing for 1st catherization, catherization technique, far infrared therapy. 4,5. . Even with those approaches detailed in some. guidelines for vascular access creation and care, the AVF stenosis rates were still far from optimal. According to a recent systemic review, the 1-year patency rates were reported 62~68%, and 2-year patency rates were 38~56% excluding primary failure6 . To overcome the difficulty, the physiological mechanisms of AVF stenosis were widely studied and proposed to be affected mainly by intimal hyperplasia and inappropriate outward remodeling 7. On the basis of these understandings, some medications possessing potential beneficial effect on AVF patency such as ACEI, AIIA, anti-platelets, anti-coagulants had been tested in clinical studies. 8-10. , but no medication had been. consistently reported to possessing beneficial effect on AVF patency. Among the candidate medicine for improving AVF stenosis, statins received special scrutiny. Statins are well known to reduce inflammation 12.

(14) and improve endothelial function beyond cholesterol lowering in end stage renal disease patients. In animal studies, statins had been demonstrated to improve blood flow, endothelial function and prevent stenosis of AVF which may result from attenuating the activity of proinflammatory genes and oxidative stress in the remodeled vasculature 11-13. . Though possessing potential protection effects on AVF patency,. statins had never been proved to be protective from AVF stenosis in a large human dialysis patients study. Some studies reported that statins were not associated with improved survival of vascular access in dialysis 8,14-16. , while one small number study showed that it was beneficial. 17. .. There is no one medication having as more discrepant results as statins between basic researches and clinical studies. To fill up the gap, one part of this thesis was aimed to evaluate the effect of statins on the AVF long term patency through a nation-wide population cohort study.. 1.2 Glomerulonephritis and Oxidative Stress Glomerulonephritis(GN) is a constellation of heterogenous renal diseases featured as a shared pathophsiology of immune mediated glomerular inflammation 18,19. Despite some of these patients may benefit from specific immunosuppressive therapies, many others who are irresponsive to the management eventually develop ESRD. 20-23. .. Glomerulonephritis is one of the leading causes of ESRD and remains a medical challenge today 24. Implementation of more interventions shown to be effective to cure or slow down the progression of GN is very important from both public health and economic point of view. Stem cells 13.

(15) had been proved the potential of self-renewal and the ability of immune modulation and provide one potential therapeutic option to the unmet need of those GN patients 25. Mesenchymal stem cells (MSC) had been demonstrated to improve renal function in animal GN models studies human lupus nephritis studies. 26,27. and. of. decreases. severity. 25. , and in some refractory. . MSC therapy improves renal function glomerulosclerosis. through. direct. differentiating into renal cells and paracrine effects. The evidence suggests that the direct contribution of transplanted cells to tissue regeneration is minimal. 28. . The paracrine effects are believed to play a. major role in the therapeutic effect to damaged kidney 19. Stem cells cross talk with target organs by secreting growth factors, cytokines and prostaglandins, which regulate anti-inflammation, anti-apoptosis, and anti-fibrosis. effect. to. enhance. cell. proliferation,. survival. and. angiogenesis to repair injuried tissue. In glomerlonephritis, increased oxidative stress contributes to the pathogenesis of mesangial proliferative GN leading to renal dysfunction 29. . MSC had been shown to possess anti-oxidative effect which plays a. role an important role in the treatment of GN anti-inflammatory,. immune. modulatory. 30. . Compared with the. effects,. the. mechanisms. underlying protection of MSC from oxidative injury in the therapeutic effect on GN remain scarcely elucidated. Hypoxic preconditioning is a promising strategy to improve efficacy of stem cell therapy. Under hypoxic condition, MSC can not only prevent senescence, increase differentiation efficiency but also enhance stem cell homing 14. 31,32. . But.

(16) there is little knowledge about how hypoxic pre-condition affect the anti-oxidative effect. The another part of the present thesis aimed to investigate the anti-oxidative stress mechanisms involved in the use of mesenchymal stem cells and hypoxic mesenchymal stem cell in the GN rat model. The growing end stage renal disease (ESRD) population and expanding cost of renal replacement therapy (RRT) have great burden on both public health and economics. Both to develop effective treatment for GN and to improve ESRD patient care are important for management of the growing problem. However, high stenosis rates of AVF and limited available treatment of GN remained medical challenges today. This thesis was to explore the medical preventive strategy of AVF stenosis and the anti-oxidative therapeutic mechanism of stem cell on GN through population based and animal model studies.. 15.

(17) Chapter 2.. Material and Method. 2.1 A population-based Method for Evaluation of Statin on AVF Stenosis Prevention Data source The data was extracted from the Taiwanese NHIRD, which contains the healthcare utilization information of about 99% of the 23 million people enrolled in the universal National Health Insurance Program. The information kept in the NHIRD includes age, gender, residency area, income, diagnosis codes, and medications. We used the International Classification of Diseases, Ninth Revision (ICD-9) to define investigated comorbid diseases. The study was approved by the institutional review board at the National Health Research Institutes. Design and Study subjects We used a population-based retrospective cohort study design to evaluate the relationship between statin use and recreation of the vascular access in patients under hemodialysis. A regular hemodialysis cohort older than 20 years who received dialysis treatment for at least 3 months during 2000 to 2008 was selected in this study. From this cohort, we identified 40 459 study subjects who survived more than 2 years after starting hemodialysis, did not shift to peritoneal dialysis within 2 years after the dialysis commencement, and received their first vascular access operation within 1 year prior to or after the first dialysis. The status of statin exposure was scrutinized for 2 years after the vascular access creation. Those who had been prescribed statins for at least 30 days within 1 year were defined as statin users (n=11 297). The 16.

(18) first date of statin prescription was assigned as the index date. For those who took statins before the vascular access creation, the index date was assigned as the date of the vascular access creation. The statin users were then matched by age and gender to their nonuser counterparts (n=29 161) in a 1:1 ratio. Finally, 9 826 statin user and nonuser pairs were identified for further analysis. Of the study subjects, 16 700 (85%) received autogenous arteriovenous fistula (AVF) and 2 952 (15%) received arteriovenous graft (AVG).The index date of the statin nonusers was assigned as the same index date for each individual pair. The study subjects were followed through until vascular access recreation (the primary outcome), kidney transplantation, death, or December 31, 2008, whichever came first. History of comorbidities and medications From the NHIRD, we collected patients’ information about age, gender, residency area (city, township and rural area), income level (low, middle and high), and pre-dialysis comorbidities defined by the ICD-9 disease or procedure coding for at least 1 hospitalization or 2 ambulatory visits within 1 year due to some important chronic illnesses. The comorbidities investigated in this study included congestive heart failure, cerebrovascular. disease,. peripheral. vascular. disease,. diabetes,. atherosclerotic heart disease, other heart disease, chronic obstructive pulmonary disease, gastrointestinal disease, liver disease, dysrhythmia, cancer, percutaneous coronary interventions, and implantable cardioverter defibrillators / cardiac resynchronization therapy with defibrillator. In addition to the statins, other pre-dialysis medications used were 17.

(19) also. controlled. in. anti-inflammatory antihypertensive. this. drugs agents. study,. which. (NSAIDs),. included. acetaminophen,. (angiotensin-converting. enzyme. non-steroid aspirin, inhibitor,. angiotensin receptor blocker, alpha-blocker, beta-blocker, acetazolamide, calcium channel blocker and diuretics), anti-diabetic drugs (biguanide, sulfonylurea, alpha glucosidase inhibitor, thiazolidinedione, meglitinide, and insulin), morphine, warfarin, clopidogrel and digoxin. Statistical analysis Baseline characteristics of the study subjects were described as the frequencies with percentages for categorical variables and means with standard deviation for continuous variables. We also used 1:1 propensity score matching to recruit the similar two groups with known confounders to balance the different baseline characteristics between statin users and nonusers that might confound the outcomes. The Cox proportional hazards model was used to estimate the hazard ratio (HR) and 95% confidence interval (CI) for the risk of permanent HD access recreation compared between the statin users and nonusers. The cumulative hazards of vascular access recreation over time were calculated using the Nelson-Aalen method to adjust the covariates adopted in the Cox proportional hazards models. We used log–log survival plots for all time-independent covariates to test the proportional hazard assumption and confirmed all assessed graphs did not violate the assumption. Study entry was defined as the index date. Observations were censored on December 31, 2008, the date patients died, or the date patients received kidney transplantation, whichever occurred first. The covariates adjusted 18.

(20) in the multivariate Cox hazards models included age, gender, residency area, income level, and pre-dialysis medication use and comorbidities. During the follow-up period, because the number of death (698 [21.9%] for statin nonusers and 769 [19.9%] for statin users) and the number of those who received kidney transplant (108 [3.4%] for statin nonusers and 220 [5.7%] for statin users) could not be ignored, we further extended the Cox hazards models for competing risk adjustment, by considering the subdistribution hazard. All p values were 2-sided, and the p value < 0.05 was considered to be a significance level. All analysis was conducted using the SAS version 9.3 (SAS Institute Inc, Cary, North Carolina).. 19.

(21) 2.2 Animal model to Exploration of the Anti-oxidative Mechanisms in Therapeutic Application of Mesenchymal Stem Cell for Glomerulonephritis Animals Female Wistar rats (220-260 g) were housed at the Experimental Animal Center, National Taiwan University. All the surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine and College of Public Health and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997).. Cell Preparations (MSC isolation, characterization, and culture) Femora from Wistar rats (BioLASCO Taiwan Co Ltd, Taipei, Taiwan), 8 to 10 weeks of age, were removed, and the soft tissues were detached aseptically. Bone marrow was extruded by inserting a 23-gauge needle into the shaft of the bone and flushed out with basal medium (α-minimal essential medium [α-MEM], Gibco-BRL, Gaithersburg, Maryland). Isolation of MSCs was performed according to similar procedures as described in a previous study 33. Briefly, mononuclear cells isolated from the bone marrow aspirates by the density gradient centrifugation method were suspended in complete culture medium (CCM: α-MEM supplemented with 16.6% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine) and seeded in plastic dishes. After 24 hours of the initial culture, nonadherent cells 20.

(22) were removed by a change of medium and irrigation of the culture. The culture typically reaches 65% to 70% confluence within 6 to 8 days and reaches subconfluence at 9 days, when the cells (passage 0) were harvested for further subcultures. Starting from passage 1, the cells were seeded at 100 cells/cm2 and grown in CCM with a medium change twice per week. For hypoxic cultures, cells were cultured in a gas mixture composed of 94% N2, 5% CO2, and 1% O2. 34. . For maintenance of the. hypoxic gas mixture, an incubator with 2 air sensors, one for CO2 and the other for O2, was used; O2 concentration was achieved and maintained using delivery of N2 gas from a tank containing pure N2. If the O2 concentration rose above the desired level, N2 gas was automatically injected into the system to displace the excess O2.. Experimental Model and Design We injected 0.2 mL of phosphate-buffered saline containing 250 μg anti-Thy1 (CD90) monoclonal antibody（Cedarlane，Ontario，Canada） to rats via a jugular vein under sodium pentobarbital anesthesia (50 mg/kg, i.p.) at day 0 and gave 0.2 mL of saline injection to the jugular vein as a control group. This method for induction of acute glomerulonephritis had been reported previously. 35. . All the rats had an. elevated urinary protein level, mesangial cell lysis and proliferation and slerosis appearance after day 5 to day 7 of injury. For direct MSC or HMSC delivery, an intrarenal arterial catheter was performed via the left femoral artery. Under avertin anesthesia (400 mg/kg, ACROS ORGANICS, NJ, USA), one PE10 tubing was introduced into the left 21.

(23) renal artery from the left femoral artery via the aorta. 1-5x105 MSC or HMSC were administered via this catheter. The grouping and experimental design were shown in Figure 1.. Tracking of Intrarenal Arterial Injected ASCs in Rat Kidneys To ascertain the MSC expression in the kidney, we infused MSCs containing a green fluorescent protein (GFP) into the left kidney and examined the GFP expression in rat kidneys one hour later. The sections were examined under UV light for the detection of fluorescence around the glomeruli, arterial lining cells and tubular cells. Immunochemical stains with primary antibodies against MSC CD44 (MCA643GA, Serotec) were also performed for identification of MSCs in kidneys.. Measurements of proteinuria 24-hour urine samples were collected from all experimental rat with free access to water. Urinary protein concentration was determined by Bio-Rad protein assay (Bio-Rad Laboratories, München, Germany).. Renal Pathology for Evaluation of Glomerulonephritis Severity Renal histology was evaluated using H&E, periodic acid-Schiff and Masson-stained 5-µm paraffin sections based on at least 50 glomeruli per kidney. section.. PAS-stained. sections. were. examined. for. glomerulosclerosis. One hundred glomeruli per section were randomly selected and the degree of glomerular damage assessed using a semi-quantitative scoring method: grade 0, normal glomeruli; grade 1, 22.

(24) sclerotic area up to 25% (minimal sclerosis); grade 2, sclerotic area 25 to 50% (moderate sclerosis); grade 3, sclerotic area 50 to 75% (moderate-severe sclerosis); grade 4, sclerotic area 75 to 100% (severe sclerosis). The glomerulosclerotic index (GSI) was calculated using the following formula: GSI = (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)/(n0 + n1 + n2 + n3 + n4), where nx is the number of glomeruli in each grade of glomerulosclerosis 36. Immunohistochemistry assessing intrarenal inflammation, stress index protein, autophagy, apoptosis and pyroptosis Immunohistochemical staining was performed on formalin fixed, paraffin-embedded kidney sections with ED-1 (clone ED-1, Serotec, Oxford, UK), primary antibodies against MSC CD44 (MCA643GA, Serotec), GRP-78 (1:500; Santa Cruz Biotechnology, Texas, USA), LC3-II (1:1000; MBLI Corporation, Woburn, USA), caspase 1 (. Epitomics,. transferase. California,. –mediated. USA),. terminal. digoxigenin-deoxyuridine. deoxynucleotidyl nick-end. labeling. (TUNEL, BioVision, California, USA), collagen IV (Abcam, Cambridge, UK) and 4-hydroxynonenal (4HNE, Bioss, Massachusetts, USA) . Briefly, paraffin sections of kidneys were deparaffinized with xylene and rehydrated in an alcohol series and water. Kidney sections were subjected to antigen retrieval and were blocked with a peroxidase-blocking reagent. Sections were incubated with the primary antibody overnight at 4°C. After washing, the kidney sections were incubated with Envision system-horseradish. peroxidase-labeled. polymer. (Dako,. Glostrup,. Denmark) for 1 hour at room temperature. The sections were visualized 23.

(25) with. 3,3’-diaminobenzidine. tetrahydrochloride. (Dako,. Glostrup,. Denmark) and counterstained with hematoxylin. Apoptotic cells in the kidney were identified by TUNEL staining. The TUNEL method for the in situ apoptotic assay was performed according to the method of Gavrieli et al. 37. with minor modifications. The number of positive ED1 and. TUNEL stained cells was evaluated by counting stained cells per high power field (× 400) in at least 20 randomly selected fields. The percentage of positive stained area in the GRP-78, LC3-II, caspase1 and collagen VI assays was analyzed by Adobe Photoshop 7.0.1 imaging software analysis.. Western Blot and nuclear extraction for Elucidate the Effects of MSCs and HMSCs on the Nrf2/Keap1 System and intrinsic anti-oxidant reponsive elements Western blot analysis was performed on whole tissue to detect the levels of renal anti-oxidant responsive element proteins, and nuclear extractions were done to detect nuclear factor (erythroid-derived 2)-like 2(Nrf2), nuclear factor kappa B (NFkB) expressions. Briefly, tissues were grinded to powder in liquid nitrogen. Then the tissue powder was lysed in RIPA Buffer (Bio Basic, NY, USA) supplemented with a protease inhibitor (Roche, Basel, Switzerland) for 10 minutes at 4 oC. The tissue homogenate was centrifuged at 14,000 rpm for 30 minutes. After centrifugation, supernatant was collected into a new eppendorf tube. The concentration of protein was measured by a BCA protein assay kit (Thermo Scientific, Rockford, USA). A protein sample (80 μg) was 24.

(26) mixed with 1×sample buffer and was boiled for 3 minutes. Protein samples were resolved in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, Billerica, USA). The blot was blocked with Hyblock (Hycell, Taipei, Taiwan) for 1 minute, and incubated with primary antibodies overnight at 4oC. After washing three times with TBST, the blot was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 hour. Detection of signals was performed by Western Lightning plus-ECL (PerkinElmer, Waltham, USA). Nuclear extracts were obtained using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific) according to the manufacturer’s instructions. Primary antibodies included Mn-superoxide dismutase (MnSOD, 1:1000; Enzo Life Sciences, NY, USA), Cu/Zn-SOD (1:500;Millipore, Billerica, USA), catalase (Assay Designs, Ann Arbor, USA), heme oxygenase 1 (BioVision, Milpitas, California, USA), Nrf2 (Cayman, Ann Arbor, USA), NFkB (Santa Cruz, Dallas, USA), glutamate-cysteine ligase catalytic subunit (GCLC, Abcam, Cambridge, UK ), glutamate-cysteine ligase modifier subunit (GCLM, Abcam, Cambridge, UK ), glutathione peroxidase 1 (GPX1, Abcam, Cambridge, UK ) ,β-actin (1:5000; Sigma-Aldrich, Missouri, USA), γ-tubulin (Abcam, Cambridge, UK) and Lamin A/C (Abcam, Cambridge, UK) as a control for nuclear extraction . Secondary antibodies included HRP-conjugated goat anti-mouse IgG, HRP-conjugated rabbit anti-goat IgG, and HRP-conjugated goat anti-rabbit IgG (all at 1:10000; all from Southern Biotech Laboratories, Birmingham, USA). 25.

(27) Statistic analysis The soft Scion Image β3b（Scion Corporation，Frederick，MD）was used to quantify western blot density. We used one-way ANOVA and Bonferroni SPSS/Windows SPSS Inc. ， Chicago ， Il ） to analyze experimental data. The difference from the experimental was assessed by one way ANOVA. GraphPad PRISM® 3.0（GraphPad Software Inc.，San Diego，CA）and Sigma Plot 8.0 were used for figures preparation.. 26.

(28) Chapter 3. Results. 3.1 Therapeutic Effect of Statin on AVF Stenosis 3.1.1 Study subjects’ characteristics The comparisons of demographic characteristics, comorbid diseases and medication exposure between the statin users and non-users are listed on Table 1. The mean age of statin users was 60.1±14.8 years, and 57.7% were women. The matched non-users had almost identical mean age and gender percentages. A significant difference found between statin users and non-users is that a higher percentage of the control group lived in rural areas. Compared with statin non-users, the statin users were more likely to be co-morbid with congestive heart failure, cerebral vascular accident, peripheral vascular disease, diabetes and atherosclerotic heart disease. However, they were less likely to have had liver disease and cancers. Statin users were also more likely to be treated with aspirin, clopidogrel, warfarin, anti-diabetic medicine, and all categories of antihypertensives. 3.1.2 Association between statin use and vascular access recreation After a mean follow-up period of 3.34 years in statin user group and 3.79 years in non-user group, the incidence rates of VA recreation were 9.83 and 10.23 per 100 person-years, respectively, resulting in a crude hazard ratio of 0.98 (95% CI, 0.93 to 1.03) and an adjusted hazard ratio of 0.79 ( 95% CI, 0.74 to 0.84; P < 0.001). The effect of statins on VA recreation was calculated using Cox regression model and is demonstrated on Figure 2. As for the AVFs, the crude and adjusted hazard ratios of VA recreation were 0.94 (95% CI, 0.89 to 27.

(29) 0.99; P < 0.05) and 0.74 (95% CI, 0.69 to 0.80; P < 0.001). Meanwhile, for the patients with arteriovenous graft (AVG), the crude and adjusted hazard ratios were 0.99 (95% CI, 0.90 to 1.09) and 0.94 (95% CI, 0.83 to 1.07). The hazard ratios of vascular access recreation for statin users and non-users are listed on Table 2. To further verify if the statins’ protective effect met the dose-response relation, prescription days were used as a dose indicator; statin users were then stratified into quartiles by prescription days. The adjusted hazard ratios for VA recreation in the AVF patients from Q1 to Q4 were 0.82 (95% CI, 0.75 to 0.90), 0.72 (95% CI, 0.65 to 0.80), 0.72 (95% CI, 0.65 to 0.80), and 0.67 (95% CI, 0.60 to 0.75). In AVG patients, the hazard ratios were 1.02 (95% CI, 0.86 to 1.21), 1.13 (95% CI, 0.96 to 1.33), 0.85 (95% CI, 0.72 to 1.01), and 0.74 (95% CI, 0.62 to 0.89). 3.1.3 Other factors associated with vascular access recreation Using a multivariate survival model, the hazard ratios of other factors including age, sex, comorbid diseases, and other medicines prescribed before the index date are listed on Table 3. Older age, female, peripheral vascular disease and gastrointestinal diseases were linked to increased risk for VA recreation in the AVF patients. Of all the medicines covered in this study, ARBs were associated with a reduced risk (HR, 0.92, 95%CI, 0.87 to 0.98; P<0.05); however, loop diuretics, thiazides, alpha glucosidase inhibitors and digoxin were associated with increased risks for VA recreation in AVF patients. In AVG patients, thiazides and insulin were associated with increased 28.

(30) risks for VA recreation. 3.1.4 The protective effect on VA recreation among various statins To determine whether the protective effects on vascular access recreation differ across the various statins, a subgroup analysis was performed. Four out of six statins showed significant protective effects on vascular access, but lovastatin 0.90 (95% CI, 0.71 to 1.14) and fluvastatin 0.84 (95% CI, 0.70 to 1.02) failed to demonstrate statistically significant protective effects. The crude and adjusted hazard ratios for vascular access recreation across different statins are shown on Table 4. 3.1.5 Propensity score matched cohort, time dependent analysis and competing risk survival analysis To minimize potential bias in the differences between statin users and non-users, we created a propensity score matched cohort, containing 3,864 matched pairs. The baseline characteristics of the matched cohort are shown on Table 5. The R square of logistic regression model used for creating propensity scores is 0.4254 by Cox & Snell R-square or 0.6254 by Nagelkerke R-square. The cohort was then used for the following 3 analyses to test the protective effect of statins on permanent HD access. A Cox regression model was used to calculate the statin-users’ hazard ratios for permanent HD access recreation. As shown in Table 6, the hazard ratios for statin-users were 0.88 (95%CI; 0.80-0.97) for AVF, 1.09 (95% CI; 0.93-1.29) for AVG, and 0.94 (95% CI; 0.86-1.01) for across both types (Table 7). For further clarification on the association of statin use and permanent HD 29.

(31) access recreation, a time-dependent Cox analysis was performed and the results are provided online in supplementary Table 8. The hazard ratios for permanent HD access recreation for statin-users were 0.80 (95%CI; 0.72-0.90) for AVF, 0.84 (95% CI; 0.70-1.00) for AVG and 0.80 (95% CI; 0.73-0.88) for across both types. Lastly, competing risks adjusted survival analysis was done and the results are shown online in supplementary Table 9. Death and renal transplantation were taken as the competing risks. The adjusted hazard ratios for permanent HD access recreation for statin-users were 0.87 (95%CI; 0.79-0.96) for AVF, 1.07 (95% CI; 0.90-1.25) for AVG and 0.92 (95% CI; 0.85-1.00) for across both types.. 3.2 Theapeutic Effects of Mesenchymal Stem Cell on GN 3.2.1 Recruitment of MSCs and HMSCs into diseased kidneys Detection of fluorescence in the kidneys after intrarenal arterial administration of fluorescent MSCs is illustrated in Figure 3 A. High levels of GFP were visualized under UV light and the fluorescence was found in the diseased kidney sections but not in the normal kidney (Figure 3 B-2C). Fluorescence cells were found at glomeruli in the MSC treated groups. CD44 positive cells were found in renal proximal tubule (PT), distal tubule (DT) and glomeruli of MSC or HMSC treated kidneys, but not in the control kidney (Figure 3D-F). These results confirm the recruitment of MSCs and HMSCs in injured kidneys. There was no significant difference in trafficking CD44 positive cell numbers between the MSC and HMSC groups. 30.

(32) 3.2.2 Administration of MSCs or HMSCs ameliorates disease severity in the rat GN model The success of acute glomerulonephritis induction in the rat model was confirmed by elevated urine protein concentrations and typical characteristics in the histopathologic examination of kidneys, including mesangial cell proliferation, mesangialysis and sclerosis appearance 5 to 7 days after injury. The evaluation of the severity of glomerulonephritis and proteinuria among the experimental groups are shown in Figure 4. In H&E stains of both MSC and HMSC treated kidneys, the severity of glomerulosclerosis was attenuated and decreased numbers of inflammatory cells in glomeruli were found (Figure 4 A). The glomerulosclerosis index by PAS stains sections (Figure 4 B) are 0.01 for the control, 0.5 for anti-Thy1 with placebo, 0.19 for 1×105 MSC treated group, 0.16 for 2×105 MSC treated group, 0.03 for 5×105 MSC treated group, 0.03 for 1×105 HMSC treated group, and 0 for both 2 and 5 ×105 MSC treated groups (Figure 4 E). The severity of glomeruli fibrosis was also ameliorated by MSC and HMSC administration, which is shown in the Masson stains (Figure 4 C). Both normoxic and hypoxic MSC reduced the severity of proteinuria in GN rats. The level of proteinuria reduction did not differ among 1×, 2×, 5×105 MSC infusion, but there exists a trend in HMSCs with a higher cell number having a lower proteinuria (Figure 4 D).. 31.

(33) 3.2.3 MSCs and HMSCs demonstrate a consistent reduction in macrophage/monocyte. infiltration,. stress. index. protein. accumulation, autophagy, apoptosis and pyroptosis index proteins The representative pictures of immunohistochemical stained sections and semi-quantitative analyses of ED1, GRP78, LC3-II, caspase 1, TUNEL and collagen IV in the study groups are presented in Figure 4. With the semi-quantitative analysis of the histochemical stained kidney sections for ED-1, anti-Thy1.1 administration markedly increased the numbers of ED-1positive cells in the glomeruli. Infusion of MSCs reduced the number of ED-1 positive cells that infiltrated in the kidneys, and infusion of HMSCs had a better reduction of the ED1 positive cell number (Figure 5 A1-A5) than MSCs. Similar results were found for GRP78, LC3 II, caspase 1, TUNEL and collagen IV. These sections show that anti-Thy1 .1 administration increased stress index protein (GRP78) accumulation (Figure 5 B1-B5), autophagy index protein (LC3II) detection (Figure 5 C1-C5), pyroptosis (caspase 1+. cells (Figure 5 D1-D5), apoptotic (TUNEL+) cells (Figure 5. E1-E5), and collagen IV accumulation (Figure 5 F1-F5). MSC infusion ameliorated the increase in GRP78, LC3 II, caspase 1, TUNEL and collagen IV in kidneys after anti-Thy1.1 infusion. HMSC infusion had a greater effect on ameliorating inflammatory cell infiltration, stress protein accumulation, apoptotic cells and autophagy in glomeuli than MSC.. 3.2.4 HMSCs promote nuclear Nrf-2 expression, reduce NF-kB 32.

(34) expression, rescue ROS enzymatic scavengers and elevate anti-oxidative response element proteins Figure 6 demonstrates the ROS injury index and the intrinsic anti-oxidative mechanisms expression among the experimental groups. The accumulation of ROS by 4 HNE histochemical stain among the groups are demonstrated in Figure 6 A1-A4, the semi-quantitative analyses of these sections revealed that anti-Thy1.1 infusion greatly increased ROS. In the treatment groups, only a higher HMSC cell number ameliorated the ROS accumulation (Figure 6 A5). ROS enzymatic scavenger expressions including MnSOD, Cu/ZnSOD and catalase were significantly reduced by antiThy-1.1 infusion. These enzymes were rescued by HMSC transplantation, but not by MSCs (Figure 6 B1-B3). As for Nrf2 signaling, the master regulator of ROS injury, the results showed that nuclear Nrf2 expression was not changed, while NF-kB expression was elevated by anti-Thy1.1 infusion. Infusion of HMSCs significantly increased nuclear Nrf2 and reduced NK-kB expressions in anti-Thy1.1 treated rat kidneys. Infusion of MSCs had a lesser effect on nuclear Nrf2 elevation and NF-kB expression. However, the effect did not reach statistical significance (Figure 6 C1-C3). Semi-quantitative detection of anti-oxidative response element (ARE) protein expression (GCLC, GCLM and GPX) by Western blotting in the kidneys are shown (Figure 6 D1-D3). These proteins had a consistent expression trend in the experimental groups. Briefly, the anti-Thy1.1 antibody infusion reduced ARE protein expressions, and hypoxic preconditioning MSCs rescued these protein expressions. Administration of MSCs did not significantly 33.

(35) increase protein expression, though it tended to increase their levels.. 34.

(36) Chapter 4. Discussion and Conclusion. 4.1 Statins Possess a Dose-responsive Effect of Protecting AVF from Stenosis The study demonstrates that statins are associated with an overall 21% risk reduction for recreation of vascular access for long term hemodialysis. The risk reduction effect of statins was found in the autogenous AVF group (HR 0.74, 95% CI, 0.74 to 0.84), but not generally in the AVG group (HR 0.94, 95% CI, 0.83 to 1.07). The protective effect in AVF was found to respond to a dose-index, surrogated by days of prescription. While a significant protective effect was found in the AVG patients with the highest range of prescription days. It is reasonable to assume that statins could also exert a milder beneficial effect on vascular access in AVG patients than in AVF patients. The finding of such a beneficial effect of statins is first reported on the basis of a large scale nation-wide population study. The randomized controlled clinical trial is the gold standard for confirming the efficacy of an intervention.. However, a large. observational study provides a unique opportunity to study possible effects of a pharmacological intervention, often without the sample size and ethical limitations of a clinical trial. The potential confounding factors are usually problematic and should be collected and adjusted in such an analysis. In the present study, we further recruited a propensity score matched cohort and re-calculated the hazard ratios by multivariate Cox regression to minimize the potential bias resulting from baseline differences. The results indicate that statins prevent AVF 35.

(37) from permanent. HD. access. recreation.. Mortality. and. renal. transplantation cases were censored in our survival analysis of permanent HD access, which could result in potential bias from these competing risks. The results of competing risk analysis also approve the protective effect of statins on AVF. Furthermore, the statin users are more frequently co-morbid with peripheral vascular disease and diabetes, which may increase risk of AVF failure. The protective effect is thus potentially underestimated in this non-intentional study. Taken these findings together, we conclude that statin users had a lower permanent HD access recreation hazard for AVF. Statins have been repeatedly shown to possess beneficial effects on the prevention of AVF stenosis in animal and cellular studies. Simvastatin had been shown to reduce venous neo-intimal hyperplasia and vascular smooth muscle proliferation by decreasing the expression of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase 9 (MMP-9)13. Rosuvastatin had been demonstrated to increase the blood flow in the venous limb of AVF in diabetic rats, which was associated with the anti-inflammatory effect and resulting from endothelial function improvement12. Atovastatin has been shown to decrease proliferation, migration, and the passage of human smooth muscle cells (HSMC) across a matrix barrier38. Pravastatin was reported to reduce intimal hyperplasia in mice that was associated with decreased vascular smooth muscle cell (VSMC) proliferation and platelet-derived growth factor-induced VSMC migration and inhibited macrophage migration39. Statins have been almost uniformly 36.

(38) demonstrated to be beneficial for vascular access patency in basic research but surprisingly this has not been supported by clinical studies. The present study provides a pharmaco-epidemiologic link between basic research and clinical evidence. Several previous studies have debated the relationship between statins and outcomes of VA for hemodialysis. Righette et al showed that statins possess a beneficial effect on AVF survival but it was not clear about the generalizability of this small-scale study which was conducted in a single center. 17. . Saran et al evaluated the association. between specific medicines and AVF outcomes 8. The study concluded that statins are not associated with better VA outcomes based on Dialysis Outcomes and Practice Patterns Study (DOPPS), but the study was confined to United States patients. Another study by Andreucci et al based on DOPPS using US, European and Japanese data found the futility of statins on VA patency. 40. . Yevzlin et al evaluated the. relationship between medication use and VA patency using the cohort of US Renal Data System Dialysis Mortality and Morbidity Wave II study, and also reported that statins were not associated with better vascular access patency. 10. . These hemodialysis cohort studies had. similar design to the present study but failed to demonstrate the beneficial effect of statins. Apart from the ethnic and sampling differences, the critical difference between the current study and others is the definition of statin users. Contradictory to previous studies, the current study is based on a national, large population dataset and defines statin users by post VA creation statin prescription. By 37.

(39) demonstrating a significant risk reduction effect and dose responsive relationship between post VA statin use and VA recreation, our findings provide solid evidence that statins exert a beneficial effect on AVF patency and suggest the needs of further clarification of the target patients who would benefit most. Four out of six statins investigated in the current study demonstrated a significant risk reduction effect on the AVF failure rate. Meanwhile, lovastatin and fluvastatin showed a risk lowering trend but not one that was statistically significant. Both of them are categorized as moderate to low potency 41. This finding suggests that the beneficial effect on VA outcomes is a universal but that the effect depends on the statin’s potency categorization. The differential effects among individual statin should be clarified in future studies. Older age and being female are demographic features associated with increased VA recreation for AVF in this study. Older age was uniformly recognized as one risk factor of unassisted and assisted VA failure in the previous studies 42-44. Female patients were shown to have higher AVF failure rates in several recent studies. 45. , but not in other. studies 4,8,14,16. One meta-analysis reported that female gender was not a significant risk factor of VA failure. 46. . We found that peripheral. vascular disease was associated with increased VA recreation, which is compatible with previous studies. 4,43-45. . However, congestive heart. failure, cerebral vascular disease and diabetes were not identified as significant risk factors of AVF or AVG failure in this analysis. These results may indicate that only the more severe cardiovascular disease is 38.

(40) an important risk factor of vascular access stenosis. Future application of statins for AVF protection should take age, gender and the severity of cardiovascular disease into consideration. Regarding to VA protection, angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor antagonists (ARB) are also drugs of choice for their pleiotrophic effects. Saran’s study based on DOPPS reported that ACEIs were associated with better assisted patency in AVFs, that calcium channel blockers (CCBs) were associated with better unassisted patency in AVGs and that aspirin was associated with better assisted patency in AVGs 8. Our findings suggest a benefit of ARBs in AVF patients. No other antihypertensive agents including ACEI and CCB were found to be beneficial for reducing VA failure. In another retrospective study, ARB was also noted to be beneficial for AVF patency in ACE DD genotype patients. 47. .. Anti-platelets were reported by a meta-analysis to increase the short term patency of AVFs and AVGs 48. In our analysis, neither aspirin nor clopidogrel was related to a decreased risk of VA recreation. To elucidate the relationships between these medications and long term VA outcomes mandates further laboratory and clinical studies. In summary, the findings suggest that statins possess a dose-responsive effect of protecting AVF from stenosis for patients undertaking long term hemodialysis. The beneficial effect on VA outcomes is a universal class effect but the effect size is dependent on the statins’ potency. Prescribing statin therapy for the hemodialysis patients may be one of the most convenient interventions to prevent 39.

(41) their vascular access from failure, which will greatly promote the patients’ quality of care and reduce health cost. These results have important therapeutic implications for future prospective randomized control. studies.. Further. investigations. towards. exploring. anti-inflammatory and anti-oxidative effect of statins on the prevtion of AVF stensosis should be addressed.. 4.2 Hypoxic Preconditioning Promote the Ability of Stem Cell to Activate Intrinsic Anti-oxidative Defense Systems In the present study, we demonstrated that intra renal arterial administration of either MSCs or HMSCs ameliorates the severity of glomerulosclerosis and levels of proteinuria in the anti-Thy-1.1 induced rat GN model. HMSCs showed a better therapeutic effect than MSCs on the amelioration of glomerulosclerosis, inflammatory cell infiltration, endoplasmic reticulum stress and three types of programmed cell death, apoptosis, autophagy and pyroptosis. In addition, hypoxic preconditioning enabled MSCs to activate nuclear Nrf2 expression and rescued ROS scavengers in kidneys after Thy-1.1 lesion. Our results indicate that hypoxic preconditioning enhances the therapeutic effects of MSCs through multiple mechanisms including increasing intra-nuclear Nrf2 expression in the target organ. Several studies have demonstrated that stem cells derived from various origins ameliorate kidney injury in GN animal models. In addition, MSCs have the ability to cause anti-inflammation, antifibrosis and inhibition of cell death and this is the basis for cell therapies 40. 49-55. ..

(42) In this study, both MSCs and HMSCs showed anti-inflammatory effects by decreasing macrophage/monocyte infiltration in glomeruli of treated kidneys and by inhibiting NF-κB translocation into nucleus. A significant further decrease in numbers was found in the HMSC treated group, indicating that hypoxic preconditioning is an effective strategy to promote the anti-inflammatory effect. The results of Masson stains confirm the therapeutic effect of MSCs on anti-fibrosis, and this too was. further. enhanced by hypoxic preconditioning. We also. demonstrated that intra-glomerular cell apoptosis and autophagy were decreased by MSC infusion, and a further reduction was noted in the HMSC treat group. The therapeutic anti-inflammation, anti-fibrosis, anti-apoptosis and antipyrotosis mechanisms of MSCs are compatible to previous studies. 56,57. . Hypoxic preconditioning showed a consistent. enhancement of these therapeutic mechanisms seen in MSCs. Another important finding in this study is that hypoxic preconditioning enabled MSCs to increase nuclear Nrf2 and decrease NF-κB expression. Reactive oxygen species (ROS) generated by the immune reaction is believed to be one of the crucial mechanisms that cause injuries to glomeruli in the GN. The Keap1–Nrf2 (Kelch-like ECH-associated protein 1) pathway signaling and the anti-oxidant responsive elements play a central role in protection against oxidative stresses. In this study, anti-Thy1.1 lesion was found to suppress the ROS scavenger system by decreasing ROS scavenger expression and elevating nuclear NF-κB, which contributed to inflammatory cytokine cascades. The nuclear Nrf2 expression remained unchanged after 41.

(43) anti-Thy1.1 lesion, indicating that the master intrinsic anti-oxidative regulator, Nrf2/Keap1 pathway, was not triggered. Nrf2 expression levels increased with MSC transplantation to GN rats. However, it failed to reach a significant difference. At the same time, ROS enzymatic scavengers and other ARE proteins were not rescued. With HMSC transplantation, nuclear Nrf2 expression increased and ROS scavengers and ARE proteins were rescued in diseased kidneys. From these results it seems that conventional MSC transplantation may not trigger enough Nrf2 pathway signaling activity to enhance ARE protein expressions. Therefore, hypoxic preconditioning enabled MSCs to activate the Nrf2 pathway signaling and to rescue the ROS scavengers in kidneys which were suppressed by the anti-Thy1.1 infusion. Our findings provide evidence supporting the viewpoint of Ezquer et al.’s study 58, in which MSCs were believed to possess the main enzymatic mechanisms to detoxify the reactive species and to prevent oxidative damage in rat nephritis based on some in vitro cellular studies 59,60. The major challenges that underlie the application of stem cell therapy to GN patients are safety concerns and efficacy issues. Enhancing the anti-oxidative effect of stem cells is one promising strategy to promote their efficacy for inflammatory or oxidative stress related disease such as GN. Nrf2 is a crucial regulator of the antioxidant defense system and governs the expression of genes associated with redox homeostasis. The beneficial effects of targeting the Nrf2 pathway for nephritis have been demonstrated in animal studies through tranduction of the OR1 gene to enhance antioxidation 61 42.

(44) or via Keap 1gene knockout to activate the Nrf2 system. 62,63. .. Bardoxolone, an Nrf2 activator, has been shown to improve renal functions in type 2 diabetes with CKD patients in a human phase 2 trial 63. , though it had cardiovascular safety issues. 64. . The current study. shows that HMSC transplantation is an effective measure to enhance Nrf2 pathway signaling and therapeutic effects in damaged kidneys.Hypoxic preconditioning, i.e. stem cell cultured in an ischemic condition which mimics the bone marrow niche environment, is a common way to preserve stemness, enhance homing and increase efficacy. of. stem. cell. therapy.. In. cellular. studies,. hypoxic. preconditioning has enhanced stemness and expanded cell numbers. 65. .. Hypoxic mimetic preconditioning enhances MSC migration and prolongs kidney retention through promoting CXCR4 expression. 66. .. The results of this study link hypoxic preconditioning to anti-oxidative injury. We demonstrated that HMSCs promote Nrf2 signaling and resultant ARE protein elevation. The cytokines involved in the Nrf2 pathway signaling activation promoted by HMSC need to be further investigated. Stem cell homing to injured tissue is crucial because the therapeutic application of stem cell therapy is predicated on the transplanted cells migrating and participating in tissue repair. Enhancing the homing capabilities of stem cells can promote their therapeutic efficacy. In. the present study, intrarenal arterial. administration of MSCs or HMSCs leaded to CD44 staining in the glomeruli. The advantage of intrarenal arterial administration can 43.

(45) demonstrate the direct delivery and location of stem cells to the kidney and prevent the risk of stem cells trapping in the lung or other non-target tissue/organ by systemically intravenous administration. The average stem cell numbers trafficked in glomeruli increased with hypoxic preconditioning and higher infused cell numbers. In previous studies, hypoxic precondition have been associated with increased CXCR4, CX3CR1 expression in a cellular study. 31. . CXCR4 and. CX3CR1 respond to SDF-1α, activate the Akt signal pathway elevate MMPs. 68. 67. and. contributing to transmigration. Our findings confirm. that hypoxic preconditioning is an effective strategy to enhance the homing effect of MSCs in the rat GN model. There are some limitations to the current study. First, we only investigated the Nrf2 pathway and ARE expressions in the regulation of stem cell antioxidant status. Functions of other oxidative stress-related pathways, such as PI3K/Akt and FoxO/TXNIP need further elucidation. Secondly, mechanisms that influence the enhanced repairing efficacy of stem cells after transplantation were not fully elucidated. Further studies focusing on the cytokines involved in the anti-oxidant enhancement are needed. Third, we used an acute GN model in this study. Whether these results can be applied to chronic GN, mandates further investigations. As well as enhancing the anti-inflammatory, anti-endoplasmic reticulum stress, anti-fibrosis, anti-apoptosis, anti-autophagy and anti-pyroptosis properties, anti-oxidative mechanisms also play a role in. the. therapeutic. effect. of 44. mesenchymal. stem. cells. on.

(46) glomerulonephritis. Hypoxic preconditioning is one effective strategy to activate intrinsic anti-oxidative defense systems by promoting the Nrf2 pathway signaling, rescue ROS scavengers and increase anti-oxidative responsive element proteins. The results of this thesis provide evidence supporting therapeutic options to medical intervention for permanent hemodialysis vascular access failure and cell therapy for glomerulonephritis.. 45.

(47) Table 1 Demographic characteristics, comorbid diseases and medications exposure between statin users and nonusers. Follow up time (year) Means (SD) Age (year) Means (SD) Sex (n, %) Male Female Location (n, %) City Township Rural area Comorbidity CHF CVA PVD DM ASHD Other heart disease COPD GI disease Liver disease Cancer Medication use NSAID Aspirin Clopidogrel Warfarin ACEI ARB Beta-blocker Non-DHP CCB DHP CCB Biguanide. Statin nonusers. Statin users. P value. N=9,826. N=9,826. 3.79. (2.76). 3.34. (2.34). <.0001. 60.0. (11.6). 60.1. (11.5). 0.8989 1.0000. 4157 5669. (42.3) (57.7). 4157 5669. (42.3) (57.7) <.0001. 2398 3001 4427. (24.4) (30.5) (45.1). 2779 3328 3719. (28.3) (33.9) (37.8). 774. (7.9). 1061. (10.8). <.0001. 1286 629 3941 2284 1155 1450 2884 1424 695. (13.1) (6.4) (40.1) (23.2) (11.8) (14.8) (29.4) (14.5) (7.1). 2152 985 7113 3593 1256 1436 2874 1033 549. (21.9) (10.0) (72.4) (36.6) (12.8) (14.6) (29.3) (10.5) (5.6). <.0001 <.0001 <.0001 <.0001 0.0281 0.7778 0.8755 <.0001 <.0001. 6970 2676 436 217 3558 3213 4675 1476 7110. (70.9) (27.2) (4.4) (2.2) (36.2) (32.7) (47.6) (15.0) (72.4). 7228 4607 1335 264 4244 5189 6123 1904 8278. (73.6) (46.9) (13.6) (2.7) (43.2) (52.8) (62.3) (19.4) (84.3). <.0001 <.0001 <.0001 0.0300 <.0001 <.0001 <.0001 <.0001 <.0001. 890. (9.1). 1920. (19.5). <.0001. 46.

(48) Sulfonylurea Alpah glucosidase inhibitor Thiazolidinedione Meglitinide Insulin Statin. 1918 373 314 859 2572 0. (19.5) (3.8) (3.2) (8.7) (26.2) (0.0). 3956 1375 1299 2320 4840 5075. (40.3) (14.0) (13.2) (23.6) (49.3) (51.7). <.0001 <.0001 <.0001 <.0001 <.0001 <.0001. Abbreviations: CHF, congestive heartfailure; CVA, cerebral vascular accident; PVD, peripheral vascular disease; DM, diabetes mellitus; ASHD, atherosclerotic heart disease; COPD, chronic obstructive pulmonary disease; GI, gastrointestinal; NSAID, non steroid anti-inflammatory drug; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor blocker; DHP, dihydropyridine; CCB, calcium channel blocker.. 47.

(49) Table 2 Crude and adjusted hazard ratio of vascular access recreation for statin users and nonusers. Overall Statin nonusers Statin users AVF Statin nonusers Statin users AVG Statin nonusers Statin users. N. Event. Person-years Incidence (%). Hazard ratio. Adjusted hazard ratio. 9826 9826. 3665 3355. 37275.01 32780.10. 9.83 10.23. 1.0 0.98 (0.93-1.03). 1.0 0.79 (0.74-0.84)**. 8519. 2882. 33823.15. 8.52. 1.0. 1.0. 8181. 2431. 28727.13. 8.46. 0.94 (0.89-0.99)*. 0.74 (0.69-0.80)**. 1307 1645. 783 924. 3451.86 4052.97. 22.68 22.80. 1.0 0.99 (0.90-1.09). 1.0 0.94 (0.83-1.07). *p<0.05; **p<0.001 ※Covariates adjusted in multivariate models included income, age, sex, area, CHF, CVA, PVD, DM, ASHD, cardiac, COPD, GI, liver disease, dysrhythmia, CABG, PCI, ICD, drugs prescribed before index date （ included NSAID, aspirin, acetamonphen, ACEI, ARB, beta_blocker, non_DHP and DHP CCB, acetazolamide, thiazides, loop, potassium sparing diuretics, alpah blocker, biguanides, sulfonylureas, alpha glucosidase inhibitors, thiazolidinediones, meglitinides, insulin, DPP4 inhibitors, statins, morphine, warfarin, clopidogrel, digoxin）. 48.

(50) Table 3 Risk factors of vascular access recreation by multivariate Cox proportional hazards model, by different types of vascular access Overall. AVF. HR. 95% CI. HR. 95% CI. HR. 0.79. (0.74-0.84)**. 0.74. (0.69-0.80)**. 0.94 (0.83-1.07). 1.03 1.14 1.29. (0.90-1.17) (1.01-1.30)* (1.14-1.46)**. 0.99 1.09 1.20. (0.85-1.14) (0.95-1.25) (1.05-1.38)*. 1.20 (0.83-1.74) 1.18 (0.84-1.67) 1.31 (0.93-1.85). 1.49 0.82. (1.31-1.70)** (0.78-0.86)**. 1.36 0.86. (1.18-1.56)** (0.82-0.91)**. 1.47 (1.04-2.08)* 0.94 (0.84-1.05). 1.07 1.03 1.09 1.06 1.07 1.05. (0.98-1.15) (0.97-1.10) (1.01-1.18)* (0.99-1.14) (1.01-1.13)* (0.99-1.12). 1.09 0.99 1.13 1.06 1.06 1.03. (0.99-1.20) (0.92-1.07) (1.03-1.25)* (0.98-1.15) (0.99-1.14) (0.95-1.11). 0.90 0.99 0.89 1.00 1.10 1.09. 1.10 0.99 1.12. (1.04-1.15)** (0.92-1.06) (1.02-1.23)*. 1.11 0.99 1.10. (1.04-1.18)** (0.91-1.07) (0.98-1.23). 0.95 (0.86-1.05) 0.90 (0.78-1.04) 0.97 (0.82-1.14). 1.08 1.05 0.96 0.93 0.99. (1.02-1.14)* (0.99-1.11) (0.92-1.02) (0.88-0.98)* (0.94-1.05). 1.04 1.04 0.99 0.92 1.00. (0.97-1.11) (0.97-1.11) (0.94-1.05) (0.87-0.98)* (0.95-1.07). 1.12 0.97 0.91 0.96 0.94. (0.98-1.27) (0.87-1.08) (0.82-1.01) (0.86-1.07) (0.84-1.04). Non DHP CCB DHP CCB Thiazides Loop diuretics Potassium sparing Alpha blocker Biguanides Sulfonylureas AGI TZD. 0.96 0.98 1.09 1.11 1.04 1.03 1.06 1.02 1.10 0.96. (0.90-1.02) (0.92-1.05) (1.03-1.15)* (1.05-1.18)** (0.96-1.13) (0.97-1.09) (0.98-1.15) (0.95-1.09) (1.01-1.20)* (0.88-1.06). 0.96 0.99 1.09 1.12 1.05 1.05 1.06 1.03 1.13 0.91. (0.89-1.04) (0.92-1.07) (1.02-1.16)* (1.05-1.20)** (0.95-1.15) (0.98-1.11) (0.97-1.16) (0.95-1.12) (1.01-1.25)* (0.82-1.02). 1.00 0.96 1.15 0.93 1.08 0.98 1.10 1.04 1.00 1.13. (0.88-1.14) (0.83-1.10) (1.03-1.29)* (0.82-1.05) (0.91-1.28) (0.87-1.10) (0.95-1.29) (0.91-1.19) (0.84-1.18) (0.95-1.34). Meglitinides. 0.97. (0.90-1.04). 0.97. (0.89-1.06). 0.96 (0.84-1.10). Statin Age (ref=20-39) 40-49 50-59 60-69 ≧70 Male Comorbidity CHF CVA PVD DM ASHD COPD GI disease Liver disease Cancer Drug NSAID Aspirin ACEI ARB Beta blocker. 49. AVG 95% CI. (0.78-1.06) (0.88-1.11) (0.76-1.04) (0.87-1.15) (0.99-1.24) (0.96-1.24).

(51) Insulin Warfarin Clopidogrel Digoxin. 1.09 1.18 0.94 1.13. (1.03-1.16)* (1.03-1.37)* (0.86-1.03) (1.04-1.24)*. 1.05 1.07 0.90 1.15. (0.98-1.13) (0.89-1.30) (0.80-1.01) (1.04-1.28)*. 1.25 0.99 1.03 1.09. (1.11-1.41)** (0.79-1.24) (0.87-1.21) (0.91-1.30). *p<0.05; **p<0.001 Abbreviations: CHF, congestive heart failure; CVA, cerebral vascular accident; PVD, peripheral vascular disease; DM, diabetes mellitus; ASHD, atherosclerotic heart disease; COPD, chronic obstructive pulmonary disease; GI, gastrointestinal; NSAID, non steroid anti-inflammatory drug; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor blocker; DHP, dihydropyridine; CCB, calcium channel blocker; AGI, alpha glucosidase inhibitor; TZD, thiazolidinediones.. 50.

(52) Table 4 Crude and adjusted hazard ratio of vascular access recreation for different statins N. event. Person-years Incidence (%). HR. Adjused HR. 4693 4693. 1739 1612. 17723.87 15521.13. 9.81 10.39. 1.0 0.99 (0.92-1.06). 1.0 0.79 (0.72-0.87)**. 621. 214. 2387.40. 8.96. 1.0. 1.0. Users Simvastatin Nonusers Users Rosuvastatin. 621. 232. 2265.60. 10.24. 1.13 (0.94-1.36). 0.90 (0.71-1.14). 1616 1616. 602 566. 6321.88 6050.86. 9.52 9.35. 1.0 0.96 (0.86-1.08). 1.0 0.72 (0.61-0.84)**. Nonusers Users Fluvastatin Nonusers. 1115 1115. 415 313. 4224.19 2676.11. 9.82 11.70. 1.0 0.95 (0.82-1.11). 1.0 0.74 (0.59-0.93)*. 1106. 421. 4098.16. 10.27. 1.0. 1.0. Users Pravastatin Nonusers Users. 1106. 386. 3791.23. 10.18. 0.95 (0.83-1.09). 0.84 (0.70-1.02). 582 582. 243 221. 2172.62 2181.54. 11.18 10.13. 1.0 0.90 (0.75-1.08). 1.0 0.72 (0.56-0.92)*. Atorvastatin Nonusers Users Lovastatin Nonusers. *p<0.05; **p<0.001 ※Covariates adjusted in multivariate models included income, age, sex, area, CHF, CVA, PVD, DM, ASHD, COPD, GI disease, liver disease, dysrhythmia, CABG, PCI, ICD, drugs prescribed before index date（included NSAID, aspirin, acetaminophen, ACEI, ARB, beta-blocker, non-DHP and DHP CCB, acetazolamide, thiazides, loop, potassium sparing diuretics, alpha-blocker, biguanides, sulfonylureas, alpha glucosidase inhibitors, thiazolidinediones, meglitinides, insulin, DPP4 inhibitors, statins, morphine, warfarin, clopidogrel, digoxin）. 51.

(53) Table 5 Demographic characteristics, comorbid diseases and medications exposure between statin users and nonusers in propensity-score matched cohort. Follow up time (year) Means (SD) Age (year) Means (SD) Sex (n, %) Male Female Location (n, %) City Township Rural area. Statin nonusers. Statin users. P value. N=3,181. N=3,864. 3.74. (2.61). 3.59. (2.47). 0.0109. 59.3. (13.1). 59.8. (12.4). 0.1068 0.9891. 1279 1902. (40.2) (59.8). 1553 2311. (40.2) (59.8) 0.4252. 865 1095 1221. (27.2) (34.4) (38.4). 1046 1281 1537. (27.1) (33.1) (39.8). 691 502. (21.7) (15.8). 873 615. (22.6) (15.9). 0.3817 0.8774. PVD DM ASHD Other heart disease COPD GI disease Liver disease Cancer Medication use. 198 1613 841 340 402 816 295 178. (6.2) (50.7) (26.4) (10.7) (12.6) (25.7) (9.3) (5.6). 264 1992 1054 440 517 1070 379 244. (6.8) (51.6) (27.3) (11.4) (13.4) (27.7) (9.8) (6.3). 0.3051 0.4799 0.4292 0.3523 0.3572 0.0544 0.4477 0.2057. NSAID Aspirin Clopidogrel Warfarin ACEI ARB Beta-blocker Non-DHP CCB DHP CCB Biguanide. 2076 889 127 36 1207 1186 1708 468 2484 516. (65.3) (28.0) (4.0) (1.1) (37.9) (37.3) (53.7) (14.7) (78.1) (16.2). 2552 1142 170 41 1497 1477 2101 616 3035 637. (66.1) (29.6) (4.4) (1.1) (38.7) (38.2) (54.4) (15.9) (78.6) (16.5). 0.4908 0.1382 0.3974 0.7766 0.4930 0.4177 0.5688 0.1546 0.6432 0.7655. Sulfonylurea. 929. (29.2). 1122. (29.0). 0.8777. Comorbidity CHF CVA. 52.

(54) Alpah glucosidase inhibitor Thiazolidinedione Meglitinide Insulin Statin. 195 159 329 836 51. (6.1) (5.0) (10.3) (26.3) (1.6). 253 202 435 1080 57. (6.6) (5.2) (11.3) (28.0) (1.5). 0.4749 0.6640 0.2190 0.1171 0.668. Abbreviations: CHF, congestive heart failure; CVA, cerebral vascular accident; PVD, peripheral vascular disease; DM, diabetes mellitus; ASHD, atherosclerotic heart disease; COPD, chronic obstructive pulmonary disease; GI, gastrointestinal; NSAID, non steroid anti-inflammatory drug; ACEI, angiotensin converting enzyme inhibitor; ARB, angiotensin II receptor blocker; DHP, dihydropyridine; CCB, calcium channel blocker.. 53.

(55) Table 6 Crude and adjusted hazard ratio of vascular access recreation for statin users and nonusers in propensity-score matched cohort. Overall Statin nonusers Statin users AVF Statin nonusers Statin users AVG Statin nonusers Statin users. N. Event. Person-years Event Rate Hazard ratio (per 100 pearson-ye ars). Adjusted hazard ratio. 3,181 3,864. 1,028 1,144. 11908.13 13864.40. 8.63 8.25. 1.0 0.94 (0.86-1.01). 1.0 0.92 (0.84-0.99)*. 2,736 3,267. 786 815. 10762.77 12474.59. 7.30 6.53. 1.0 0.88 (0.80-0.97)*. 1.0 0.87 (0.79-0.96)*. 445. 242. 1145.36. 21.13. 1.0. 1.0. 597. 329. 1389.81. 23.67. 1.09 (0.93-1.29). 1.11 (0.94-1.32). *p<0.05; **p<0.001 ※Covariates adjusted in multivariate models included income, age, sex, area, CHF, CVA, PVD, DM, ASHD, COPD, GI disease, liver disease, dysrhythmia, CABG, PCI, ICD, drugs prescribed before index date（included NSAID, aspirin, acetaminophen, ACEI, ARB, beta-blocker, non-DHP and DHP CCB, acetazolamide, thiazides, loop, potassium sparing diuretics, alpah blocker, biguanides, sulfonylureas, alpha glucosidase inhibitors, thiazolidinediones, meglitinides, insulin, DPP4 inhibitors, statins, morphine, warfarin, clopidogrel, digoxin）. 54.

(56) Table 7 Hazard ratio of permanent hemodialysis access recreation for statin users in propensity-score matched cohort. Overall Statin nonusers Statin users AVF Statin nonusers Statin users AVG Statin nonusers Statin users. N. Event. Person-years Event Rate Hazard ratio (per 100 pearson-years). 3,181 3,864. 1,028 1,144. 11908.13 13864.40. 8.63 8.25. 1.0 0.94 (0.86-1.01). 2,736 3,267. 786 815. 10762.77 12474.59. 7.30 6.53. 1.0 0.88 (0.80-0.97)*. 445 597. 242 329. 1145.36 1389.81. 21.13 23.67. 1.0 1.09 (0.93-1.29). *p<0.05; **p<0.001. 55.