Chapter 4 Discussion and Conclusion
4.2 Hypoxic Preconditioning Promote the Ability of Stem Cell to
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 49-55.
41
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
42
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
43
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
44
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 67 and elevate MMPs 68 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 mesenchymal stem cells on
45
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.
46
Table 1 Demographic characteristics, comorbid diseases and medications exposure
between statin users and nonusers
47
Sulfonylurea 1918 (19.5) 3956 (40.3) <.0001
Alpah glucosidase inhibitor 373 (3.8) 1375 (14.0) <.0001
Thiazolidinedione 314 (3.2) 1299 (13.2) <.0001
Meglitinide 859 (8.7) 2320 (23.6) <.0001
Insulin 2572 (26.2) 4840 (49.3) <.0001
Statin 0 (0.0) 5075 (51.7) <.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.
48
Table 2 Crude and adjusted hazard ratio of vascular access recreation for statin users and nonusers
N Event Person-years Incidence (%)
Hazard ratio Adjusted hazard ratio Overall
Statin nonusers 9826 3665 37275.01 9.83 1.0 1.0
Statin users 9826 3355 32780.10 10.23 0.98 (0.93-1.03) 0.79 (0.74-0.84)**
AVF
Statin nonusers 8519 2882 33823.15 8.52 1.0 1.0
Statin users 8181 2431 28727.13 8.46 0.94 (0.89-0.99)* 0.74 (0.69-0.80)**
AVG
Statin nonusers 1307 783 3451.86 22.68 1.0 1.0
Statin users 1645 924 4052.97 22.80 0.99 (0.90-1.09) 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）
49
Table 3 Risk factors of vascular access recreation by multivariate Cox proportional hazards model, by different types of vascular access
Overall AVF AVG
50
Insulin 1.09 (1.03-1.16)* 1.05 (0.98-1.13) 1.25 (1.11-1.41)**
Warfarin 1.18 (1.03-1.37)* 1.07 (0.89-1.30) 0.99 (0.79-1.24) Clopidogrel 0.94 (0.86-1.03) 0.90 (0.80-1.01) 1.03 (0.87-1.21) Digoxin 1.13 (1.04-1.24)* 1.15 (1.04-1.28)* 1.09 (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.
51
Table 4 Crude and adjusted hazard ratio of vascular access recreation for different statins
N event Person-years Incidence (%) 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）
52
Table 5 Demographic characteristics, comorbid diseases and medications exposure between statin users and nonusers in propensity-score matched cohort
Statin nonusers Statin users P value
53
Alpah glucosidase inhibitor 195 (6.1) 253 (6.6) 0.4749
Thiazolidinedione 159 (5.0) 202 (5.2) 0.6640
Meglitinide 329 (10.3) 435 (11.3) 0.2190
Insulin 836 (26.3) 1080 (28.0) 0.1171
Statin 51 (1.6) 57 (1.5) 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.
54
Table 6 Crude and adjusted hazard ratio of vascular access recreation for statin users and nonusers in propensity-score matched cohort
N Event Person-years Event Rate (per 100 pearson-ye ars)
Hazard ratio Adjusted hazard ratio
Overall
Statin nonusers 3,181 1,028 11908.13 8.63 1.0 1.0
Statin users 3,864 1,144 13864.40 8.25 0.94 (0.86-1.01) 0.92 (0.84-0.99)*
AVF
Statin nonusers 2,736 786 10762.77 7.30 1.0 1.0
Statin users 3,267 815 12474.59 6.53 0.88 (0.80-0.97)* 0.87 (0.79-0.96)*
AVG
Statin nonusers 445 242 1145.36 21.13 1.0 1.0
Statin users 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）
55
Table 7 Hazard ratio of permanent hemodialysis access recreation for statin users in propensity-score matched cohort
N Event Person-years Event Rate (per 100 pearson-years)
Hazard ratio
Overall
Statin nonusers 3,181 1,028 11908.13 8.63 1.0
Statin users 3,864 1,144 13864.40 8.25 0.94 (0.86-1.01) AVF
Statin nonusers 2,736 786 10762.77 7.30 1.0
Statin users 3,267 815 12474.59 6.53 0.88 (0.80-0.97)*
AVG
Statin nonusers 445 242 1145.36 21.13 1.0
Statin users 597 329 1389.81 23.67 1.09 (0.93-1.29)
*p<0.05; **p<0.001
56
Table 8 Crude and adjusted hazard ratio of vascular access recreation for statin users by time-dependent Cox regression models
HR aHR
Overall 0.80 (0.73-0.88)** 0.80 (0.72-0.88)**
AVF 0.80 (0.72-0.90)** 0.80 (0.71-0.89)**
AVG 0.84 (0.70-1.00) 0.84 (0.70-1.01)
*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, drug use before index date（included NSAID, aspirin, Acetaminophen, ACEI, ARB, beta-blocker, non-DHP, DHP, Aetazolamide, Thiazides, Loop, Potassium_sparing, alpah_blocker, Biguanides, Sulfonylureas, alpha-glucosidase_inhibitors, Thiazolidinediones, Meglitinides,
Table 9 Crude and competing-risk adjusted hazard ratio of vascular access recreation for statin users
HR aHR
Overall 0.92 (0.85-1.00) 0.91 (0.84-0.99)*
AVF 0.87 (0.79-0.96)* 0.86 (0.78-0.95)*
AVG 1.07 (0.90-1.25) 1.08 (0.91-1.27)
*p<0.05; **p<0.001
※ Death and renal transplantation are the two competing risks in the analysis
57
Figure 1 The experimental grouping and design were displayed in the eight groups.
58
Figure 2 Cumulative incidence of vascular access recreation for statin users and nonusers by the multivariate Nelson-Aalen method in (A) overall pairs (B) arteriovneous fistula pairs (C) arteriovenous graft pairs.
59
60
Figure 4 Mesenchymal stem cell transplantation ameliorated disease
activity in a rat
anti-Thy1.1-induced GN model.
Normoxic and hypoxic MSCs
markedly reduced the
inflammatory cell infiltration in the glomeruli in H&E stains (A). The severity of glomerulosclerosis was attenuated in both normoxic and hypoxic MSCs treated rats in PAS stains (B) and in Masson stains (C). Both normoxic and hypoxic MSCs reduced urinary protein levels (D). Glomerular sclerotic index calculated by the PAS sections among the experimental groups are presented in E.
61
Figure 5 Immunohistochemical stains for ED1, GRP78, LC3-II, Caspase1, TUNEL and Collagen IV in the study groups. Detection of ED1+ cell in the study groups are demonstrated in A1-A4.
Semiquantitative assessment revealed infusion of MSCs and showed a reduction in numbers of macrophage/monocyte infiltration (ED1+ cell). HMSCs possessed a better reduction effect (A5).
Representative pictures of sections for stress index protein acculumation (B1-B4), autophagy by LC3-II (C1-C4), apoptotic cells by caspase 1(D1-D4) and TUNEL (E1-E4) and collagen deposition (F1-F4) in glomeruli. The semiquantitative assessment of these sections revealed anti-Thy1 .1 administration increased stress index protein (GRP78) accumulation, autophagy index protein
62
(LC3II) detection, apoptotic (TUNEL, caspase 1) cells, and collagen IV accumulation. 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 greater therapeutic reduction effects.
63
Figure 6 The index of ROS, ROS enzymatic scavenger expression, master anti-oxidative injury regulator Nrf2 expression, and anti-oxidative response protein expressions among the experimental groups. The accumulation of ROS by 4 HNE stain among the groups are demonstrated from A1-A4. The semi-quantitative analyses of these sections revealed that anti-Thy1.1 infusion greatly increased ROS, and HMSCs ameliorated the ROS accumulation (A5). ROS enzymatic scavengers including MnSOD, Cu/ZnSOD and catalase were significantly reduced by antiThy-1 infusion. These enzymes were rescued by HMSCs (B1-B3). The nuclear
64
Nrf2 expression is not triggered by anti-Thy 1.1 infusion and is enhanced in the HMSC treated group. Significantly elevated nuclear NFkB expression is noted in anti-Thy1.1 group, which is suppressed in HMSCs treated group (C1-3). Downstream antioxidative response element protein expression including GCLC, GCLM, GPX are suppressed by anti-Thy 1.1 infusion. These proteins are rescued by HMSC transplantation (D1-D3).
65
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