| Cardiorenal syndrome is a complex pathophysiologic disorder of the heart and the kidney whereby acute or chronic dysfunction of one organ may cause acute or chronic dysfunction of the other. Despite of the rapid development of diagnostic and treatment technologies for renal and cardiovascular diseases, the prevalence of cardiorenal syndrome is still growing. Above all, cardiorenal syndrome has become a significant challenge to public health.A close relationship exists between CKD and CVD. The prevalence of CVD in patients with CKD, associated with the renal dysfunction, is higher than that in general population. CKD is the major cause (about40-50%) of death in dialysis patients. Conversely, CKD is one of the major risk factors for CAD. Both the albuminuria and the reduction of GFR are recognized as independent risk factors for CAD. Results from animal studies also support the concept that CKD could accelerate the progression of chronic heart failure (CHF) and atherosclerosis. However, the mechanisms underlying the CKD associated CVD are not fully understanding.Indoxyl sulfate (IS) and p-cresy sulfate (PCS) are protein-bound uremic toxins resulting from bacterial metabolism of food protein in the colon. In the setting of CKD, IS and PCS are accumulated in circulation due to injured renal function, exerting detrimental effects on the kidney, heart, vessel and other organs. The plasma levels of IS and PCS are both reported to be associated with CVD, major adverse cardiovascular event, cardiovascular and all-cause mortality, and the progression of CKD. Thus, IS and PCS may be important contributors to the progression of both CKD and CVD. However, the exact mechanism remains unclear.Glomerulosclerosis, cardiac remodeling and atherosclerosis are the major pathophysiological changes in CKD, CHF and CAD, respectively. Further understanding of the effects of IS and PCS on the development of glomerulosclerosis, cardiac remodeling and atherosclerosis is important to the prevention and treatment for cardiorenal syndrome. Thus, the questions below are urgently required to be answered.(1) Renal fibrosis, characterized by glomerulosclerosis and tubulointerstitial fibrosis, is the final common manifestation of a wide variety of CKD and one of the main contributors to kidney failure. Most of the studies about IS and PCS were focused on their effects on renal tubular cells and tubulointerstitial fibrosis but not glomerulosclerosis. Both IS and PCS could stimulate cell damage, senescence, and the expression of inflammatory factors in renal tubular cells in vitro, and are associated with renal interstitial fibrosis in vivo. The pathogenesis of glomerulosclerosis is characterized by an excessive accumulation and deposition of extracellular matrix (ECM) components, which are mostly synthesized by mesangial cells in glomerulus. However, whether IS or PCS could contribute to glomerulosclerosis via promoting mesangial cell ECM synthesis is still unknown.(2) Cardiac remodeling is the final common manifestation of a wide variety of CVD and plays an important role in heart failure. Cardiac remodeling is characterized by cardiac myocyte hypertrophy and cardiac fibrosis. Our recent study have showed that IS significantly increased neonatal rat cardiac fibroblast collagen synthesis and cardiac myocyte hypertrophy. However, whether PCS could contribute to cardiac remodeling via increasing cardiac fibroblast collagen synthesis and cardiac myocyte hypertrophy like IS is still unknown.(3) Coronary artery atherosclerosis is the principal cause of CAD. Atherosclerosis is currently considered as a chronic inflammatory disease. Monocyte/macrophages play an important role in the development of atherosclerosis. In particular, uptake of oxidized low-density lipoprotein (Ox-LDL) and subsequent foam cell forming by macrophages is a key step in early atherogenesis. IS is reported to play an important role in the progression of CKD-associated atherosclerosis. Recent studies have found that IS are associated with expression of inflammatory factors in monocyte, and vascular smooth muscle cell proliferation and migration in vitro. However, whether IS could contribute to the progression of atherosclerosis via increasing uptake of Ox-LDL by macrophages is still unknown.To answer the above question and to further determine the mechanism underlying cardiorenal syndrome,3part studies below were conducted in this thesis.Part1Effects of indoxyl sulfate and p-cersyl sulfate on mesangial cell collagen synthesis1. ObjectTo determine the effects of IS and PCS on collagen synthesis in cultured mesangial cells, and the involvement of OAT1/3(organic anion transporter1/3), ASK1(apoptosis signal-regulating kinase1), p38, ERK1/2and NFκB in the effects.2. MethodsRat kidney mesangial cell (RMC cell line) was cultured. Collagen synthesis and cell viability in RMCs were then determined by3H-proline incorporation and MTT analyses, respectively. Briefly,2h after pre-treatment with or without0.1-100μM OAT1/2antagonist probenecid (Pro),0.03-1.0μM ASK1MAPK inhibitor G2261818A (G226),0.1-3μM p38MAPK inhibitor RWJ-67657(RWJ) or0.03-1.0μM ERK1/2inhibitor U0126, IS or PCS at a concentration range from0.1nM to300μM were added to RMCs for48hour incubation. For western blot analysis, NCMs were pre-treated with or without100μM Pro,1μM G226,3μM RWJ, or1μM U0126for2h prior to10μM IS or100μM PCS stimulation for15min.Statistical analysis was performed using GraphPad Prism software version6. When homogeneity of variance was satisfied, One-way ANOVA with Neuman-Keul post hoc test were used for comparison among all groups using; otherwise, Kruskal-Wallis test with Dunn’s post hoc test were used. Data are presented as means±SD. A two-tailed P-value of less than0.05was considered statistically significant. 3. Results3.1IS0.0001μM to300μM significantly stimulated RMC collagen synthesis. Probenecid0.1μM to100μM, G2260.03μM to1.0μM, RWJ0.1μM to1.0μM, and U01260.03μM to1.0μM suppressed IS (10μM)-stimulated collagen synthesis, respectively.3.210μM IS activated ASK1, P38MAPK, ERK1/2MAPK, and NFκB pathways by significantly increasing their phosphorylation levels in RMCs. In the presence of100μM probenecid or1μM G226, the increase in the phosphorylation levels of ASK1, p38, ERK1/2and NFκB was abolished. Similarly,3μM RWJ abolished the increase in phosphorylation levels of p38and NFκB increased by IS. And1μM U0126abolished the increase in phosphorylation levels of ERK1/2and NFκB increased by IS. In addition, IS treatment did not affect the levels of total p38, total ERK1/2, toal NFκB and Pan-actin protein even in the presence of probenecid, G226, RWJ, or U0126, indicating that IS did not affect protein phosphorylation by changing the protein levels in RMCs.3.3PCS0.0001μM to300μM significantly stimulated RMC collagen synthesis. Probenecid0.1μM to100μM, G2260.03μM to1.0μM, RWJ0.1μM to1.0μM, and U01260.03μM to1.0μM suppressed PCS (100μM)-stimulated collagen synthesis, respectively.3.4PCS100μM stimulated ASK1, p38MAPK, ERK1/2MAPK, and NFκB pathways by significantly increasing their phosphorylation levels in RMCs. In the presence of100μM probenecid or1μM G226, the increase in the phosphorylation levels of ASK1, p38, ERK1/2and NFκB was abolished. Similarly,3μM RWJ abolished the increase in phosphorylation levels of p38and NFκB increased by PCS. And1μM U0126abolished the increase in phosphorylation levels of ERK1/2and NFκB increased by PCS. PCS treatment did not affect the levels of total p38, total ERK1/2, toal NFκB and Pan-actin protein even in the presence of probenecid, G226, RWJ, or U0126, indicating that IS did not affect protein phosphorylation by changing the protein levels in RMCs. 3.5IS and PCS co-culture at a dose of up to300μM did not affect cell viability of RMCs, respectively. Additionally, the presence of probenecid, G226, RWJ or U0126did not affect cell viability of RMCs.4. ConclusionThis study has, for the first time, demonstrated that both IS and PCS could stimulate RMC collagen synthesis at least partly via activation of the ASK1, p38, ERK1/2, and NFκB pathways after OAT1/3-mediated entry into cells. OAT1/3-ASK1-MAPK (p38, ERK1/2)-NFκB pathway may play an important role in the pro-fibrosis effects of both IS and PCS in RMCs.Part2Effects of p-cresyl sulfate on neonatal rat cardiac myocyte hypertrophy and cardiac fibroblast collagen synthesis1. ObjectTo determine the effects of IS and PCS on neonatal rat cardiac myocyte hypertrophy and cardiac fibroblast collagen synthesis, and the involvement of OAT1/3, ASK1, p38, ERK1/2and NFκB in the effects.2. MethodsNeonatal Sprague-Dawley cardiac myocytes (NCMs) and cardiac fibroblasts (NCFs) were isolated from1-to2-day-old pups with enzymatic digestion. NCM hypertrophy, NCF collagen synthesis and cell viability were then determined by H-leucine incorporation, H-proline incorporation and MTT analyses, respectively. After serum starving for48hours, NCFs were stimulated with0.1nM to300PCS for48hours, while NCMs were treated with0.1nM to300μM PCS for48hours in the presence or absence of2hour pretreatment with0.1-100μM OAT1/2antagonist probenecid (Pro),0.03-1.0μM ASK1MAPK inhibitor G2261818A (G226),0.1-3μM p38MAPK inhibitor RWJ-67657(RWJ) or0.03-1.0μM ERK1/2inhibitor U0126. For western blot analysis, NCMs were pre-treated with or without100μM Pro,1μM G226,3μM RWJ, or1μM U0126for2h prior to100μM PCS stimulation for15min. Statistical analysis was performed as described in Part1. 3. Results3.1PCS0.0001μM to300μM significantly stimulated NCMs hypertrophy. Probenecid0.1μM to100μM, G2260.03μM to1.0μM, RWJ0.1μM to1.0μM, and U01260.03μM to1.0μM suppressed PCS (100μM)-stimulated NCM hypertrophy, respectively.3.2PCS0.0001μM to300μM did not affect NCFs collagen synthesis.3.3PCS100μM stimulated ASK1, p38MAPK and ERK1/2MAPK pathways by significantly increasing their phosphorylation levels in NCMs. In the presence of100μM probenecid or1μM G226, the increase in the phosphorylation levels of ASK1, p38, and ERK1/2was abolished. Similarly,3μM RWJ abolished the increase in phosphorylation levels of p38increased by PCS. And1μM U0126abolished the increase in phosphorylation levels of ERK1/2increased by PCS. In addition, PCS treatment did not affect the levels of total p38, total ERK1/2, phosphorylated and toal NFκB protein even in the presence of probenecid, G226, RWJ, or U0126, indicating that IS did not affect protein phosphorylation by changing the protein levels in NCMs.3.4PCS co-culture at a dose of up to300μM did not affect cell viability of both NCMs and NCFs. Additionally, the presence of probenecid, G226, RWJ or U0126did not affect cell viability of NCMs.4. ConclusionThis study has, for the first time, demonstrated that PCS could stimulate NCMs hypertrophy at least partly via activation of the ASK1, p38, ERK1/2, but not NFκB pathways after OAT1/3-mediated entry into cells, while it could not affect NCFs collagen synthesis at a dose up to300μM. In addition, OAT1/3-ASK1-MAPK (p38, ERK1/2) pathway may play an important role in the pro-hypertrophy effect of PCS in NCMs.Part3Effects of indoxyl sulfate on uptake of Ox-LDL by THP-1derived macrophages1. Object To determine the effects of indoxyl sulfate on uptake of Ox-LDL by THP-1derived macrophages, and the involvement of ERK1/2pathway in the effects.2. Methods2.1THP-1monocytes were differentiated into macrophages by pre-incubation with PMA (160nM) for72hours.2.2THP-1derived macrophages were grouped as described below for different purposes. The uptake of Ox-LDL and the cell viability were then determined by Dil-Ox-LDL incorporation and CCK-8analyses, respectively. In the uptake assay, THP-1derived macrophages were exposed to Dil-Ox-LDL in the last4hours incubation.(1) Dose-dependent effect of IS:Control group, IS (1-500μM) groups, U0126(5μM, pre-treatment for2hours)+IS (250μM) group;(2) Time-dependent effect of IS (250μM)-. Control group, IS (12-48hours) groups.2.3Cells were pre-treated with or without U0126(5μM) for2hours prior to IS (250μM) stimulation for15min. Protein expressions of ERK1/2signaling pathways in THP-1derived macrophages were then determined by western blot analyses.3. Results3.1The concentration range of IS tested with THP-1derived macrophages for24hours incubation was from1to500μM. At concentrations of100to500μM, IS significantly increased uptake of Dil-Ox-LDL by macrophages in a dose-dependent manner at24hours.5μM U0126significantly inhibited the increase of uptake of Dil-ox-LDL stimulated by IS.3.2250μM IS significantly increased uptake of Dil-Ox-LDL by macrophages from12hours up to48hours in a time-dependent manner.3.3250μM IS stimulated ERK1/2pathway by significantly increasing it phosphorylation level in THP-1derived macrophages. In the presence of5μM U0126, the increase in the phosphorylation levels of ERK1/2was abolished. In addition, IS treatment did not affect the levels of total ERK1/2even in the presence of U0126, indicating that IS did not affect protein phosphorylation by changing the protein levels in THP-1derived macrophages.3.4IS co-culture at a dose of up to500μ.M did not affect cell viability of THP-1derived macrophages. Additionally, the presence of U0126did not affect cell viability of THP-1derived macrophages.4. ConclusionThis study has, for the first time, demonstrated that IS could stimulate uptake of Ox-LDL by THP-1derived macrophages in both dose-and time-dependent manners at least partly via activation of ERK1/2pathway. |
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