Cardiotoxicity of Uremic Toxins: A Driver of Cardiorenal Syndrome

Cardiovascular disease (CVD) is highly prevalent in the setting of chronic kidney disease (CKD). Such coexistence of CVD and CKD—the so-called “cardiorenal or renocardiac syndrome”—contributes to exponentially increased risk of cardiovascular (CV) mortality. Uremic cardiomyopathy is a characteristic cardiac pathology commonly found in CKD. CKD patients are also predisposed to heart rhythm disorders especially atrial fibrillation. Traditional CV risk factors as well as known CKD-associated CV risk factors such as anemia are insufficient to explain CV complications in the CKD population. Accumulation of uremic retention solutes is a hallmark of impaired renal excretory function. Many of them have been considered inert solutes until their biological toxicity is unraveled and they become accepted as “uremic toxins”. Direct cardiotoxicity of uremic toxins has been increasingly demonstrated in recent years. This review offers a mechanistic insight into the pathological cardiac remodeling and dysfunction contributed by uremic toxins with a main focus on fibroblastic growth factor-23, an emerging toxin playing a central role in the chronic kidney disease–mineral bone disorder, and the two most investigated non-dialyzable protein-bound uremic toxins, indoxyl sulfate and p-cresyl sulfate. Potential therapeutic strategies that could address these toxins and their relevant mediated pathways since pre-dialysis stages are also discussed

Cardiorenal syndrome: pathophysiology, role of protein-bound uraemic toxins

Cardiorenal syndrome is a condition in which there exists a close bi-directional relationship between renal impairment and cardiac dysfunction. Failure of one organ can induce dysfunction of the other, eventually leading to failure of both. Despite optimal current therapy, morbidity and mortality in the population with cardiorenal syndrome remains unacceptably high. Importantly, pathophysiology of this condition is still not completely understood. This thesis therefore aimed to further explore this condition. The first part of the thesis evaluated how the diseased kidney affects the heart. In the population with chronic kidney disease (CKD), cardiovascular (CV) diseases are the most common cause of mortality. One of the most distinct cardiac abnormalities in the setting of CKD is the so-called uraemic cardiomyopathy, comprising left ventricular hypertrophy and cardiac fibrosis. One under-explored contributor is the family of uraemic toxins. Removal of protein-bound uraemic toxins in particular is not sufficient using current conventional dialysis. Indoxyl sulfate (IS) is one of those insufficiently dialysed protein-bound uraemic toxins; its adverse renal effects have been extensively studied. Specifically, IS has been demonstrated to be association with progressive renal pathological changes especially renal fibrosis, CKD progression and vascular pathology. However, adverse cardiac effects of IS have not been well-studied. We hypothesized that IS had direct adverse cardiac effects which may contribute to uraemic cardiomyopathy. The in vitro IS study was initially conducted to test IS on cardiac and THP-1 (human leukaemia monocytic cell line) cells. This study demonstrated for the first time that IS had direct adverse cardiac effects, comprised of pro-fibrotic, pro-hypertrophic and pro-inflammatory effects on cardiac fibroblasts, cardiac myocytes and THP-1 cells, respectively. These effects were likely mediated via p42/44 and p38 mitogen-activated protein kinases and nuclear factor-kappa B (NF-κB) pathways. Thus, IS may be implicated at least in part in the development of uraemic cardiomyopathy. These findings were confirmed in vivo using a renal failure model (induced by 5/6-subtotal nephrectomy; 5/6-STNx) with AST-120, an IS-lowering treatment. A significant increase in cardiac fibrosis and cardiomyocyte cross-sectional area (a surrogate of cardiac hypertrophy) was demonstrated in the 5/6-STNx model routinely used in our lab prior to conducting the STNx AST-120 study (uraemic cardiomyopathy–STNx retrospective study). In the in vivo STNx AST-120 study, increased serum IS levels, impaired renal function determined by glomerular filtration rate (GFR), serum creatinine, and 24-hour urine total protein, and increased tail-cuff blood pressure (BP) as well as diastolic dysfunction and increased cardiac fibrosis were observed in 5/6-STNx animals at 12 weeks post-op. Treatment with AST-120 significantly reduced serum IS, improved serum creatinine and reduced 24-hour urine total protein without change in BP. A significant reduction in cardiac fibrosis in association with a reduction in cardiac transforming growth factor-beta (TGF-β) and phosphorylated-NF-κB protein over-expression was observed in 5/6-STNx animals treated with AST-120. The extent of cardiac fibrosis was positively correlated with serum IS levels (and change in levels) independently of BP and renal function parameters. In conclusion, this study demonstrated that reducing serum IS levels with AST-120 might represent a possible adjunctive therapeutic strategy in the management of cardiac fibrosis associated with CKD. The second part of this thesis assessed how the diseased heart affects the kidney. Decline in renal function following myocardial infarction (MI) and heart failure is not uncommon. Renal impairment is one of the strongest predictors of poor clinical outcomes in patients with CV diseases. However little is known about its pathophysiology. We therefore aimed to systematically identify post-MI renal changes (functional, histological and molecular) over time (1, 4, 8, 12 and 16 weeks) in a rat MI model, and to examine potential mechanisms underlying these changes (in vivo MI time-course study). Left ventricular systolic dysfunction was demonstrated in MI animals at these points. There was no difference in infarct size across all time points. Post-MI renal dysfunction determined by GFR was observed in a biphasic pattern. Reduction in GFR occurred at 1 week post-MI was associated with a significant decrease in tail-cuff BP. Normalisation of BP was seen at 4 weeks post-MI whilst GFRs were back to sham levels at 8 and 12 weeks. At 1 week, renal macrophage (ED-1 positive cells) infiltration, renal cortical TGF-β protein expression and renal cortical interleukin-6 mRNA expression were significantly increased in MI animals. Progressive renal interstitial fibrosis with persistently activated renal phosphorylated-Smad2 protein expression was observed in MI animals from 4 to 16 weeks in association with the second decline in GFR at 16 weeks (p=0.052). This study therefore demonstrated that renal impairment occured early post-MI and was associated with hemodynamic and renal structural changes possibly via activation of the Smad2 signaling pathway. Increased circulating IS levels in association with decline renal function and increased renal interstitial fibrosis were observed in MI animals at 16 weeks in the in vivo MI time-course study. This suggested another hypothesis i.e. that reduction in serum IS had renal and cardiac beneficial effects in this MI model with renal impairment. In a follow-on in vivo MI study with AST-120 treatment (in vivo MI AST-120 study), MI animals showed a significant reduction in GFR and a significant increase in serum IS at 16 weeks post-MI. AST-120-treated MI animals showed a significant decrease in serum IS levels. A significant decrease in renal interstitial fibrosis was observed in AST-120-treated MI animals despite no renal functional improvement. AST-120 reduced cardiac gene over-expression of TGF-β1 and tumor necrosis factor-alpha in non-infarct myocardium in MI animals, without improvement on cardiac systolic dysfunction and cardiac fibrosis. In conclusion, these findings suggested that reducing IS levels by AST-120 had beneficial effects on both heart and kidney in the post-MI setting with accompanying renal impairment. Taken together, this thesis has demonstrated adverse cardiac effects of IS, whilst reducing IS levels by AST-120 treatment improved cardiac pathological changes in the setting of CKD. In the setting of MI, renal dysfunction occurred early, and change in renal structure (i.e. interstitial fibrosis) appeared to be persistent and progressive. AST-120 treatment demonstrated cardiorenal beneficial effects in the post-MI setting with concomitant renal dysfunction

Cardiorenal syndrome: pathophysiology, role of protein-bound uraemic toxins

Cardiorenal syndrome is a condition in which there exists a close bi-directional relationship between renal impairment and cardiac dysfunction. Failure of one organ can induce dysfunction of the other, eventually leading to failure of both. Despite optimal current therapy, morbidity and mortality in the population with cardiorenal syndrome remains unacceptably high. Importantly, pathophysiology of this condition is still not completely understood. This thesis therefore aimed to further explore this condition. The first part of the thesis evaluated how the diseased kidney affects the heart. In the population with chronic kidney disease (CKD), cardiovascular (CV) diseases are the most common cause of mortality. One of the most distinct cardiac abnormalities in the setting of CKD is the so-called uraemic cardiomyopathy, comprising left ventricular hypertrophy and cardiac fibrosis. One under-explored contributor is the family of uraemic toxins. Removal of protein-bound uraemic toxins in particular is not sufficient using current conventional dialysis. Indoxyl sulfate (IS) is one of those insufficiently dialysed protein-bound uraemic toxins; its adverse renal effects have been extensively studied. Specifically, IS has been demonstrated to be association with progressive renal pathological changes especially renal fibrosis, CKD progression and vascular pathology. However, adverse cardiac effects of IS have not been well-studied. We hypothesized that IS had direct adverse cardiac effects which may contribute to uraemic cardiomyopathy. The in vitro IS study was initially conducted to test IS on cardiac and THP-1 (human leukaemia monocytic cell line) cells. This study demonstrated for the first time that IS had direct adverse cardiac effects, comprised of pro-fibrotic, pro-hypertrophic and pro-inflammatory effects on cardiac fibroblasts, cardiac myocytes and THP-1 cells, respectively. These effects were likely mediated via p42/44 and p38 mitogen-activated protein kinases and nuclear factor-kappa B (NF-κB) pathways. Thus, IS may be implicated at least in part in the development of uraemic cardiomyopathy. These findings were confirmed in vivo using a renal failure model (induced by 5/6-subtotal nephrectomy; 5/6-STNx) with AST-120, an IS-lowering treatment. A significant increase in cardiac fibrosis and cardiomyocyte cross-sectional area (a surrogate of cardiac hypertrophy) was demonstrated in the 5/6-STNx model routinely used in our lab prior to conducting the STNx AST-120 study (uraemic cardiomyopathy–STNx retrospective study). In the in vivo STNx AST-120 study, increased serum IS levels, impaired renal function determined by glomerular filtration rate (GFR), serum creatinine, and 24-hour urine total protein, and increased tail-cuff blood pressure (BP) as well as diastolic dysfunction and increased cardiac fibrosis were observed in 5/6-STNx animals at 12 weeks post-op. Treatment with AST-120 significantly reduced serum IS, improved serum creatinine and reduced 24-hour urine total protein without change in BP. A significant reduction in cardiac fibrosis in association with a reduction in cardiac transforming growth factor-beta (TGF-β) and phosphorylated-NF-κB protein over-expression was observed in 5/6-STNx animals treated with AST-120. The extent of cardiac fibrosis was positively correlated with serum IS levels (and change in levels) independently of BP and renal function parameters. In conclusion, this study demonstrated that reducing serum IS levels with AST-120 might represent a possible adjunctive therapeutic strategy in the management of cardiac fibrosis associated with CKD. The second part of this thesis assessed how the diseased heart affects the kidney. Decline in renal function following myocardial infarction (MI) and heart failure is not uncommon. Renal impairment is one of the strongest predictors of poor clinical outcomes in patients with CV diseases. However little is known about its pathophysiology. We therefore aimed to systematically identify post-MI renal changes (functional, histological and molecular) over time (1, 4, 8, 12 and 16 weeks) in a rat MI model, and to examine potential mechanisms underlying these changes (in vivo MI time-course study). Left ventricular systolic dysfunction was demonstrated in MI animals at these points. There was no difference in infarct size across all time points. Post-MI renal dysfunction determined by GFR was observed in a biphasic pattern. Reduction in GFR occurred at 1 week post-MI was associated with a significant decrease in tail-cuff BP. Normalisation of BP was seen at 4 weeks post-MI whilst GFRs were back to sham levels at 8 and 12 weeks. At 1 week, renal macrophage (ED-1 positive cells) infiltration, renal cortical TGF-β protein expression and renal cortical interleukin-6 mRNA expression were significantly increased in MI animals. Progressive renal interstitial fibrosis with persistently activated renal phosphorylated-Smad2 protein expression was observed in MI animals from 4 to 16 weeks in association with the second decline in GFR at 16 weeks (p=0.052). This study therefore demonstrated that renal impairment occured early post-MI and was associated with hemodynamic and renal structural changes possibly via activation of the Smad2 signaling pathway. Increased circulating IS levels in association with decline renal function and increased renal interstitial fibrosis were observed in MI animals at 16 weeks in the in vivo MI time-course study. This suggested another hypothesis i.e. that reduction in serum IS had renal and cardiac beneficial effects in this MI model with renal impairment. In a follow-on in vivo MI study with AST-120 treatment (in vivo MI AST-120 study), MI animals showed a significant reduction in GFR and a significant increase in serum IS at 16 weeks post-MI. AST-120-treated MI animals showed a significant decrease in serum IS levels. A significant decrease in renal interstitial fibrosis was observed in AST-120-treated MI animals despite no renal functional improvement. AST-120 reduced cardiac gene over-expression of TGF-β1 and tumor necrosis factor-alpha in non-infarct myocardium in MI animals, without improvement on cardiac systolic dysfunction and cardiac fibrosis. In conclusion, these findings suggested that reducing IS levels by AST-120 had beneficial effects on both heart and kidney in the post-MI setting with accompanying renal impairment. Taken together, this thesis has demonstrated adverse cardiac effects of IS, whilst reducing IS levels by AST-120 treatment improved cardiac pathological changes in the setting of CKD. In the setting of MI, renal dysfunction occurred early, and change in renal structure (i.e. interstitial fibrosis) appeared to be persistent and progressive. AST-120 treatment demonstrated cardiorenal beneficial effects in the post-MI setting with concomitant renal dysfunction