Rat Models of Cardiorenal Syndrome and Methods for Functional Assessment.

Cardiorenal syndrome (CRS) is a clinical disorder involving combined heart and kidney dysfunction, which leads to poor clinical outcomes. To understand the complex pathophysiology and mechanisms that lie behind this disease setting, and design/evaluate appropriate treatment strategies, suitable animal models are required. Described here are the protocols for establishing surgically induced animal models of CRS including important methods to determine clinically relevant measures of cardiac and renal function, commonly used to assess the degree of organ dysfunction in the model and treatment efficacy when evaluating novel therapeutic strategies.

Aryl Hydrocarbon Receptor Inhibition Restores Indoxyl Sulfate-Mediated Endothelial Dysfunction in Rat Aortic Rings.

The uremic toxin indoxyl sulfate (IS), elevated in chronic kidney disease (CKD), is known to contribute towards progressive cardiovascular disease. IS activates the aryl hydrocarbon receptor (AhR) mediating oxidative stress and endothelial dysfunction via activation of the CYP1A1 pathway. The present study examines AhR inhibition with the antagonist, CH223191, on IS-mediated impairment of vascular endothelial function and disruption of redox balance. The acute effects of IS on endothelium-dependent relaxation were assessed in aortic rings from Sprague Dawley rats exposed to the following conditions: (1) control; (2) IS (300 µM); (3) IS + CH223191 (1 µM); (4) IS + CH223191 (10 µM). Thereafter, tissues were assessed for changes in expression of redox markers. IS reduced the maximum level of endothelium-dependent relaxation (Rmax) by 42% (p < 0.001) compared to control, this was restored in the presence of increasing concentrations of CH223191 (p < 0.05). Rings exposed to IS increased expression of CYP1A1, nitro-tyrosine, NADPH oxidase 4 (NOX4), superoxide, and reduced eNOS expression (p < 0.05). CH223191 (10 µM) restored expression of these markers back to control levels (p < 0.05). These findings demonstrate the adverse impact of IS-mediated AhR activation on the vascular endothelium, where oxidative stress may play a critical role in inducing endothelial dysfunction in the vasculature of the heart and kidneys. AhR inhibition could provide an exciting novel therapy for CVD in the CKD setting.

NP202 treatment improves left ventricular systolic function and attenuates pathological remodelling following chronic myocardial infarction.

AIMS: Myocardial injury is a major contributor to left ventricular (LV) remodelling activating neurohormonal and inflammatory processes that create an environment of enhanced oxidative stress. This results in geometric and structural alterations leading to reduced LV systolic function. In this study we evaluated the efficacy of NP202, a synthetic flavonol, on cardiac remodelling in a chronic model of myocardial infarction (MI). MAIN METHODS: A rat model of chronic MI was induced by permanent surgical ligation of the coronary artery. NP202 treatment was commenced 2 days post-MI for 6 weeks at different doses (1, 10 and 20 mg/kg/day) to determine efficacy. Cardiac function was assessed by echocardiography prior to treatment and at week 6, and pressure-volume measurements were performed prior to tissue collection. Tissues were analysed for changes in fibrotic and inflammatory markers using immunohistochemistry and gene expression analysis. KEY FINDINGS: Rats treated with NP202 demonstrated improved LV systolic function and LV geometry compared to vehicle treated animals. Furthermore, measures of hypertrophy and interstitial fibrosis were attenuated in the non-infarct region of the myocardium with NP202 at the higher dose of 20 mg/kg (P < 0.05). At the tissue level, NP202 reduced monocyte chemoattractant protein-1 expression (P < 0.05) and tended to attenuate active caspase-3 expression to similar levels observed in sham animals (P = 0.075). SIGNIFICANCE: Improved LV function and structural changes observed with NP202 may be mediated through inhibition of inflammatory and apoptotic processes in the MI setting. NP202 could therefore prove a useful addition to standard therapy in patients with post-MI LV dysfunction.

The effect of dihydroceramide desaturase 1 inhibition on endothelial impairment induced by indoxyl sulfate.

Protein-bound uremic toxins (PBUTs) have adverse effects on vascular function, which is imperative in the progression of cardiovascular and renal diseases. The role of sphingolipids in PBUT-mediated vasculo-endothelial pathophysiology is unclear. This study assessed the therapeutic potential of dihydroceramide desaturase 1 (Des1) inhibition, the last enzyme involved in de novo ceramide synthesis, to mitigate the vascular effects of the PBUT indoxyl sulfate (IS). Rat aortic rings were isolated and vascular reactivity was assessed in organ bath experiments followed by immunohistochemical analyses. Furthermore, cultured human aortic endothelial cells were assessed for phenotypic and mechanistic changes. Inhibition of Des1 by a selective inhibitor CIN038 (0.1 to 0.3 µM) improved IS-induced impairment of vasorelaxation and modulated immunoreactivity of oxidative stress markers. Des1 inhibition also reversed IS-induced reduction in endothelial cell migration (1.0 µM) by promoting the expression of angiogenic cytokines and reducing inflammatory and oxidative stress markers. These effects were associated with a reduction of TIMP1 and the restoration of Akt phosphorylation. In conclusion, Des1 inhibition improved vascular relaxation and endothelial cell migration impaired by IS overload. Therefore, Des1 may be a suitable intracellular target to mitigate PBUT-induced adverse vascular effects.

Cost-effectiveness of dapagliflozin in chronic heart failure: an analysis from the Australian healthcare perspective.

AIM: To assess the cost-effectiveness of dapagliflozin in addition to standard care versus standard care alone in patients with chronic heart failure and reduced ejection fraction. METHODS: A Markov model was constructed based on the Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure trial to assess the clinical outcomes and costs of 1000 hypothetical subjects with established heart failure and reduced ejection fraction. The model consisted of three health states: 'alive and event-free', 'alive after non-fatal hospitalisation for heart failure' and 'dead'. Costs and utilities were estimated from published sources. The main outcome was the incremental cost-effectiveness ratio per quality-adjusted life-year gained. An Australian public healthcare perspective was employed. All outcomes and costs were discounted at a rate of 5% annually. RESULTS: Over a lifetime horizon, the addition of dapagliflozin to standard care in patients with heart failure and reduced ejection fraction prevented 88 acute heart failure hospitalisations (including readmissions) and yielded an additional 416 years of life and 288 quality-adjusted life-years (discounted) at an additional cost of A$3,692,440 (discounted). This equated to an incremental cost-effectiveness ratio of A$12,482 per quality-adjusted life-year gained, well below the Australian willingness-to-pay threshold of A$50,000 per quality-adjusted life-year gained. Subanalyses in subjects with and without diabetes resulted in similar incremental cost-effectiveness ratios of A$13,234 and A$12,386 per quality-adjusted life-year gained, respectively. CONCLUSION: Dapagliflozin is likely to be cost-effective when used as an adjunct therapy to standard care compared with standard care alone for the treatment of chronic heart failure and reduced ejection fraction.

Sphingolipid imbalance and inflammatory effects induced by uremic toxins in heart and kidney cells are reversed by dihydroceramide desaturase 1 inhibition.

Non-dialysable protein-bound uremic toxins (PBUTs) contribute to the development of cardiovascular disease (CVD) in chronic kidney disease (CKD) and vice versa. PBUTs have been shown to alter sphingolipid imbalance. Dihydroceramide desaturase 1 (Des1) is an important gatekeeper enzyme which controls the non-reversible conversion of sphingolipids, dihydroceramide, into ceramide. The present study assessed the effect of Des1 inhibition on PBUT-induced cardiac and renal effects in vitro, using a selective Des1 inhibitor (CIN038). Des1 inhibition attenuated hypertrophy in neonatal rat cardiac myocytes and collagen synthesis in neonatal rat cardiac fibroblasts and renal mesangial cells induced by the PBUTs, indoxyl sulfate and p-cresol sulfate. This is at least attributable to modulation of NF-κB signalling and reductions in ß-MHC, Collagen I and TNF-α gene expression. Lipidomic analyses revealed Des1 inhibition restored C16-dihydroceramide levels reduced by indoxyl sulfate. In conclusion, PBUTs play a critical role in mediating sphingolipid imbalance and inflammatory responses in heart and kidney cells, and these effects were attenuated by Des1 inhibition. Therefore, sphingolipid modifying agents may have therapeutic potential for the treatment of CVD and CKD and warrant further investigation.

The Preventable Productivity Burden of Kidney Disease in Australia.

BACKGROUND: Kidney disease is associated with impaired work productivity. However, the collective effect of missed work days, reduced output at work, and early withdrawal from the workforce is rarely considered in health-economic evaluations. METHODS: To determine the effect on work productivity of preventing incident cases of kidney disease, using the novel measure "productivity-adjusted life year" (PALY), we constructed a dynamic life table model for the Australian working-age population (aged 15-69 years) over 10 years (2020-2029), stratified by kidney-disease status. Input data, including productivity estimates, were sourced from the literature. We ascribed a financial value to the PALY metric in terms of gross domestic product (GDP) per equivalent full-time worker and assessed the total number of years lived, total PALYs, and broader economic costs (GDP per PALY). We repeated the model simulation, assuming a reduced kidney-disease incidence; the differences reflected the effects of preventing new kidney-disease cases. Outcomes were discounted by 5% annually. RESULTS: Our projections indicate that, from 2020 to 2029, the estimated number of new kidney-disease cases will exceed 161,000. Preventing 10% of new cases of kidney disease during this period would result in >300 premature deaths averted and approximately 550 years of life and 7600 PALYs saved-equivalent to a savings of US$1.1 billion in GDP or US$67,000 per new case avoided. CONCLUSIONS: Pursuing a relatively modest target for preventing kidney disease in Australia may prolong years of life lived and increase productive life years, resulting in substantial economic benefit. Our findings highlight the need for investment in preventive measures to reduce future cases of kidney disease.

The impact of coronary heart disease prevention on work productivity: a 10-year analysis.

AIMS: To determine the impact of preventing new (incident) cases of coronary heart disease (CHD) on years of life and productivity, using the novel measure 'productivity-adjusted life year' (PALY), over the next 10 years. METHODS AND RESULTS: A dynamic life table model was constructed for the total Australian working-age population (15-69 years) over 10 years (2020-2029), separated by CHD status. Productivity estimates were sourced from the literature. The PALY was ascribed a financial value in terms of gross domestic product (GDP) per equivalent full-time worker. The total number of years lived, PALYs, and economic burden (in terms of GDP per PALY) were estimated. The model simulation was repeated assuming incidence was reduced, and the differences represented the impact of CHD prevention. All outcomes were discounted by 5% per annum. Over 10 years, the total projected years lived and PALYs in the Australian working-age population (with and without CHD) were 133 million and 83 million, respectively, amounting to A$17.2 trillion in GDP. We predicted more than 290 000 new (incident) CHD cases over the next 10 years. If all new cases of CHD could be prevented during this period, a total of 4 000 deaths could be averted, resulting in more than 8 000 years of life saved and 104 000 PALYs gained, equivalent to a gain of nearly A$21.8 billion (US$14.8 billion) in GDP. CONCLUSION: Prevention of CHD will prolong years of life lived and productive life years, resulting in substantial economic benefit. Policy makers and employers are encouraged to engage in preventive measures addressing CHD.

Apoptosis signal-regulating kinase 1 inhibition reverses deleterious indoxyl sulfate-mediated endothelial effects.

AIMS: Indoxyl sulfate (IS), a protein-bound uremic toxin, is implicated in endothelial dysfunction, which contributes to adverse cardiovascular events in chronic kidney disease. Apoptosis signal regulating kinase 1 (ASK1) is a reactive oxygen species-driven kinase involved in IS-mediated adverse effects. This study assessed the therapeutic potential of ASK1 inhibition in alleviating endothelial effects induced by IS. MAIN METHODS: IS, in the presence and absence of a selective ASK1 inhibitor (GSK2261818A), was assessed for its effect on vascular reactivity in rat aortic rings, and cultured human aortic endothelial cells where we evaluated phenotypic and mechanistic changes. KEY FINDINGS: IS directly impairs endothelium-dependent vasorelaxation and endothelial cell migration. Mechanistic studies revealed increased production of reactive oxygen species-related markers, reduction of endothelial nitric oxide synthase and increased protein expression of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). IS also increases angiopoietin-2 and tumour necrosis factor α gene expression and promotes transforming growth factor ß receptor abundance. Inhibition of ASK1 ameliorated the increase in oxidative stress markers, promoted autocrine interleukin 8 pro-angiogenic signalling and decreased anti-angiogenic responses at least in part via reducing TIMP1 protein expression. SIGNIFICANCE: ASK1 inhibition attenuated vasorelaxation and endothelial cell migration impaired by IS. Therefore, ASK1 is a viable intracellular target to alleviate uremic toxin-induced impairment in the vasculature.

Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction.

AIMS: To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction. METHODS AND RESULTS: Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs. CONCLUSIONS: Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

Inhibition of apoptosis signal-regulating kinase 1 ameliorates left ventricular dysfunction by reducing hypertrophy and fibrosis in a rat model of cardiorenal syndrome.

BACKGROUND: Cardiorenal syndrome (CRS) is a major health burden worldwide in need of novel therapies, as current treatments remain suboptimal. The present study assessed the therapeutic potential of apoptosis signal-regulating kinase 1 (ASK1) inhibition in a rat model of CRS. METHODS: Adult male Sprague-Dawley rats underwent surgery for myocardial infarction (MI) (week 0) followed by 5/6 subtotal nephrectomy (STNx) at week 4 to induce to induce a combined model of heart and kidney dysfunction. At week 6, MI + STNx animals were randomized to receive either 0.5% carboxymethyl cellulose (Vehicle, n = 15, Sham = 10) or G226 (15 mg/kg daily, n = 11). Cardiac and renal function was assessed by echocardiography and glomerular filtration rate (GFR) respectively, prior to treatment at week 6 and endpoint (week 14). Haemodynamic measurements were determined at endpoint prior to tissue analysis. RESULTS: G226 treatment attenuated the absolute change in left ventricular (LV) fractional shortening and posterior wall thickness compared to Vehicle. G226 also attenuated the reduction in preload recruitable stroke work. Increased myocyte cross sectional area, cardiac interstitial fibrosis, immunoreactivity of cardiac collagen-I and III and cardiac TIMP-2 activation, were significantly reduced following G226 treatment. Although we did not observe improvement in GFR, G226 significantly reduced renal interstitial fibrosis, diminished renal collagen-I and -IV, kidney injury molecule-1 immunoreactivity as well as macrophage infiltration and SMAD2 phosphorylation. CONCLUSION: Inhibition of ASK1 ameliorated LV dysfunction and diminished cardiac hypertrophy and cardiorenal fibrosis in a rat model of CRS. This suggests that ASK1 is a critical pathway with therapeutic potential in the CRS setting.

Cardiorenal syndrome: Multi-organ dysfunction involving the heart, kidney and vasculature.

Cardiorenal syndrome (CRS) is a multi-organ disease, encompassing heart, kidney and vascular system dysfunction. CRS is a worldwide problem, with high morbidity, mortality, and inflicts a significant burden on the health care system. The pathophysiology is complex, involving interactions between neurohormones, inflammatory processes, oxidative stress and metabolic derangements. Therapies remain inadequate, mainly comprising symptomatic care with minimal prospect of full recovery. Challenges include limiting the contradictory effects of multi-organ targeted drug prescriptions and continuous monitoring of volume overload. Novel strategies such as multi-organ transplantation and innovative dialysis modalities have been considered but lack evidence in the CRS context. The adjunct use of pharmaceuticals targeting alternative pathways showing positive results in preclinical models also warrants further validation in the clinic. In recent years, studies have identified the involvement of gut dysbiosis, uraemic toxin accumulation, sphingolipid imbalance and other unconventional contributors, which has encouraged a shift in the paradigm of CRS therapy.

Cardiomyocyte Functional Etiology in Heart Failure With Preserved Ejection Fraction Is Distinctive-A New Preclinical Model.

BACKGROUND: Among the growing numbers of patients with heart failure, up to one half have heart failure with preserved ejection fraction (HFpEF). The lack of effective treatments for HFpEF is a substantial and escalating unmet clinical need-and the lack of HFpEF-specific animal models represents a major preclinical barrier in advancing understanding of HFpEF. As established treatments for heart failure with reduced ejection fraction (HFrEF) have proven ineffective for HFpEF, the contention that the intrinsic cardiomyocyte phenotype is distinct in these 2 conditions requires consideration. Our goal was to validate and characterize a new rodent model of HFpEF, undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology. METHODS AND RESULTS: The selectively inbred Hypertrophic Heart Rat (HHR) strain exhibits adult cardiac enlargement (without hypertension) and premature death (40% mortality at 50 weeks) compared to its control strain, the normal heart rat. Hypertrophy was characterized in vivo by maintained systolic parameters (ejection fraction at 85%-90% control) with marked diastolic dysfunction (increased E/E'). Surprisingly, HHR cardiomyocytes were hypercontractile, exhibiting high Ca2+ operational levels and markedly increased L-type Ca2+ channel current. In HHR, prominent regions of reparative fibrosis in the left ventricle free wall adjacent to the interventricular septum were observed. CONCLUSIONS: Thus, the cardiomyocyte remodeling process in the etiology of this HFpEF model contrasts dramatically with the suppressed Ca2+ cycling state that typifies heart failure with reduced ejection fraction. These findings may explain clinical observations, that treatments considered appropriate for heart failure with reduced ejection fraction are of little benefit for HFpEF-and suggest a basis for new therapeutic strategies.

Angiotensin receptor neprilysin inhibition provides superior cardioprotection compared to angiotensin converting enzyme inhibition after experimental myocardial infarction.

BACKGROUND: Angiotensin receptor neprilysin inhibitor (ARNi) enhances beneficial natriuretic peptides by inhibiting their breakdown through neprilysin. Although the first-in-class ARNi sacubitril/valsartan (LCZ696) reduced mortality and morbidity in heart failure (HF) with reduced ejection fraction (EF) compared to angiotensin converting enzyme inhibitor (ACEi), mechanistic data on ARNi are scarce. ARNi may be superior to ACEi in attenuating adverse cardiac remodeling and dysfunction post-myocardial infarction (MI). METHODS: Rats randomized at 1 week post-MI were administered LCZ696 (60 mg/kg, N = 12), the ACEi perindopril (2 mg/kg, N = 11) or vehicle (corn oil, N = 13), orally for 4 weeks. Sham rats received vehicle (corn oil, N = 9). Echocardiography was assessed before and after treatment, prior to invasive hemodynamics using pressure-volume analysis. Hypertrophy and fibrosis was evaluated by histochemical staining, and analysis of myocardial gene and protein expression using real-time quantitative PCR and Western blot. RESULTS: Compared to Sham, MI groups had large infarcts (>40%) and reduced left ventricular (LV) EF. LCZ696 improved LVEF and end systolic pressure-volume relationship compared to perindopril (P < 0.05). LCZ696 but not perindopril reduced lung weight and LV filling pressures post-MI. Reductions in cardiac hypertrophy and fibrosis were similar, however gene expression of hypertrophic markers, ANP and ßMHC were reduced with LCZ696 versus perindopril. LCZ696 versus perindopril reduced myocardial TIMP2 gene expression with a trend (P = 0.067) to lowering collagen I. CONCLUSION: LCZ696 attenuated adverse cardiac remodeling and dysfunction and reduced pulmonary congestion and hypertrophic markers after MI compared to perindopril. This study supports clinical evaluation of ARNi versus ACEi in targeting post-MI cardiac dysfunction and remodeling.

Chronic kidney disease with comorbid cardiac dysfunction exacerbates cardiac and renal damage.

To address the pathophysiological mechanisms underlying chronic kidney disease with comorbid cardiac dysfunction, we investigated renal and cardiac, functional and structural damage when myocardial infarction (MI) was applied in the setting of kidney injury (induced by 5/6 nephrectomy-STNx). STNx or Sham surgery was induced in male Sprague-Dawley rats with MI or Sham surgery performed 4 weeks later. Rats were maintained for a further 8 weeks. Rats (n = 36) were randomized into four groups: Sham+Sham, Sham+MI, STNx+Sham and STNx+MI. Increased renal tubulointerstitial fibrosis (P < 0.01) and kidney injury molecule-1 expression (P < 0.01) was observed in STNx+MI compared to STNx+Sham animals, while there were no further reductions in renal function. Heart weight was increased in STNx+MI compared to STNx+Sham or Sham+MI animals (P < 0.05), despite no difference in blood pressure. STNx+MI rats demonstrated greater cardiomyocyte cross-sectional area and increased cardiac interstitial fibrosis compared to either STNx+Sham (P < 0.01) or Sham+MI (P < 0.01) animals which was accompanied by an increase in diastolic dysfunction. These changes were associated with increases in ANP, cTGF and collagen I gene expression and phospho-p38 MAPK and phospho-p44/42 MAPK protein expression in the left ventricle. Addition of MI accelerated STNx-induced structural damage but failed to significantly exacerbate renal dysfunction. These findings highlight the bidirectional response in this model known to occur in cardiorenal syndrome (CRS) and provide a useful model for examining potential therapies for CRS.

Protein-bound uremic toxins: a long overlooked culprit in cardiorenal syndrome.

Protein-bound uremic toxins (PBUTs) accumulate once renal excretory function declines and are not cleared by dialysis. There is increasing evidence that PBUTs exert toxic effects on many vital organs, including the kidney, blood vessels, and heart. It has been suggested that PBUTs are likely to be a potential missing link in cardiorenal syndrome, based on the high incidence of cardiovascular events and mortality in the dialysis population, which are dramatically reduced in successful kidney transplant recipients. These data have led the call for more effective dialysis or additional adjunctive therapy to eradicate these toxins and their adverse biological effects. Indoxyl sulfate and p-cresyl sulfate are the two most problematic PBUTs, conferring renal and cardiovascular toxicity, and are derived from dietary amino acid metabolites by colonic microbial organisms. Therefore, targeting the colon where these toxins are initially produced appears to be a potential therapeutic alternative for patients with chronic kidney disease. This strategy, if approved, is likely to be applicable to predialysis patients, thereby potentially preventing progression of chronic kidney disease to end-stage renal disease as well as preventing the development of cardiorenal syndrome.