Sphingolipid imbalance and inflammatory effects induced by uremic toxins in heart and kidney cells are reversed by dihydroceramide desaturase 1 inhibition
Introduction
Dyslipidaemia is a key process involved in atherosclerotic disease development and metabolic derangement in cardiovascular disease (CVD) and chronic kidney disease (CKD) (Dincer et al., 2019; Liu et al., 2014). The role of low- and high-density lipoprotein imbalance in dyslipidaemia is well described in these patients (Bianchi et al., 2011; Napoli et al., 2011). Sphingolipid imbalance, however, is a rather new consideration. De novo synthesis of sphingolipids is characterised by the non-reversible conversion of dihydroceramide (dhCer) into ceramide (Cer) mediated by the enzyme dihydroceramide desaturase 1 (Des1) (Rodriguez-Cuenca et al., 2015). Cer is an important precursor of other bioactive sphingolipids. Increased Cer level is implicated in cardiac and renal lipotoxicity (Rodriguez-Cuenca et al., 2015; Zager et al., 1997) and in worsening of cardiac and renal ischemia reperfusion injury (Baranowski and Gorski, 2011; Zager et al., 1997). On the contrary, the role of dhCer in diseases remain largely obscure (Magaye et al., 2018).
Non-dialysable protein-bound uremic toxins (PBUTs) are important contributors to CVD development in CKD patients. Indoxyl sulfate (IS) and p-cresol sulfate (PCS) are highly clinically relevant PBUTs due to their high protein-binding affinity (>90 %, especially towards albumin), leading to poor removal rate via conventional dialysis methods and their subsequent accumulation (Savira et al., 2019). IS and PCS contribute to cardiac hypertrophy and cardiorenal fibrosis in vitro and in vivo (Savira et al., 2017). Furthermore, there is emerging evidence of the role of PBUTs in dyslipidaemia. PCS administration to healthy mice induces CKD-like insulin resistance and fat loss, and prompts ectopic lipid storage in muscle and liver (Koppe et al., 2013), while IS is involved in the oxidation of low density lipoprotein (Praschberger et al., 2014). In terms of sphingolipids, IS was demonstrated to elevate eryptosis via a Cer-mediated increase in cytosolic Ca2+ activity (Ahmed et al., 2013a, b). These studies suggest PBUT could alter the lipid profile in diseased states, denoting a novel mechanism by which PBUTs can cause adverse effects. Therefore, this proof-of-concept study sought to assess 1) whether sphingolipid imbalance is involved in PBUT-mediated cardiorenal effects, and 2) whether Des1 inhibition could reverse PBUT-mediated adverse effects in heart and kidney cells.
Section snippets
Materials
IS and PCS were obtained from Sigma. The selective Des1 inhibitor CIN038 was developed in house at Monash Institute of Pharmaceutical Science. Our group had previously identified 4-((5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (CIN038) as a potent and selective inhibitor of Des1 (IC50 = 0.55 μM) (Aurelio et al., 2016; Flynn et al., 2015). All cell culture media and supplements were obtained from GIBCO (ThermoFisher Scientific, Waltham, MA, USA). Other reagents were purchased
The effect of Des1 inhibition on uremic toxin-induced hypertrophy
IS (10 μM) and PCS (100 μM) modestly increased NCM hypertrophy by 14.2 % and 14.7 % vs. control, respectively (p < 0.01, Fig. 1), similar to our previous reports (Lekawanvijit et al., 2010; Savira et al., 2017). The selective Des1 inhibitor, CIN038, dose dependently reduced these effects at a concentration range of 0.01–1.0 μM, reaching significance at the highest concentration (p < 0.001).
The effect of Des1 inhibition on uremic toxin-induced collagen synthesis
Collagen synthesis was 20.6 ± 2.9 % and 19.5 ± 1.7 % higher in IS- and PCS-stimulated NCF compared to
Discussion
The present study assessed Des1 inhibition in the setting of PBUT-induced adverse cardiac and renal cellular effects. The principal findings include: 1) direct inflammatory and cellular remodelling effects of PBUT are at least partially attributable to the reduction of C16-dhCer (d34:0) levels and increased NF-κB protein expression, and 2) Des1 inhibition mitigates PBUT-mediated inflammatory responses, cellular remodelling and restored SL imbalance (Fig. 8).
Our phenotypic data suggest that Des1
Conclusion
This preliminary, proof-of-concept study suggest Des1 inhibition can attenuate PBUT-mediated adverse cardiac and renal cellular effects by restoring sphingolipid imbalance and attenuating inflammatory responses, with few notable differences in mechanism of action between IS and PCS. These results demonstrate that sphingolipid modifying agents may have a therapeutic role in CVD and CKD, and thus warrant further investigation.
Funding
The work described here was funded by a National Health and Medical Research Council of Australia (Program Grant ID#1092642 and Project Grant ID#1087355).
Author contributions
Conceptualisation; B.H.W., R.M., F.S. Data curation; F.S. Formal analysis; F.S. Funding acquisition; B.H.W., D.L., C.R. Investigation; F.S., Y.H., R.M., X.X., D.A. Methodology; B.H.W., D.J.C, D.A. Project administration; B.H.W., L.H. Resources; B.H.W., B.L.F., C.V.S., S.M.P. Software; B.H.W., D.C. Supervision; B.H.W., A.R.K. Validation; F.S., R.M., Y.H. X.X., A.R.K, B.L.F., C.V.S., S.M.P, D.K. Visualisation; F.S., R.M., X.X. Roles/Writing - original draft; F.S. Writing - review & editing: R.M.,
Declaration of Competing Interest
B.L.F. is CEO and S.M.P. is CSO of Cincera Therapeutics Pty Ltd (cinceratx.com), which is developing Des1 inhibitors for the treatment of a variety of human diseases. All other authors have nothing to declare.
Acknowledgements
F.S. and R.M. are supported by Monash International Postgraduate Research Scholarship and Monash Graduate Scholarship. F.S. is supported by a Postgraduate Publication Award. S.M.P. was supported by a National Health and Medical Research Council of Australia Senior Research Fellowship (ID#1156693). Lipidomics analysis was performed at the Monash Proteomics and Metabolomics Facility.
References (48)
- et al.
Nrf2 signaling pathway: pivotal roles in inflammation
Biochim. Biophys. Acta Mol. Basis Dis.
(2017) - et al.
Inhibitory kappa-B kinase-β inhibition prevents adaptive left ventricular hypertrophy
J. Surg. Res.
(2012) - et al.
The varying faces of IL-6: from cardiac protection to cardiac failure
Cytokine
(2015) - et al.
Downregulation of adipose triglyceride lipase promotes cardiomyocyte hypertrophy by triggering the accumulation of ceramides
Arch. Biochem. Biophys.
(2015) - et al.
Elevated circulating levels of interleukin-6 in patients with chronic renal failure
Kidney Int.
(1991) - et al.
Sphingolipid signaling in renal fibrosis
Matrix Biol.
(2018) - et al.
Dihydroceramide is a key metabolite that regulates autophagy and promotes fibrosis in hepatic steatosis model
Biochem. Biophys. Res. Commun.
(2017) - et al.
Subtotal nephrectomy accelerates pathological cardiac remodeling post-myocardial infarction: implications for cardiorenal syndrome
Int. J. Cardiol.
(2013) - et al.
Integrated targeted sphingolipidomics and transcriptomics reveal abnormal sphingolipid metabolism as a novel mechanism of the hepatotoxicity and nephrotoxicity of triptolide
J. Ethnopharmacol.
(2015) - et al.
Dihydroceramide desaturase 1, the gatekeeper of ceramide induced lipotoxicity
Biochim. Biophys. Acta
(2015)
Molecular mechanisms of protein-bound uremic toxin-mediated cardiac, renal and vascular effects: underpinning intracellular targets for cardiorenal syndrome therapy
Toxicol. Lett.
Vitamin E γ-tocotrienol inhibits cytokine-stimulated NF-κB activation by induction of anti-inflammatory A20 via stress adaptive response due to modulation of sphingolipids
J. Immunol.
p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase
Kidney Int.
Indoxyl sulfate induces oxidative stress and hypertrophy in cardiomyocytes by inhibiting the AMPK/UCP2 signaling pathway
Toxicol. Lett.
Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats
Life Sci.
Altered ceramide and sphingosine expression during the induction phase of ischemic acute renal failure
Kidney Int.
Triggering of suicidal erythrocyte death by uremic toxin indoxyl sulfate
BMC Nephrol.
The uremic toxin acrolein promotes suicidal erythrocyte death
Kidney Blood Press. Res.
Short-term magnesium deficiency upregulates ceramide synthase in cardiovascular tissues and cells: cross-talk among cytokines, Mg2+, NF-κB, and de novo ceramide
Am. J. Physiol. Heart Circ. Physiol.
From sphingosine kinase to dihydroceramide desaturase: a structure-activity relationship (SAR) study of the enzyme inhibitory and anticancer activity of 4-((4-(4-chlorophenyl)thiazol-2-yl)amino)phenol (SKI-II)
J. Med. Chem.
Heart sphingolipids in health and disease
Adv. Exp. Med. Biol.
Decreased expression of natriuretic peptides associated with lipid accumulation in cardiac ventricle of obese mice
Endocrinology
Statins and lipid-lowering strategies in cardiorenal patients
Contrib. Nephrol.
Indoxyl sulfate, a uremic toxin, downregulates renal expression of Nrf2 through activation of NF-kappaB
BMC Nephrol.
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