Indoxyl sulphate (IS) accumulates during chronic kidney disease (CKD) leading to CKD- related myocardial fibrosis. We previously reported that IS activates cardiac fibroblasts (Fib) to a fibrotic/inflammatory phenotype [Ruggeri C, SIRC 2018]. Previous authors proposed the inhibition of Aryl hydrocarbon Receptor (AhR) activity to counteract IS effects. Thus, we tested the AhR inhibitor CH-223191 (CH) against IS effects in neonatal cardiac mouse Fib (nmFib). Then, we focused on the interplay between AhR and its cognate transcriptor factor nuclear erythroid 2- related factor 2-(Nrf2). CH hampered the myoFib transition of neonatal mouse Fib (nmFib) treated with 50 μM IS, keeping the same proliferation rate of control cells and counteracting a-SMA and Periostin overexpression. Unexpectedly, CH upregulated and didn't prevent IS- induced mRNA of Renin- Angiotensin System (R.A.S.) elements. IS increased TLR4, MCP1 mRNA and collagen1 deposition; CH upregulated TLR4 expression and collagen deposition, both in coadministration with IS or alone. Mouse neonatal cardiomyocytes incubated with IS-treated nmFib conditioned media overexpressed TNFa, Angiotensin type 1 Receptor, Neprilysin and Myostatin; such effects were enhanced with media from CH + IS-treated nmFib. CH counteracted Nrf2 mRNA and nuclear translocation induced by IS in nmFib; Nrf2 loss-of- function obtained with Nrf2 Dominant Negative (DN) transfection, enhanced IS-induced TLR4 mRNA and decreased that of NQO1, a Nrf2-downstream gene. IS induced Nrf2 perinuclear localization in control cells while Nrf2 DN in nmFib restrained the Nrf2 signal in the cytoplasm and enhanced TLR4 immunofluorescence. In agreement with CH + IS treated nmFib, Nrf2 DN increased IS-induced RAS elements, MCP1 and TNFa mRNA. Concluding, IS caused Fib-to-myoFib transition through a AhR/Nrf2 balance. AhR inhibition in nmFib counteracted IS-induced fibrosis but hampered Nrf2 activity and upregulated TLR4, the R.A. S. and the release of mediators with inflammatory paracrine effects on cardiomyocytes. These results provide hints to design multiple targeting pharmacological strategies for protection from IS- induced heart fibrosis.
Effects of the AhR inhibitor CH-223191 on the indoxyl sulphateinduced cardiac fibroblast activation. Role of Nrf2
Barisione C;Garibaldi S;Verzola D;Altieri P;Ghigliotti G;Furfaro A;Nitti M;Ameri P
2022-01-01
Abstract
Indoxyl sulphate (IS) accumulates during chronic kidney disease (CKD) leading to CKD- related myocardial fibrosis. We previously reported that IS activates cardiac fibroblasts (Fib) to a fibrotic/inflammatory phenotype [Ruggeri C, SIRC 2018]. Previous authors proposed the inhibition of Aryl hydrocarbon Receptor (AhR) activity to counteract IS effects. Thus, we tested the AhR inhibitor CH-223191 (CH) against IS effects in neonatal cardiac mouse Fib (nmFib). Then, we focused on the interplay between AhR and its cognate transcriptor factor nuclear erythroid 2- related factor 2-(Nrf2). CH hampered the myoFib transition of neonatal mouse Fib (nmFib) treated with 50 μM IS, keeping the same proliferation rate of control cells and counteracting a-SMA and Periostin overexpression. Unexpectedly, CH upregulated and didn't prevent IS- induced mRNA of Renin- Angiotensin System (R.A.S.) elements. IS increased TLR4, MCP1 mRNA and collagen1 deposition; CH upregulated TLR4 expression and collagen deposition, both in coadministration with IS or alone. Mouse neonatal cardiomyocytes incubated with IS-treated nmFib conditioned media overexpressed TNFa, Angiotensin type 1 Receptor, Neprilysin and Myostatin; such effects were enhanced with media from CH + IS-treated nmFib. CH counteracted Nrf2 mRNA and nuclear translocation induced by IS in nmFib; Nrf2 loss-of- function obtained with Nrf2 Dominant Negative (DN) transfection, enhanced IS-induced TLR4 mRNA and decreased that of NQO1, a Nrf2-downstream gene. IS induced Nrf2 perinuclear localization in control cells while Nrf2 DN in nmFib restrained the Nrf2 signal in the cytoplasm and enhanced TLR4 immunofluorescence. In agreement with CH + IS treated nmFib, Nrf2 DN increased IS-induced RAS elements, MCP1 and TNFa mRNA. Concluding, IS caused Fib-to-myoFib transition through a AhR/Nrf2 balance. AhR inhibition in nmFib counteracted IS-induced fibrosis but hampered Nrf2 activity and upregulated TLR4, the R.A. S. and the release of mediators with inflammatory paracrine effects on cardiomyocytes. These results provide hints to design multiple targeting pharmacological strategies for protection from IS- induced heart fibrosis.
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