bay-11-7082 and Renal-Insufficiency--Chronic

Vascular calcification (VC) is highly prevalent in patients with chronic kidney disease (CKD) and contributes to their high rate of cardiovascular mortality. Indoxyl sulfate (IS) is a representative protein-bound uremic toxin in CKD patients, which has been recognized as a major risk factor for VC. Recent studies have demonstrated that nuclear factor-kappa B (NK-κB) is highly activated in the chronic inflammation conditions of CKD patients and participated in the pathogenesis of VC. However, whether NK-κB is involved in the progression of IS-induced VC remains without elucidation. Here, we showed that NK-κB activity was increased in the IS-induced calcification of human aortic smooth muscle cells (HASMCs). Blocking the NK-κB with a selective inhibitor (Bay-11-7082) significantly relieved the osteogenic transdifferentiation of HASMCs, characterized by the downregulation of early osteogenic-specific marker, core-binding factor alpha subunit 1 (Cbfα1), and upregulation of smooth muscle α-actin (α-SMA), a specific vascular smooth muscle cell marker. Besides, IS stimulated the activation of PI3K/Akt signaling. Furthermore, LY294002, a specific inhibitor of PI3K/Akt pathway, attenuated the activation of NK-κB and osteogenic differentiation of HASMCs. Together, these results suggest that PI3K/Akt/NK-κB signaling plays an important role in the pathogenesis of osteogenic transdifferentiation induced by IS.

Topics: Actins; Cell Line; Chromones; Core Binding Factor Alpha 1 Subunit; Down-Regulation; Humans; Indican; Morpholines; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Renal Insufficiency, Chronic; Signal Transduction; Sulfones; Up-Regulation; Vascular Calcification

Vascular calcification is common in chronic kidney disease (CKD) patients, while erythropoietin (EPO) is widely used in the treatment of renal anemia in CKD patients, whether there is a link between the two is still not clear.. The primary rat vascular smooth muscle cells (VSMCs) and CKD rats were treated with EPO and the calcium deposition was observed by alizarin red staining, von Kossa staining and calcium quantification. Activation of JAK2/STAT3/BMP-2 axis and NF-κB signaling pathways was investigated by Western blotting.. EPO-induced calcium deposition in VSMCs and significantly potentiated calcification in CKD rats. Furthermore, EPO activated JAK2/STAT3/BMP-2 axis, NF-κB pathway and the pro-calcification effect of EPO was partially blocked by the STAT3 inhibitor (Cryptotanshinone) or NF-κB inhibitor (BAY 11-7082), respectively, in vitro.. EPO could promote VSMCs calcification in vitro and in vivo and this effect may be achieved through the JAK2/STAT3/BMP-2 axis and NF-κB pathway.

Topics: Alkaline Phosphatase; Anemia; Animals; Bone Morphogenetic Protein 2; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Erythropoietin; Gene Expression; Janus Kinase 2; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NF-kappa B; Nitriles; Phenanthrenes; Phosphorylation; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Signal Transduction; STAT3 Transcription Factor; Sulfones; Up-Regulation; Vascular Calcification

The Na/K-ATPase is an important membrane ion transporter and a signaling receptor that is essential for maintaining normal cell function. The current study examined the role of Na/K-ATPase signaling in regulating miR-29b-3p, an anti-fibrotic microRNA, in a mouse chronic kidney disease (CKD) model (5/6th partial nephrectomy or PNx). The results showed that CKD induced significant reduction of miR-29b-3p expression in the heart tissue by activation of Src and NFκB signaling in these animals. To demonstrate the role of Na/K-ATPase signaling, we also performed the PNx surgery on Na/K-ATPase α1 heterozygous (α1+/-) mice, which expresses ~40% less Na/K-ATPase α1 compared to their wild type littermates (WT) and exhibits deficiency in Na/K-ATPase signaling. We found that CKD did not significantly change the miR-29b-3p expression in heart tissue from the α1+/- animals. We also found that CKD failed to activate Src and NFκB signaling in these animals. Using isolated cardiac fibroblasts from α1+/- mice and their WT littermates, we showed that ouabain, a specific Na/K-ATPase ligand, induces decreased miR-29b-3p expression in fibroblasts isolated from WT mice, but had no effect in cells from α1+/- mice. Inhibition of NFκB by Bay11-7082 prevented ouabain-induced miR-29b-3p reduction in WT fibroblasts. To further confirm the in vivo effect of Na/K-ATPase signaling in regulation of miR-29b-3p and cardiac fibrosis in CKD animals, we used pNaKtide, a Src inhibiting peptide derived from the sequence of Na/K-ATPase, to block the activation of Na/K-ATPase signaling. The result showed that pNaKtide injection significantly increased miR-29b-3p expression and mitigated the CKD-induced cardiac fibrosis in these animals. These results clearly demonstrated that Na/K-ATPase signaling is an important mediator in CKD that regulates miR-29b-3p expression and cardiac fibrosis, which provides a novel target for regulation of miR-29b-3p in CKD. We also demonstrate that antagonizing Na/K-ATPase signaling by pNaKtide can reduce organ fibrosis through the stimulation of tissue miR-29b-3p expression.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Down-Regulation; Fibrosis; Humans; Male; Mice; MicroRNAs; Myocardium; Nitriles; Ouabain; Renal Insufficiency, Chronic; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Sulfones