No abstract

Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G(1) phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic kidney disease (CKD). This study aimed to determine if IS induces epithelial-to-mesenchymal transition (EMT) in the kidneys of hypertensive rats and human proximal tubular cells (HK-2). Methods: EMT was evaluated by immunohistochemistry, reverse transcription-polymerase chain reaction and immunoblotting of the epithelial markers E-cadherin and zonula occludens-1 (ZO-1), and the mesenchymal marker α-smooth muscle actin (α-SMA). Rat groups consisted of (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive IS-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive IS-administered rats (DH+IS). HK-2 cells were incubated with or without IS. Results: In kidneys, DH rats showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA compared with DN rats. DN+IS and DH+IS rats showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA compared with DN and DH rats, respectively. DH+IS and DH rats showed increased Masson’s trichrome-positive fibrosis areas compared with DH and DN, respectively. IS-treated HK-2 cells showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA. Conclusion: IS induces EMT in the kidneys of hypertensive rats and in human proximal tubular cells.

Background/Aim: Klotho, an anti-aging gene, is expressed in the kidneys, and its renal expression is decreased in chronic kidney disease (CKD). The present study aimed to examine whether renal expression of Klotho is regulated by indoxyl sulfate, a uremic toxin, using rat kidneys and human proximal tubular cells (HK-2). Methods: The effect of indoxyl sulfate on renal expression of Klotho was examined using (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). The effects of indoxyl sulfate, inhibitors of nuclear factor-ĸB (NF-ĸB) and an antioxidant on the expression of Klotho in HK-2 cells were examined. Results: DH+IS and DN+IS rats showed decreased expression of Klotho mRNA in the kidneys as compared with DH and DN rats, respectively. Indoxyl sulfate suppressed the expression of Klotho mRNA and protein in HK-2 cells, whereas an antioxidant, N-acetylcysteine, and NF-ĸB inhibitors, pyrrolidine dithiocarbamate and isohelenin, alleviated these effects. Conclusions: Indoxyl sulfate downregulates Klotho expression in kidneys through production of reactive oxygen species and activation of NF-ĸB in proximal tubular cells. Indoxyl sulfate may be involved in reduced renal expression of Klotho in CKD.

In chronic kidney disease (CKD), indoxyl sulfate, a uremic toxin, accumulates in serum, and the expression of angiotensinogen (AGT) is upregulated in renal proximal tubular cells. The present study aimed to determine the relationship between indoxyl sulfate and the upregulation of AGT expression in proximal tubular cells. Indoxyl sulfate induced expression of AGT in rat renal cortex and in cultured human proximal tubular cells (HK-2). In proximal tubular cells, indoxyl sulfate induced phosphorylation of cAMP response element-binding protein (CREB) on Ser-133, and small interfering RNA (siRNA) specific to CREB inhibited indoxyl sulfate-induced AGT expression. Our previous study demonstrated that indoxyl sulfate activated nuclear factor-κB (NF-κB) through reactive oxygen species (ROS) production. NF-κB inhibitors (pyrrolidine dithiocarbamate and isohelenin), NF-κB p65 siRNA, an antioxidant [N-acetylcysteine (NAC)], and a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor [diphenyleneiodonium (DPI)] suppressed indoxyl sulfate-induced AGT expression. Both NAC and DPI suppressed indoxyl sulfate-induced expression of NF-κB p65 and CREB. CREB siRNA suppressed indoxyl sulfate-induced NF-κB p65 expression, whereas both NF-κB inhibitors and NF-κB p65 siRNA prevented indoxyl sulfate-induced CREB expression. Furthermore, we focused on the expression of NADPH oxidase 4 (NOX4), because indoxyl sulfate induced NOX4 expression in vascular smooth muscle cells and vascular endothelial cells. Indoxyl sulfate induced the expression of NOX4 in proximal tubular cells, which was suppressed by NAC, DPI, NF-κB inhibitors, NF-κB p65 siRNA, and CREB siRNA. Taken together, CREB, NF-κB, and NOX4 coordinately upregulate indoxyl sulfate-induced AGT expression in proximal tubular cells.