Indoxyl sulfate down-regulates SLCO4C1 transporter through up-regulation of GATA3.

The accumulated uremic toxins inhibit the expression of various renal transporters and this inhibition may further reduce renal function and subsequently cause the accumulation of uremic toxins. However, the precise mechanism of the nephrotoxicity of uremic toxins on renal transport has been poorly understood. Here we report that indoxyl sulfate, one of the potent uremic toxins, directly suppresses the renal-specific organic anion transporter SLCO4C1 expression through a transcription factor GATA3. The promoter region of SLCO4C1 gene has several GATA motifs, and indoxyl sulfate up-regulated GATA3 mRNA and subsequently down-regulated SLCO4C1 mRNA. Overexpression of GATA3 significantly reduced SLCO4C1 expression, and silencing of GATA3 increased SLCO4C1 expression vice versa. Administration of indoxyl sulfate in rats reduced renal expression of slco4c1 and under this condition, plasma level of guanidinosuccinate, one of the preferable substrates of slco4c1, was significantly increased without changing plasma creatinine. Furthermore, in 5/6 nephrectomized rats, treatment with oral adsorbent AST-120 significantly decreased plasma indoxyl sulfate level and conversely increased the expression of slco4c1, following the reduction of plasma level of guanidinosuccinate. These data suggest that the removal of indoxyl sulfate and blocking its signal pathway may help to restore the SLCO4C1-mediated renal excretion of uremic toxins in CKD

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism

Schematic representation of putative pathological role of IS as GATA(s) inducer.

<p>IS enhances the expression of GATA factors and causes reduction of SLCO4C1 expression, followed by the further accumulation of uremic toxins, which causes the exacerbation of CKD. Oral absorbent AST-120, kidney specific GATA inhibition and up-regulation of SLCO4C1 by statins would be hopeful therapeutic strategies for CKD. UTs, uremic toxins.</p

Negative regulation of GATA3 on SLCO4C1 expression.

<p>(A) Overexpression of GATA3. (B and C) Knockdown of GATA3. (B) Time-dependent alteration of the GATA3 and SLCO4C1 mRNA expression levels. (C) Western blot analysis of GATA3 (left) and SLCO4C1 (right). The bands were quantified by densitometry and normalized to the level of GAPDH. A representative band is shown. **<i>P</i><0.01 versus control (n = 3 per group).</p

Effect of AST-120 on slco4c1 expression in the renal failure <i>in vivo</i>.

<p>(A) Plasma IS concentration after 4 weeks of treatment of AST-120. (B) mRNA level of rat slco4c1 in the kidney after AST-120 administration. (C) Plasma concentrations of creatinine, creatinine clearance (Ccr) and representative substrates for slco4c1 transporter, GSA, ADMA and <i>trans</i>-aconitate. *<i>P</i><0.05; **<i>P</i><0.01; ^#P<0.01 versus control.</p

Effects of IS on SLCO4C1 expression.

<p>(A) The mRNA expression levels of human SLCO4C1 24 h after treatment with various uremic solutes in ACHN are shown. IS, indoxyl sulfate; SDMA, symmetric demethylarginine; ADMA, asymmetric dimethylarginine; GSA, guanidinosuccinate; t-Aco, <i>trans</i>-aconintate; c-Aco, <i>cis</i>-aconitate; TMAO, trimethylamine <i>N</i>-oxide; IAA, indole-3-acetate. <u>All the concentrations were 1.0 mM.</u> (B) Effect of IS on SLCO4C1 mRNA expression in HK-2 cells. HK-2 cells were incubated with IS for 24 h. **<i>P</i><0.01 versus control (n = 3–4 per group).</p