Characterization of human SLCO4C1 TG rats is shown. (A) Three different smaller sizes of mRNA by alternative splicing were found and mutated to avoid unusual splicing (AGGT to AGGG). (B) The mutated human SLCO4C1 cDNA was inserted into a plasmid under the proximal tubule–specific promoter. (C) Expression of human SLCO4C1 in rat organs and microdissected renal tubules examined by reverse transcriptase–PCR. Gl, glomerulus; S1, proximal tubule S1 segment; S2, proximal tubule S2 segment; S3, proximal tubule S3 segment; mTAL, medullary thick ascending limb; cTAL, cortical thick ascending limb; CCD, cortical collecting duct; IMCD, inner medulla collecting duct. (D) Immunohistochemical analysis. The human SLCO4C1 immunostains were abolished by peptide absorption. Bars = 100 μm.

Metabolome analysis and characterization of uremic toxins are shown. (A through E and G) The plasma concentration of creatinine (A), indoxyl sulfate (B), ADMA (C), GSA (D), trans-aconitate (E), and citrulline (G) before and 3 wk after five-sixths Nx (n = 4 to 5 per group). (F) The mRNA expression level of DDAH1 and DDAH2 in the kidney 3 wk after five-sixths Nx (n = 5 per group). (H) BP after intraperitoneal injection of trans-aconitate (400 mg/kg; n = 5 per group). (I) Trans-aconitate–induced superoxide production in HK-2 cells. *P < 0.05.

Transcriptional analysis and ligand screening are shown. (A) The 5′ region of human SLCO4C1. Potential cis-acting sequences are indicated. Met, first methionine. (B) Promoter activity of human SLCO4C1. Deletion constructs of the human SLCO4C1 promoter region were analyzed with 3-MC (5 μM). *P < 0.05 (n = 3 to 4 per group). (C) Enhancement of promoter activity of human SLCO4C1 with various compounds (concentration as indicated, μM). Quer, quercetin; t-BHQ, tert-butylhydroquinone; I3C, indole-3-carbinole; IS, indoxyl sulfate; Tauro, taurocholic acid; Prava, pravastatin; Fluva, fluvastatin. *P < 0.05 versus DMSO; ^#P < 0.05 versus H₂O (n = 3 to 4 per group). (D) Effect of fluvastatin (10 μM) on human SLCO4C1 transcription. Deletion constructs and loss-of-function mutation construct in XRE motifs of human SLCO4C1 were examined. *P < 0.05 (n = 3 to 4 per group). (E) ChIP assay and Western blotting of 3-MC or fluvastatin-treated cells. (Top) After application of 3-MC (1 μM) or fluvastatin (10 μM), fixed cell extract was analyzed by mouse cyp1a1 XRE or human SLCO4C1 XRE PCR. (Bottom) Western blotting of nuclear and cytoplasmic fractions from HEK293T cells were stained with antibodies against AhR, Lamin B, or α-tubulin antibodies. Cy, cytosolic fraction; Nu, nuclear fraction. (F) Enhancement of human SLCO4C1 promoter activity with various statins (10 μM) using the minimal promoter region (−129). *P < 0.05; ^#P < 0.01 (n = 3 to 4 per group).

Effects of pravastatin in vivo. (A) BP in control and pravastatin-treated (0.1 mg/ml drinking water) rats after five-sixths Nx (n = 6 to 7 per group). (B) The mRNA expression of rat slco4c1 in the kidney after pravastatin administration (n = 11 per group). (C through F) Renal clearance of creatinine (C), ADMA (D), trans-aconitate (E), and GSA (F) 3 wk after five-sixths Nx (n = 5 to 7 per group). (G) Thickness of the interventricular septum (IVSTd) and left ventricular posterior wall at end-diastole (LVPWTd) before and after five-sixths Nx (n = 6 to 7 per group). *P < 0.05.

Phenotype of human SLCO4C1 TG rats. (A) BP and heart rate of TG(−)Nx and TG(+)Nx rats. *P < 0.05 versus TG(−)Nx rats (n = 4 to 6 per group). (B) Thickness of the interventricular septum (IVSTd) and left ventricular posterior wall at end-diastole (LVPWTd) were measured by echocardiogram before and 3 wk after five-sixths Nx. *P < 0.05; ^#P < 0.01 (n = 4 to 9 per group). (C) CD68 staining in the rat kidney before and 3 wk after five-sixths Nx. CD68⁺ cell number counts were performed before and 3 wk after five-sixths Nx. *P < 0.05 versus TG(−) rats (n = 6 to 9 per group). Bars = 100 μm.

Relation between uremic toxins and eGFR as well as plasma creatinine in 41 patients with CKD is shown. (A through C) Correlations between eGFR and the plasma ADMA (A), GSA (B), and trans-aconitate (C) in patients with CKD. (D through F) Concentrations between plasma creatinine (Cr) and plasma ADMA (D), GSA (E), and trans-aconitate (F).

Effects of statins on SLCO4C1 expression and function in vitro. (A) Real-time PCR of SLCO4C1 in ACHN cells with fluvastatin (10 μM) or pravastatin (100 μM; n = 3 per group). (B) The uptake of T₃ by ACHN cells treated with fluvastatin (10 μM) and pravastatin (100 μM). *P < 0.05 (n = 3).

Uremic toxins and SLCO4C1 transporter in renal failure. ADMA is formed by protein arginine N-methyltransferase (PRMT) from arginine and degrades to citrulline by DDAH. Note that SLCO4C1 facilitates the excretion of GSA, ADMA, and trans-aconitate and that statins increase the expression and the function of SLCO4C1, resulting in reductions of the uremic toxins and BP. Trans-aconitase inhibits aconitase activity and induces reactive oxygen species (ROS). Aco, aconitase.