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As a final common pathway of renal injuries, renal fibrosis leads to chronic kidney disease (CKD). Currently, there is no safe and effective therapy to prevent the progression of renal fibrosis to CKD. Inhibition of transforming growth factor-β1 (TGF-β1) pathway is proposed as one of the most promising approaches for anti-renal fibrosis therapies. This study aimed to identify novel anti-fibrotic agents using the TGF-β1-induced fibrosis in renal proximal tubule epithelial cells (RPTEC) and characterize their mechanism of action as well as in vivo efficacy. By screening 362 natural product-based compounds for their ability to reduce collagen accumulation assessed by picro-sirius red (PSR) staining in RPTEC cells, a chalcone derivative AD-021 was identified as an anti-fibrotic agent with IC50 of 14.93 μM. AD-021 suppressed TGF-β1-induced collagen production, expression of pro-fibrotic proteins (fibronectin and α-smooth muscle actin (αSMA)), and Smad-dependent and Smad-independent signaling pathways via suppression of TGF-β receptor II (TGFβRII) phosphorylation in RPTEC cells. Furthermore, TGF-β1-induced mitochondrial fission in RPTEC cells was ameliorated by AD-021 via mechanisms involving inhibition of Drp1 phosphorylation. In a mouse model of unilateral ureteral obstruction (UUO)-induced renal fibrosis, AD-021 reduced plasma TGF-β1, ameliorated renal fibrosis and improved renal function. Collectively, AD-021 represents a novel class of natural product-based anti-fibrotic agent that has therapeutic potential in the prevention of fibrosis-associated renal disorders including CKD.

Colistin is a last-resort polypeptide antibiotic widely used to treat against multidrug-resistant Gram-negative bacterial infections. However, this treatment is associated with nephrotoxicity. The aim of this study was to examine the potential protective effect of panduratin A, a bioactive compound of Boesenbergia rotunda, on colistin-induced nephrotoxicity in both in vivo and in vitro models. Intraperitoneal injection of 15 mg/kg colistin for 7 days markedly promoted renal tubular degeneration, increased blood urea nitrogen (BUN) levels, and upregulated the expression of renal injury biomarker and apoptosis proteins. In addition, treatment with colistin increased oxidative stress and apoptosis in mice kidney tissues. Interestingly, these defects were attenuated when co-administered of colistin with panduratin A (2.5 or 25 mg/kg). The underlying mechanisms of panduratin A attenuating colistin toxicity was investigated in human renal proximal tubular cells (RPTEC/TERT1). The mechanisms by which colistin-triggered cytotoxicity was determined by analysis of cell death, reactive oxygen species (ROS) levels, mitochondria function as well as the expression of proteins related to apoptosis pathway. Colistin treatment (200 µg/ml) significantly increased cell apoptosis, elevated ROS production, reduced mitochondrial membrane potential, and decreased anti-apoptotic protein (Bcl-2) expression. These effects were notably suppressed by co-treatment with panduratin A (5 μM). Collectively, panduratin A exerts as a novel nephroprotective agent to protect against colistin-induced renal injury by attenuating mitochondrial damage and renal cell apoptosis.

The purpose of this study was to create novel urate under-excretion animal models using pyrazinamide and to evaluate whether dihydropyridine calcium channel blockers (CCBs) have uricosuric effects in vivo.Adult male ICR mice were treated with pyrazinamide, vehicle (dimethyl sulfoxide: DMSO), or tap water. Thirty minutes later, pyrazinamide-treated mice were given benzbromarone, losartan, nilvadipine, nitrendipine, nifedipine or azelnidipine. Six hours after the second administration, urine (by urinary bladder puncture) and plasma were collected to measure uric acid and creatinine levels, and fractional excretion of uric acid (FEUA) and creatinine clearance (Ccr) were calculated and evaluated. There was no significant difference in the levels of plasma uric acid, plasma creatinine, Ccr, urinary N-acetyl-β-d-glucosaminidase (NAG) and urinary NAG-creatinine ratio between water, DMSO, and pyrazinamide-treated mice. But the FEUA of pyrazinamide-treated mice was significantly lower than water mice. The FEUA was significantly higher in mice taking the dihydropyridine CCBs (nilvadipine, nitrendipine, nifedipine, and high-dose azelnidipine) than in pyrazinamide-treated mice. There was no significant difference in Ccr.Thus, a novel animal model created with PZA administration was useful as a urate under-excretion animal model that was probably URAT1-mediated, and the uricosuric effects of dihydropyridine CCBs were confirmed in vivo. Keywords: Uric acid, Urate, Fractional excretion of uric acid, Urate under-excretion animal model, Calcium channel blocker

L-type amino acid transporter 1 (LAT1, SLC7A5) incorporates essential amino acids into cells. Recent studies have shown that LAT1 is a predominant transporter in various human cancers. However, the function of LAT1 in thymic carcinoma remains unknown. Here we demonstrate that LAT1 is a critical transporter for human thymic carcinoma cells. LAT1 was strongly expressed in human thymic carcinoma tissues. LAT1-specific inhibitor significantly suppressed leucine uptake and growth of Ty82 human thymic carcinoma cell lines, suggesting that thymic carcinoma takes advantage of LAT1 as a quality transporter and that LAT1-specific inhibitor might be clinically beneficial in therapy for thymic carcinoma.

The Coix lacryma-jobi L. or Adlay seed has been used as a healthy food and herb in many Asian countries. A previous in vitro study found that it has an inhibitory effect on xanthine oxidase (XOD) activity but its use as a traditional medicine in the treatment of hyperuricemia and gout has a lacked scientific evidence. This study aimed to investigate the effects of Adlay seed extract on the level of plasma uric acid (Pua), reduction in fractional excretion of urate (%FEua), decreasing creatinine clearance (Ccr), and increasing plasma creatinine and urea nitrogen in hyperuricemic mice. The steps of the study comprised collecting and inducing the sample mice by giving potassium oxonate and uric acid and treating the hyperuricemic mice by giving Adlay seed extract at doses of 0.1 to 100 mg/kg for 10 days. The results showed that Adlay seed extract significantly reduced Pua in a dose-dependent manner, increased %FEua and Ccr to normal level at doses of 10 and 100 mg/kg and also inhibited XOD activity in liver and plasma in hyperuricemic mice. From this results indicated that Adlay seed extract has hypouricemia and nephroprotective action in hyperuricemic mice.

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l- type amino acid transporter-1, a Na + - independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l -type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l -type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [ 14 C] l -leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l -type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue–stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal’s body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l -type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

We analyzed the functional properties of five nonsynonymous single nucleotide polymorphisms (SNPs) in the sodium-phosphate transporter NPT4 gene (SLC17A3) using the Xenopus oocyte expression system. NPT4 variants carrying SNP V257F, G279R, or P378L exhibited reduced transport of [14C]para-aminohippurate, [3H]bumetanide, [3H]estrone sulfate, and [14C]urate, when each variant clone was expressed in the plasma membrane of oocytes. This study suggests the possibility that the genetic variation of NPT4 contributes to inter-individual differences in disposition of anionic drugs such as diuretics as well as certain endogenous organic anions such as urate. Keywords:: single nucleotide polymorphism, diuretic, urate

Abstract.: In the present study, we investigated the transport of nephrotoxic mycotoxin ochratoxin A (OTxA) by a novel human organic anion transporter hNPT4 using the Xenopus oocyte expression system. hNPT4 mediated time- and concentration-dependent uptake of OTxA (Km: 802.8 μM) in a pH- and voltage-sensitive manner. Cis-inhibition experiments suggest that the substrate selectivity of hNPT4 is similar to that of hOAT4. The fact that the Km of OTxA for the efflux transporter hNPT4 was much higher than those for the uptake transporters hOAT1 and hOAT3 may favor the accumulation of OTxA in the tubular cell and lead to nephrotoxicity. Keywords:: ochratoxin A, organic anion transporter, nephrotoxicity

Endothelial cell proliferation supporting angiogenesis requires sufficient nutrient supply because of facilitated intracellular metabolism. However, little is known about the mechanism for the promotion of nutrient incorporation in proliferating endothelial cells. Here we show that L-type amino acid transporter 1 (LAT1) is a major transporter of essential amino acids in human umbilical vein endothelial cells (HUVECs). Growing HUVECs express a certain level of LAT1. A LAT1-specific inhibitor suppressed leucine uptake, cell proliferation, and tube formation of HUVECs. Therefore, LAT1 acts to support effective uptake of amino acids, which is critical for the optimal function of HUVECs for angiogenesis. Keywords:: LAT1, amino acid transporter, HUVEC

The mycotoxin, ochratoxin A (OTA), is thought to be responsible for Balkan endemic nephropathy. OTA accumulates in several tissues, especially in the kidneys and liver. The excretion of OTA into urine is thought to be mainly by tubular secretion, presumably via the organic anion transport system. Recently, several families of multispecific organic anion transporters have been identified: organic anion transporters (OATs), organic anion-transporting polypeptides (OATPs), oligopeptide transporters (PEPTs), and ATP-binding cassette (ABC) transporters, such as MRP2 and BCRP. These renal transporters mediate the transmembrane transport of OTA and play a pivotal role in the development of OTA-induced nephrotoxicity.

Hyperuricemia is currently recognized as a risk factor for cardiovascular diseases. It has been reported that the angiotensin II–receptor blocker (ARB) losartan decreases serum uric acid level. In this study, the effects of another ARB, irbesartan, on [14C]uric acid–transport activity of renal uric acid reabsorptive transporters URAT1 and URATv1 were examined with Xenopus oocytes expressing each transporter. The results showed that irbesartan (100 – 500 μM) inhibited the uptake of uric acid via both transporters. The inhibitory effects of irbesartan exceeded those of losartan and other ARBs, and the results suggest that irbesartan can reduce serum uric acid level. Keywords:: angiotensin II–receptor blocker, transporter, uric acid

Renal organic anion transporters play an important role in the handling of a number of endogenous and exogenous anionic substances in the kidney. In this study, we investigated prostaglandin E2 (PGE2) transport properties and intrarenal localization of mouse organic anion transporter 3 (mOat3). When expressed in Xenopus oocytes, mOat3 mediated the time- and concentration-dependent transport of PGE2 (Km: 1.48 µM). PGE2 transport mediated by mOat3 was trans-stimulated by intracellular glutarate injected into the oocytes. PGE2 efflux via mOat3 was also trans-stimulated by extracellular glutarate. Thus, mOat3 was shown to mediate the bidirectional transport of PGE2, partly coupled to the dicarboxylate exchange mechanism. Immunohistochemical study revealed that mOat3 protein was localized at the basolateral membrane of renal proximal and distal tubules. Furthermore, diffuse expression of mOat3, including expression in the basolateral membrane in macula densa (MD) cells, was observed. These results indicate that mOat3 plays an important role as a basolateral transport pathway of PGE2 in the distal nephron including MD cells that may constitute one of the indispensable steps for renin release and regulation of the tubuloglomerular feedback mechanism. Keywords:: organic anion transporter, OAT, prostaglandin E2, glutarate, macula densa

Human organic anion transporter OAT4 is expressed in the kidney and placenta and mediates high-affinity transport of estrone-3-sulfate (E1S). Because a previous study demonstrated no trans-stimulatory effects by E1S, the mode of organic anion transport via OAT4 remains still unclear. In the present study, we examined the driving force of OAT4 using mouse proximal tubular cells stably expressing OAT4 (S2 OAT4). OAT4-mediated E1S uptake was inhibited by glutarate (GA) (IC50: 1.25 mM) and [14C]GA uptake via S2 OAT4 was significantly trans-stimulated by unlabeled GA (5 mM) (P

Idiopathic renal hypouricaemia is an inherited form of hypouricaemia, associated with abnormal renal handling of uric acid. There is excessive urinary wasting of uric acid resulting in hypouricaemia. Patients may be asymptomatic, but the persistent urinary abnormalities may manifest as renal stone disease, and hypouricaemia may manifest as exercise induced acute kidney injury. Here we have identified Macedonian and British patients with hypouricaemia, who presented with a variety of renal symptoms and signs including renal stone disease, hematuria, pyelonephritis and nephrocalcinosis. We have identified heterozygous missense mutations in SLC22A12 encoding the urate transporter protein URAT1 and correlate these genetic findings with functional characterization. Urate handling was determined using uptake experiments in HEK293 cells. This data highlights the importance of the URAT1 renal urate transporter in determining serum urate concentrations and the clinical phenotypes, including nephrolithiasis, that should prompt the clinician to suspect an inherited form of renal hypouricaemia.

Controlled release of proteins, such as growth factors, from biocompatible silk fibroin (SF) hydrogel is valuable for its use in tissue engineering, drug delivery, and other biological systems. To achieve this, we introduced silk fibroin-mimetic peptides (SFMPs) with the repeating unit (GAGAGS) n . Using green fluorescent protein (GFP) as a model protein, our results showed that SFMPs did not affect the GFP function when conjugated to it. The SFMP-GFP conjugates incorporated into SF hydrogel did not change the gelation time and allowed for controlled release of the GFP. By varying the length of SFMPs, we were able to modulate the release rate, with longer SFMPs resulting in a slower release, both in water at room temperature and PBS at 37 °C. Furthermore, the SF hydrogel with the SFMPs showed greater strength and stiffness. The increased β-sheet fraction of the SF hydrogel, as revealed by FTIR analysis, explained the gel properties and protein release behavior. Our results suggest that the SFMPs effectively control protein release from SF hydrogel, with the potential to enhance its mechanical stability. The ability to modulate release rates by varying the SFMP length will benefit personalized and controlled protein delivery in various systems., (© 2024. The Author(s).)

N-Substituted 2-iodobenzamides and enaminones undergo cascade transformations to achieve quinazolinones via a copper-catalyzed Ullmann-type coupling, a Michael addition and a retro-Mannich reaction. A unique stereochemical feature of this domino process was that Z-enaminones reacted without external ligands, whereas E-enaminones required the assistance of ligands.

Objective: To study the expression and function of L-type amino acid transporter 1 (LAT1), a major catalytic subunit of system L that is responsible for the transport of large neutral amino acids, including most essential amino acids, in concert with the covalently bound 4F2 heavy chain, and is implicated in tumorigenesis., Methods: Human glioma cell lines and tumor specimens were analyzed for LAT1 expression using Western blotting and reverse transcription polymerase chain reaction analysis. The rate of neutral amino acid uptake was measured using L-[C]leucine. The proliferation and apoptosis rates were analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated nick end-labeling assays, respectively, on inhibition of system L by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. The effects on proliferation and tumor growth caused by exogenously overexpressed LAT1 were similarly analyzed., Results: LAT1 was expressed in most human high-grade gliomas and glioma cell lines at various levels, with more ubiquitous expression of 4F2 heavy chain. Glioma cells with high LAT1 expression exhibited a marked increase in the uptake rate of L-[C]leucine. 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid treatment not only suppressed deoxyribonucleic acid synthesis in association with the up-regulation of the cyclin-dependent kinase inhibitor p21 but also enhanced apoptosis with caspase activation, thereby exerting both cytostatic and cytocidal effects in glioma cells with high LAT1 expression levels. Furthermore, overexpression of LAT1 in glioma cells with low endogenous LAT1 expression significantly enhanced the rates of tumor cell growth in athymic mice., Conclusion: LAT1, the major transporter of system L, is frequently expressed at higher levels in high-grade gliomas than in low-grade gliomas and brain tissues, and it may play an important role in enhancing the rates of tumor cell proliferation and growth in vivo.