Seok Ho Cha

Role of Organic Anion Transporters in the Tubular Transport of Indoxyl Sulfate and the Induction of its Nephrotoxicity

In uremic patients, various uremic toxins are accumulated and exert various biologic effects on uremia. Indoxyl sulfate (IS) is one of uremic toxins that is derived from dietary protein, and serum levels of IS are markedly increased in both uremic rats and patients. It has been previously reported that the accumulation of IS promotes the progression of chronic renal failure (CRF). This study demonstrates the role of rat organic anion transporters (rOATs) in the transport of IS and the induction of its nephrotoxicity. The administration of IS to 5/6-nephrectomized rats caused a faster progression of CRF, and immunohistochemistry revealed that IS was detected in the proximal and distal tubules where rOAT1 (proximal tubules) and/or rOAT3 (proximal and distal tubules) were also shown to be localized. In in vitro study, the proximal tubular cells derived from mouse that stably express rOAT1 (S2 rOAT1) and rOAT3 (S2 rOAT3) were established. IS inhibited organic anion uptake by S2 rOAT1 and S2 rOAT3, and the Ki values were 34.2 and 74.4 microM, respectively. Compared with mock, S2 rOAT1 and S2 rOAT3 exhibited higher levels of IS uptake, which was inhibited by probenecid and cilastatin, organic anion transport inhibitors. The addition of IS induced a decrease in the viability of S2 rOAT1 and S2 rOAT3 as compared with the mock, which was rescued by probenecid. These results suggest that rOAT1 and rOAT3 play an important role in the transcellular transport of IS and the induction of its nephrotoxicity.

Expression of a system L neutral amino acid transporter at the blood-brain barrier

Amino acid transport system L has been proposed to be one of the major nutrient transport systems at the blood–brain barrier. Using immunohistochemical analyses, a system L transporter LAT1 was shown to be expressed in the brain capillary endothelial cells in rats. Because LAT1 was co-expressed with 4F2 heavy chain which brings LAT1 to the plasma membrane, LAT1 is proposed to be functional in the plasma membrane of brain capillary endothelial cells. Both LAT1 and 4F2hc immunoreactivities were detected in a double line appearance surrounding endothelial cell nuclei, suggesting both proteins are present in the luminal and abluminal membranes. LAT1 is, thus, a blood–brain barrier system L transporter responsible for the permeation of aromatic or branched-chain amino acids and amino acid-related drugs such as L-DOPA.

Cloning, functional characterization, and localization of a rat renal Na^+ -dicarboxylate transporter

We report here the isolation, functional characterization, tissue distribution, and membrane localization of rat renal Na+-dicarboxylate transporter (rNaDC-1). rNaDC-1 consists of 2,245 nucleotides, and the deduced amino acid sequence showed 73% and 75% identity to rabbit and human NaDC-1, respectively. When expressed in Xenopus laevis oocytes, rNaDC-1 mediated sodium-dependent uptake of di- and tricarboxylates. Substrates of rNaDC-1 evoked inward currents in oocytes expressed with rNaDC-1; succinate, α-ketoglutarate, and glutarate were relatively high-affinity substrates, and citrate was a low-affinity substrate of rNaDC-1. The coupling ratio of citrate to charge was determined to be 1:1 at pH 7.4; influx of one positive charge per citrate molecule suggests a symport of three Na+with a divalent citrate. Expression of rNaDC-1 mRNA was detected in the kidney and the small and large intestines. Immunohistochemistry using polyclonal antibodies raised against the 14 amino acids at the COOH terminus of rNaDC-1 revealed that rNaDC-1 is localized exclusively in the luminal membrane of S2 and S3.

Transport properties of a system y+L neutral and basic amino acid transporter: Insights into the mechanisms of substrate recognition

The properties of system y+L-mediated transport were investigated on rat system y+L transporter, ry+LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopusoocytes. ry+LAT1-mediated transport of basic amino acids was Na+-independent, whereas that of neutral amino acids, although not completely, was dependent on Na+, as is typical of system y+ L-mediated transport. In the absence of Na+, lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H+, besides Na+ and Li+, is capable of supporting neutral amino acid transport. Na+ and H+ augmented leucine transport by decreasing the apparent K m values, without affecting the V max values. We demonstrate that although ry+LAT1-mediated transport of [14C]l-leucine was accompanied by the cotransport of 22Na+, that of [14C]l-lysine was not. The Na+ to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na+. ry+LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry+LAT1 and 4F2 heavy chain in the absence of Na+, but not in the presence of physiological concentrations of Na+, indicating that cotransport of H+ with leucine occurred in the absence of Na+. Therefore, for the substrate recognition by ry+LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na+ and H+, which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry+LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.

P2 purinoceptor localization along rat nephron and evidence suggesting existence of subtypes P2Y1 and P2Y2

Effects of extracellular ATP on intracellular free calcium concentration ([Ca2+]i) were examined in rat single nephron segments using the fura 2-AM. ATP (10 μM) induced a significant transient increase in [Ca2+]iin the glomerulus, the early proximal convoluted tubule (S1), the cortical collecting tubule (CCT), and the outer medullary collecting tubule (OMCT). The magnitude of the response was the greatest in the OMCT among four segments. ATP induced an increase in the [Ca2+]iin a dose-dependent manner in S1 and OMCT. In the OMCT, ATP caused a biphasic increase in [Ca2+]iconsisting of an initial rapid rise and a sustained phase. Removal of calcium from the medium resulted in an attenuation of the sustained phase of [Ca2+]iand an ∼30% reduction in the height of the initial [Ca2+]ipeak in response to 10 μM ATP. Effects of ATP, its analogs, and its metabolites were tested in the S1 and OMCT. ATP, 2-methylthio-ATP (2-MeS-ATP), ADP, and UTP increased [Ca2+]idose dependently. AMP and adenosine did not affect [Ca2+]iin the S1 and OMCT. The ATP- or 2-MeS-ATP-induced [Ca2+]iincrease was inhibited by the pretreatment of the S1 and OMCT with suramin or reactive blue 2. Neomycin, a phospholipase C inhibitor, attenuated the ATP-induced [Ca2+]iincrease. To investigate the hormonelike action of ATP in OMCT, a heterologous cross desensitization was performed. The pretreatment of OMCT with ATP inhibited increases in vasopressin-, ANG II-, endothelin-1-, or bradykinin-induced [Ca2+]iincrease. These findings suggest that ATP might affect the above peptidyl agonist-activated calcium mobilizations.

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells.

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.

Induction of glioma apoptosis by microglia-secreted molecules: The role of nitric oxide and cathepsin B.

Microglia contributes significantly to brain tumor mass, particularly in astrocytic gliomas. Here, we examine the cytotoxic effects of soluble components secreted from microglia culture on glioma cells. Microglia conditioned culture medium (MCM) actively stimulated apoptotic death of glioma cells, and the effects of MCM prepared from LPS- or IFN-gamma-activated microglia were more pronounced. The cytotoxic effects were glioma-specific in that primary cultured rat astrocytes were not affected by MCM. A donor of peroxynitrite induced glioma-specific cell death. In addition, NO synthase inhibitor suppressed glioma cell death induced by activated MCM, indicating that NO is one of the key molecules responsible for glioma cytotoxicity mediated by activated MCM. However, since unstimulated resting microglia produces low or very limited level of NO, MCM may contain other critical molecule(s) that induce glioma apoptosis. To identify the proteins secreted in MCM, proteomic analysis was performed on control or activated medium. Among over 200 protein spots detected by Coomassie blue staining, we identified 26 constitutive and 28 LPS- or IFN-gamma-regulated MCM proteins. Several cathepsin proteases were markedly expressed, which were reduced upon activation. In particular, suppression of cathepsin B by the chemical inhibitors significantly reversed MCM-induced glioma cell death, implying a critical role of this protease in cytotoxicity. Our findings provide evidence on the functional implications of specific microglial-secreted proteins in glioma cytotoxicity, as well as a basis to develop a proteomic databank of both basal and activation-related proteins in microglia.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney.

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OATs functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1s function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Evidence for heme oxygenase-1 association with caveolin-1 and -2 in mouse mesangial cells

The interaction of heme oxygenase‐1 (HO‐1) and caveolin in the cultured mouse mesangial cells (MMC) was investigated. In normal MMCs, high levels of caveolin‐2 and low level of caveolin‐1 at mRNA and protein level were observed without any detectable expression of caveolin‐3. Upon treating the MMCs either with cadmium (Cd) or spermine NONOate (SPER/NO), expression of HO‐1 mRNA and protein was increased. Caveolae rich membranous fractions from the MMCs treated with Cd or SPER/NO contained both HO‐1 and caveolin‐1 or caveolin‐2. The experiments of immuno‐precipitation showed complex formation between the HO‐1 and caveolin‐1 or caveolin‐2 in the Cd treated MMCs. Confocal microscopic results also support co‐localization of HO‐1 and caveolin‐1 or caveolin‐2 at the plasma membrane. Co‐localization of caveolins with HO‐1 in caveolae suggested that caveolin could also play an important role in regulating the function of HO‐1. IUBMB Life, 55: 525‐532, 2003

Dual blockade of the renin-angiotensin system with angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in chronic kidney disease.

Despite the renoprotective effects of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs), many patients with chronic kidney disease develop end-stage kidney disease. Combination treatment with an ACEI and an ARB is a recently introduced approach to obtain more complete blockade of the renin–angiotensin system, based on the different mechanisms of action of the two classes of drug. To assess the shortcomings of single treatment with ACEIs and ARBs, and the potential benefits of combination treatment, we reviewed the experimental and clinical evidence suggesting that combination treatment offers more complete blockade of the renin–angiotensin system and identified areas in which further research is necessary to confirm the benefits of combination treatment. The available data suggest that combination treatment with an ACEI and an ARB has a greater renoprotective effect than either drug alone. In addition, more recent data have shown that combination treatment is more potent in suppressing renal fibrosis, and is well tolerated in patients with advanced chronic kidney disease. Clinical trials with rigorous endpoints are needed to further establish the benefits of combination treatment in renal protection.