Hiroki Miyazaki

Human Vascular Smooth Muscle Cells Express a Urate Transporter

An elevated serum uric acid is associated with the development of hypertension and renal disease. Renal regulation of urate excretion is largely controlled by URAT1 (SLC22A12), a member of the organic anion transporter superfamily. This study reports the specific expression of URAT1 on human aortic vascular smooth muscle cells, as assessed by reverse transcription-PCR and Western blot analysis. Expression of URAT1 was localized to the cell membrane. Evidence that the URAT1 transporter was functional was provided by the finding that uptake of 14C-urate was significantly inhibited in the presence of probenecid, an organic anion transporter inhibitor. It is proposed that URAT1 may provide a mechanism by which uric acid enters the human vascular smooth muscle cell, a finding that may be relevant to the role of uric acid in cardiovascular disease.

Modulation of Renal Apical Organic Anion Transporter 4 Function by Two PDZ Domain–Containing Proteins

Human organic anion transporter 4 (OAT4) is an apical organic anion/dicarboxylate exchanger in the renal proximal tubules and mediates high-affinity transport of steroid sulfates such as estrone-3-sulfate (E1S) and dehydroepiandrosterone sulfate. Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteins PDZK1 and NHERF1 were examined as interactors of OAT4 by a yeast two-hybrid assay. These interactions require the extreme C-terminal region of OAT4 and the first and fourth PDZ domains of PDZK1 and the first PDZ domain of NHERF1. These interactions were confirmed by surface plasmon resonance assays (K(D): 36 nM, 1.2 microM, and 41.7 microM, respectively). In vitro binding assays and co-immunoprecipitation studies revealed that the OAT4 wild-type but not a mutant lacking the PDZ motif interacted directly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1 proteins were shown to be localized at the apical membrane of renal proximal tubules. The association with PDZK1 or NHERF1 enhanced OAT4-mediated E1S transport activities in HEK293 cells (1.2- to 1.4-fold), and the deletion of the OAT4 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by alteration in V(max) of E(1)S transport via OAT4 and was associated with the increased surface expression level of OAT4 protein. This study indicates that the functional activity of OAT4 is modulated through the PDZ interaction with the network of PDZK1 and NHERF1 and suggests that OAT4 is involved in the regulated apical organic anion handling in the renal proximal tubules, provided by the PDZ scaffold.

Interactions of urate transporter URAT1 in human kidney with uricosuric drugs.

Aim:  Hyperuricaemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. The kidney plays a dominant role in maintaining plasma urate levels through the excretion process. Human renal urate transporter URAT1 is thought to be an essential molecule that mediates the reabsorption of urate on the apical side of the proximal tubule. In this study the pharmacological characteristics and clinical implications of URAT1 were elucidated.

Vasopressin regulates the renin-angiotensin-aldosterone system via V1a receptors in macula densa cells

The neuropeptide hormone arginine-vasopressin (AVP) is well known to exert its antidiuretic effect via the vasopressin V2 receptor (V2R), whereas the role of the vasopressin V1a receptor (V1aR) in the kidney remains to be clarified. Previously, we reported decreased plasma volume and blood pressure in V1a receptor-deficient (V1aR-/-) mice (Koshimizu T, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mori T. Proc Natl Acad Sci USA 103: 7807-7812, 2006). In this study, we investigated the role of V1aR in urine concentration, renal function, and the renin-angiotensin system (RAS) using V1aR-/- mice. Urine volume of V1aR-/- mice was greater than that of wild-type mice, particularly when water was loaded, while the glomerular filtration rate (GFR), urinary NaCl excretion, AVP-dependent cAMP generation, V2R, and aquaporin 2 (AQP2) expression in the kidney were lower, indicating that the diminished GFR and V2R-AQP2 system led to impaired urinary concentration in V1aR-/- mice. Since the GFR and V2R-AQP2 system are regulated by RAS, we analyzed renin and angiotensin II in V1aR-/- mice and found that the plasma renin and angiotensin II were decreased. The expression of renin in granule cells was decreased in V1aR-/- mice, which led to a decreased level of plasma renin. In addition, the expression of renin stimulators such as neuronal nitric oxide synthase and cyclooxygenase-2 in macula densa (MD) cells, where V1aR was specifically expressed, was decreased in V1aR-/- mice. These data indicate that AVP regulates body fluid homeostasis and GFR via the V1aR in MD cells by activating RAS and subsequently the V2R-AQP2 system.

Functional and Immunochemical Characterization of a Novel Organic Anion Transporter Oat8 (Slc22a9) in Rat Renal Collecting Duct

In this study, we demonstrate that a putative membrane unknown solute transporter 1 of the rat kidney (UST1r; Slc22a9) is a multispecific transporter of organic anions (OAs). When expressed in Xenopus oocytes, UST1r mediated uptake of ochratoxin A (OTA; Km = 1.0 µM) and sulfate conjugates of steroids, such as estrone-3-sulfate (ES; Km = 3.1 µM) and dehydroepiandrosterone sulfate (DHEAS; Km = 2.1 µM) in a sodium-independent manner. We herein propose that UST1r be renamed OA transporter 8 (rOat8). rOat8 interacted with chemically heterogenous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, probenecid, taurocholate, and methotrexate, but not with the organic cation tetraethylammonium. The rOat8-mediated ES transport was: a) cis-inhibited by 4-methylumbelliferyl sulfate and β-estradiol sulfate, but not by glucuronide conjugates of these compounds, b) cis-inhibited by four- and five- carbon (C4/C5) dicarboxylates (succinate and glutarate (GA)), and c) trans-stimulated by GA, whereas the efflux of GA was significantly trans-stimulated by ES. By RT-PCR, rOat8 mRNA was expressed in proximal convoluted tubules and cortical and outer medullary collecting ducts, whereas in immunochemical studies, Oat8 was identified as the ñ58 kDa protein that in the collecting duct colocalized with the V-ATPase in plasma membranes and intracellular vesicles in various subtypes of intercalated cells. Molecular identification of Oat8 in these cells indicates a possible novel role of OAT family in the renal secretion/reabsorption of OA and acids and bases via affecting the V-ATPase-dependent functions.

Acute and chronic metabolic acidosis interferes with aquaporin-2 translocation in the rat kidney collecting ducts

Renal aquaporin-2 (AQP2) expression plays a key role in urine concentration. However, it is not known whether metabolic acidosis affects urine-concentrating ability through AQP2 expression in the kidney and urine. We examined urinary excretion and renal expression of AQP2 in control and acidosis rats, using RT-competitive PCR, immunoblot and immunocytochemistry. Urinary excretion of AQP2 is decreased by 92% even with the increase in AQP2 mRNA and protein expressions in the collecting ducts by metabolic acidosis in rats. Urine osmolality in control rats was 1670±198 mOsm per kg H2O, and immunocytochemistry revealed the presence of AQP2 in the apical plasma membrane of the principal cells in the collecting ducts. Urine osmolality in acidosis rats was lower than that in control (1397±243 mOsm per kg H2O), and immunocytochemistry showed the diffuse presence of AQP2 in the cytoplasm of the principal cells. Differential centrifugation-coupled immunoblot showed a significant decrease in the ratio of AQP2 in plasma membrane-enriched fraction to that in intracellular vesicle-enriched fraction by metabolic acidosis. In summary, AQP2 translocation is largely decreased by metabolic acidosis even with increased expression in the collecting ducts. A disorder of AQP2 translocation in the collecting ducts with acidosis may be responsible for the diuresis in patients with chronic renal failure.