Toshiyuki Fukutomi

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis.

Bordetella pertussis, the causative agent of whooping cough, is highly adapted to cause human infection. The production of virulence factors, such as adhesins and toxins, is just part of an array of mechanisms by which B. pertussis causes infection. The stringent response is a global bacterial response to nutritional limitation that is mediated by the accumulation of cellular ppGpp and pppGpp [termed together as (p)ppGpp]. Here, we demonstrate that production of (p)ppGpp was controlled by RelA and SpoT proteins in B. pertussis, and that mutation-induced loss of both proteins together caused deficiencies in (p)ppGpp production. The (p)ppGpp-deficient mutants also exhibited defects in growth regulation, decreases in viability under nutritionally limited conditions, increases in susceptibility to oxidative stress and defects in biofilm formation. Analysis of the secreted proteins and the respective transcripts showed that lack of (p)ppGpp led to decreased expression of fim3 and bsp22, which encode a fimbrial subunit and the self-polymerizing type III secretion system tip protein, respectively. Moreover, electron microscopic analysis also indicated that (p)ppGpp regulated the formation of filamentous structures. Most virulence genes - including fim3 and bsp22 - were expressed in the Bvg(+) phase during which the BvgAS two-component system was activated. Although fim3 and bsp22 were downregulated in a (p)ppGpp-deficient mutant, normal expression of fhaB, cyaA and ptxA persisted. Lack of coherence between virulence gene expression and (p)ppGpp production indicated that (p)ppGpp did not modulate the Bvg phase. Taken together, our data indicate that (p)ppGpp may govern an as-yet-unrecognized system that influences B. pertussis pathogenicity.

Urate Transport: Regulators of Serum Urate Levels in Humans

Urate (uric acid) is the final product of purine metabolism, and its antioxidant capacity has drawn attention recently for its protective role against oxidative stress. However, hyperuricemia has a known association with onset of illnesses such as gout and cardiovascular diseases. Renal urate transport mechanisms are known to be major determinants of serum urate levels, but the molecular mechanisms involved have not yet been fully elucidated. Molecular identification of a kidney-specific urate transporter SLC22A12 (URAT1) by our research group in 2002 marked the start of a subsequent compilation of information on several different molecules contributing to urate transport by the kidneys. In addition, recent genome-wide association (GWA) studies have contributed to the detection of novel candidate genes related to uric acid metabolism such as SLC2A9 (GLUT9/URATv1), ABCG2 (BCRP), SLC17A1 (NPT1), SLC17A3 (NPT4) and PDZK1. Furthermore, use of urate transporter gene-modified mice for Slc22a12 and Slc2a9 may provide clues for understanding the physiological role of each transporter. Results of recent studies on urate transport with emphasis on the relation to serum urate disorders are described in this report.

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

UNLABELLED Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro, and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg(+) mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. IMPORTANCE Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis experiments. As predicted, the T3SS was induced by glutamate limitation. These results are suggestive of the importance of regulation by nutrient conditions and in the pathogenicity of B. pertussis.

Comprehensive proteome analysis of brush border membrane fraction of ileum of ezrin knockdown mice.

Ezrin is an actin binding protein which cross-links membrane proteins with cytoskeleton directly or indirectly via PDZ domain-containing scaffold proteins. It is mainly expressed at the brush border membrane (BBM) of gastrointestinal tracts, and is involved in the construction of microvilli structure and the functional expression of membrane protein complexes at the cell surface. To precisely study the roles of ezrin on the expression of membrane proteins at the cell surface, here we prepared the BBM fractions of ileums from the wild-type and ezrin-knockdown (Vil2(kd/kd)) mice, analyzed them by mass spectrometry, and compared their proteomic patterns. Totally 313 proteins were identified in the BBM fractions. Several transport proteins, cytoskeleton-associated proteins, and trafficking proteins were up- or down-regulated in the BBM fraction of the ileum in the Vil2(kd/kd) mice. Among them, the expressions of i) Na(+)/H(+) exchanger regulatory factor 1 (a PDZ domain-containing scaffold protein), ii) sodium monocarboxylate transporter 1, which contains a PDZ domain-binding motif at their carboxy-terminal, and iii) chloride intracellular channel protein 5 were down-regulated at the BBM fraction of the ileum in the Vil2(kd/kd) mice, suggesting that ezrin is involved in their expression in the BBM.

Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion

ABSTRACT Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pylori. IMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB. This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

Paracellular route is the major urate transport pathway across the blood‐placental barrier

Urate, the final oxidation product of purine metabolism, is excreted into urine in humans. Clinically, increased serum urate levels are indicative of pregnancy‐induced hypertension (PIH). However, how urate is handled in the placenta is still largely unknown. In this study, we compared maternal serum urate levels with those of umbilical cord blood and investigated urate transport mechanisms in BeWo cells, a trophoblast‐derived cell line. The maternal and umbilical cord blood samples and placentas were collected from patients undergoing cesarean section at Kyorin University Hospital after obtaining informed consents. There were no significant differences in serum urate levels between maternal blood and umbilical cord blood, and between umbilical cord vein and arterial blood, suggesting that urate is freely movable at the placenta and that fetus is not a major source of urate production. RT‐PCR and immunohistochemistry showed that urate transporters including OAT4, OAT10, GLUT9/URATv1 and ABCG2 were expressed in the syncytiotrophoblast cells in the placenta as well as BeWo cells. Despite expressing aforementioned urate transporters BeWo cells did not take up urate. After confirming the formation of tight junctions of these cells cultured on the transwell, urate transport between upper and lower chambers was measured. Urate moved through BeWo cell monolayers with nonsaturation kinetics and this movement was observed even when the cells were incubated at 4°C, suggesting that urate moves through the paracellular route by simple diffusion.

VAMP7 Regulates Autophagosome Formation by Supporting Atg9a Functions in Pancreatic β-Cells From Male Mice

Dysfunctional mitochondria are observed in β-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic β-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient β-cells and β-cell-derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a-resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

Wolf-Hirschhorn syndrome candidate 1-like 1 epigenetically regulates nephrin gene expression

Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes, and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte-specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-κB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.