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.Urate is the end product of purine metabolism in humans and certain primates as a result of uricase genetic loss (urate oxidase degrades urate to allantoin) (1). Two independent nonsense mutations in this gene, found in human, chimpanzee, and orangutan but not in the gibbon, indicate that this loss had evolutionary advantages for early primates (2). Because urate has powerful antioxidant properties, uricase loss resulting in elevated serum urate may have been beneficial to early primate survival (1). In addition, Watanabe et al. (3) hypothesized that elevated serum urate levels provided a survival advantage by helping to maintain blood pressure under the low salt dietary conditions that prevailed during the middle to late Miocene period. Despite its beneficial role and given the fact that more than half of uricase-deficient mice die from urate nephropathy within 4 weeks of age, elevation in serum urate level produces a burden on the body (4). To circumvent this problem, the human body had to develop a urate excretion system.The kidney plays a dominant role in maintaining serum urate levels (1, 5). Renal urate excretion is a function of the balance between reabsorption and secretion. Recently it was demonstrated that luminal urate is taken up by a urate-anion exchanger (URAT1; SLC22A12) 3 (6) into the renal proximal tubular cell and that intracellular urate exits the cell into the interstitium/blood ...

Serum proteins/peptides reflect physiological or pathological states in humans and are an attractive target for the discovery of disease biomarkers. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make any quantitative analysis of low-abundance proteins/peptides challenging. Furthermore, analyses of peptides, including the cleaved fragments of proteins, are difficult because of carrier protein binding. Here, we developed a differential solubilization (DS) method to extract low-molecular-weight proteins/peptides in serum with good reproducibility and yield as compared to typical peptide-extraction methods such as organic solvent precipitation and ultrafiltration. Using the DS method combined with reverse-phase HPLC fractionation followed by MALDI-TOF-MS, we performed high-quality comparative analyses of more than 1500 peptides from 1 microL of serum samples, including low-abundance peptides in the subnanomolar range and containing many peptides bound to carrier proteins such as albumin. We applied this method and successfully discovered four new biomarker candidates of colon cancer, none of which have previously been observed in serum and one of which is a fragment of the protein zyxin that possibly originated from tumor cells. Our results indicate that serum peptide analyses based on the DS method should greatly contribute to the discovery of novel low-abundance biomarkers.

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 selfpolymerizing 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.

Abstract. In this study, we have elucidated that propionate, one of the short chain fatty acids (SCFAs), is the transport substrate for murine organic anion transporter 2 (mOat2), which is expressed in the kidneys and the liver. When expressed in Xenopus oocytes, mOat2-mediated [ 3 H]PGE 2 transport was inhibited by three-to five-carbon SCFAs (C3 to C5). Among the SCFAs tested, propionate (3-carbon SCFA) was transported by mOat2 in a time-dependent manner. Since propionate is a potent glucogenic compound, Oat2 may be involved in the regulation of cellular metabolism through the transport of these metabolites in the kidneys and the liver.