Shunsuke Kotani

p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-β1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

Lewis base-catalyzed conjugate reduction and reductive aldol reaction of α,β-unsaturated ketones using trichlorosilane

Lewis bases such as Ph3P=O and HMPA catalyze the 1,4-reduction of alpha,beta-unsaturated ketones with trichlorosilane, and because the 1,2-reduction of aldehydes scarcely proceeded under the conditions, one-pot reductive aldol reactions with aldehydes were successfully achieved; preliminary studies using a chiral Lewis base revealed a high potential for enantioselective catalysis.

Diastereo‐ and Enantioselective Reductive Aldol Reaction with Trichlorosilane Using Chiral Lewis Bases as Organocatalysts

The catalytic enantioselective tandem reaction is an efficient synthetic methodology in which optically active compounds are assembled from simple prochiral substrates via two (or more) distinct catalytic processes taking place under the same conditions. The synthetic efficiency is enhanced by avoiding the time-intensive and yield-reducing isolation and purification of synthetic intermediates and by decreasing the amounts of chemicals and solvents used. The asymmetric catalytic reductive aldol reaction is an efficient tandem transformation involving conjugate reduction of a,b-unsaturated carbonyl compounds followed by aldol reaction of the enolate intermediate with aldehydes or ketones. Chiral transition-metal catalysts have been used to control the stereochemistry of these transformations. We recently reported that achiral phosphorus oxides function as Lewis base organocatalysts to promote both the conjugate reduction of enones with trichlorosilane and the reductive aldol reaction of enones with aldehydes. Herein we report that enantioselective catalysis of this tandem reaction by chiral Lewis bases provides good to high diastereoand enantioselectivities. Scheme 1 outlines the current catalytic method. Our previous study had shown that the Lewis base catalyzed conjugate reduction with trichlorosilane proceeds via a six-membered transition state with an enone in the s-cis conformation to give the (Z)-trichlorosilyl enolate exclusively. Therefore, high syn selectivity is expected for the subsequent aldol process, assuming that the reaction proceeds through a chair-like cyclic transition state. Moreover, high enantioselectivity could also be achieved by judicious selection of chiral Lewis base catalysts (LB*). We first examined various chiral Lewis base catalysts (Figure 1) for the reductive aldol reaction of chalcone (1a) and benzaldehyde (2a) with trichlorosilane at 78 8C (Table 1). With (S)-BINAPO, the reaction in dichloromethane gave aldol adduct 3a with respectable stereoselectivities (Table 1, entry 1). By simply changing the solvent from dichloromethane to propionitrile, both the stereoselectivities and chemical yield dramatically improved (Table 1, entry 2). Other Lewis base catalysts were then examined using this solvent (Table 1, entries 3–6). (R,R)-DIOPO showed a comparable activity to BINAPO to afford similar enantioselectivity with a slight loss of diastereoselectivity (Table 1, entry 3). Although structurally similar to BINAPO, (S)[a] Prof. Dr. M. Sugiura, N. Sato, Y. Sonoda, Prof. Dr. M. Nakajima Graduate School of Pharmaceutical Sciences Kumamoto University 5-1 Oe-honmachi, Kumamoto 862-0973 (Japan) Fax: (+81)96-362-7692 E-mail : msugiura@kumamoto-u.ac.jp (M. Sugiura) nakajima@gpo.kumamoto-u.ac.jp (M. Nakajima) [b] Prof. Dr. S. Kotani Priority Organization for Innovation and Excellence Kumamoto University 5-1 Oe-honmachi, Kumamoto 862-0973 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.200900450. Scheme 1. The enantioselective reductive aldol reaction with trichlorosilane catalyzed by a chiral Lewis base catalyst.

Organic anion transporters play an important role in the uptake of p -cresyl sulfate, a uremic toxin, in the kidney

BACKGROUND p-Cresyl sulfate (PCS), a recently identified anionic uremic toxin, is the main circulating metabolite of p-cresol. In cases of chronic kidney disease (CKD), it might be associated with cardiovascular outcomes and the progression of CKD. However, the renal excretion pathway of PCS is currently unknown. The objective of the present study was to determine whether organic anion transporters (OATs), which are renal tubular basolateral membrane transporters, play an important role in this process. METHODS The uptake of PCS was investigated using rat renal cortical slices and human proximal tubular cells (HK-2). The active uptake velocity was calculated by subtracting the uptake velocity at 4°C (nonspecific uptake) from that at 37°C. RESULTS As evidenced by renal cortical slice experiments, the uptake of PCS was saturable with a mean K(m) of 231.6 μM, indicating that the active transport is involved in the basolateral uptake of PCS. Similar results were also observed in HK-2 cells. The active transport of PCS was significantly suppressed by inhibitors of OATs, such as probenecid, benzylpenicillin, p-aminohippuric acid and estrone sulfate. Similar inhibitions were observed in the presence of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropionate, OATs substrates among uremic toxins. In contrast, digoxin and tetraethylammonium that did not interact with OATs had little inhibitory effect. CONCLUSIONS The findings of the present study strongly suggest that PCS serves as a substrate for OATs, is preferentially recognized by OAT3 and plays a key role in the renal tubular secretion process.

Interaction between Two Sulfate-Conjugated Uremic Toxins, p-Cresyl Sulfate and Indoxyl Sulfate, during Binding with Human Serum Albumin

Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 105 M−1 (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.

Lithium acetylides as alkynylating reagents for the enantioselective alkynylation of ketones catalyzed by lithium binaphtholate

Chiral lithium binaphtholate effectively catalyzed the enantioselective alkynylation of ketones using lithium acetylide as an alkynylating agent. This is the first example of the catalytic enantioselective addition of lithium acetylide to carbonyl compounds without the aid of other metal sources.

Trichlorosilyl triflate-mediated enantioselective directed cross-aldol reaction between ketones using a chiral phosphine oxide as an organocatalyst

Trichlorosilyl triflate-promoted directed cross-aldol reaction between ketones in the presence of a chiral phosphine oxide as an organocatalyst is described. This is the first enantioselective cross-aldol reaction between simple ketones with good enantioselectivity.

Lithium Binaphtholate-Catalyzed Asymmetric Addition of Lithium Acetylides to Carbonyl Compounds

The asymmetric addition of lithium acetylides to carbonyl compounds in the presence of a chiral lithium binaphtholate catalyst was developed. A procedure involving the slow addition of carbonyl compounds to lithium acetylides improved the enantioselectivity. This reaction afforded diverse chiral secondary and tertiary propargylic alcohols in high yields and with good to high enantioselectivities.

Stereoselective Synthesis of Multiple Stereocenters by Using a Double Aldol Reaction

Asymmetric multicomponent reactions have attracted much attention in organic synthesis because they both simplify synthetic processes and permit the construction of multiple chiral centers in a single operation. Although several asymmetric aldol reactions have been developed, relatively few examples of sequential aldol reactions which lead to the formation of multiple carbon–carbon bonds with chiral centers are available. Among the various sequential aldol reactions, double aldol reactions involving one aldol donor and two aldol acceptors have two types of reaction modes (Scheme 1): a) two aldol reactions may occur at a single a-

p‐Cresyl sulfate, a uremic toxin, causes vascular endothelial and smooth muscle cell damages by inducing oxidative stress

The major cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease. Here, p‐Cresyl sulfate (PCS), a uremic toxin, is considered to be a risk factor for cardiovascular disease in CKD. However, our understanding of the vascular toxicity induced by PCS and its mechanism is incomplete. The purpose of this study was to determine whether PCS enhances the production of reactive oxygen species (ROS) in vascular endothelial and smooth muscle cells, resulting in cytotoxicity. PCS exhibited pro‐oxidant properties in human umbilical vein endothelial cells (HUVEC) and aortic smooth muscle cells (HASMC) by enhancing NADPH oxidase expression. PCS also up‐regulates the mRNA levels and the protein secretion of monocyte chemotactic protein‐1 (MCP‐1) in HUVEC. In HASMC, PCS increased the mRNA levels of alkaline phosphatase (ALP), osteopontin (OPN), core‐binding factor alpha 1, and ALP activity. The knockdown of Nox4, a subunit of NADPH oxidase, suppressed the cell toxicity induced by PCS. The vascular damage induced by PCS was largely suppressed in the presence of probenecid, an inhibitor of organic anion transporters (OAT). In PCS‐overloaded 5/6‐nephrectomized rats, plasma MCP‐1 levels, OPN expression, and ALP activity of the aortic arch were increased, accompanied by the induction of Nox4 expression. Collectively, the vascular toxicity of PCS can be attributed to its intracellular accumulation via OAT, which results in an enhanced NADPH oxidase expression and increased ROS production. In conclusion, we found for the first time that PCS could play an important role in the development of cardiovascular disease by inducing vascular toxicity in the CKD condition.