N-methyl-2-pyridone-5-carboxamide (N-Me-2PY) has potent anti-fibrotic and anti-inflammatory activity in a fibrotic kidney model: is it an old uremic toxin?

BACKGROUND: Uremic toxins accumulate in renal tissues and cells due to chronic kidney disease (CKD). Abnormalities in nicotinamide adenine dinucleotide (NAD +) metabolism lead to the progression of CKD. NAD + metabolites, such as N-methyl-2-pyridone-5-carboxamide (N-Me-2PY) and N-methyl-4-pyridone-5-carboxamide (N-Me-4PY), have been recognized as uremic toxins. However, no reports have validated whether they are actually harmful to the body. Therefore, we focused on the structural similarity of these metabolites to the anti-fibrotic drug pirfenidone and evaluated their effects on renal fibrosis. METHODS: Each NAD + metabolite was treated with TGFß1 to kidney fibroblasts or tubular epithelial cells, and quantitative RT-PCR and Western blot analysis were conducted. N-Me-2PY was orally administered to a ligated murine kidney fibrosis model (UUO) to evaluate its anti-fibrotic and toxic effects on the body. RESULTS: N-Me-2PY, N-Me-4PY, and nicotinamide N-oxide (NNO) inhibited TGFß1-induced fibrosis and inflammatory gene expression in kidney fibroblasts. N-Me-2PY strongly suppressed the expression of types I and III collagen, αSMA, and IL-6. N-Me-2PY also suppressed TGFß1-induced type I collagen and IL-6 expression in renal tubular epithelial cells. No toxic effect was observed with N-Me-2PY treatment, while attenuating renal fibrosis and tubular dilation in UUO mice. Suppression of various fibrosis- and inflammation-related genes was also observed. N-Me-2PY did not inhibit TGFß1-induced Smad3 phosphorylation but inhibited Akt phosphorylation, suggesting that N-Me-2PY exerts anti-fibrotic and anti-inflammatory effects through Akt inhibition, similar to pirfenidone. CONCLUSIONS: NAD + metabolites, such as N-Me-2PY, are not uremic toxins but are potential therapeutic agents that have anti-fibrotic effects in CKD.

Differences between group X and group V secretory phospholipase A(2) in lipolytic modification of lipoproteins.

Secretory phospholipases A(2) (sPLA(2)s) are a diverse family of low molecular mass enzymes (13-18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA(2) (sPLA(2)-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA(2) (sPLA(2)-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA(2)-X in several respects. Although sPLA(2)-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA(2)-X. In addition, the requirement of Ca(2+) for the lipolysis of LDL was about 10-fold higher for sPLA(2)-V than sPLA(2)-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA(2)-V in the presence of sodium citrate, which contrasted with the potent response to sPLA(2)-X. Moreover, sPLA(2)-X, but not sPLA(2)-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA(2)-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (K(d) = 3.1 nM) in the presence of Ca(2+). Selective interaction of sPLA(2)-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.

Group V and X secretory phospholipase A(2)s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions.

The quantitative or qualitative decline of high-density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis because of its antiatherogenic functions, including the mediation of reverse cholesterol transport from the peripheral cells to the liver. We have recently shown that group X secretory phospholipase A(2) (sPLA(2)-X) is involved in the pathogenesis of atherosclerosis via potent lipolysis of low-density lipoprotein (LDL) leading to macrophage foam cell formation. We demonstrate here that sPLA(2)-X as well as group V secretory PLA(2) (sPLA(2)-V), another group of sPLA(2) that can potently hydrolyze phosphatidylcholine (PC), also possess potent hydrolytic potency for PC in HDL linked to the production of a large amount of unsaturated fatty acids and lysophosphatidylcholine (lysoPC). In contrast, the classical types of group IB and IIA secretory PLA(2)s evoked little, if any, lypolytic modification of HDL. Treatment with sPLA(2)-X or -V also caused an increase in the negative charge of HDL with no oxidation and little modification of apolipoprotein AI (apoAI). Modification with sPLA(2)-X or -V resulted in significant decrease in the capacity of HDL to cause cellular cholesterol efflux from lipid-loaded macrophages. Immunohistochemical analysis revealed significant expression of sPLA(2)-X in foam cell lesions in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) rabbit. These findings suggest that lipolytic modification of HDL by sPLA(2)-X or -V causes drastic change of HDL in terms of the production of a large amount of unsaturated fatty acids and lysoPC linked to the reduction of its antiatherogenic functions. These sPLA(2)-mediated modifications of plasma lipoproteins might be relevant to the pathogenesis of atherosclerosis.

Role of group IIA phospholipase A2 in rat colitis induced by dextran sulfate sodium.

Although group IIA phospholipase A(2) has been suggested to be implicated in inflammatory bowel disease, its pathophysiological role in colitis remains unclear. We investigated whether group IIA phospholipase A(2) had pro-inflammatory roles in dextran sulfate sodium-induced colitis in the rat. Secretory phospholipase A(2) activity was markedly increased in the distal colon with two peaks. Strong immunostaining for group IIA phospholipase A(2) was found in subepithelial tissue and lamina propria at early stage and in deeper tissues of the erosion area at later stage. Treatment with a specific group IIA phospholipase A(2) inhibitor, S-3013/LY333013 (methyl[[3-(aminooxoacetyl)-2-ethyl-1-(phenylmethyl)]-1H-indol-4yl]oxy) acetate), reduced erosion area, shortening of colon and colonic inflammation, and strongly inhibited the increase in secretory phospholipase A(2) activity and moderately reduced myeloperoxidase activity in the colon in vivo, while eicosanoid levels were not affected. Marked group IIA phospholipase A(2) expression in the erosion area and the improvement of colitis by the group IIA phospholipase A(2) inhibitor strongly suggest that this enzyme plays pro-inflammatory roles in this colitis model.

Characterization of a novel inhibitor of cytosolic phospholipase A2alpha, pyrrophenone.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha), one of the three subtypes of cPLA(2) (alpha, beta and gamma), is thought to be a rate-limiting enzyme in eicosanoid biosynthesis. We developed a novel and potent cPLA(2)alpha inhibitor with an optically active pyrrolidine, termed pyrrophenone, and characterized this compound in detail using enzyme and cellular assay systems. Pyrrophenone, which shows strong inhibition of cPLA(2)alpha activity, is one of the most potent cPLA(2)alpha inhibitors reported to date. Similar inhibitory potencies for cPLA(2)alpha were obtained from three different assays. The inhibitory activity of pyrrophenone is two or three orders of magnitude more potent than arachidonyl trifluoromethyl ketone (AACOCF(3)) under the same assay conditions. Pyrrophenone shows reversible inhibition of cPLA(2)alpha and displays no characteristics of the slow-binding inhibition observed for AACOCF(3). Pyrrophenone also inhibited the esterase and lysophospholipase activities of cPLA(2)alpha. However, the inhibition by pyrrophenone of 14 kDa secretory PLA(2)s, types IB and IIA, was over two orders of magnitude less potent than that for cPLA(2)alpha. Pyrrophenone strongly inhibited arachidonic acid release in calcium ionophore (A23187)-stimulated human monocytic cells (THP-1 cells) in a dose-dependent manner with an IC(50) value of 0.024 microM, followed by suppression of eicosanoid synthesis, and also showed dose-dependent inhibition for interleukin-1-induced prostaglandin E(2) synthesis in human renal mesangial cells with an IC(50) value of 0.0081 microM. The mechanism of inhibition of eicosanoid synthesis in these cell-based assays was due to inhibition of only one step of arachidonic acid release without any effect on cyclo-oxygenase or lipoxygenase pathways. These results suggest that pyrrophenone could be a potential therapeutic agent for inflammatory diseases.

Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation.

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.

Effect of a selective inhibitor of secretory phospholipase A2, S-5920/LY315920Na, on experimental acute pancreatitis in rats.

We investigated the efficacy of a potent inhibitor of secretory phospholipase A2 (sPLA2), S-5920/LY315920Na, in an experimental model of acute pancreatitis in rats. Combined intraductal injection of sodium taurocholate (5 mg/rat) and porcine pancreatic sPLA2-IB (300 microg/rat) caused severe hemorrhagic necrotizing pancreatitis resulting in high mortality, along with rapid increases of catalytic PLA2 and lipase activities in plasma and ascites and with gradual increases of plasma amylase and aspartate aminotransferase levels over 9 h after the pancreatitis. Prophylactic intravenous treatment with S-5920/LY315920Na significantly reduced mortality at 7 days, and strongly abrogated PLA2 activities in both plasma and ascites along with significant reduction of lipase activity, amylase, aspartate aminotransferase, and hemorrhage at 6 h. It also significantly reduced histological damage such as edema and parenchymal and fat necroses of the pancreatic tissue. This sPLA2 inhibitor could become an effective agent for the treatment of severe acute pancreatitis.

Synthesis and activities of oxidative metabolites of the anti-arthritic drug candidate S-2474.

We have synthesized and characterized some oxidative metabolites of S-2474. In this study, we discovered a novel skeleton, the 2,3-dihydrobenzofuran derivative, which inhibited PGE(2) production at a very low concentration and was effective in the anti-carrageenin footpad edema assay.