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We have previously demonstrated that KS-133 is a specific and potent antagonist of vasoactive intestinal peptide receptor 2 (VIPR2). We have also shown that vasoactive intestinal peptide-VIPR2 signaling affects the polarity and activation of tumor-associated macrophages, which is another strategy for cancer immunotherapy apart from the activation of effector T cells. In this study, we aimed to examine whether the selective blockade of VIPR2 by KS-133 changes the polarization of macrophages and induces anti-tumor effects. In the presence of KS-133, genetic markers indicative of tumor-aggressive M1-type macrophages were upregulated, and conversely, those of tumor-supportive M2-type macrophages were downregulated. Daily subcutaneous administration of KS-133 tended to suppress the growth of CT26 tumors (murine colorectal cancer-derived cells) implanted subcutaneously in Balb/c mice. To improve the pharmacological efficacy and reduce the number of doses, we examined a nanoformulation of KS-133 using the US Food and Drug Administration-approved pharmaceutical additive surfactant Cremophor® EL. KS-133 nanoparticles (NPs) were approximately 15 nm in size and stable at 4°C after preparation. Meanwhile, KS-133 was gradually released from the NPs as the temperature was increased. Subcutaneous administration of KS-133 NPs once every 3 days had stronger anti-tumor effects than daily subcutaneous administration of KS-133. Furthermore, KS-133 NPs significantly enhanced the pharmacological efficacy of an immune checkpoint-inhibiting anti-PD-1 antibody. A pharmacokinetic study suggested that the enhancement of anti-tumor activity was associated with improvement of the pharmacokinetic profile of KS-133 upon nanoformulation. Our data have revealed that specific blockade of VIPR2 by KS-133 has therapeutic potential for cancer both alone and in combination with immune checkpoint inhibitors.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease with no decisive treatment. The sodium glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin was developed as a new oral hypoglycemic drug, which can improve NASH via an insulin-independent glucose-lowering effect by inhibiting glucose reabsorption in the renal proximal tubules. However, ipragliflozin appears to modulate steatosis or inflammation via different pathways. To elucidate the new mechanism of ipragliflozin for the treatment of NASH, we evaluated its effects in a NASH mouse model (STAM mice) with beta cell depletion, and compared the expression of microRNAs (miRNAs) in STAM mice treated with or without ipragliflozin (16.7 μg/day for 5 weeks). Ipragliflozin reduced aspartate transaminase and alanine aminotransferase levels, along with reduced hepatic steatosis, hepatocyte ballooning, lobular inflammation, and liver fibrosis. In addition, ipragliflozin upregulated mitochondrial transport-related and antioxidant defensive system-related genes in the liver. Among 2555 mouse miRNA probes, miR-19b-3p was commonly differentially expressed with ipragliflozin treatment for 5 weeks in both the liver and serum but in different directions, with a decrease in the liver and increase in the serum. Therefore, ipragliflozin can improve NASH development likely through the antioxidative stress pathway and by regulating miR-19b-3p.

Caloric restriction (CR) is one of the most important behavioral interventions to reduce excessive abdominal adiposity, which is a risk factor for the development of insulin resistance. Previous metabolomics studies have characterized substrate metabolism during healthy conditions; however, the effects of CR and subsequent mass recovery on shifts in substrate metabolism during insulin resistance (IR) have not been widely investigated. To assess the effects of acute CR and the subsequent mass recovery on shifts in substrate metabolism, a cohort of 15-week old Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats were calorie restricted (CR: 50% × 10 days) with or without partial body mass recovery (PR; 73% x 7 days), along with their respective ad libitum controls. End-of-study plasma samples were analyzed for primary carbon metabolites by gas chromatography (GC) time-of-flight (TOF) mass spectrometry (MS) data acquisition. Data analysis included PCA, Pearson correlation vs previously reported variables (adipose and body masses, and insulin resistance index, IRI), and metabolomics maps (MetaMapp) generated for the most significant group comparisons. All treatments elicited a significant group differentiation in at least one principal component. CR improved TCA cycle in OLETF, and increased lipolysis and proteolysis. These changes were reversed after PR except for gluconeogenesis. Plasma lipid concentrations were inversely correlated to IRI in LETO, but not OLETF. These shifts in substrate metabolism suggest that the CR-induced decreases in adipose may not be sufficient to more permanently alter substrate metabolism to improve IR status during metabolic syndrome.

To date, good experimental animal models of renal anemia are not available. Therefore, the purpose of this study was to establish a novel approach to induce chronic kidney disease (CKD) with severe anemia by oral administration of adenine in rodents. Adenine was administered to 6-week-old male C57BL/6 mice (25 and 50 mg/kg body weight) by oral gavage daily for 28 days. Serum creatinine and BUN as well as hematocrit, hemoglobin (Hb) and plasma erythropoietin (EPO) levels were monitored to assess renal function and anemia, respectively. Adenine at 25 mg/kg for 28 days slightly increased plasma creatinine levels, but did not induce anemia. In contrast, 50 mg/kg of adenine daily for 28 days showed severe renal dysfunction (plasma creatinine 1.9 ± 0.10 mg/dL) and anemia (hematocrit 36.5 ± 1.0% and EPO 28 ± 2.4 pg/mL) as compared with vehicle-treated mice (0.4 ± 0.02 mg/dL, 49.6 ± 1.6% and 61 ± 4.0 pg/mL, respectively). At the end of experiment, level of Hb also significantly reduced in 50 mg/kg adenine administration group. Remarkable histological changes of kidney tissues characterized by interstitial fibrosis and cystic appearance in tubules were observed in 50 mg/kg of adenine treatment group. These results have demonstrated that oral dosing with adenine at 50 mg/kg for 28 days is suitable to induce a stable anemia associated with CKD in mice.

High glucose has been demonstrated to induce angiotensinogen (AGT) synthesis in the renal proximal tubular cells (RPTCs) of rats, which may further activate the intrarenal renin-angiotensin system (RAS) and contribute to diabetic nephropathy. This study aimed to investigate the effects of high glucose on AGT in the RPTCs of human origin and identify the glucose-responsive transcriptional factor(s) that bind(s) to the DNA sequences of AGT promoter in human RPTCs. Human kidney (HK)-2 cells were treated with normal glucose (5.5 mM) and high glucose (15.0 mM), respectively. Levels of AGT mRNA and AGT secretion of HK-2 cells were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Consecutive 5'-end deletion mutant constructs and different site-directed mutagenesis products of human AGT promoter sequences were respectively transfected into HK-2 cells, followed by AGT promoter activity measurement through dual luciferase assay. High glucose significantly augmented the levels of AGT mRNA and AGT secretion of HK-2 cells, compared with normal glucose treatment. High glucose also significantly augmented AGT promoter activity in HK-2 cells transfected with the constructs of human AGT promoter sequences, compared with normal glucose treatment. Hepatocyte nuclear factor (HNF)-5 was found to be one of the glucose-responsive transcriptional factors of AGT in human RPTCs, since the mutation of its binding sites within AGT promoter sequences abolished the above effects of high glucose on AGT promoter activity as well as levels of AGT mRNA and its secretion. The present study has demonstrated, for the first time, that high glucose augments AGT in human RPTCs through HNF-5, which provides a potential therapeutic target for diabetic nephropathy.

Cardiac rupture is an important cause of death in the acute phase after myocardial infarction (MI). Macrophages play a pivotal role in cardiac remodeling after MI. Apoptosis inhibitor of macrophage (AIM) is secreted specifically by macrophages and contributes to macrophage accumulation in inflamed tissue by maintaining survival and recruiting macrophages. In this study, we evaluated the role of AIM in macrophage accumulation in the infarcted myocardium and cardiac rupture after MI.Wild-type (WT) and AIM‒/‒ mice underwent permanent left coronary artery ligation and were followed-up for 7 days. Macrophage accumulation and phenotypes (M1 pro-inflammatory macrophage or M2 anti-inflammatory macrophage) were evaluated by immunohistological analysis and RT-PCR. Matrix metalloproteinase (MMP) activity levels were measured by gelatin zymography. The survival rate was significantly higher (81.1% vs. 48.2%, P

In patients with diabetes, albuminuria is a risk marker of end-stage renal disease and cardiovascular events. An increased renin-angiotensin system activity has been reported to play an important role in the pathological processes in these conditions. We compared the effect of aliskiren, a direct renin inhibitor (DRI), with that of angiotensin receptor blockers (ARBs) on albuminuria and urinary excretion of angiotensinogen, a marker of intrarenal renin-angiotensin system activity.We randomly assigned 237 type 2 diabetic patients with high-normal albuminuria (10 to

A growing body of evidence indicates that renal tissue injuries are reversible. We investigated whether dietary salt reduction with the combination therapy of angiotensin II type 1 receptor blocker (ARB) plus calcium channel blocker (CCB) reverses renal tissue injury in Dahl salt-sensitive (DSS) hypertensive rats. DSS rats were fed a high-salt diet (HS; 4% NaCl) for 4 weeks. Then, DSS rats were given one of the following for 10 weeks: HS diet; normal-salt diet (NS; 0.5% NaCl), NS + an ARB (olmesartan, 10 mg/kg/day), NS + a CCB (azelnidipine, 3 mg/kg/day), NS + olmesartan + azelnidipine or NS + hydralazine (50 mg/kg/day). Four weeks of treatment with HS diet induced hypertension, proteinuria, glomerular sclerosis and hypertrophy, glomerular podocyte injury, and tubulointerstitial fibrosis in DSS rats. A continued HS diet progressed hypertension, proteinuria and renal tissue injury, which was associated with inflammatory cell infiltration and increased proinflammatory cytokine mRNA levels, NADPH oxidase activity and NADPH oxidase-dependent superoxide production in the kidney. In contrast, switching to NS halted the progression of hypertension, renal glomerular and tubular injuries. Dietary salt reduction with ARB or with CCB treatment further reduced blood pressure and partially reversed renal tissues injury. Furthermore, dietary salt reduction with the combination of ARB plus CCB elicited a strong recovery from HS-induced renal tissue injury including the attenuation of inflammation and oxidative stress. These data support the hypothesis that dietary salt reduction with combination therapy of an ARB plus CCB restores glomerular and tubulointerstitial injury in DSS rats.

BACKGROUND AND OBJECTIVE: We have reported that toll-like receptor 4 (TLR4) and one of its endogenous ligands, myeloid-related protein 8 (MRP8 or S100A8), play an important role in the progression of diabetic nephropathy in mice. The aim of this study was to evaluate significance of kidney MRP8 expression in patients with obesity- or type 2 diabetes-associated kidney diseases. METHODS: In diabetic, obese or control subjects, MRP8 mRNA and protein expression levels in renal biopsy samples were determined by real-time RT-PCR and immunohistochemistry (n = 28 and 65, respectively), and their associations with baseline and prognostic parameters were analyzed. Effects of MRP8 upon pro-inflammatory gene expressions were examined using macrophages. RESULTS: Kidney MRP8 gene and protein expression levels were elevated in obese or diabetic groups compared to control group. Among all subjects, by univariate linear regression analysis, glomerular MRP8-positive cell count and tubulointerstitial MRP8-positive area at baseline were both, respectively, correlated not only with various known risk factors for diabetic nephropathy (such as systolic blood pressure, proteinuria and serum creatinine) but also with extent of glomerulosclerosis and tubulointerstitial fibrosis. Independent factors predicting urinary protein levels a year later were examined by multivariate analysis, and they included glomerular MRP8-positive cell count (β = 0.59, P

Recent clinical trials have demonstrated that combination therapy with renin-angiotensin system inhibitors plus calcium channel blockers (CCBs) elicits beneficial effects on cardiovascular and renal events in hypertensive patients with high cardiovascular risks. In the present study, we hypothesized that CCB enhances the protective effects of an angiotensin II type 1 receptor blocker (ARB) against diabetic cerebrovascular-renal injury. Saline-drinking type 2 diabetic KK-A(y) mice developed hypertension and exhibited impaired cognitive function, blood-brain barrier (BBB) disruption, albuminuria, glomerular sclerosis and podocyte injury. These brain and renal injuries were associated with increased gene expression of NADPH oxidase components, NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Treatment with the ARB, olmesartan (10 mg/kg/day) reduced blood pressure in saline-drinking KK-A(y) mice and attenuated cognitive decline, BBB disruption, glomerular injury and albuminuria, which were associated with a reduction of NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Furthermore, a suppressive dose of azelnidipine (3 mg/kg/day) exaggerated these beneficial effects of olmesartan. These data support the hypothesis that a CCB enhances ARB-associated cerebrovascular-renal protective effects through suppression of NADPH oxidase-dependent oxidative stress in type 2 diabetes.

Mineralocorticoid receptor (MR) antagonists attenuate renal injury in salt-sensitive hypertensive rats with low plasma aldosterone levels. We hypothesized that oxidative stress causes MR activation in high-salt-fed Dahl salt-sensitive rats. Furthermore, we determined if MR activation persisted and induced renal injury, even after switching from a high- to a normal-salt diet.High-salt feeding for 4 weeks increased dihydroethidium fluorescence (DHE, an oxidant production marker), p22phox (a NADPH oxidase subunit) and serum and glucocorticoid-regulated kinase-1 (SGK1, an MR transcript) in glomeruli, compared with normal-salt feeding, and these changes persisted 4 weeks after salt withdrawal. Tempol treatment (0.5 mmol/L) during high-salt feeding abolished the changes in DHE fluorescence, p22phox and SGK1. Dietary salt reduction after a 4-week high-salt diet decreased both blood pressure and proteinuria, but was associated with significantly higher proteinuria than in normal control rats at 4 weeks after salt reduction. Administration of tempol during high-salt feeding, or eplerenone, an MR antagonist (100 mg/kg/day), started after salt reduction, recovered proteinuria to normal levels at 4 weeks after salt reduction. Paraquat, a reactive oxygen species generator, enhanced MR transcriptional activity in cultured rat mesangial cells and mouse podocytes.These results suggest that oxidative stress plays an important role in glomerular MR activation in Dahl salt-sensitive rats. Persistent MR activation even after reducing salt intake could limit the beneficial effects of salt restriction.

Some anti-cancer drugs, including those that alter microtubule dynamics target mitotic cells and induce apoptosis in some cell types. However, such drugs elicit protective responses in other cell types allowing cells to escape from drug-induced mitotic inhibition. Cells with a faulty protective mechanism undergo defective mitosis, leading to genome instability. Brd4 is a double bromodomain protein that remains on chromosomes during mitosis. However, Brd4 is released from mitotic chromosomes when cells are exposed to anti-mitotic drugs including nocodazole. Neither the mechanisms, nor the biological significance of drug-induced Brd4 release has been fully understood. We found that deletion of the internal C-terminal region abolished nocodazole induced Brd4 release from mouse P19 cells. Furthermore, cells expressing truncated Brd4, unable to dissociate from chromosomes were blocked from mitotic progression and failed to complete cell division. We also found that pharmacological and peptide inhibitors of the c-jun-N-terminal kinases (JNK) pathway, but not inhibitors of other MAP kinases, prevented release of Brd4 from chromosomes. The JNK inhibitor that blocked Brd4 release also blocked mitotic progression. Further supporting the role of JNK in Brd4 release, JNK2-/- embryonic fibroblasts were defective in Brd4 release and sustained greater inhibition of cell growth after nocodazole treatment. In sum, activation of JNK pathway triggers release of Brd4 from chromosomes upon nocodazole treatment, which mediates a protective response designed to minimize drug-induced mitotic stress.

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8⁻/⁻Irf4⁻/⁻ mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8⁻/⁻ mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4⁻/⁻ mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis.