Sodium glucose cotransporter 2 inhibitor suppresses renal injury in rats with renal congestion.

Renal congestion is an issue of cardiorenal syndrome in patients with heart failure. Recent clinical and basic studies suggest a renoprotective potential of sodium-glucose cotransporter (SGLT) 2 inhibitors. However, the effect on renal congestion and its mechanism is not fully understood. Thus, we aimed to clarify the effect of SGLT inhibition in a renal congestion model. Renal congestion was induced in the left kidney of male Sprague-Dawley rats by ligation of the inferior vena cava between the renal veins. The SGLT2 inhibitor tofogliflozin or vehicle was orally administered daily from the day before IVC ligation until two days after surgery. On the third postoperative day, both the right control kidney and the left congested kidney were harvested and analyzed. Kidney weight and water content was increased, and renal injury and fibrosis were observed in the left congested kidney. Kidney weight gain and hydration were improved with tofogliflozin treatment. Additionally, this treatment effectively reduced renal injury and fibrosis, particularly in the renal cortex. SGLT2 expression was observed in the congested kidney, but suppressed in the damaged tubular cells. Molecules associated with inflammation were increased in the congested kidney and reversed by tofogliflozin treatment. Mitochondrial dysfunction provoked by renal congestion was also improved by tofogliflozin treatment. Tofogliflozin protects against renal damage induced by renal congestion. SGLT2 inhibitors could be a candidate strategy for renal impairment associated with heart failure.

Inhibition of platelet-derived growth factor pathway suppresses tubulointerstitial injury in renal congestion.

OBJECTIVE: Increased central venous pressure in congestive heart failure is responsible for renal dysfunction, which is mediated by renal venous congestion. Pericyte detachment from capillaries after renal congestion might trigger renal fibrogenesis via pericyte-myofibroblast transition (PMT). Platelet-derived growth factor receptors (PDGFRs), which are PMT indicators, were upregulated in our recently established renal congestion model. This study was designed to determine whether inhibition of the PDGFR pathway could suppress tubulointerstitial injury after renal congestion. METHODS: The inferior vena cava between the renal veins was ligated in male Sprague-Dawley rats, inducing congestion only in the left kidney. Imatinib mesylate or vehicle were injected intraperitoneally daily from 1 day before the operation. Three days after the surgery, the effect of imatinib was assessed by physiological, morphological and molecular methods. The inhibition of PDGFRs against transforming growth factor-ß1 (TGFB1)-induced fibrosis was also tested in human pericyte cell culture. RESULTS: Increased kidney weight and renal fibrosis were observed in the congested kidneys. Upstream inferior vena cava (IVC) pressure immediately increased to around 20 mmHg after IVC ligation in both the imatinib and saline groups. Although vasa recta dilatation and pericyte detachment under renal congestion were maintained, imatinib ameliorated the increased kidney weight and suppressed renal fibrosis around the vasa recta. TGFB1-induced elevation of fibrosis markers in human pericytes was suppressed by PDGFR inhibitors at the transcriptional level. CONCLUSION: The activation of the PDGFR pathway after renal congestion was responsible for renal congestion-induced fibrosis. This mechanism could be a candidate therapeutic target for renoprotection against renal congestion-induced tubulointerstitial injury.

Hydrochlorothiazide ameliorates polyuria caused by tolvaptan treatment of polycystic kidney disease in PCK rats.

BACKGROUND: Tolvaptan is an effective treatment for polycystic kidney disease (PKD), but also causes unfortunate polyuria. Hydrochlorothiazide (HCTZ) has been shown to reduce urine volume in nephrogenic diabetes insipidus, raising the possibility that HCTZ could also be effective in reducing tolvaptan-induced polyuria. In this study, we examined the combined administration of HCTZ and tolvaptan. METHODS: Male PCK rats were divided into four groups of normal chow (Cont), normal chow plus tolvaptan, gavage HCTZ treatment, and tolvaptan + HCTZ. Biochemical examinations of the plasma and urine were performed as well as histological and molecular (mRNA and protein expression) analyses. RESULTS: Groups treated with tolvaptan had significantly higher 24 h urine excretion, which was significantly reduced in the tolvaptan + HCTZ group after 2 weeks. Cyst size, pERK protein expression, and Cyclin D1 mRNA expression were all significantly reduced in both the tolvaptan and tolvaptan + HCTZ groups, indicating that HCTZ did not affect the beneficial functions of tolvaptan. Notably, aquaporin 2 redistribution from the apical to intracellular domains was observed in tolvaptan-treated rats and was partially reversed in the tolvaptan + HCTZ group. The renal glomerular filtration rate was reduced in the tolvaptan + HCTZ group. Significantly lowered mRNA expression of neuronal nitric oxide synthase, prostaglandin E synthase 2 and renin were also found in the medulla, but not in the cortex. CONCLUSION: HCTZ reduces tolvaptan-induced polyuria without altering its beneficial effects on PKD. This novel therapeutic combination could potentially lead to better PKD treatments and improved quality of life for the affected patients.

The relationship between the effect of matured hop extract and physical activity on reducing body fat: re-analysis of data from a randomized, double-blind, placebo-controlled parallel group study.

BACKGROUND: We recently reported that successive ingestion of matured hop extract (MHE), produced by oxidation of hops, results in a reduction of body fat in healthy overweight participants. A combined effect of MHE and physical activity on body fat has not been investigated. Thus, we re-analyzed data from the previous study to explore the relationship between the effect of MHE and walking as an index of physical activity. METHODS: This analysis uses existing data from a randomized, double-blind, placebo-controlled parallel group study in which MHE (active) or placebo was given for 12 w to 200 healthy overweight Japanese, from May to December 2014. Correlation between the change in abdominal fat areas at 12 w and the number of steps taken per day was tested by Spearman's correlation coefficient test. The subjects were stratified using the average number of steps per day of Japanese into walking less and walking more subgroups (WL and WM, respectively) as follows: placebo (WL, n = 43; WM, n = 44) and active (WL, n = 49; WM, n = 42). Reductions in total, visceral, and subcutaneous fat area (TFA, VFA and SFA, respectively) were evaluated. The interaction effect between ingestion (active/placebo) and walking (WL/WM) was analyzed using two-way analysis of variance (ANOVA). RESULTS: There was a significant negative correlation between the change in VFA and daily steps taken in the active group (r = - 0.208, P = 0.048). No significant correlation in TFA or SFA. Although the interaction effect in TFA was not significant, the main effect of ingestion was significant (P = 0.045). In contrast, the interaction effect in VFA was suggested to be synergistic (P = 0.055). CONCLUSION: The results suggested that MHE ingestion combined with light intensity exercise would induce a greater reduction in VFA which would be beneficial for obese or overweight individuals in reducing obesity and obesity-related diseases. TRIAL REGISTRATION: UMIN-CTR UMIN000014185 registered 6 June 2014.

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A.

mab21-l3 regulates cell fate specification of multiciliate cells and ionocytes.

Cell fate specifications of multiciliate cells (MCCs) and ionocytes are commonly suppressed by the Notch pathway in developing epithelia, but are governed by different master regulators, suggesting the existence of a common regulator linking the Notch pathway to both MCC and ionocyte specifications. Here we show that a mab21 family gene, mab21-l3, represents the missing link. In Xenopus embryonic epidermis, mab21-l3 expression is specifically found in MCCs and ionocytes and is downregulated by the Notch pathway. Knockdown of mab21-l3 in Xenopus downregulates both MCC-specific and ionocyte-specific master genes, resulting in drastic loss of MCCs and ionocytes. In mouse tracheal epithelial cells, mab21-l3 expression is also downregulated by the Notch pathway and is required for MCC differentiation. Moreover, conditional gain of function of mab21-l3 rescues Notch-induced loss of MCCs and ionocytes in Xenopus. These results indicate that mab21-l3 acts downstream of the Notch pathway in cell fate specifications of MCCs and ionocytes.

Identification of significant amino acids in multiple transmembrane domains of human transient receptor potential ankyrin 1 (TRPA1) for activation by eudesmol, an oxygenized sesquiterpene in hop essential oil.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca(2+) measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03-0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by ß-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.

Estimation of the prevalence of latent abnormalities in the lung and the accuracy of indirect chest x-ray examination among Japanese workers of different age groups.

It is considered that annual chest x-ray (CXR) examination can be restricted to workers by age; however, adequate epidemiological evidence is needed regarding the difference in the effectiveness of CXR examination between younger and older age groups. This study attempted to estimate the prevalence of latent abnormalities (P) in the lung and the false negative rate (F) and false positive rate (G) of examinations among workers of different age groups. 112,482 subjects who were screened for three consecutive years with no findings at the first screening, were selected. The proportion of positive findings at the second screening and the proportion of negative findings at the third screening were incorporated into a stochastic model, and 5-yr-age-specific P, F, and G were estimated. P tended to increase with increasing age. F was 40-45% in <45-yr-old subjects and was only 10% in the older age groups. G showed a steady increase with increasing age. This study indicates that the nature of findings differs between younger and older age groups, and this difference affects the accuracy of examination. In Japan, the age of workers should be considered when a CXR examination is performed.

Molecular cloning and expression analysis of feline α1-microglobulin.

Full-length cDNA that encodes feline α1-microglobulin (Feα1m)-bikunin was obtained from a feline liver and cloned using an oligo-capping method. The Feα1m-bikunin cDNA was found to contain 1284 nucleotides, and Feα1m was found to include an open reading frame encoding a polypeptide of 201 amino acids. The deduced amino acid sequence of Feα1m showed varying amino acid identity when compared with the published sequences of the related α1-m of other species, ranging from 71.1 to 82.1%. Feα1m mRNA expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analysis in the cerebrum, cerebellum, lung, heart, liver, spleen, pancreas, kidney, adrenal gland, and testicle. The highest Feα1m mRNA level was found in the liver.

Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L.

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ~95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.

Molecular cloning, expression in Escherichia coli, and development of monoclonal antibodies to feline cystatin C.

Cystatin C is a member of the cystatin superfamily of low-molecular-weight proteins that inhibit the activity of cysteine protease. The full-length cDNA that encodes feline cystatin C (FeCysC) has been cloned from feline white blood cells by the oligo-capping method. The cDNA consists of 796 nucleotides and includes an open reading frame encoding a polypeptide of 146 amino acids. Next, we developed several mouse anti-FeCysC monoclonal antibodies (mAbs). The recombinant FeCysC (rFeCysC) was expressed in Escherichia coli (E. coli) BL21 and used as an antigen to immunise mice. The reactivity of the mAbs to native FeCysC was examined by western blot analysis against the urinary protein from cats with chronic kidney disease (CKD). The three anti-rFeCysC mAbs (3-9G, 7-7C, and 9-12F) were able to recognise native FeCysC.

Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843.

The nucleotide sequence of the complete genome of a cyanobacterium, Microcystis aeruginosa NIES-843, was determined. The genome of M. aeruginosa is a single, circular chromosome of 5,842,795 base pairs (bp) in length, with an average GC content of 42.3%. The chromosome comprises 6312 putative protein-encoding genes, two sets of rRNA genes, 42 tRNA genes representing 41 tRNA species, and genes for tmRNA, the B subunit of RNase P, SRP RNA, and 6Sa RNA. Forty-five percent of the putative protein-encoding sequences showed sequence similarity to genes of known function, 32% were similar to hypothetical genes, and the remaining 23% had no apparent similarity to reported genes. A total of 688 kb of the genome, equivalent to 11.8% of the entire genome, were composed of both insertion sequences and miniature inverted-repeat transposable elements. This is indicative of a plasticity of the M. aeruginosa genome, through a mechanism that involves homologous recombination mediated by repetitive DNA elements. In addition to known gene clusters related to the synthesis of microcystin and cyanopeptolin, novel gene clusters that may be involved in the synthesis and modification of toxic small polypeptides were identified. Compared with other cyanobacteria, a relatively small number of genes for two component systems and a large number of genes for restriction-modification systems were notable characteristics of the M. aeruginosa genome.