Comparison of fecal secretory IgA concentrations and bacterial diversity between BD patients and normal individuals.

<p>(A) We evaluated the secretory IgA (sIgA) concentrations of fecal supernatants using ELISA. We observed a significant increase in sIgA concentrations of patients with BD (BD) compared with those of normal individuals (NI). (B) We counted OTU numbers (annotated species numbers) and estimated alpha diversity score (Chao 1 and Shannon indexes) of each sample. We compared the titers between BD patients and normal individuals. We did not find significant differences in the parameters between BD patients and normal individuals. These biological parameters of BD patients and normal individuals were displayed with dot plots. A box-plot and a mean level (green line) of each group of BD patients and normal individuals were indicated. (C) We estimated beta diversity between BD patients and normal individuals using PCoA of QIIME software with linear conversion formulas. We visualized the PCoA plots in a three dimensional structure where three axes and each contribution ratio (principal coordinate, PC1–3, %) were depicted. We calculated the distance between the distribution of BD patients and that of normal individuals using a two-sided Student's two-sample t-test as an exploratory analysis. We obtained a significant P value of the test of beta diversity between BD patients and normal individuals.</p

Direct comparison of bacterial taxonomic abundance between BD patients and normal individuals.

<p>We compared directly the relative abundance (expressed as parts per unit) between BD patients (BD) and normal individuals (NI). We conducted Wilcoxon rank sum test for the differences in every taxon, followed by Tukey’s honestly significant difference (HSD) test. We considered that bacterial taxa increased or decreased significantly in patients with BD as compared with those in normal individuals by fulfilling the following criteria simultaneously; #1: bacterial taxa showing significant differences by the Wilcoxon rank sum test, and #2: bacterial taxa showing positive Tukey’s HSD values. We found that there were significant differences in 11 bacterial taxa (also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153746#pone.0153746.g002" target="_blank">Fig 2</a> with asterisks). “p__”, “c__”, “o__”, “f__” and “g__” indicate phylum, class, order, family and genus, respectively. Relative abundance of bacterial taxa in BD patients and normal individuals were displayed with dot plots. A box-plot and a mean level (green line) of each group of BD patients and normal individuals were indicated.</p

Comparison of the taxa showing different abundance values in BD patients and normal individuals.

<p>We analyzed the metagenomic data of bacterial taxa using LEfSe to detect major differences between BD patients (BD) and normal individuals (NI). The LEfSe provides us with cladograms of six-level (from kingdom to genus). Significantly enriched bacterial taxa in samples obtained from BD patients were demonstrated using red circles and shadings. Significantly enriched bacterial taxa in samples obtained from normal individuals were demonstrated using green circles and shadings. In patients with BD, the phylum <i>Actinobacteria</i>, namely the classes <i>Actinobacteria</i> and <i>Coriobacteria</i>, the orders <i>Bifidobacteriales</i> and <i>Coriobacteriales</i>, and the genera <i>Bifidobacterium</i>, <i>Eggerthella</i> and <i>Atopobium</i> had large effect sizes. The phylum <i>Firmicutes</i>, the class <i>Clostridia</i>, the order <i>Clostridiales</i>, the family <i>Veillonellaceae</i>, and the genera <i>Megamonas</i> and <i>Phascolarctobacterium</i> had large effect sizes in normal individuals.</p

Demographical and clinical characteristics of patients with Behcet’s disease and normal individuals.

<p>Demographical and clinical characteristics of patients with Behcet’s disease and normal individuals.</p

Renal papillary tip extract stimulates BNP production and excretion from cardiomyocytes

<div><p>Background</p><p>Brain natriuretic peptide (BNP) is an important biomarker for patients with cardiovascular diseases, including heart failure, hypertension and cardiac hypertrophy. It is also known that BNP levels are relatively higher in patients with chronic kidney disease and no heart disease; however, the mechanism remains unclear.</p><p>Methods and results</p><p>We developed a BNP reporter mouse and occasionally found that this promoter was activated specifically in the papillary tip of the kidneys, and its activation was not accompanied by <i>BNP</i> mRNA expression. No evidence was found to support the existence of BNP isoforms or other nucleotide expression apart from BNP and tdTomato. The pBNP-tdTomato-positive cells were interstitial cells and were not proliferative. Unexpectedly, both the expression and secretion of BNP increased in primary cultured neonatal cardiomyocytes after their treatment with an extract of the renal papillary tip. Intraperitoneal injection of the extract of the papillary tips reduced blood pressure from 210 mmHg to 165 mmHg, the decrease being accompanied by an increase in serum BNP and urinary cGMP production in stroke-prone spontaneously hypertensive (SHR-SP) rats. Furthermore the induction of BNP by the papillary extract from rats with heart failure due to myocardial infarction was increased in cardiomyocytes.</p><p>Conclusions</p><p>These results suggested that the papillary tip express a substance that can stimulate BNP production and secretion from cardiomyocytes.</p></div

Characterization of papillary extracts by proteolysis and molecular weight fractionation.

<p>A, Papillary extracts were digested with 0.3 μg/ml Proteinase K for each indicated time and loaded into 10% and 15% gel. The pictures are representative of three experiments. B, BNP mRNA expression was examined by Northern blot analysis in cardiomyocytes treated with control buffer (MOCK) or each digested extract of the papillary tip. *P < 0.05 vs. cardiomyocytes treated with the buffer alone (MOCK). n = 4. C, Papillary extracts were fractionated according to their molecular weight range. The pictures are representative of three experiments. D, BNP mRNA expression was examined by Northern blot analysis in cardiomyocytes treated with control buffer or each molecular-weight-fractionated extract of the papillary tip. *P < 0.05 vs. cardiomyocytes treated with the buffer alone (MOCK). n = 3.</p

Mild activation of the BNP promoter in the heart, with no activation in the other organs, of pBNP-tdTomato Tg mice.

<p>The pictures are representative of three mice.</p

tdTomato was expressed specifically in cardiomyocytes from pBNP-tdTomato transgenic mice.

<p>A, Transgenic (Tg) vector of pBNP-tdTomato. B, Isolated cardiomyocytes expressing tdTomato and mixed fibroblasts with no expression of tdTomato in a BNP reporter mouse. The pictures are representative of five mice.</p

Effects of extracts of the papillary tip (●) and inner medulla (○) of the kidneys on stroke-prone spontaneously hypertensive rats (SHR-SP).

<p>A, Each extract was intraperitoneally administered to SHR-SP rats and their systolic blood pressure was followed for 24 h using the tail-cuff method. *P < 0.05 vs. basal blood pressure (at 0 h). n = 4 to 6. B, Measurement of serum BNP (n = 4 to 8) and C, cGMP in the urine 4h and 24 h after intraperitoneal injection of buffer alone or the extract of the papillary tip or the inner medulla. *P < 0.05 or n.s. = no significant difference vs. serum from SHR-SP rats treated with buffer alone (MOCK). n = 4 to 8. D, The ratio of urine volume to MOCK 4h and 24h after the injection. n.s. = no significant difference vs. the urine from SHR-SP rats treated with buffer alone (MOCK). n = 4 to 8. E, urine sodium excretion for 4h (0-4h) and 20h (4–24 h) after the injection of control buffer or extract of the papillary tip or the inner medulla into SHR-SP rats. n.s. = no significant difference vs. the urine from SHR-SP rats treated with buffer alone (MOCK). n = 4 to 8. For all figures, data indicate the mean +/- standard error of the mean.</p

Activation of the BNP promoter in the papillary tip from the kidneys of pBNP-tdTomato Tg mice with no expression of <i>BNP</i> mRNA.

<p>A, Upper panels, whole imaging (bright-field) of a kidney stained with hematoxylin-eosin (HE), endogenous signal of tdTomato and merged images of frozen sections. Magnifications of the top panels are shown in the middle and lower panels. The pictures are representative of six mice. B, Northern blot analysis of BNP mRNA expression in the heart, inner medulla in the upper and lower poles and the papillary tip of the kidneys (n = 4).</p