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

IL-1α and IL-1β trigger fat tissue remodeling.

<p><b>A</b>) KCASP1Tg mice were treated once-a-week with an intra-peritoneal injection of 10 µg anti-IL-1α and/or IL-1β neutralizing antibodies between 4 to 24 weeks of age. PBS-treated KCASP1Tg littermates were used as controls. The body weight loss was ameliorated by either anti-IL-1α, anti-IL-1β or anti-IL-1α/β administration, (n = 7, each group). <b>B</b>) Emaciation was reproduced by administering 1 µg of recombinant IL-1α or IL-1β protein 3 times per week from 6 to 16 weeks of age to wild type mice compared to PBS-injected mouse controls from 6 to 16 weeks (n = 6, each group). <b>C</b>) H&E staining of abdominal adipose tissue revealed that the adipocytes were large and plump in shape in normal control and 6-months old KIL-18Tg(−) mice; they were small and round, however, in 6-month-old KCASP1Tg and 18-months old KIL-18Tg(+) mice. The number of infiltrating mononuclear cells was similar among these groups (n = 7, each group). <b>D</b>) Cytokine levels in the skin culture supernatant were measured by flow cytometry. IL-1α and IL-1β were detected in conditioned medium from the skin culture of normal control mice and KIL-18Tg(−) mice, but was significantly higher in medium from skin culture of KCASP1Tg mice (n = 7, each group). <b>E</b>) Mouse adipose cells cultured in regular medium contained abundant lipid drops on day 14. The addition of supernatant from normal skin culture revealed a decrease in the number of plump adipocytes containing lipids as stained with oil red O, which was reversed by supplementing with supernatant from KCASP1Tg mice skin culture. The pretreatment of KCASP1Tg mice skin culture medium with anti-IL-1α or anti-IL-1β neutralizing antibodies partially ameliorated the inhibitory effects on adipose cells, which were almost abrogated by simultaneous treatment with both antibodies (n = 7, each group).</p

KCASP1Tg and KIL-18Tg(+) mice showed emaciation and altered lipid metabolism in addition to dermatitis.

<p><b>A</b>) KCASP1Tg mice had erosive dermatitis at week 8, which spread across the entire face and trunk when mice were 5-months old. KIL-18Tg mice showed no dermatitis at 6 months of age, KIL-18Tg(−). Weight loss began at 10 weeks in KCASP1Tg mice, but not in age matched KIL-18Tg mice (n = 10, each group). The left Y-axis shows body weight and the right Y-axis shows the percentage of dermatitis. KIL-18Tg mice developed dermatitis at 1-year old, followed by weight loss, KIL-18Tg(+) (n = 7, each group) (*p<0.05, **p<0.001, ***p<0.0001). <b>B</b>) CT scan of KCASP1Tg mice at 6 months of age revealed a dramatic decrease in visceral fat as shown in yellow compared to normal control or KIL-18Tg(−) mice. Eighteen-year old KIL-18Tg(+) showed decreased visceral fat. Subcutaneous fat (in orange color) was also decreased in KCASP1Tg and KIL-18Tg(+). <b>C</b>) A comparison of the somatic fat ratio across the three groups determined by CT scan at 2, 4 and 6 months of age, and a decrease was observed in KCASP1Tg mice compared to the other two groups (n = 6, each group). <b>D</b>) Six-month-old KCASP1Tg mice showed decreased plasma HDL cholesterol and leptin levels, as well as increased triglyceride levels. LDL cholesterol and adiponectin levels remained normal. Eighteen-months old KIL-18Tg(+) mice showed decreased leptin levels. No significant change was identified in the triglyceride, HDL and LDL cholesterol, and adiponectin levels in KIL-18Tg(+) mice. <b>E</b>) Plasma IL-1α and β levels were elevated in 6-months old KCASP1Tg mice. IL-1 levels were under the detection limit in KIL-18Tg(−) mice, but were elevated in 18-months old KIL-18Tg(+) mice. Plasma IL-18 levels were increased in both KCASP1Tg and KIL-18Tg mice (n = 10).</p

Cardiovascular findings in KCASP1Tg and KIL-18Tg(+).

<p><b>A</b>) Six-months old KCASP1Tg mice had significantly heavier hearts compared to normal control and KIL-18Tg(−) mice. The heart weight of 18-months old KIL-18Tg(+) mice was also increased (n = 6, each group). <b>B</b>) The maximal tension produced in an 1 mm ring segment obtained from normal and KCASP1Tg mice is shown. The strength of the snapping point of the aorta ring from KCASP1Tg mice was significantly lower than in those from control mice (n = 12, each group). <b>C</b>) Thermography showed deterioration of peripheral blood circulation in the lower limbs and tail in 6-months old KCASP1Tg and 18-months old KIL-18Tg(+) mice. <b>D</b>) Both systolic and diastolic pressures were significantly lower in KCASP1Tg and KIL-18Tg(+) mice, (n = 10, each group) compared to control or KIL-18Tg(−) mice.</p

KCASP1Tg and KIL-18Tg(+) mice developed amyloidosis in the liver, kidney and spleen.

<p><b>A</b>) Histological analyses showed loss of normal architecture: hepatocytes were replaced by dense deposits in the liver, the glomeruli and renal tubules were damaged in the kidney, and lymph-follicles were absent in the spleen. Dense amyloid deposition was detected in KCASP1Tg and KIL-18Tg(+) mice by Congo-red staining. <b>B</b>) KCASP1Tg mice showed mild liver and kidney dysfunction while renal function had significantly deteriorated in KIL-18Tg(+) mice (n = at least 7).</p