The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Measures of the adipokine chemerin are elevated in multiple cardiovascular diseases, including hypertension, but little mechanistic work has been done to implicate chemerin as being causative in such diseases. The chemerin knockout (KO) rat was created to test the hypothesis that removal of chemerin would reduce pressure in the normal and hypertensive state. Western analyses confirmed loss of chemerin in the plasma and tissues of the KO vs. wild-type (WT) rats. Chemerin concentration in plasma and tissues was lower in WT females than in WT males, as determined by Western analysis. Conscious male and female KO rats had modest differences in baseline measures vs. the WT that included systolic, diastolic, mean arterial and pulse pressures, and heart rate, all measured telemetrically. The mineralocorticoid deoxycorticosterone acetate (DOCA) and salt water, combined with uninephrectomy as a hypertensive stimulus, elevated mean and systolic blood pressures of the male KO higher than the male WT. By contrast, all pressures in the female KO were lower than their WT throughout DOCA-salt treatment. These results revealed an unexpected sex difference in chemerin expression and the ability of chemerin to modify blood pressure in response to a hypertensive challenge.-Watts, S. W., Darios, E. S., Mullick, A. E., Garver, H., Saunders, T. L., Hughes, E. D., Filipiak, W. E., Zeidler, M. G., McMullen, N., Sinal, C. J., Kumar, R. K., Ferland, D. J., Fink, G. D. The chemerin knockout rat reveals chemerin dependence in female, but not male, experimental hypertension.

Fenfluramine-induced PVAT-dependent contraction depends on norepinephrine and not serotonin.

Perivascular adipose tissue (PVAT) modulates vascular tone and altered PVAT function is observed in vascular diseases such as hypertension and atherosclerosis. We discovered that the PVAT surrounding rat thoracic aorta (RA) and the superior mesenteric artery (SMA) contain significant amounts of 5-hydroxytryptamine (5-HT). We hypothesized that the 5-HT contained within the PVAT is functional and vasoactive. Isolated tissue baths were used for isometric contractility studies and high performance liquid chromatography was used to quantitatively measure amines in the PVAT and release studies. The 5-HT releaser fenfluramine (10 nM-100 µM) was tested for its ability to contract arteries with and without PVAT. Contraction was reported as a percentage of the initial contraction to 10 µM phenylephrine. The RA with PVAT contracted to fenfluramine to a greater maximum (98 ± 10%) than RA without PVAT (24 ± 4%), while no difference in contraction of SMA to maximum fenfluramine with (78 ± 2%) and without (75 ± 6%) PVAT was observed. Contradicting our hypothesis, the maximum contraction of RA with PVAT to fenfluramine was diminished by the alpha-1 adrenoreceptor antagonist prazosin (100 nM; vehicle: 71 ± 4%, prazosin: 24 ± 2%) and the norepinephrine transporter (NET) inhibitor nisoxetine (1 µM; vehicle: 71 ± 4%, nisoxetine: 25 ± 4%) but not the 5-HT2A/2C receptor antagonist ketanserin (10 nM) or serotonin specific reuptake inhibitor fluoxetine (10 µM). To test if fenfluramine caused release of 5-HT or NE from PVAT, PVAT from RA was incubated with vehicle or fenfluramine (10 µM-10 mM), and amines released into the incubating buffer were quantified. A pronounced concentration-dependent NE-release (more than 5-HT) was observed. Collectively, this research illustrates the pharmacology of fenfluramine to primarily stimulate NE release (better than 5-HT) in a NET-dependent manner, leading to vasoconstriction. This adds additional support to PVAT as being an important reservoir of amines.

Targeting Phospholipase D4 Attenuates Kidney Fibrosis.

Phospholipase D4 (PLD4), a single-pass transmembrane glycoprotein, is among the most highly upregulated genes in murine kidneys subjected to chronic progressive fibrosis, but the function of PLD4 in this process is unknown. Here, we found PLD4 to be overexpressed in the proximal and distal tubular epithelial cells of murine and human kidneys after fibrosis. Genetic silencing of PLD4, either globally or conditionally in proximal tubular epithelial cells, protected mice from the development of fibrosis. Mechanistically, global knockout of PLD4 modulated innate and adaptive immune responses and attenuated the upregulation of the TGF-ß signaling pathway and α1-antitrypsin protein (a serine protease inhibitor) expression and downregulation of neutrophil elastase (NE) expression induced by obstructive injury. In vitro, treatment with NE attenuated TGF-ß-induced accumulation of fibrotic markers. Furthermore, therapeutic targeting of PLD4 using specific siRNA protected mice from folic acid-induced kidney fibrosis and inhibited the increase in TGF-ß signaling, decrease in NE expression, and upregulation of mitogen-activated protein kinase signaling. Immunoprecipitation/mass spectrometry and coimmunoprecipitation experiments confirmed that PLD4 binds three proteins that interact with neurotrophic receptor tyrosine kinase 1, a receptor also known as TrkA that upregulates mitogen-activated protein kinase. PLD4 inhibition also prevented the folic acid-induced upregulation of this receptor in mouse kidneys. These results suggest inhibition of PLD4 as a novel therapeutic strategy to activate protease-mediated degradation of extracellular matrix and reverse fibrosis.

RNA Sequencing Identifies Novel Translational Biomarkers of Kidney Fibrosis.

CKD is the gradual, asymptomatic loss of kidney function, but current tests only identify CKD when significant loss has already happened. Several potential biomarkers of CKD have been reported, but none have been approved for preclinical or clinical use. Using RNA sequencing in a mouse model of folic acid-induced nephropathy, we identified ten genes that track kidney fibrosis development, the common pathologic finding in patients with CKD. The gene expression of all ten candidates was confirmed to be significantly higher (approximately ten- to 150-fold) in three well established, mechanistically distinct mouse models of kidney fibrosis than in models of nonfibrotic AKI. Protein expression of these genes was also high in the folic acid model and in patients with biopsy-proven kidney fibrosis. mRNA expression of the ten genes increased with increasing severity of kidney fibrosis, decreased in response to therapeutic intervention, and increased only modestly (approximately two- to five-fold) with liver fibrosis in mice and humans, demonstrating specificity for kidney fibrosis. Using targeted selected reaction monitoring mass spectrometry, we detected three of the ten candidates in human urine: cadherin 11 (CDH11), macrophage mannose receptor C1 (MRC1), and phospholipid transfer protein (PLTP). Furthermore, urinary levels of each of these three proteins distinguished patients with CKD (n=53) from healthy individuals (n=53; P<0.05). In summary, we report the identification of urinary CDH11, MRC1, and PLTP as novel noninvasive biomarkers of CKD.

Naïve, Regulatory, Activated, and Memory Immune Cells Co-exist in PVATs That Are Comparable in Density to Non-PVAT Fats in Health.

Perivascular adipose tissue (PVAT), the fat surrounding peripheral blood vessels, is protective and reduces the contraction of blood vessels in health. PVAT is composed of adipocytes, stromal cells, and immune cells. Recent work supports eosinophils as one of the cell types key to the anti-contractile nature of PVAT in health. Hence, we hypothesized that there exists a basally activated immune cell community in healthy PVAT that is distinctly different from non-PVAT fats. PVATs were from around mesenteric resistance vessels (MRPVAT - white fat) and thoracic aorta (APVAT - brown fat). Non-PVATs included retroperitoneal (RP fat - white fat) and subscapular (SS fat - brown fat) while the spleen was a positive control. Tissues were harvested from adult male and female Sprague Dawley rats. Six primary immune cell types were identified in PVATs. T cells (CD4 and CD8), B cells, natural killer (NK) cells, macrophages, mast cells, and neutrophils in the stromal vascular fraction of each fat were identified using nine-color flow cytometry. PVATs contained a higher number of total immune cells vs. their respective non-PVAT fats in females. Females had a higher number of T cells in MRPVAT vs. males. Females also had a greater number of T cells and total immune cells in APVAT vs. males. Further, activation, differentiation, and/or polarization of various immune cell types were similarly determined by flow cytometry. PVATs were similar to their respective non-PVAT fats in density of recently activated B cells (B220+ CD25+). However, MRPVAT in females had a higher number of naïve CD4 T cells vs. MRPVAT in males and APVAT in females. MRPVAT also had denser naïve CD8 T cells vs. APVAT in females. Overall, this research for the first time has identified a community of discrete populations of immune cells (naive/recently activated/regulatory/memory) in healthy PVATs. Contrary to our hypothesis, PVATs are more similar than different in density to their respective non-PVAT fats.

Renal perivascular adipose tissue: Form and function.

Renal sympathetic activity affects blood pressure in part by increasing renovascular resistance via release of norepinephrine (NE) from sympathetic nerves onto renal arteries. Here we test the idea that adipose tissue adjacent to renal blood vessels, i.e. renal perivascular adipose tissue (RPVAT), contains a pool of NE which can be released to alter renal vascular function. RPVAT was obtained from around the main renal artery/vein of the male Sprague Dawley rats. Thoracic aortic PVAT and mesenteric PVAT also were studied as brown-like and white fat comparators respectively. RPVAT was identified as a mix of white and brown adipocytes, because of expression of both brown-like (e.g. uncoupling protein 1) and white adipogenic genes. All PVATs contained NE (ng/g tissue, RPVAT:524 ±â€¯68, TAPVAT:740 ±â€¯16, MPVAT:96 ±â€¯24). NE was visualized specifically in RPVAT adipocytes by immunohistochemistry. The presence of RPVAT (+RPVAT) did not alter the response of isolated renal arteries to NE compared to responses of arteries without RPVAT (-RPVAT). By contrast, the maximum contraction to the sympathomimetic tyramine was ~2× greater in the renal artery +PVAT versus -PVAT. Tyramine-induced contraction in +RPVAT renal arteries was reduced by the α1-adrenoceptor antagonist prazosin and the NE transporter inhibitor nisoxetine. These results suggest that tyramine caused release of NE from RPVAT. Renal denervation significantly (>50%) reduced NE content of RPVAT but did not modify tyramine-induced contraction of +RPVAT renal arteries. Collectively, these data support the existence of a releasable pool of NE in RPVAT that is independent of renal sympathetic innervation and has the potential to change renal arterial function.

Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

Secreted modular calcium-binding protein 2 (SMOC2) belongs to the secreted protein acidic and rich in cysteine (SPARC) family of matricellular proteins whose members are known to modulate cell-matrix interactions. We report that SMOC2 is upregulated in the kidney tubular epithelial cells of mice and humans following fibrosis. Using genetically manipulated mice with SMOC2 overexpression or knockdown, we show that SMOC2 is critically involved in the progression of kidney fibrosis. Mechanistically, we found that SMOC2 activates a fibroblast-to-myofibroblast transition (FMT) to stimulate stress fiber formation, proliferation, migration, and extracellular matrix production. Furthermore, we demonstrate that targeting SMOC2 by siRNA results in attenuation of TGFß1-mediated FMT in vitro and an amelioration of kidney fibrosis in mice. These findings implicate that SMOC2 is a key signaling molecule in the pathological secretome of a damaged kidney and targeting SMOC2 offers a therapeutic strategy for inhibiting FMT-mediated kidney fibrosis - an unmet medical need.