Vanesa D. Ramseyer

Tumor necrosis factor-α: regulation of renal function and blood pressure

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine that becomes elevated in chronic inflammatory states such as hypertension and diabetes and has been found to mediate both increases and decreases in blood pressure. High levels of TNF-α decrease blood pressure, whereas moderate increases in TNF-α have been associated with increased NaCl retention and hypertension. The explanation for these disparate effects is not clear but could simply be due to different concentrations of TNF-α within the kidney, the physiological status of the subject, or the type of stimulus initiating the inflammatory response. TNF-α alters renal hemodynamics and nephron transport, affecting both activity and expression of transporters. It also mediates organ damage by stimulating immune cell infiltration and cell death. Here we will summarize the available findings and attempt to provide plausible explanations for such discrepancies.

Tumor necrosis factor α decreases nitric oxide synthase type 3 expression primarily via Rho/Rho kinase in the thick ascending limb.

Inappropriate Na+ reabsorption by thick ascending limbs (THALs) induces hypertension. NO produced by NO synthase type 3 (NOS3) inhibits NaCl reabsorption by THALs. Tumor necrosis factor &agr; (TNF-&agr;) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-&agr; on THAL NOS3 and the signaling cascade are unknown. TNF-&agr; activates several signaling pathways, including Rho/Rho kinase (ROCK), which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-&agr; decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L of TNF-&agr; for 24 hours, and NOS3 expression was measured by Western blot. TNF-&agr; decreased NOS3 expression by 51±6% (P<0.002) and blunted stimulus-induced NO production. A 10-minute treatment with TNF-&agr; stimulated RhoA activity by 60±23% (P<0.04). Inhibition of Rho GTPase with 0.05 &mgr;g/mL of C3 exoenzyme blocked TNF-&agr;–induced reductions in NOS3 expression by 30±8% (P<0.02). Inhibition of ROCK with 10 &mgr;mol/L of H-1152 blocked TNF-&agr;–induced decreases in NOS3 expression by 66±15% (P<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases, and Rac-1 were also not involved in TNF-&agr;–induced decreases in NOS3 expression. We conclude that TNF-&agr; decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be attributed to the mitigation of TNF-&agr;–induced reduction in NOS3 expression.

Adrenergic regulation of cellular plasticity in brown, beige/brite and white adipose tissues

ABSTRACT The discovery of brown adipose tissue in adult humans along with the recognition of adipocyte heterogeneity and plasticity of white fat depots has renewed the interest in targeting adipose tissue for therapeutic benefit. Adrenergic activation is a well-established means of recruiting catabolic adipocyte phenotypes in brown and white adipose tissues. In this article, we review mechanisms of brown adipocyte recruitment by the sympathetic nervous system and by direct β-adrenergic receptor activation. We highlight the distinct modes of brown adipocyte recruitment in brown, beige/brite, and white adipose tissues, UCP1-independent thermogenesis, and potential non-thermogenic, metabolically beneficial effects of brown adipocytes.

Angiotensin II-mediated hypertension impairs nitric oxide-induced NKCC2 inhibition in thick ascending limbs.

In thick ascending limbs (THALs), nitric oxide (NO) decreases NaCl reabsorption via cGMP-mediated inhibition of Na-K-2Cl cotransporter (NKCC2). In angiotensin (ANG II)-induced hypertension, endothelin-1 (ET-1)-induced NO production by THALs is impaired. However, whether this alters NOs natriuretic effects and the mechanisms involved are unknown. In other cell types, ANG II augments phosphodiesterase 5 (PDE5)-mediated cGMP degradation. We hypothesized that NO-mediated inhibition of NKCC2 activity and stimulation of cGMP synthesis are blunted via PDE5 in ANG II-induced hypertension. Sprague-Dawley rats were infused with vehicle or ANG II (200 ng·kg-1·min-1) for 5 days. ET-1 reduced NKCC2 activity by 38 ± 13% (P < 0.05) in THALs from vehicle-treated rats but not from ANG II-hypertensive rats (Δ: -9 ± 13%). A NO donor yielded similar results as ET-1. In contrast, dibutyryl-cGMP significantly decreased NKCC2 activity in both vehicle-treated and ANG II-hypertensive rats (control: Δ-44 ± 15% vs. ANG II Δ-41 ± 10%). NO increased cGMP by 2.08 ± 0.36 fmol/μg protein in THALs from vehicle-treated rats but only 1.06 ± 0.25 fmol/μg protein in ANG II-hypertensive rats (P < 0.04). Vardenafil (25 nM), a PDE5 inhibitor, restored NOs ability to inhibit NKCC2 activity in THALs from ANG II-hypertensive rats (Δ: -60 ± 9%, P < 0.003). Similarly, NOs stimulation of cGMP was also restored by vardenafil (vehicle-treated: 1.89 ± 0.71 vs. ANG II-hypertensive: 2.02 ± 0.32 fmol/μg protein). PDE5 expression did not differ between vehicle-treated and ANG II-hypertensive rats. We conclude that NO-induced inhibition of NKCC2 and increases in cGMP are blunted in ANG II-hypertensive rats due to PDE5 activation. Defects in the response of THALs to NO may enhance NaCl retention in ANG II-induced hypertension.

Angiotensin II-induced hypertension blunts thick ascending limb NO production by reducing NO synthase 3 expression and enhancing threonine 495 phosphorylation

Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (ANG II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites, respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic ANG II infusion, due to reduced NOS3 expression, increased phosphorylation of T495, and decreased phosphorylation of S1177. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 1 and 5 days. ANG II infusion for 5 days decreased NOS3 expression by 40 ± 12% (P < 0.007; n = 6) and increased T495 phosphorylation by 147 ± 26% (P < 0.008; n = 6). One-day ANG II infusion had no significant effect. NO production in response to endothelin-1 was blunted in thick ascending limbs from ANG II-infused animals [ANG II -0.01 ± 0.06 arbitrary fluorescence units (AFU)/min vs. 0.17 ± 0.02 AFU/min in controls; P < 0.01]. This was not due to reduced endothelin-1 receptor expression. Phosphatidylinositol 3,4,5-triphosphate (PIP3)-induced NO production was also reduced in ANG II-infused rats (ANG II -0.07 ± 0.06 vs. 0.13 ± 0.04 AFU/min in controls; P < 0.03), and this correlated with an impaired ability of PIP3 to increase S1177 phosphorylation. We conclude that in ANG II-induced hypertension NO production by thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation.

Role of endothelin in thick ascending limb sodium chloride transport

The thick ascending limb of the loop of Henle reabsorbs 20-30% of filtered sodium chloride (NaCl) and generates the osmotic gradient necessary for water absorption in the distal nephron. It is second only to the collecting duct as a source of renal endothelin (ET)-1, which inhibits NaCl reabsorption in the thick ascending limb by reducing NaCl entry into the cell via the furosemide-sensitive Na(+)/K(+)/2 Cl(-) cotransporter. The mechanism by which this occurs appears to be due to activation of ET(B) receptors, phosphatidylinositol 3 kinase and Akt, and enhanced nitric oxide production by nitric oxide synthase 3. ET-1 may inhibit thick ascending limb NaCl absorption in either an autocrine or paracrine fashion. High-salt intake elevates ET-1 release by thick ascending limbs, although the molecular mechanism involved is unknown. Enhanced ET-1 release and inhibition of thick ascending limb NaCl absorption are thought to be among the mechanisms required to eliminate a salt load without increasing blood pressure. However, we still have much to learn about how ET-1 inhibits thick ascending limb NaCl absorption, how release and processing of ET-1 are regulated, and the receptors involved.

Vacuolar Protein Sorting 13C is a novel lipid droplet protein that inhibits lipolysis in brown adipocytes

Objective Brown adipose tissue (BAT) thermogenesis depends on the mobilization and oxidation of fatty acids from intracellular lipid droplets (LD) within brown adipocytes (BAs); however, the identity and function of LD proteins that control BAT lipolysis remain incomplete. Proteomic analysis of mouse BAT subcellular fractions identified vacuolar protein sorting 13C (VPS13C) as a novel LD protein. The aim of this work was to investigate the role of VPS13C on BA LDs. Methods Biochemical fractionation and high resolution confocal and immuno-transmission electron microscopy (TEM) were used to determine the subcellular distribution of VPS13C in mouse BAT, white adipose tissue, and BA cell culture. Lentivirus-delivered shRNA was used to determine the role of VPS13C in regulating lipolysis and gene expression in cultured BA cells. Results We found that VPS13C is highly expressed in mouse BAT where it is targeted to multilocular LDs in a subspherical subdomain. In inguinal white adipocytes, VPS13C was mainly observed on small LDs and β3-adrenergic stimulation increased VPS13C in this depot. Silencing of VPS13C in cultured BAs decreased LD size and triglyceride content, increased basal free fatty acid release, augmented the expression of thermogenic genes, and enhanced the lipolytic potency and efficacy of isoproterenol. Mechanistically, we found that BA lipolysis required activation of adipose tissue triglyceride lipase (ATGL) and that loss of VPS13C greatly increased the association of ATGL to LDs. Conclusions VPS13C is present on BA LDs where is targeted to a distinct subdomain. VPS13C limits the access of ATGL to LD and loss of VPS13C elevates lipolysis and promotes oxidative gene expression.

Deconstructing Adipogenesis Induced by β3-Adrenergic Receptor Activation with Single-Cell Expression Profiling

Recruitment of brown/beige adipocytes (BAs) in white adipose tissue (WAT) involves proliferation and differentiation of adipocyte stem cells (ASCs) in concert with close interactions with resident immune cells. To deconvolve stromal cell heterogeneity in a comprehensive and unbiased fashion, we performed single-cell RNA sequencing (scRNA-seq) of >33,000 stromal/vascular cells from epididymal WAT (eWAT) and inguinal WAT (iWAT) under control conditions and during β3-adrenergic receptor (ADRB3) activation. scRNA-seq identified distinct ASC subpopulations in eWAT and iWAT that appeared to be differentially poised to enter the adipogenic pathway. ADRB3 activation triggered the dramatic appearance of proliferating ASCs in eWAT, whose differentiation into BAs could be inferred from a single time point. scRNA-seq identified various immune cell types in eWAT, including a proliferating macrophage subpopulation that occupies adipogenic niches. These results demonstrate the power of scRNA-seq to deconstruct adipogenic niches and suggest novel functional interactions among resident stromal cell subpopulations.

461 ANGIOTENSIN II-HYPERTENSION IMPAIRS ENDOTHELIN-1-INDUCED INHIBITION OF NKCC2 ACTIVITY BY THE THICK ASCENDING LIMB

Background: Thick ascending limbs (THALs) absorb 25% of the filtered NaCl. Endothelin-1 (ET-1) inhibits NaCl absorption in THALs by increasing NO which reduces Na/K/2Cl cotransporter (NKCC2) activity. In contrast, superoxide augments NKCC2 activity. THAL superoxide production and NaCl absorption are enhanced while NO production is reduced in angiotensin II (Ang II)-hypertensive rats. Hypothesis: inhibition of NKCC2 by ET-1 is blunted in Ang II-induced hypertension due to reduced NO and elevated superoxide levels. Methods: Rats were infused with vehicle or Ang II 200 ng/kg/min. At day 5 we measured NKCC2 activity in perfused THALs by fluorescence microscopy. Results: In vehicle-treated rats ET-1 reduced NKCC2 activity from 1.58 ± 0.14 to 0.96 ± 0.16 AU/s (p < 0.04) whereas in Ang II-hypertensive rats ET-1 did not significantly decrease NKCC2 activity (control: 1.46 ± 0.24 vs ET-1: 1.23 ± 0.21 AU/s). Scavenging superoxide did not restore the effect of ET-1 on NKCC2 activity in Ang II-hypertensive rats (tempol: 1.03 ± 0.18 vs tempol+ET-1: 1.15 ± 0.20 AU/s). In vehicle-treated rats 100 &mgr;M spermine NONOate (NO donor) reduced NKCC2 activity from 1.67 ± 0.31 to 1.11 ± 0.21 AU/s (p < 0.04). In Ang II-hypertensive rats Spermine NONOate did not significantly decrease NKCC2 activity (control: 1.13 ± 0.17 vs NO: 1.32 ± 0.24 AU/s). Conclusions: Inhibition of NKCC2 activity by ET-1 is blunted in Ang II-induced hypertension due to reduced NO production and a defect in the signaling cascade downstream NO. These factors likely contribute to the enhanced NaCl absorption seen in this model of hypertension.