Mariela Mendez

PPARγ Inhibition of Cyclooxygenase-2, PGE2 Synthase, and Inducible Nitric Oxide Synthase in Cardiac Myocytes

Abstract—Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They regulate lipid metabolism, glucose homeostasis, cell proliferation, and differentiation and modulate inflammatory responses. We examined whether PPAR&ggr; is functional in cultured neonatal ventricular myocytes and studied its role in inflammation. Western blots revealed PPAR&ggr; in myocytes. When myocytes were transfected with a PPAR response element reporter plasmid (PPRE-TK-luciferase), the PPAR&ggr; activator 15-deoxy-&Dgr;12,14-prostaglandin J2 (15dPGJ2) increased promoter activity, whereas cotransfection of a dominant negative PPAR&ggr; inhibited it. To determine the role of 15dPGJ2 in expression of proinflammatory genes, we tested its effect on interleukin-1&bgr; induction of cyclooxygenase-2 (COX-2). 15dPGJ2 decreased interleukin-1&bgr; stimulation of COX-2 by 40% and PGE2 production by 73%. We next questioned whether 15dPGJ2 was modulating the expression of inducible prostaglandin E2 synthase (PGES) and found that it completely blocked interleukin-1&bgr; induction of PGES. Use of a second PPAR&ggr; agonist, troglitazone, and the selective PPAR&ggr; antagonist GW9662 demonstrated that the effects seen were PPAR&ggr;-dependent. In addition, we found that 15dPGJ2 blocked interleukin-1&bgr; stimulation of inducible nitric oxide synthase (iNOS). We concluded that 15dPGJ2 may play an anti-inflammatory role in a PPAR&ggr;-dependent manner, decreasing COX-2, PGES, and PGE2 production, as well as iNOS expression.

Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP3 pathway and the ryanodine receptor

The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

Vesicle-associated membrane protein 2 (VAMP2) but Not VAMP3 mediates cAMP-stimulated trafficking of the renal Na+-K+-2Cl- co-transporter NKCC2 in thick ascending limbs.

Background: Exocytic delivery of the renal co-transporter NKCC2 to the cell surface is a major mechanism for NaCl reabsorption. Results: We describe a mechanism that mediates cAMP-stimulated NKCC2 delivery in renal cells. Conclusion: Vesicle fusion protein VAMP2 interacts with NKCC2 and mediates cAMP-stimulated NKCC2 exocytic delivery. Significance: The molecular mechanism mediating NKCC2 exocytic delivery could provide new targets for treatment of hypertension. In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.

Vesicle-associated Membrane Protein 3 (VAMP3) Mediates Constitutive Trafficking of the Renal Co-transporter NKCC2 in Thick Ascending Limbs ROLE IN RENAL FUNCTION AND BLOOD PRESSURE

Renal cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl− co-transporter NKCC2. Trafficking of NKCC2 to the apical surface regulates NKCC2-mediated NaCl absorption and blood pressure. The molecular mechanisms by which NKCC2 reaches the apical surface and their role in renal function and maintenance of blood pressure are poorly characterized. Here we report that NKCC2 interacts with the vesicle fusion protein VAMP3, and they co-localize at the TAL apical surface. We observed that silencing VAMP3 in vivo blocks constitutive NKCC2 exocytic delivery, decreasing the amount of NKCC2 at the TAL apical surface. VAMP3 is not required for cAMP-stimulated NKCC2 exocytic delivery. Additionally, genetic deletion of VAMP3 in mice decreased total expression of NKCC2 in the TAL and lowered blood pressure. Consistent with these results, urinary excretion of water and electrolytes was higher in VAMP3 knock-out mice, which produced more diluted urine. We conclude that VAMP3 interacts with NKCC2 and mediates its constitutive exocytic delivery to the apical surface. Additionally, VAMP3 is required for normal NKCC2 expression, renal function, and blood pressure.

Renin release: role of SNAREs

Little is known about the molecular mechanism mediating renin granule exocytosis and the identity of proteins involved. We previously showed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAREs), a family of proteins required for exocytosis, mediate the stimulated release of renin from juxtaglomerular cells. This minireview focuses on the current knowledge of the proteins that facilitate renin-granule exocytosis. We discuss the identity of potential candidates that mediate the signaling and final steps of exocytosis of the renin granule.

CBP and p300 in renin homeostasis: can they drive the fate?

renin is the rate-limiting enzyme in the renin-angiotensin system that is responsible for the control of blood pressure and fluid homeostasis. In the adult mammalian kidney, renin is synthesized, stored, and released by the juxtaglomerular (JG) cells, which are confined to the vascular wall of the afferent arteriole at the entrance of the glomerulus. To reach its final differentiated state, a renin-expressing cell “travels” a long way, leaving its memory imprinted: in the embryo, renin precursor cells are present in the metanephric mesenchyme and give rise to JG cells and to arteriolar smooth muscle cells even before vessel development has occurred and before the hemodynamic role of renin is required. Later, in the fetus, renin-containing cells are present in the large intrarenal arteries and in the glomerular interstitium. As maturation continues, the number of renin-containing cells diminishes to a few into the classical adult JG distribution. When homeostasis is imbalanced or threatened, as in the case of a low salt intake, the number of renin-expressing cells along the preglomerular arteries increases, resembling the fetal pattern. This phenomenon is usually called recruitment, and it is thought to provide additional plasma renin to maintain salt balance and blood pressure. The work of Sequeira Lopez, Gomez, et al. (9) and other investigators (2) established that smooth muscle cells in the afferent arteriole retain the plasticity to synthesize renin during the recruitment process. The signaling and molecular mechanisms involved in recruitment of renin-expressing cells have remained obscure. It is unequivocally established that cAMP plays an important role in renin regulation. Increases in intracellular cAMP induce renin release from the JG cells (6), stabilize renin mRNA (1), and increase the expression of renin (3). The reacquisition of renin expression may be, in part, mediated by the second messenger cAMP. Recently, Pentz, Gomez, et al. (8) began to identify the mechanism and role of cAMP in governing the fate of renin-containing cell recruitment. They generated mouse models in which cells of the renin lineage that expressed renin during development were labeled with cyan fluorescent protein (CFP), while cells actively expressing renin were labeled with yellow fluorescent protein (YFP). They elegantly demonstrated that cAMP stimulation of preglomerular smooth muscle cells from the renin cell lineage (CFP-positive cells) results in reexpression of renin (YPF-positive cells). This finding shows that the smooth muscle cells that retain the plasticity to reexpress renin are those that expressed renin earlier during development. Importantly, they also showed that cAMP-induced recruitment of smooth muscle cells into renin-expressing cells requires histone acetylation and chromatin remodeling of the cAMP responsive element (CRE) region of the renin gene promoter. The CRE region in the renin promoter is known to be essential in renin gene transcription (7). In other cells, the regulation of this promoter region by cAMP involves the complex interaction of CRE-binding protein with coactivators Cre-binding protein (CBP) and/or p300, which facilitate access of the transcription factor TFIIB and initiate transcription (4). In addition, the coactivators CBP and p300 have been described to possess histone acetyltransferase activity and, therefore, may play a role in chromatin remodeling (5). On the basis of their previous results, Gomez et al. (1a) extended their finding implicating the cAMP pathway in the recruitment process and maintenance of JG cell identity in vitro to an in vivo mouse model. Gomez et al. very elegantly demonstrate that JG cell CBP and p300 are required for maintenance of JG cell identity, renin expression, and overall kidney development. They generated transgenic mice, where CBP, p300, or both are deleted, specifically in the JG cells, by crossing floxed CBP and/or floxed p300 mice with their previously described renin cre-recombinase mice (9). They report that renin expression and kidney structure are not altered by homozygous deletion of CBP (CBPfl/fl;Rencre/+) or p300 (p300fl/fl;Rencre/+) individually. However, dual deletion of these coactivators from renin-expressing cells (double-homozygous CBPfl/fl;p300fl/fl;Rencre/+) severely decreased renin expression and the number of renin-positive JG cells and caused severe disruption of kidney development. This was reflected by reduced kidney growth, extensive fibrosis, and significant structural alteration of the kidneys. In addition, reduced kidney growth and altered morphology were accentuated in the triple-homozygous (CBPfl/fl;p300fl/fl;Rencre/cre) animals, where both renin alleles are deleted, in addition to CBP and p300. Taken together, these data show an essential role of CBP and p300 in renin expression and JG cell development in vivo. The data also emphasize the essential role of the cAMP-dependent regulation of the CRE promoter region, which drives renin expression during development and also during adult life. The data show, for the first time, the important function of CBP and p300 in the development of kidney function in vivo, since whole animal knockout of any of these genes is lethal. All good science leads to novel and exciting questions. The previous work by Pentz, Gomez, et al. (8) suggests that renin expression per se in a population of cells during development is linked to the acquisition of renin cell memory and plasticity. However, the molecular mechanisms that drive the fate for JG cell identity are unclear. Since the triple-homozygous (CBPfl/fl;p300fl/fl;Rencre/cre) animals presumably did not express renin during development, can CBP/p300-dependent regulation of gene expression be sufficient to predestine the acquisition of renin cell memory? If so, a threat to homeostasis (that would induce recruitment in wild-type mice) should be impaired in double-homozygous (CBPfl/fl;p300fl/fl;Rencre/+) animals, emphasizing the relevance of CBP/p300 and cAMP signaling in renin cell lineage conditioning. In my opinion, Gomez et al. (1a) have challenged the philosophical thought that “fate” cannot be changed.