Alexis A. Gonzalez

Angiotensin II increases fibronectin and collagen I through the β-catenin-dependent signaling in mouse collecting duct cells

The contribution of angiotensin II (ANG II) to renal and tubular fibrosis has been widely reported. Recent studies have shown that collecting duct cells can undergo mesenchymal transition suggesting that collecting duct cells are involved in interstitial fibrosis. The Wnt/β-catenin signaling pathway plays an essential role in development, organogenesis, and tissue homeostasis; however, the dysregulation of this pathway has been linked to fibrosis. In this study, we investigated whether AT1 receptor activation induces the expression of fibronectin and collagen I via the β-catenin pathway in mouse collecting duct cell line M-1. ANG II (10(-7) M) treatment in M-1 cells increased mRNA, protein levels of fibronectin and collagen I, the β-catenin target genes (cyclin D1 and c-myc), and the myofibroblast phenotype. These effects were prevented by candesartan, an AT1 receptor blocker. Inhibition of the β-catenin degradation with pyrvinium pamoate (pyr; 10(-9) M) prevented the ANG II-induced expression of fibronectin, collagen I, and β-catenin target genes. ANG II treatment promoted the accumulation of β-catenin protein in a time-dependent manner. Because phosphorylation of glycogen synthase kinase-3β (GSK-3β) inhibits β-catenin degradation, we further evaluated the effects of ANG II and ANG II plus pyr on p-ser9-GSK-3β levels. ANG II-dependent upregulation of β-catenin protein levels was correlated with GSK-3β phosphorylation. These effects were prevented by pyr. Our data indicate that in M-1 collecting duct cells, the β-catenin pathway mediates the stimulation of fibronectin and collagen I in response to AT1 receptor activation.

Renal medullary cyclooxygenase-2 and (pro)renin receptor expression during angiotensin II-dependent hypertension

The (pro)renin receptor [(P)RR] upregulates cyclooxygenase-2 (COX-2) in inner medullary collecting duct (IMCD) cells through ERK1/2. Intrarenal COX-2 and (P)RR are upregulated during chronic ANG II infusion. However, the duration of COX-2 and (P)RR upregulation has not been determined. We hypothesized that during the early phase of ANG II-dependent hypertension, membrane-bound (P)RR and COX-2 are augmented in the renal medulla, serving to buffer the hypertensinogenic and vasoconstricting effects of ANG II. In Sprague-Dawley rats infused with ANG II (0.4 μg·min(-1)·kg(-1)), systolic blood pressure (BP) increased by day 7 (162 ± 5 vs. 114 ± 10 mmHg) and continued to increase by day 14 (198 ± 15 vs. 115 ± 13 mmHg). Membrane-bound (P)RR was augmented at day 3 coincident with phospho-ERK1/2 levels, COX-2 expression, and PGE2 in the renal medulla. In contrast, membrane-bound (P)RR was reduced and COX-2 protein levels were not different from controls by day 14. In cultured IMCD cells, ANG II increased secretion of the soluble (P)RR. In anesthetized rats, COX-2 inhibition decreased the glomerular filtration rate (GFR) and renal blood flow (RBF) during the early phase of ANG II infusion without altering BP. However, at 14 days of ANG II infusions, COX-2 inhibition decreased mean arterial BP (MABP), RBF, and GFR. Thus, during the early phase of ANG II-dependent hypertension, the increased (P)RR and COX-2 expression in the renal medulla may contribute to attenuate the vasoconstrictor effects of ANG II on renal hemodynamics. In contrast, at 14 days the reductions in RBF and GFR caused by COX-2 inhibition paralleled the reduced MABP, suggesting that vasoconstrictor COX-2 metabolites contribute to ANG II hypertension.

Angiotensin II increases the expression of (pro)renin receptor during low-salt conditions.

Background:Evidence indicates that chronic angiotensin II (AngII) infusion increases (pro)renin receptor ((P)RR) expression in renal inner medullary collecting duct (IMCD) cells. Recently, it has been shown that renal (P)RR expression is augmented during a low-salt (LS) diet. However, the role of AngII in mediating the stimulation of (P)RR during LS conditions is unknown. We hypothesized that AngII mediates the increased expression of (P)RR during low-salt conditions in IMCDs. Methods:(P)RR expression and AngII levels were evaluated in Sprague-Dawley rats fed a LS diet (0.03% NaCl) and normal salt (NS; 0.4% NaCl) for 7 days. We examined the effects of sodium reduction (130 mM NaCl) and AngII on (P)RR expression in IMCDs isolated in hypertonic conditions (640 mOsmol/L with 280 mM NaCl). Results:Plasma renin activity in LS rats was significantly higher than rats fed with NS (28.1 ± 2.2 versus 6.7 ± 1.1 ng AngI·mL−1·hr−1; P < 0.05), as well as renin content in renal cortex and medulla. The (P)RR mRNA and protein levels were higher in medullary tissues from LS rats but did not change in the cortex. Intrarenal AngII was augmented in LS compared with NS rats (cortex: 710 ± 113 versus 277 ± 86 fmol/g, P < 0.05; medulla: 2093 ± 125 versus 1426 ± 126 fmol/g, P < 0.05). In cultured IMCDs, (P)RR expression was increased in response to LS or AngII treatment and potentiated by both treatments (both at 640 mOsmol/L). Conclusions:These data indicate that (P)RR is augmented in medullary collecting ducts in response to LS and that this effect is further enhanced by the increased intrarenal AngII content.

Renin and the (Pro)renin Receptor in the Renal Collecting Duct:: Role in the Pathogenesis of Hypertension

The intrarenal renin–angiotensin system (RAS) plays a critical role in the pathogenesis and progression of hypertension and kidney disease. In angiotensin (Ang) II‐dependent hypertension, collecting duct renin synthesis and secretion are stimulated despite suppression of juxtaglomerular (JG) renin. This effect is mediated by the AngII type I receptor (AT1R), independent of blood pressure. Although the regulation of JG renin has been extensively studied, the mechanisms by which renin is regulated in the collecting duct remain unclear. The augmentation of renin synthesis and activity in the collecting duct may provide a pathway for additional generation of intrarenal and intratubular AngII formation due to the presence of angiotensinogen substrate and angiotensin‐converting enzyme in the nephron. The recently described (pro)renin receptor ((P)RR) binds renin or prorenin, enhancing renin activity and fully activating the biologically inactive prorenin peptide. Stimulation of (P)RR also activates intracellular pathways related to fibrosis. Renin and the (P)RR are augmented in renal tissues of AngII‐dependent hypertensive rats. However, the functional contribution of the (P)RR to enhanced renin activity in the collecting duct and its contribution to the development of hypertension and kidney disease have not been well elucidated. This review focuses on recent evidence demonstrating the mechanism of renin regulation in the collecting ducts and its interaction with the (P)RR. The data suggest that renin–(P)RR interactions may induce stimulation of intracellular pathways associated with the development of hypertension and kidney disease.

Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct

Renin is synthesized in the principal cells of the collecting duct (CD), and its production is increased via cAMP in angiotensin (ANG) II-dependent hypertension, despite suppression of juxtaglomerular (JG) renin. Vasopressin, one of the effector hormones of the renin-angiotensin system (RAS) via the type 2-receptor (V2R), activates the cAMP/PKA/cAMP response element-binding protein (CREB) pathway and aquaporin-2 expression in principal cells of the CD. Accordingly, we hypothesized that activation of V2R increases renin synthesis via PKA/CREB, independently of ANG II type 1 (AT1) receptor activation in CD cells. Desmopressin (DDAVP; 10(-6) M), a selective V2R agonist, increased renin mRNA (∼3-fold), prorenin (∼1.5-fold), and renin (∼2-fold) in cell lysates and cell culture media in the M-1 CD cell line. Cotreatment with DDAVP+H89 (PKA inhibitor) or CREB short hairpin (sh) RNA prevented this response. H89 also blunted DDAVP-induced CREB phosphorylation and nuclear localization. In 48-h water-deprived (WD) mice, prorenin-renin protein levels were increased in the renal inner medulla (∼1.4- and 1.8-fold). In WD mice treated with an ACE inhibitor plus AT1 receptor blockade, renin mRNA and prorenin protein levels were still higher than controls, while renin protein content was not changed. In M-1 cells, ANG II or DDAVP increased prorenin-renin protein levels; however, there were no further increases by combined treatment. These results indicate that in the CD the activation of the V2R stimulates renin synthesis via the PKA/CREB pathway independently of RAS, suggesting a critical role for vasopressin in the regulation of renin in the CD.

Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg-1·min-1), the increases in systolic BP and diastolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CDPRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments.

Roles of collecting duct renin and (pro)renin receptor in hypertension: mini review.

In angiotensin (Ang)-II-dependent hypertension, collecting duct renin synthesis and secretion are stimulated despite suppression of juxtaglomerular (JG) renin. This effect is mediated by Ang II type 1 (AT1) receptor independent of blood pressure. Although the regulation of JG renin is known, the mechanisms by which renin is regulated in the collecting duct are not completely understood. The presence of renin activity in the collecting duct may provide a pathway for intratubular Ang II formation since angiotensinogen substrate and angiotensin converting enzyme are present in the distal nephron. The recently named new member of the renin–angiotensin system (RAS), the (pro)renin receptor [(P)RR], is able to bind renin and the inactive prorenin, thus enhancing renin activity and fully activating prorenin. We have demonstrated that renin and (P)RR are augmented in renal tissues from rats infused with Ang II and during sodium depletion, suggesting a physiological role in intrarenal RAS activation. Importantly, (P)RR activation also causes activation of intracellular pathways associated with increased cyclooxygenase 2 expression and induction of profibrotic genes. In addition, renin and (P)RR are upregulated by Ang II in collecting duct cells. Although the mechanisms involved in their regulation are still under study, they seem to be dependent on the intrarenal RAS activation. The complexities of the mechanisms of stimulation also depend on cyclooxygenase 2 and sodium depletion. Our data suggest that renin and (P)RR can interact to increase intratubular Ang II formation and the activation of profibrotic genes in renal collecting duct cells. Both pathways may have a critical role in the development of hypertension and renal disease.

The prorenin receptor in the cardiovascular system and beyond

Since the prorenin receptor (PRR) was first reported, its physiological role in many cellular processes has been under intense scrutiny. The PRR is currently recognized as a multifunctional receptor with major roles as an accessory protein of the vacuolar-type H+-ATPase and as an intermediary in the Wnt signaling pathway. As a member of the renin-angiotensin system (RAS), the PRR has demonstrated to be of relevance in cardiovascular diseases (CVD) because it can activate prorenin and enhance the enzymatic activity of renin, thus promoting angiotensin II formation. Indeed, there is an association between PRR gene polymorphisms and CVD. Independent of angiotensin II, the activation of the PRR further stimulates intracellular signals linked to fibrosis. Studies using tissues and cells from a variety of organs and systems have supported its roles in multiple functions, although some remain controversial. In the brain, the PRR appears to be involved in the central regulation of blood pressure via activation of RAS- and non-RAS-dependent mechanisms. In the heart, the PRR promotes atrial structural and electrical remodeling. Nonetheless, animals overexpressing the PRR do not exhibit cardiac injury. In the kidney, the PRR is involved in the development of ureteric bud branching, urine concentration, and regulation of blood pressure. There is great interest in the PRR contributions to T cell homeostasis and to the development of visceral and brown fat. In this mini-review, we discuss the evidence for the pathophysiological roles of the PRR with emphasis in CVD.

Prostaglandin E2 Induces Prorenin-Dependent Activation of (Pro)renin Receptor and Upregulation of Cyclooxygenase-2 in Collecting Duct Cells☆

Background: Prostaglandin E2 (PGE2) regulates renin expression in renal juxtaglomerular cells. PGE2 acts through E‐prostanoid (EP) receptors in the renal collecting duct (CD) to regulate sodium and water balance. CD cells express EP1 and EP4, which are linked to protein kinase C (PKC) and PKA downstream pathways, respectively. Previous studies showed that the presence of renin in the CD, and that of PKC and PKA pathways, activate its expression. The (pro)renin receptor (PRR) is also expressed in CD cells, and its activation enhances cyclooxygenase‐2 (COX‐2) through extracellular signal–regulated kinase (ERK). We hypothesized that PGE2 stimulates prorenin and renin synthesis leading to subsequent activation of PRR and upregulation of COX‐2. Methods: We used a mouse M‐1 CD cell line that expresses EP1, EP3 and EP4 but not EP2. Results: PGE2 (10−6 M) treatment increased prorenin and renin protein levels at 4 and 8 hours. No differences were found at 12‐hour after PGE2 treatment. Phospho‐ERK was significantly augmented after 12 hours. COX‐2 expression was decreased after 4 hours of PGE2 treatment, but increased after 12 hours. Interestingly, the full‐length form of the PRR was upregulated only at 12 hours. PGE2‐mediated phospho‐ERK and COX‐2 upregulation was suppressed by PRR silencing. Conclusions: Our results suggest that PGE2 induces biphasic regulation of COX‐2 through renin‐dependent PRR activation via EP1 and EP4 receptors. PRR‐mediated increases in COX‐2 expression may enhance PGE2 synthesis in CD cells serving as a buffer mechanism in conditions of activated renin‐angiotensin system.

Role of Collecting Duct Renin in the Pathogenesis of Hypertension

The presence of renin production by the principal cells of the collecting duct has opened new perspectives for the regulation of intrarenal angiotensin II (Ang II). Angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) are present in the tubular fluid coming from the proximal tubule and collecting duct. All the components needed for Ang II formation are present along the nephron, and much is known about the mechanisms regulating renin in juxtaglomerular cells (JG); however, those in the collecting duct remain unclear. Ang II suppresses renin via protein kinase C (PKC) and calcium (Ca2+) in JG cells, but in the principal cells, Ang II increases renin synthesis and release through a pathophysiological mechanism that increases further intratubular Ang II de novo formation to enhance distal Na+ reabsorption. Transgenic mice overexpressing renin in the collecting duct demonstrate the role of collecting duct renin in the development of hypertension. The story became even more interesting after the discovery of a specific receptor for renin and prorenin: the prorenin receptor ((P)RR), which enhances renin activity and fully activates prorenin. The interactions between (P)RR and prorenin/renin may further increase intratubular Ang II levels. In addition to Ang II, other mechanisms have been described in the regulation of renin in the collecting duct, including vasopressin (AVP), bradykinin (BK), and prostaglandins. Current active investigations are aimed at elucidating the mechanisms regulating renin in the distal nephron segments and understand its role in the pathogenesis of hypertension.