Lucienne S. Lara

Soluble Form of the (Pro)Renin Receptor Is Augmented in the Collecting Duct and Urine of Chronic Angiotensin II–Dependent Hypertensive Rats

Renin synthesis and secretion by principal cells of the collecting duct are enhanced in angiotensin (Ang) II–dependent hypertension. The presence of renin/(pro)renin and its receptor, the (pro)renin receptor ([P]RR), in the collecting duct may provide a pathway for Ang I generation with further conversion to Ang II. To assess whether (P)RR activation occurs during Ang II–dependent hypertension, we examined renal (P)RR levels and soluble (P)RR excretion in the urine of chronic Ang II–infused rats (80 ng/min; for 2 weeks; n=10) and sham-operated rats (n=10). Systolic blood pressure and Ang II levels in the plasma and kidney were increased whereas plasma renin activity was suppressed in Ang II–infused rats. Renal (P)RR transcripts were upregulated in the cortex and medulla of Ang II–infused rats. (P)RR immunoreactivity in collecting duct cells and the protein levels of the full-length form (37-kDa band) were significantly decreased in the medulla of Ang II–infused rats. The soluble (P)RR (28-kDa band) was detected in the renal medulla and urine samples of Ang II–infused rats, which also showed increases in urinary renin content. To determine whether the soluble (P)RR could stimulate Ang I formation, urine samples were incubated with recombinant human (pro)renin. Urine samples of Ang II–infused rats exhibited increased Ang I formation compared with sham-operated rats. Thus, in chronic Ang II–infused rats, the catalytic activity of the augmented renin produced in the collecting duct may be enhanced by the intraluminal soluble (P)RR and cell-surface located (P)RR, thus contributing to enhanced intratubular Ang II formation.

AT1 receptor-mediated augmentation of angiotensinogen, oxidative stress, and inflammation in ANG II-salt hypertension

Augmentation of intrarenal angiotensinogen (AGT) synthesis, secretion, and excretion is associated with the development of hypertension, renal oxidative stress, and tissue injury during ANG II-dependent hypertension. High salt (HS) exacerbates hypertension and kidney injury, but the mechanisms remain unclear. In this study, we determined the consequences of HS intake alone compared with chronic ANG II infusion and combined HS plus ANG II on the stimulation of urinary AGT (uAGT), renal oxidative stress, and renal injury markers. Sprague-Dawley rats were subjected to 1) a normal-salt diet [NS, n = 5]; 2) HS diet [8% NaCl, n = 5]; 3) ANG II infusion in NS rats [ANG II 80 ng/min, n = 5]; 4) ANG II infusion in HS rats [ANG II+HS, n = 5]; and 5) ANG II infusion in HS rats treated with ANG II type 1 receptor blocker (ARB) [ANG II+HS+ARB, n = 5] for 14 days. Rats fed a HS diet alone did not show changes in systolic blood pressure (SBP), proteinuria, cell proliferation, or uAGT excretion although they did exhibit mesangial expansion, collagen deposition, and had increased NADPH oxidase activity accompanied by increased peroxynitrite formation in the kidneys. Compared with ANG II rats, the combination of ANG II infusion and a HS diet led to exacerbation in SBP (175 ± 10 vs. 221 ± 8 mmHg; P < 0.05), proteinuria (46 ± 7 vs. 127 ± 7 mg/day; P < 0.05), and uAGT (1,109 ± 70 vs.. 7,200 ± 614 ng/day; P < 0.05) associated with greater collagen deposition, mesangial expansion, interstitial cell proliferation, and macrophage infiltration. In both ANG II groups, the O(2)(-) levels were increased due to increased NADPH oxidase activity without concomitant increases in peroxynitrite formation. The responses in ANG II rats were prevented or ameliorated by ARB treatment. The results indicate that HS independently stimulates ROS formation, which may synergize with the effect of ANG II to limit peroxynitrite formation, leading to exacerbation of uAGT and greater injury during ANG II salt hypertension.

Angiotensin II Stimulates Renin in Inner Medullary Collecting Duct Cells via Protein Kinase C and Independent of Epithelial Sodium Channel and Mineralocorticoid Receptor Activity

Collecting duct (CD) renin is stimulated by angiotensin (Ang) II, providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates the epithelial sodium channel via the Ang II type 1 receptor and increases mineralocorticoid receptor activity attributed to increased aldosterone release. Our objective was to determine whether CD renin augmentation is mediated directly by Ang II type 1 receptor or via the epithelial sodium channel and mineralocorticoid receptor. In vivo studies examined the effects of epithelial sodium channel blockade (amiloride; 5 mg/kg per day) on CD renin expression and urinary renin content in Ang II–infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure, medullary renin mRNA, urinary renin content, and intrarenal Ang II levels. Amiloride cotreatment did not alter these responses despite a reduction in the rate of progression of systolic blood pressure. In primary cultures of inner medullary CD cells, renin mRNA and (pro)renin protein levels increased with Ang II (100 nmol/L), and candesartan (Ang II type 1 receptor antagonist) prevented this effect. Aldosterone (10−10 to 10−7 mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II–treated cells. However, inhibition of protein kinase C with calphostin C prevented the Ang II–mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, protein kinase C activation with phorbol 12-myristate 13-acetate increased renin expression to the same extent as Ang II. These data indicate that an Ang II type 1 receptor–mediated increase in CD renin is induced directly by Ang II via the protein kinase C pathway and that this regulation is independent of mineralocorticoid receptor activation or epithelial sodium channel activity.

Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1–7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats

Alterations in the balance between ANG II/ACE and ANG 1-7/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG 1-7 and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG 1-7 contents, angiotensin-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K1C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K1C rats (n = 11) compared with sham rats (n = 9). Renal medullary angiotensin peptide levels were increased in 2K1C rats [ANG I: (CK = 171 ± 4; NCK = 251 ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± 79; NCK = 328 ± 18 vs. sham = 94 ± 7 pg/g protein; P < 0.001)]; and ANG 1-7 levels decreased (CK = 18 ± 2; NCK = 19 ± 2 pg/g vs. sham = 63 ± 10 pg/g; P < 0.001). In renal medullas of both kidneys of 2K1C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K1C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K1C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG 1-7.

Angiotensin-(1–7) modulates the ouabain-insensitive Na+-ATPase activity from basolateral membrane of the proximal tubule

Angiotensin-(1-7) (Ang-(1-7)) modulates the Na+-ATPase, but not the Na+,K+-ATPase activity present in pig kidney proximal tubules. The Na+-ATPase, insensitive to ouabain, but sensitive to furosemide, is stimulated by Ang-(1-7) (68% by 10(-9) M), in a dose-dependent manner. This effect is due to an increase in Vmax, while the apparent affinity of the enzyme for Na+ is not modified. Saralasin, a general angiotensin receptor antagonist, abolishes the stimulation, demonstrating that the Ang-(1-7) effect is mediated by receptor. The Ang-(1-7) stimulatory effect is not changed by either PD 123319, an AT2 receptor antagonist, or A779, an Ang-(1-7) receptor antagonist. On the other hand, increasing the concentration of the AT1 receptor antagonist losartan from 10(-11) to 10(-9) M, reverses the Ang(1-7) stimulation completely. A further increase to 10(-3) M losartan reverses the Na+-ATPase activity to a level similar to that obtained with Ang-(1-7) (10(-9) M) alone. The stimulatory effect of Ang-(1-7) at 10(-9) M is similar to the effect of angiotensin II (AG II) alone. However, when the two peptides are both present, Na+-ATPase activity is restored to control values. These data suggest that Ang-(1-7) selectively modulates the Na+-ATPase activity present in basolateral membranes of kidney proximal tubules through a losartan-sensitive receptor. This receptor is probably different from the receptor involved in the stimulation of the Na+-ATPase activity by angiotensin II.

Angiotensin-(1–7) reverts the stimulatory effect of angiotensin II on the proximal tubule Na+-ATPase activity via a A779-sensitve receptor

Abstract Recently, we demonstrated that the stimulatory effect of Ang II on the Na+-ATPase activity in proximal tubules is reversed, in a dose-dependent manner, by Ang-(1–7) [Biochim. Biophys. Acta 1467 (2000) 189]. In the present paper, we characterized the receptor involved in this phenomenon. The preincubation of the Na+-ATPase with 10−8 M Ang II increases the enzyme activity from 7.50±0.02 (control) to 12.40±1.50 nmol Pi mg−1 min−1 (p

Angiotensin II stimulates renal proximal tubule Na + -ATPase activity through the activation of protein kinase C

Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.

Angiotensin II activates the ouabain-insensitive Na+-ATPase from renal proximal tubules through a G-protein

Angiotensin II (AG II) stimulates the ouabain-insensitive, furosemide- sensitive Na+-ATPase present in the basolateral membrane of pig renal proximal tubules in a dose dependent manner. Maximum effect was obtained with 10-8 M AG II, which corresponded to an activity 134% higher than control. Half of the maximum effect was observed between 10-11 M and 10-10 M, corresponding to physiological hormone levels. Saralasin, an AG II peptide analogue receptor antagonist, abolished the phenomenon, demonstrating that AG II interacts with specific sites in pig proximal tubules. The AG II stimulatory effect was also prevented by dithiothreitol (DTT), a reducing compound, and by 10 nM losartan, a non-peptide antagonist highly specific for AT1 receptors, characterizing AG II binding to AT1 receptors. GTPgammaS, a non-hydrolysable GTP analogue, increased by 159% the enzyme activity as compared to the control values. The simultaneous addition of 10-5 M GTPgammaS and 10-8 M AG II did not have additive effects. Furthermore, the stimulatory action of AG II was completely abolished by 0.1 microM GDPbetaS, a non-hydrolysable GDP analogue. Two microgram ml-1 pertussis toxin, an inhibitor of Gi-protein, did not modulate the AG II stimulatory effect. On the other hand, the Na+-ATPase activity was enhanced 100% in the presence of cholera toxin and 85% in the presence of both AG II and cholera toxin. Taken together, these data suggest that AG II activates the Na+-ATPase activity through AT1 receptors coupled to a pertussis-insensitive and cholera-sensitive G-protein.

Renin-angiotensin system in the kidney: What is new?

The renin-angiotensin system (RAS) has been known for more than a century as a cascade that regulates body fluid balance and blood pressure. Angiotensin II(Ang II) has many functions in different tissues; however it is on the kidney that this peptide exerts its main functions. New enzymes, alternative routes for Ang IIformation or even active Ang II-derived peptides have now been described acting on Ang II AT1 or AT2 receptors, or in receptors which have recently been cloned, such as Mas and AT4. Another interesting observation was that old members of the RAS, such as angiotensin converting enzyme (ACE), renin and prorenin, well known by its enzymatic activity, can also activate intracellular signaling pathways, acting as an outside-in signal transduction molecule or on the renin/(Pro)renin receptor. Moreover, the endocrine RAS, now is also known to have paracrine, autocrine and intracrine action on different tissues, expressing necessary components for local Ang II formation. This in situ formation, especially in the kidney, increases Ang II levels to regulate blood pressure and renal functions. These discoveries, such as the ACE2/Ang-(1-7)/Mas axis and its antangonistic effect rather than classical deleterious Ang II effects, improves the development of new drugs for treating hypertension and cardiovascular diseases.

Modulation of ouabain-insensitive Na+-ATPase activity in the renal proximal tubule by Mg2+, MgATP and furosemide

In addition to the (Na(+)+K(+))ATPase another P-ATPase, the ouabain-insensitive Na(+)-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg(2+), MgATP and furosemide on the Na(+)-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na(+)-ATPase at pH 7.0 are: (a) K(0.5) for Na(+)=8.9+/-2.2mM; (b) K(0.5) for MgATP=1.8+/-0.4mM; (c) two sites for free Mg(2+): one stimulatory (K(0.5)=0.20+/-0.06 mM) and other inhibitory (I(0.5)=1.1+/-0.4 mM); and (d) I(0.5) for furosemide=1.1+/-0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na(+) (K(0.5)=19.5+/-0.4) and MgATP (K(0.5)=3.4+/-0.3 mM) but increases the apparent affinity for furosemide (0.18+/-0.02 mM) and Mg(2+) (0.05+/-0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na(+) (K(0.5)=18.7+/-1.5 mM) and furosemide (I(0.5)=3.04+/-0.57 mM) but does not change the apparent affinity to MgATP and Mg(2+). The data presented in this paper indicate that the modulation of the Na(+)-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg(2+) and MgATP complex may play an important role in the Na(+)-ATPase physiological regulatory mechanisms.