Renin synthesis and secretion by principal cells of the collecting duct (CD) is enhanced in angiotensin (Ang) II-dependent hypertension. The presence of renin/(pro)renin and its receptor, the (pro)renin receptor [(P)RR], in the CD may provide a pathway for Ang I generation with further conversion to Ang II. To assess if (P)RR activation occurs during Ang II-dependent hypertension, we examined renal (P)RR levels and soluble (P)RR (s(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 while 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 CD 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 if the s(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 to sham-operated rats. Thus, in chronic Ang II-infused rats the catalytic activity of the augmented renin produced in the CD may be enhanced by the intraluminal s(P)RR and cell-surface located (P)RR, thus contributing to enhanced intratubular Ang II formation.

Lara LS, McCormack M, Semprum-Prieto LC, Shenouda S, Majid DS, Kobori H, Navar LG, Prieto MC. AT1 receptormediated augmentation of angiotensinogen, oxidative stress, and inflammation in ANG II-salt hypertension. Am J Physiol Renal Physiol 302: F85-F94, 2012. First published September 7, 2011 doi:10.1152/ajprenal.00351.2011.-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 ϭ 5]; 2) HS diet [8% NaCl, n ϭ 5]; 3) ANG II infusion in NS rats [ANG II 80 ng/min, n ϭ 5]; 4) ANG II infusion in HS rats [ANG IIϩHS, n ϭ 5]; and 5) ANG II infusion in HS rats treated with ANG II type 1 receptor blocker (ARB) [ANG IIϩHSϩARB, n ϭ 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 Ϯ 10 vs. 221 Ϯ 8 mmHg; P Ͻ 0.05), proteinuria (46 Ϯ 7 vs. 127 Ϯ 7 mg/day; P Ͻ 0.05), and uAGT (1,109 Ϯ 70 vs.. 7,200 Ϯ 614 ng/day; P Ͻ 0.05) associated with greater collagen deposition, mesangial expansion, interstitial cell proliferation, and macrophage infiltration. In both ANG II groups, the O 2 Ϫ 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. renin-angiotensin system; reactive oxygen species; kidney injury; macrophage infiltration; dihydroethidium staining THE ENHANCEMENT OF INTRARENAL angiotensin II (ANG II) content contributing to hypertension and kidney damage is supported by the presence of all components of the renin-angiotensin system (RAS) in the kidney (27-29). During ANG II-dependent hypertension, intrarenal RAS activation is characterized by increased tissue levels of ANG II due to an augmented ANG II type 1 receptor (AT 1 R) binding with concomitant internalization of circulating ANG II (5, 40) and by de novo formation of ANG II (30). In response to chronic ANG II infusions, there are 1) enhancement of angiotensinogen (AGT) synthesis and secretion by...

Abstract-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 Ϫ10 to 10 Ϫ7 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. Key Words: angiotensin II-dependent hypertension Ⅲ collecting duct renin Ⅲ renin gene expression Ⅲ protein kinase C Ⅲ cell signaling pathway R egulation of renin expression in the kidney juxtaglomerular (JG) apparatus has been widely studied; however, the mechanisms regulating expression of renin in collecting ducts (CDs) and its augmentation in animal models of hypertension remain unclear. 1,2 Increased renin synthesis is mediated by the angiotensin II (Ang II) type 1 receptor (AT 1 R) independent of changes in blood pressure. 3,4 In vitro studies also suggest that CD renin synthesis and secretion can be directly increased by Ang II 5 ; however, the mechanisms involved are poorly understood.Ang II is present at high levels in the kidneys of hypertensive animals, 6 and an important fraction of this Ang II is generated by de novo synthesis 7 as a consequence of augmented angiotensinogen expression in proximal tubule cells, leading to augmented angiotensinogen excretion in urine. 8,9 Along with the expression of angiotensin-converting enzyme in the distal nephron, 10 increases in CD renin expression and activity may have a key role in the intrarenal Ang II content, contributing to additional Ang II generation in distal ne...

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.

Voltage-gated Kv7 (KCNQ) channels underlie important K + currents in many different types of cells, including the neuronal M current, which is thought to be modulated by muscarinic stimulation via depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We studied the role of modulation by angiotensin II (angioII) of M current in controlling discharge properties of superior cervical ganglion (SCG) sympathetic neurons and the mechanism of action of angioII on cloned Kv7 channels in a heterologous expression system. In SCG neurons, which endogenously express angioII AT1 receptors, application of angioII for 2 min produced an increase in neuronal excitability and a decrease in spike-frequency adaptation that partially returned to control values after 10 min of angioII exposure. The increase in excitability could be simulated in a computational model by varying only the amount of M current. Using Chinese hamster ovary (CHO) cells expressing cloned Kv7.2 + 7.3 heteromultimers and AT1 receptors studied under perforated patch clamp, angioII induced a strong suppression of the Kv7.2/7.3 current that returned to near baseline within 10 min of stimulation. The suppression was blocked by the phospholipase C inhibitor edelfosine. Under whole-cell clamp, angioII moderately suppressed the Kv7.2/7.3 current whether or not intracellular Ca 2+ was clamped or Ca 2+ stores depleted. Co-expression of PI(4)5-kinase in these cells sharply reduced angioII inhibition, but did not augment current amplitudes, whereas co-expression of a PIP 2 5 -phosphatase sharply reduced current amplitudes, and also blunted the inhibition. The rebound of the current seen in perforated-patch recordings was blocked by the PI4-kinase inhibitor, wortmannin (50 μM), suggesting that PIP 2 re-synthesis is required for current recovery. High-performance liquid chromatographic analysis of anionic phospholipids in CHO cells stably expressing AT1 receptors revealed that PIP 2 and phosphatidylinositol 4-phosphate levels are to be strongly depleted after 2 min of stimulation with angioII, with a partial rebound after 10 min. The results of this study establish how angioII modulates M channels, which in turn affects the integrative properties of SCG neurons.

Prenatal malnutrition is responsible for the onset of alterations in renal Na(+) transport in the adult offspring. Here we investigated the molecular mechanisms by which increased formation of reactive oxygen species during prenatal malnutrition affects the pathways that couple angiotensin II (Ang II) receptors (AT(1)R and AT(2)R) to kidney Na(+)-ATPase in adulthood, and how maternal treatment with α-tocopherol can prevent alterations in the main regulatory cascade of the pump. The experiments were carried out on the adult progeny of control and malnourished dams during pregnancy that did or did not receive α-tocopherol during lactation. Malnutrition during pregnancy increased maternal hepatic and adult offspring renal malondialdehyde levels, which returned to control after supplementation with α-tocopherol. In the adult offspring, placental malnutrition programmed: decrease in Na(+)-ATPase activity, loss of the physiological stimulation of this pump by Ang II, up-regulation of AT(1)R and AT(2)R, decrease in membrane PKC activity, selective decrease of the PKCε isoform expression, and increase in PKA activity with no change in PKA α-catalytic subunit expression. These alterations were reprogrammed to normal levels by α-tocopherol during lactation. The influence of α-tocopherol on the signaling machinery in adult offspring indicates selective non-antioxidant effects at the gene transcription and protein synthesis levels.

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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 Ⅱ formation 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. Key words: Renin-angiotensin system; Angiotensin Ⅱ; Kidney; Hypertension treatment; Receptor Core tip: Activation of the angiotensin converting enzyme (ACE)/ Angiotensin Ⅱ (Ang Ⅱ)/AT1 axis leads to vasoconstriction, anti-diuresis, anti-natriuresis, release of aldosterone and anti-diuretic hormone, which can result in hypertension, renal and cardiovascular diseases. Inhibition of renin and ACE or blocking AT1 receptor is the most used therapies for heart failure and hypertension. Nevertheless, the discovery of local Ang Ⅱ synthesis, new Ang Ⅱ metabolites, receptors and axis of this system, makes possible the development of new drugs and strategies for renal and cardiovascular diseases treatment, such as activation of ACE2/Ang-(1-7)/ Mas axis, which presents opposite effects of AT1 activation by Ang Ⅱ.