The Triad Na+ Activated Na+ Channel (Nax)-Salt Inducible KINASE (SIK) and (Na+ + K+)-ATPase: Targeting the Villains to Treat Salt Resistant and Sensitive Hypertension.

The Na+-activated Na+ channel (Nax) and salt-inducible kinase (SIK) are stimulated by increases in local Na+ concentration, affecting (Na+ + K+)-ATPase activity. To test the hypothesis that the triad Nax/SIK/(Na+ + K+)-ATPase contributes to kidney injury and salt-sensitive hypertension (HTN), uninephrectomized male Wistar rats (200 g; n = 20) were randomly divided into 4 groups based on a salt diet (normal salt diet; NSD-0.5% NaCl-or high-salt diet; HSD-4% NaCl) and subcutaneous administration of saline (0.9% NaCl) or deoxycorticosterone acetate (DOCA, 8 mg/kg), as follows: Control (CTRL), CTRL-Salt, DOCA, and DOCA-Salt, respectively. After 28 days, the following were measured: kidney function, blood pressure, (Na+ + K+)-ATPase and SIK1 kidney activities, and Nax and SIK1 renal expression levels. SIK isoforms in kidneys of CTRL rats were present in the glomerulus and tubular epithelia; they were not altered by HSD and/or HTN. CTRL-Salt rats remained normotensive but presented slight kidney function decay. HSD rats displayed augmentation of the Nax/SIK/(Na+ + K+)-ATPase pathway. HTN, kidney injury, and kidney function decay were present in all DOCA rats; these were aggravated by HSD. DOCA rats presented unaltered (Na+ + K+)-ATPase activity, diminished total SIK activity, and augmented SIK1 and Nax content in the kidney cortex. DOCA-Salt rats expressed SIK1 activity and downregulation in (Na+ + K+)-ATPase activity in the kidney cortex despite augmented Nax content. The data of this study indicate that the (Na+ + K+)-ATPase activity response to SIK is attenuated in rats under HSD, independent of HTN, as a mechanism contributing to kidney injury and salt-sensitive HTN.

Luminal ANG II is internalized as a complex with AT1R/AT2R heterodimers to target endoplasmic reticulum in LLC-PK1 cells.

ANG II has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of ß-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of ß-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.

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.

ANG-(3-4) inhibits renal Na+-ATPase in hypertensive rats through a mechanism that involves dissociation of ANG II receptors, heterodimers, and PKA.

The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study 1)investigates whether ANG-(3-4) modulates ouabain-resistant Na(+)-ATPase resident in proximal tubule cells and 2) verifies whether its possible action on pumping activity, considered the fine tuner of Na(+) reabsorption in this nephron segment, depends on blood pressure. ANG-(3-4) inhibited Na(+)-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto (WKY) rats or on Na(+)-K(+)-ATPase. PD123319 (10(-7) M) and PKA(5-24) (10(-6) M), an AT2 receptor (AT2R) antagonist and a specific PKA inhibitor, respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10(-8) M) completely blocked stimulation of Na(+)-ATPase induced by 10(-10) M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10(-10) M ANG II and dissociate to a very low dimerization state upon addition of 10(-8) M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased urinary Na(+) concentration and urinary Na(+) excretion with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus the influence of ANG-(3-4) on Na(+) transport and its hypotensive action depend on receptor association and on blood pressure.

New frontiers in the intrarenal Renin-Angiotensin system: a critical review of classical and new paradigms.

The renin-angiotensin system (RAS) is well-recognized as one of the oldest and most important regulators of arterial blood pressure, cardiovascular, and renal function. New frontiers have recently emerged in the RAS research well beyond its classic paradigm as a potent vasoconstrictor, an aldosterone release stimulator, or a sodium-retaining hormone. First, two new members of the RAS have been uncovered, which include the renin/(Pro)renin receptor (PRR) and angiotensin-converting enzyme 2 (ACE2). Recent studies suggest that prorenin may act on the PRR independent of the classical ACE/ANG II/AT1 receptor axis, whereas ACE2 may degrade ANG II to generate ANG (1-7), which activates the Mas receptor. Second, there is increasing evidence that ANG II may function as an intracellular peptide to activate intracellular and/or nuclear receptors. Third, currently there is a debate on the relative contribution of systemic versus intrarenal RAS to the physiological regulation of blood pressure and the development of hypertension. The objectives of this article are to review and discuss the new insights and perspectives derived from recent studies using novel transgenic mice that either overexpress or are deficient of one key enzyme, ANG peptide, or receptor of the RAS. This information may help us better understand how ANG II acts, both independently or through interactions with other members of the system, to regulate the kidney function and blood pressure in health and disease.

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway.

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca(2+)-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)α protein structurally linked to AT(2)R--as revealed by their co-immunoprecipitation--mimicked the effect of Ang-(3-4) on Ca(2+)-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi(5-24) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca(2+)-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace (3)H-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca(2+)-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT1/AT2 receptors to activate SERCA and to promote Ca2+ mobilization.

ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC→DAG(PMA)→PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.

A scrutiny of the biochemical pathways from Ang II to Ang-(3-4) in renal basolateral membranes.

In a previous paper we demonstrated that Ang-(3-4) counteracts inhibition of the Ca(2+)-ATPase by Ang II in the basolateral membranes of kidney proximal tubules cells (BLM). We have now investigated the enzymatic routs by which Ang II is converted to Ang-(3-4). Membrane-bound angiotensin converting enzyme, aminopeptidases and neprilysin were identified using fluorescent substrates. HPLC showed that Plummer's inhibitor but not Z-pro-prolinal blocks Ang II metabolism, suggesting that carboxypeptidase N catalyzes the conversion Ang II--> Ang-(1-7). Different combinations of bestatin, thiorphan, Plummer's inhibitor, Ang II and Ang-(1-5), and use of short proteolysis times, indicate that Ang-(1-7)--> Ang-(1-5)--> Ang-(1-4)--> Ang-(3-4) is a major route. When Ang III was combined with the same inhibitors, the following pathway was demonstrated: Ang III--> Ang IV--> Ang-(3-4). Ca(2+)-ATPase assays with different Ang II concentrations and different peptidase inhibitors confirm the existence of these pathways in BLM and show that a prolyl-carboxypeptidase may be an alternative catalyst for converting Ang II to Ang-(1-7). Overall, we demonstrated that BLM have all the peptidase machinery required to produce Ang-(3-4) in the vicinity of the Ca(2+)-ATPase, enabling a local RAS axis to effect rapid modulation of active Ca(2+) fluxes.

Ang-(3-4) suppresses inhibition of renal plasma membrane calcium pump by Ang II.

We previously demonstrated that Ang II inhibits the renal plasma membrane Ca(2+)-ATPase. In the present work we have studied the effect of Ang II, at concentrations similar to those found in the renal interstitium, on the Ca(2+)-ATPase from proximal tubule cells. High Ang II concentration (5 x 10(-7) mol/L) led to the recovery of Ca(2+)-ATPase activity previously inhibited by 50% at low Ang II concentration (10(-10) mol/L). Reactivation occurred in parallel with: (i) formation of only two dead-end metabolites [Ang-(3-4) and Tyr] after incubation of isolated membranes with micromolar Ang II; and (ii) dissociation of constitutive AT(1)R/AT(2)R heterodimers, which are preserved with 10(-10) mol/L Ang II. When the membranes were incubated with 10(-14) mol/L Ang-(3-4), inhibition by 10(-10) mol/L Ang II was no longer observed. The counteracting effect of Ang-(3-4) was abolished by PD123319, an antagonist of AT(2)R, and mimicked by CGP42112A, an agonist of AT(2)R. Ang-(1-7) is an intermediate in the formation of Ang-(3-4) via a pathway involving angiotensin-converting enzyme (ACE), and complete dipeptide breakdown to Tyr and Val is impaired by low Ang II. We conclude that Ang-(3-4) may be a physiological regulator of active Ca(2+) fluxes in renal proximal cells by acting within the renin-angiotensin axis.