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Angiotensin II inhibits NaCl absorption in the rat medullary thick ascening limb
Abstract
NaCl reabsorption in the medullary thick ascending limb of Henle (MTALH) contributes to NaCl balance and is also responsible for the creation of medullary interstitial hypertonicity. Despite the presence of angiotensin II subtype 1 (AT(1)) receptors in both the luminal and the basolateral plasma membranes of MTALH cells, no information is available on the effect of angiotensin II on NaCl reabsorption in MTALH and, furthermore, on angiotensin II-dependent medullary interstitial osmolality. MTALHs from male Sprague-Dawley rats were isolated and microperfused in vitro; transepithelial net chloride absorption (J(Cl)) as well as transepithelial voltage (V(te)) were measured. Luminal or peritubular 10(-11) and 10(-10) M angiotensin II had no effect on J(Cl) or V(te). However, 10(-8) M luminal or peritubular angiotensin II reversibly decreased both J(Cl) and V(te). The effect of both luminal and peritubular angiotensin II was prevented by the presence of losartan (10(-6) M). By contrast, PD-23319, an AT(2)-receptor antagonist, did not alter the inhibitory effect of 10(-8) M angiotensin II. Finally, no additive effect of luminal and peritubular angiotensin II was observed. We conclude that both luminal and peritubular angiotensin II inhibit NaCl absorption in the MTALH via AT(1) receptors. Because of intrarenal angiotensin II synthesis, angiotensin II concentration in medullary tubular and interstitial fluids may be similar in vivo to the concentration that displays an inhibitory effect on NaCl reabsorption under the present experimental conditions.
... The medullary thick ascending limb (mTAL) reabsorbs a significant amount of sodium via the apical membrane NKCC2. Although all other nephron segments studied have shown that ANG II stimulates sodium transport, in vitro microperfusion studies have demonstrated that addition of ANG II to the apical or basolateral solution results in inhibition of sodium chloride absorption (20). Although in vitro microperfusion allows precise measurement of the effect of a hormone on solute transport in a specific nephron segment, it sometimes fails to replicate the in vivo condition. ...
... Adult male and female Sprague-Dawley rats weighing between 140 and 250 g were used in these studies. Rats were administered furosemide (2 mg/100 g body wt) by intraperitoneal injection ϳ5 min before receiving intraperitoneal Inactin (10 mg/100 g body wt) similar to what has been described previously in studies that examined transport in the mTAL (4,14,20). After the rats were anesthetized, a vertical incision was made and the abdomen was opened. ...
... It was thus surprising to find that there was discordance between the stimulatory effect of luminal ANG II in the proximal tubule and distal nephron and the inhibition found in the thick ascending limb (20). In the present study we validated the inhibitory effect of luminal ANG II in isolated perfused rat mTAL. ...
Angiotensin II is secreted by the proximal tubule resulting in luminal concentrations that are 100-1000 fold greater than that in the blood. Luminal angiotensin II has been shown to stimulate transport in the proximal tubule and in the distal nephron. Surprisingly, luminal angiotensin II inhibits NaCl transport in the medullary thick ascending limb (mTAL), a nephron segment responsible for a significant amount of sodium chloride absorption from the glomerular ultrafiltrate. We confirmed that addition of 10-8 M angiotensin II to the lumen inhibited mTAL chloride transport (220 + 19 to 165 + 25 pmol/mm.min, P<0.01) and examined if there was an interaction of basolateral norepinephrine to simulate the in vivo condition of an innervated tubule. We find that in the presence of 10-6 M bath norepinephrine, luminal angiotensin II stimulated mTAL chloride transport from 298 + 18 to 364 + 42 pmol/mm.min, P<0.05. Stimulation of chloride transport by luminal angiotensin II was also seen with 10-3 M bath dibutyryl cAMP in the bathing solution and bath isoproterenol. Bath 10-5 H-89 blocked the stimulation in chloride transport by bath norepinephrine and prevented the effect of luminal angiotensin II to either stimulate or inhibit chloride transport. Bath phentolamine, an α-adrenergic agonist, also prevented the decrease in mTAL chloride transport by luminal angiotensin II. Thus, luminal angiotensin II increases chloride transport with basolateral norepinephrine; an effect likely mediated by stimulation of cAMP. Alpha1 adrenergic stimulation prevents the inhibition of chloride transport by luminal angiotensin II.
... ANG II increased transcellular chloride transport and sodium transport across the epithelial sodium channel in the cortical collecting tubule (35,37). Despite the increase in sodium absorption in almost every nephron segment, ANG II was found to decrease chloride transport in the medullary thick ascending limb (mTAL), a nephron segment responsible for a substantive amount of sodium chloride absorption (25). ...
... I first examined whether 2ϫ10 Ϫ11 M ANG II affected chloride transport in the mTAL. The rate of chloride transport was 148.9 Ϯ 17.7 in the control period and 129.2 Ϯ 14.5 pmol·mm Ϫ1 ·min Ϫ1 after the addition of ANG II, confirming that the plasma concentration of ANG II in a euvolemic animal does not affect mTAL transport (25). Similarly, 10 Ϫ10 M ANG II had no effect on chloride transport (271.1 Ϯ 40.5 in control vs. 242.6 ...
... Ϯ 98.1 with 10 Ϫ10 M ANG II pmol·mm Ϫ1 ·min Ϫ1 ), while 10 Ϫ9 M ANG II inhibited chloride transport from 317.8 Ϯ 68.6 to 258.7 Ϯ 70.9 pmol·mm Ϫ1 ·min Ϫ1 (P Ͻ 0.05). The experiments shown in Fig. 1 confirmed previous findings that chloride transport was inhibited significantly by bath 10 Ϫ8 M ANG II (25). Similarly, 10 Ϫ8 M ANG II caused a reduction in the transepithelial potential difference from positive 4.6 Ϯ 0.7 to 3.7 Ϯ 0.7 mV, P Ͻ 0.01. ...
Previous studies have shown that in proximal and distal tubule nephron segments, peritubular ANG II stimulates sodium chloride transport. However, ANG II inhibits chloride transport in the medullary thick ascending limb (mTAL). Because ANG II and catecholamines are both stimulated by a decrease in extracellular fluid volume, the purpose of this study was to examine whether there was an interaction between ANG II and catecholamines to mitigate the inhibition in chloride transport by ANG II. In isolated perfused rat mTAL, 10(-8) M bath ANG II inhibited transport (from a basal transport rate of 165.6 +/- 58.8 to 58.8 +/- 29.4 pmol.mm(-1).min(-1); P < 0.01). Bath norepinephrine stimulated chloride transport (from a basal transport rate of 298.1 +/- 31.7 to 425.2 +/- 45.8 pmol.mm(-1).min(-1); P < 0.05) and completely prevented the inhibition in chloride transport by ANG II. The stimulation of chloride transport by norepinephrine was mediated entirely by its beta-adrenergic effect; however, both the beta- and alpha-adrenergic agonists isoproterenol and phenylephrine prevent the ANG II-mediated inhibition in chloride transport. In the presence of 10(-5) M propranolol, the effect of norepinephrine to prevent the inhibition of chloride transport by ANG II was still present. These data are consistent with an interaction of both alpha- and beta-catecholamines and ANG II on net chloride transport in the mTAL.
... The proposed signaling cascades activated by ANG II, which mediate its effects on Cl Ϫ reabsorption and/or NKCC2 activity, are contradictory and controversial. AT 1receptor activation has been reported to be required for inhibition of transport (14,379,632) and its stimulation (14,632). AT 2 receptors have been reported to either have no effect (379) or mediate inhibition of NKCC2 activity (292). ...
... AT 1receptor activation has been reported to be required for inhibition of transport (14,379,632) and its stimulation (14,632). AT 2 receptors have been reported to either have no effect (379) or mediate inhibition of NKCC2 activity (292). Inhibition of transport was reported to 1) be mediated by 20-HETE and not cAMP (14); 2) involve cAMP because a protein kinase A inhibitor could prevent the reduction (35); 3) be due to a decrease in cAMP (632); and 4) be mediated by NO (292). ...
The thick ascending limb plays a key role in maintaining water and electrolyte balance. The importance of this segment in regulating blood pressure is evidenced by the effect of loop diuretics or local genetic defects on this parameter. Hormones and factors produced by thick ascending limbs have both autocrine and paracrine effects, which can extend prohypertensive signaling to other structures of the nephron. In this review, we discuss the role of the thick ascending limb in the development of hypertension, not as a sole participant, but one that works within the rich biological context of the renal medulla. We first provide an overview of the basic physiology of the segment and the anatomical considerations necessary to understand its relationship with other renal structures. We explore the physiopathological changes in thick ascending limbs occurring in both genetic and induced animal models of hypertension. We then discuss the racial differences and genetic defects that affect blood pressure in humans through changes in thick ascending limb transport rates. Throughout the text, we scrutinize methodologies and discuss the limitations of research techniques that, when overlooked, can lead investigators to make erroneous conclusions. Thus, in addition to advancing an understanding of the basic mechanisms of physiology, the ultimate goal of this work is to understand our research tools, to make better use of them, and to contextualize research data. Future advances in renal hypertension research will require not only collection of new experimental data, but also integration of our current knowledge.
... For example, they are expressed on the luminal and basolateral membranes of the epithelium in the medullary thick ascending limb (287,298). However, their effects on activity of NKCC2 are inconsistent and appear to depend on local Ang II levels (4,203). At lower concentrations of Ang II, inhibition of NKCC2 may occur (4,203), whereas stimulation of NKCC2 may be seen at higher concentrations (4). ...
... However, their effects on activity of NKCC2 are inconsistent and appear to depend on local Ang II levels (4,203). At lower concentrations of Ang II, inhibition of NKCC2 may occur (4,203), whereas stimulation of NKCC2 may be seen at higher concentrations (4). Functions of AT 1 receptors in more distal segments of the nephron have also been examined. ...
The renin-angiotensin system has powerful effects in control of the blood pressure and sodium homeostasis. These actions are coordinated through integrated actions in the kidney, cardiovascular system and the central nervous system. Along with its impact on blood pressure, the renin-angiotensin system also influences a range of processes from inflammation and immune responses to longevity. Here, we review the actions of the "classical" renin-angiotensin system, whereby the substrate protein angiotensinogen is processed in a two-step reaction by renin and angiotensin converting enzyme, resulting in the sequential generation of angiotensin I and angiotensin II, the major biologically active renin-angiotensin system peptide, which exerts its actions via type 1 and type 2 angiotensin receptors. In recent years, several new enzymes, peptides, and receptors related to the renin-angiotensin system have been identified, manifesting a complexity that was previously unappreciated. While the functions of these alternative pathways will be reviewed elsewhere in this journal, our focus here is on the physiological role of components of the "classical" renin-angiotensin system, with an emphasis on new developments and modern concepts. © 2014 American Physiological Society. Compr Physiol 4:1201-1228, 2014.
... Several 10 min collections were performed over 30-60 min. Nanolitre droplets were carefully dropped off on a clean silver plate under water-saturated paraffin oil kept at room temperature as previously described (Lerolle et al. 2004) before sampling. For each collection, ion flux (J Na , J K , J Mg ) was calculated and corrected for the length of the tubule: J X = ([X] p -[X] c ) × V c /L, where ([X] p and [X] c are the concentrations of ion X in the perfusate and collectate, respectively; V c is the collection rate; L is the tubule length measured using the optical scale of the microscope. ...
Key points
An UHPLC method to measure picomole amounts of magnesium has been developed.
The method is sensitive, specific, accurate and reproducible.
The method is suitable for quantifying magnesium transport across intact epithelia.
Abstract
Magnesium is involved in many biological processes. Extracellular magnesium homeostasis mainly depends on the renal handling of magnesium, the study of which requires measurement of low concentrations of magnesium in renal tubular fluid. We developed an ultra‐high‐performance liquid chromatography method to measure millimolar concentrations of magnesium in nanolitre samples. Within‐assay and between‐assay coefficients of variation were lower than 5% and 6.6%, respectively. Measurement of magnesium concentration was linear (r² = 0.9998) over the range 0–4 mmol/l. Absolute bias ranged from −0.03 to 0.05 mmol/l. The lower limit of quantification was 0.2 mmol/l. Recovery was 97.5–100.3%. No significant interference with calcium, another divalent cation present in the same samples, was detected. The method was successfully applied to quantify transepithelial magnesium transport by medullary and cortical thick ascending limbs during ex vivo microperfusion experiments. In conclusion, ultra‐high‐performance liquid chromatography is suitable for measurement of picomole amounts of magnesium in renal tubular fluid. The method allows detailed studies of transepithelial magnesium transport across native epithelium.
... Importantly, the elevated plasma sodium observed in this study was significantly reduced by losartan following the concurrent ingestion of salt and losartan, in both the hypertensive and normotensive subjects. This is in keeping with the natriuretic effect of angiotensin II receptor blockers (ARB) that have been reported in experimental animal models [38]. Meanwhile, plasma K+ remained unchanged after salt load and after a combination of salt and losartan in this study. ...
Introduction:
Sympathetic and Renin-Angiotensin-Aldosterone systems play crucial roles in blood pressure response to increased salt intake. This study investigated the effects of angiotensin receptor blocker (ARB) and sympathetic excitation on the responses of blood pressure (BP) and peripheral vascular resistance (PVR) in salt loaded normotensive (NT) and hypertensive (HT) Nigerian subjects.
Methods:
16 NT and 14 HT participants, that were age-matched [39.9 ± 1.3 vs 44.1±2.1yrs (P= 0.10)], underwent 5 days each of oral administration of 200mmol NaCl, and 200mmol NaCl + 50mg Losartan, preceded by a baseline control condition. BP and PVR responses to 30% Maximum Voluntary Contraction (MVC) of handgrip (HG) for one minute were determined at baseline, after salt load and after salt + Losartan. Data were presented as Mean ± SEM, and analyzed with two-way ANOVA and paired t-test, with P<0.05 accepted as significant.
Results:
BP and PVR were significantly increased by HG at baseline, after salt load and after salt + Losartan in NT and HT. Salt load augmented the HG-induced SBP (P=0.04) and MABP responses (P=0.02) in HT. While Losartan attenuated the HG- induced Systolic Blood Pressure (SBP) SBP response (P=0.007) and DBP response (P=0.003) in HT and NT respectively after salt + Losartan. HG-induced PVR response was significantly accentuated after salt load in HT (P=0.005), but it was not significant in NT (P=0.38).
Conclusion:
The implication of our finding is that angiotensin II receptor blockade possibly attenuates salt-induced sympathetic nerve excitation in black hypertensive patients.
... The natriuretic effects of ARBs have been shown in experimental models. [7][8][9] Inhibition of FR Na (defined as tubular sodium reabsorption per filtered amount of sodium) in the upper tubules (e.g. proximal tubules or loop of Henle) should lead to a downstream increase in FR Na caused by the tubuloglomerular feedback (TGF) system and epithelial sodium channel (ENaC). ...
... Many factors regulate transport in the TAL, including aldosterone (24), bradykinin (51), TNF (64), vasopressin (5), insulin (33), and ATP (9). ANG II also acutely regulates transport in the TAL (2,23,32,40,41,65) as well as in other segments of the nephron (19,49). Furthermore, we have shown that chronic ANG II infusion increases net transport by the TAL (56). ...
Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na/K/2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (Ang II) infusion increases net TAL Na transport and Na apical entry; however little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by Ang II-infusion, a model of Ang II-induced hypertension. Rats were infused with 200 ng/kg/min Ang II or vehicle for 7 days and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in: 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from Ang II-infused rats was 12.8 ± 0.4 Arbitrary Fluorescent Units (AFU)/mg/min compared to only 9.9 ± 1.1 AFU /mg/min in controls (p < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (p < 0.043) greater in tubules from Ang II-treated than vehicle rats. Ang II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane or the affinity for Na. When furosemide (1.1 mg/Kg/day) was co-infused with Ang II, no increase in plasma membrane Na pump activity was observed. We concluded that in Ang II-induced hypertension Na pump activity is increased in the plasma membrane of TALs, and that this increase is caused by the chronically enhanced Na entry occurring in this model.
... This is consistent with the report that AngII infusion enhanced furosemide-sensitive oxygen consumption in the TAL. 12 However, a flux study performed in inner stripe of the outer medullary TAL demonstrated that AngII inhibited Cl absorption. 26 The cause of the discrepancies between these 2 studies was not clear. One possibility is that different segments of the medullary TAL were used (the inner stripe versus the outer stripe used in the present study). ...
Chloride channels in the basolateral membrane play a key role in Cl absorption in the thick ascending limb (TAL). The patch-clamp experiments were performed to test whether angiotensin II (AngII) increases Cl absorption in the TAL by stimulating the basolateral 10-pS Cl channels. AngII (1-100 nmol/L) stimulated the 10-pS Cl channel in the TAL, an effect that was blocked by losartan (angiotension AT1 receptor [AT1R] antagonist) but not by PD123319 (angiotension AT2 receptor [AT2R] antagonist). Inhibition of phospholipase C or protein kinase C also abolished the stimulatory effect of AngII on Cl channels. Moreover, stimulation of protein kinase C with phorbol-12-myristate-13-acetate mimicked the effect of AngII and increased Cl channel activity. However, the stimulatory effect of AngII on Cl channels was absent in the TAL pretreated with diphenyleneiodonium sulfate, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Moreover, treatment of the TAL with diphenyleneiodonium sulfate also blocked the effect of phorbol-12-myristate-13-acetate on the 10-pS Cl channel. Western blotting demonstrated that incubation of isolated TAL with AngII increased phosphorylation of p47(phox) at Ser(304), suggesting that AngII stimulates the basolateral Cl channels by increasing NADPH oxidase-dependent superoxide generation. This notion was also supported by the observation that H2O2 significantly increased 10-pS Cl channel activity in the TAL. We conclude that stimulation of AT1R increased the basolateral Cl channels by activating the protein kinase C-dependent NADPH oxidase pathway. The stimulatory effect of AngII on the basolateral Cl channel may contribute to AngII-induced increases in NaCl reabsorption in the TAL and AngII-infuse-induced hypertension.
... Others have attributed the effect of Ang II on TAL transport to AT1R rather than AT2R activation 16,17 . These two studies, by the same group, reported conflicting effects of Ang II on TAL transport in vitro. ...
NO reduces NaCl absorption by thick ascending limbs (TALs) by inhibiting the Na/K/2Cl cotransporter (NKCC2). We have shown that NO-induced inhibition of Na transport is reduced in Dahl salt-sensitive rat (SS) TALs. Angiotensin II increases NO production in TALs via angiotensin II type 2 receptor (AT(2)R). It is unknown whether AT(2)Rs regulate TAL NaCl absorption and whether this effect is reduced in SS rats. We hypothesized that AT(2)R activation decreases TAL Na transport via NO, and this effect is blunted in SS rats. In the presence of angiotensin II type 1 receptor antagonist losartan, AT(2)R activation with angiotensin II inhibited NKCC2 activity by 32±7% (P<0.03). AT(2)R antagonist PD-123319 abolished the effect of angiotensin II. Activation with the AT(2)R-selective agonist CGP42112A (10 nmol/L) decreased NKCC2 activity by 29±6% (P<0.03). The effect of CGP42112A on NKCC2 activity was blocked by PD-123319 and by NO synthase inhibitor N(G)-nitro-l-arginine methyl ester. In Dahl salt-resistant rat TALs, 1 nmol/L of CGP42112A decreased NKCC2 activity by 23±4% (P<0.01). In SS TALs, it had no effect. TAL AT(2)R mRNA did not differ in SS versus salt-resistant rats. We conclude the following: (1) TAL AT(2)R activation decreases Na absorption; (2) this effect is mediated by AT(2)R-induced stimulation of NO; (3) AT(2)R-induced reduction of NKCC2 activity is blunted in SS rats; and (4) defects in AT(2)R/NO signaling rather than decreased AT(2)R expression likely account for the blunted effect in SS TALs. Impaired AT(2)R-mediated signaling in TALs could contribute to the Na retention associated with salt-sensitive hypertension.
... 25 Studies performed in isolated perfused TALH tubules found that incubation with 10 28 M Ang II resulted in decreases in transepithelial net chloride absorption (J Cl ) as well as transepithelial voltage (V te ). 26 However, recent studies in Ang II-hypertensive rats have shown enhanced thick ascending limb sodium transportrelated oxygen consumption mediated through increases in protein kinase C a-activity. 27 These results would suggest an increase in TALH sodium transport by Ang II at high levels. ...
Kidney-specific induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II) -dependent hypertension, but the relative contribution of vascular versus tubular induction of HO-1 is unknown. To determine the specific contribution of thick ascending loop of Henle (TALH) -derived HO-1, we generated a transgenic mouse in which the uromodulin promoter controlled expression of human HO-1. Quantitative RT-PCR and confocal microscopy confirmed successful localization of the HO-1 transgene to TALH tubule segments. Medullary HO activity, but not cortical HO activity, was significantly higher in transgenic mice than control mice. Enhanced TALH HO-1 attenuated the hypertension induced by Ang II delivered by an osmotic minipump for 10 days (139 ± 3 versus 153 ±2 mmHg in the transgenic and control mice, respectively; P<0.05). The lower blood pressure in transgenic mice associated with a 60% decrease in medullary NKCC2 transporter expression determined by Western blot. Transgenic mice also exhibited a 36% decrease in ouabain-sensitive sodium reabsorption and a significantly attenuated response to furosemide in isolated TALH segments. In summary, these results show that increased levels of HO-1 in the TALH can lower blood pressure by a mechanism that may include alterations in NKCC2-dependent sodium reabsorption.
... Four different calibration solutions, titrated to 6.5, 6.9, 7.3, or 7.5, were used. Vte was measured continuously as described elsewhere (49). Measurement of pendrin and NDCBE activities in Xenopus oocytes. ...
Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.
... If delivery of tubule fluid to the loop is 12 nl/min, this contains ϳ1.7 nmol/min of Na ϩ (40% of filtered Na ϩ ), so that if 60% of this amount is reabsorbed in AHL, Na ϩ transport is 1.0 nmol/min, or 250 pmol·mm Ϫ1 ·min Ϫ1 . This can be compared with reabsorptive fluxes in vitro for Na ϩ , 66 pmol·mm Ϫ1 ·min Ϫ1 (39), and for Cl Ϫ , 48 -82 pmol·mm Ϫ1 ·min Ϫ1 (36), and as high as 142 pmol·mm Ϫ1 ·min Ϫ1 (43). In vitro, the rate of luminal proton secretion has been in the range of 10 -20% of the rate of Na ϩ reabsorption measured in vivo (17,21). ...
A mathematical model of ascending Henle limb (AHL) epithelium has been fashioned using kinetic representations of Na+-K+-2Cl- cotransporter (NKCC2), KCC4, and type 3 Na+/H+ exchanger (NHE3), with transporter densities selected to yield the reabsorptive Na+ flux expected for rat tubules in vivo. Of necessity, this model predicts fluxes that are higher than those measured in vitro. The kinetics of the NKCC and KCC are such that Na+ reabsorption by the model tubule is responsive to variation in luminal NaCl concentration over the range of 30 to 130 mM, with only minor changes in cell volume. Peritubular KCC accounts for about half the reabsorptive Cl- flux, with the remainder via peritubular Cl- channels. Transcellular Na+ flux is turned off by increasing peritubular KCl, which produces increased cytosolic Cl- and thus inhibits NKCC2 transport. In the presence of physiological concentrations of ammonia, there is a large acid challenge to the cell, due primarily to NH4+ entry via NKCC2, with diffusive NH3 exit to both lumen and peritubular solutions. When NHE3 density is adjusted to compensate this acid challenge, the model predicts luminal membrane proton secretion that is greater than the HCO3(-)-reabsorptive fluxes measured in vitro. The model also predicts luminal membrane ammonia cycling, with uptake via NKCC2 or K+ channel, and secretion either as NH4+ by NHE3 or as diffusive NH3 flux in parallel with a secreted proton. If such luminal ammonia cycling occurs in vivo, it could act in concert with luminal K+ cycling to facilitate AHL Na+ reabsorption via NKCC2. With physiological ammonia, peritubular KCl also blunts NHE3 activity by inhibiting NH4+ uptake on the Na-K-ATPase, and alkalinizing the cell.
... In all studies and experimental systems, activation of the CaR results in the inhibition of sodium transport in the distal nephron, whereas activation of the AII receptor (presumably AT1) can stimulate or inhibit transport depending on the concentration, experimental system and study [23,24,44,[48][49][50][60][61][62][63][64]. These differences between the responses of the distal nephron to calcium and AII indicate that the two receptors have distinct effects on cells despite the fact that they stimulate production of the same second messengers. ...
Extracellular calcium has profound effects on renal tubular transport, presumably via the calcium-sensing receptor, which is expressed in all nephron segments, but its effects in specific segments and the mechanism of regulation of transport are not fully understood.
Recognition that activating calcium-sensing receptor mutations result in a Bartter-like syndrome demonstrate that the transport effects of extracellular calcium are mediated by the calcium-sensing receptor. Its presence in the gills and solute and water-transporting organs of fish coupled with appropriate calcium-sensing receptor kinetics indicate that the calcium-sensing receptor was originally involved in the regulation of sodium chloride, calcium and magnesium transport. Based on its physiological effects on tubular transport and biochemical and genetic data, the calcium-sensing receptor appears to act by mechanisms that distinguish it from other G protein-coupled receptors.
The calcium-sensing receptor mediates the effects of extracellular calcium on the kidney, is an essential control point in the regulation of calcium balance and possibly the physiological regulation of sodium chloride balance. The thick ascending limb of Henle and distal convoluted tubule appear to be the nephron segments most responsible for the effects of the calcium-sensing receptor, although its mechanisms of action are not fully established.
... In the scenario shown in D, the C-terminus of another membrane protein (e.g. a channel) interacts with the C-terminus of the receptor, and the receptor binds an additional protein. [21][22][23][24][25][26][27][28][29][30][31]31]. These differences between the responses of the distal nephron to Ca and AII indicate that the two receptors have distinct effects on cells despite the fact that they stimulate production of the same second messengers. ...
Introduction
Signalling by the Ca receptor
Distinct effects of angiotensin II and Ca receptors
Receptor activity modifying proteins (RAMPS)
Filamin
Potassium channels
Other CaR‐interacting proteins
Conclusions
Abstract
Seven membrane‐spanning, or G protein‐coupled receptors were originally thought to act through het‐erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling ‘personalities’. The extracellular Ca‐sensing receptor (CaR) signals via Gα i , Gα q and Gα 12/13 , but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR‐interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein‐coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR‐based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling.
Chronic infusion of subpressor level of Angiotensin II (AngII) increases the abundance of Na ⁺ transporters along the distal nephron, balanced by suppression of Na ⁺ transporters along the proximal tubule and medullary thick ascending limb (defined as proximal nephron), which impacts K ⁺ handling along the entire renal tubule. The objective of this study was to quantitatively assess the impact of chronic AngII on the renal handling of Na ⁺ and K ⁺ in female rats, using a computational model of the female rat renal tubule. Our results indicate that downregulation of proximal nephron Na ⁺ reabsorption (T Na ), which occurs in response to AngII-triggered hypertension, involves changes in both transporter abundance and trafficking. Our model suggests that substantial (~ 30%) downregulation of active NHE3 in proximal tubule (PT) microvilli is needed to reestablish the Na ⁺ balance at 2 weeks of AngII infusion. The 35% decrease in SGLT2, a known NHE3 regulator, may contribute to this downregulation. Both depression of proximal nephron T Na and stimulation of distal ENaC raise urinary K ⁺ excretion in AngII-treated females, while K ⁺ loss is slightly mitigated by cortical NKCC2 and NCC upregulation. Our model predicts that K ⁺ excretion may be more significantly limited during AngII infusion by ROMK inhibition in the distal nephron, and/or KCC3 upregulation in the PT, which remain open questions for experimental validation. In summary, our analysis indicates that AngII hypertension triggers a series of events from distal T Na stimulation followed by compensatory reduction in proximal nephron T Na and accompanying adjustments to limit excessive K ⁺ secretion.
The thick ascending limb of the loop of Henle (TAL) is the first segment of the distal nephron, extending through the whole outer medulla and the cortex, two regions with different composition of the peritubular environment. The TAL plays a critical role in the control of NaCl, water, acid, and divalent cation homeostasis, as illustrated by the consequences of the various monogenic diseases that affect the TAL. It delivers tubular fluid to the distal convoluted tubule and thereby affects the function of the downstream tubular segments. The TAL is commonly considered as a whole. However, many structural and functional differences exist between its medullary and cortical parts. The present review summarizes the available data regarding the similarities and differences between the medullary and cortical parts of the TAL. Both sub-segments reabsorb NaCl, have a high Na,K-ATPase activity and a negligeable water permeability; however, they express distinct isoforms of NKCC2 at the apical membrane. Ammonia and bicarbonate are mostly reabsorbed in the medullary TAL, whereas calcium and magnesium are mostly reabsorbed in the cortical TAL. The peptidic hormone receptors controlling transport in the TAL are not homogeneously expressed along the cortical and medullary TAL. Besides this axial heterogeneity, structural and functional differences are also apparent between species, which underscores the link between properties and role of the TAL under various environments.
The sodium-potassium-adenosinetriphosphatase (Na⁺-K⁺-ATPase), or sodium pump, belongs to the P-type ATPases family (also called E1, E2-ATPases). This family of ATPases comprises active transporters involved in unidirectional transport or exchange of monovalent (H⁺, Na⁺, K⁺) or divalent (Ca²⁺, Cu²⁺, Mg²⁺) ions. Transient phosphorylation during the catalytic cycle is the major characteristic of the P-type ATPase family. The Na⁺-K⁺-ATPase is a heteromeric integral membrane protein made of a main catalytic subunit, the α subunit, and a smaller glycosylated subunit, the β subunit. The Na⁺-K⁺-ATPase is present on the surface of every animal cell where its main function is to pump three intracellular sodium ions (Na⁺) out of the cell and two extracellular potassium ions (K⁺) within the cell. This ion transport performed against electrochemical Na⁺ and K⁺ gradients existing across the cell membrane requires energy provided by adenosine triphosphate (ATP) hydrolysis. The ion exchange performed by Na⁺-K⁺- ATPase maintains a high intracellular KC concentration and a low intracellular Na+ concentration, and therefore participates in the resting potential of the cell. In specialized cells, Na⁺-K⁺-ATPase deserves additional major functions: (i) in excitable cells, Na⁺-K⁺-ATPase is essential for potassium ion re-uptake from the interstitial space during the repolarization phase (1) and (ii) in epithelial cells the polarized distribution of Na+-KC- ATPase energizes most reabsorption and secretion processes (2-6).
Ion motive ATPases involved in transcellular ion transport by renal epithelial cells include two molecular families: P-ATPases (Na,K-ATPase and H,K-ATPase) and V-ATPases. Na,K-ATPase is found in the plasma membrane of every vertebrate cell and exchanges three intracellular Na + for two intracellular K + for each hydrolyzed ATP molecule. Na,K-ATPase is highly expressed in the kidney tubule where it is located in the basolateral membrane and energizes Na + reabsorption. Na,K-ATPAse is highly regulated by hormones and local factors via modulation of its rate of synthesis, degradation and post translational events including regulatory phosphorylation and intracellular trafficking. Both gastric and non-gastric H,K-ATPases are expressed in the distal part of the kidney tubule where they reabsorb K + and secrete H + . Vacuaolar H-ATPases is found in intracellular compartments of every renal epithelial cell. In intercalated cells of connecting tubules and collecting ducts, it is localized in plasma membrane where it plays a key role in acid–base transport.
The renin-angiotensin system (RAS) is a master regulator of blood pressure and fluid homeostasis. This system is a multi-enzymatic cascade in which angiotensinogen, the major substrate, is processed in a two-step reaction by renin and angiotensin-converting enzyme (ACE), resulting in the sequential generation of angiotensins I and II. In recent years, several new enzymes, peptides, and receptors in this system have been identified, manifesting a complexity that was previously unappreciated. Although appropriate activation of the RAS is vital for preventing circulatory collapse and maintaining intravascular fluid balance, dysregulation and/or persistent RAS activation can lead to inappropriate blood pressure elevation, target organ damage, and even reduced survival. Accordingly, pharmacological agents that inhibit the synthesis or activity of angiotensin II are effective and widely used anti-hypertensive agents that can ameliorate morbidity and mortality in cardiovascular diseases including congestive heart failure and slow the progression of a wide range of progressive kidney diseases including diabetic nephropathy. Angiotensin receptor blockers (ARBs), which block type 1 (AT1) receptors, are similarly effective for treating these disorders. Below, we provide a comprehensive overview of the physiology of the RAS with an emphasis on actions impacting the kidney.
The effectors and receptors that make up the renin-angiotensin system (RAS) mediate a vast number of nonrenal physiologic effects throughout the body, notably in the heart, vasculature, brain, lung, and adrenal gland. Within the kidney, the RAS regulates renal hemodynamics, tubular transport, inflammation, and cell growth and differentiation. Perhaps the most well-known effect of the RAS in the kidney is the action of angiotensin (Ang II) on the renal vasculature. Ang II functions as a vasoconstrictor in the interlobular artery and both the afferent and efferent glomerular arterioles but with greater potency in the efferent arteriole. As a result, Ang II's net effect on the glomerulus is to elevate the intraglomerular capillary pressure. Ang II also constricts the glomerular mesangium and enhances the afferent arteriole response to tubuloglomerular feedback. These actions have contrasting effects on the glomerular filtration rate (GFR). The preferential vasoconstriction of the efferent arteriole and resulting increase in glomerular hydrostatic pressure tend to preserve GFR under states of RAS activation, but mesangial, afferent, and systemic vasoconstriction lower renal blood flow and therefore decrease GFR. The net result is generally an increase in filtration fraction, owing to a relatively smaller decrease in GFR than renal blood flow.
The aim of this project was to investigate the interaction between the calcium-sensing receptor (CaSR) and proton extrusion by the V-ATPase and gastric-like isoform of the H(+)/K(+)-ATPase in the mouse nephron. Biochemical activity of H(+)- ATPases was analysed using a partially purified membrane fraction of mouse cortex and outer medullary region. The V-ATPase activity (sensitive to 10(-7) mol·L(-1) bafilomycin) from the cortical and outer medullary region was significantly stimulated by increasing the [Formula: see text] (outside Ca(2+)), in a dose-dependent pattern. Gastric H(+)/K(+)-ATPase activity (sensitive to 10(-5) mol·L(-1) Schering 28080) was also sensitive to changes in [Formula: see text] levels. A significant increase in V-ATPase activity was also observed when CaSR was stimulated with agonists such as 300 μmol·L(-1) Gd(3+) and 200 μmol·L(-1) neomycin, both in the cortex and outer medulla. The cortical and outer medullary gastric H(+)/K(+)-ATPase activity was also stimulated by Gd(3+) and neomycin. Finally, cortical V-ATPase activity was significantly stimulated by 10(-9) mol·L(-1) angiotensin II, and the stimulation of CaSR in the presence of angiotensin significantly enhanced this effect, suggesting that an interaction in the intracellular signaling pathways is involved. In summary, CaSR stimulation enhances the biochemical activity of V-ATPase and gastric H(+)/K(+)-ATPase in both the cortical and outer medullary region of mouse kidney.
Dehydration and acute reductions of blood pressure increases ADH and Ang II levels. These hormones increase transport along the distal nephron. In the thick ascending limb (TAL) ADH increases transport via cAMP, while Ang II acts via superoxide (O2-). However, the mechanism of interaction of these hormones in this segment remains unclear. The aim of this study was to explore ADH/Ang II interactions on TAL transport. For this, we measured the effects of ADH/Ang II, added sequentially to TAL suspensions from Wistar rats, on oxygen consumption (QO2) -as a transport index-, cAMP and O2-. Basal QO2 was 112+-5 nmol O2/min/mg protein. Addition of ADH (1nM) increased QO2 by 227 percent. In the presence of ADH, Ang II (1nM) elicited a QO2 transient response. During an initial 3.1+-0.7 minutes after adding Ang II, QO2 decreased 58 percent (p less than 0.03 initial vs. ADH) and then rose by 188 percent (p less than 0.03 late vs initial Ang II). We found that Losartan blocked the initial effects of Ang II and the latter blocked ADH and forskolin-stimulated cAMP. The NOS inhibitor L-NAME or the AT2 receptor antagonist PD123319 showed no effect on transported related oxygen consumption. Then, we assessed the late period after adding Ang II. The O2- scavenger tempol blocked the late Ang II effects on QO2, while Ang II increased O2- production during this period. We conclude that 1) Ang II has a transient effect on ADH-stimulated transport; 2) this effect is mediated by AT1 receptors; 3) the initial period is mediated by decreased cAMP and 4) the late period is mediated by O2-.
Impaired renal sodium excretion causes sodium retention, which prevents the nocturnal dip in blood pressure (BP); thus, high BP persists until excess sodium is excreted. The authors defined "dipping time" (DT) as the duration until the nocturnal BP falls below 90% of the daytime average. Diuretic (e.g., hydrochlorothiazide [HCTZ]) and angiotensin receptor blocker (ARB) are able to eliminate sodium retention and restore the non-dipper BP rhythm. Reanalysis of two previous studies demonstrate that HCTZ and ARB shortened the DT. Shortening DT correlated directly with the increase in daytime urinary sodium excretion (Study 2). DT can be used as a preliminary indicator of sodium retention. (Author correspondence: m-fukuda@med.nagoya-cu.ac.jp ).
Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.
The results of recent outcome trials challenge hypotheses that tight control of both glycohemoglobin and blood pressure diminishes macrovascular events and survival among type 2 diabetic patients. Relevant questions exist regarding the adequacy of glycohemoglobin alone as a measure of diabetes control. Are we ignoring mechanisms of vasculotoxicity (profibrosis, altered angiogenesis, hypertrophy, hyperplasia, and endothelial injury) inherent in current antihyperglycemic medications? Is the polypharmacy for lowering cholesterol, triglyceride, glucose, and systolic blood pressure producing drug interactions that are too complex to be clinically identified? We review angiotensin-aldosterone mechanisms of tissue injury that magnify microvascular damage caused by hyperglycemia and hypertension. Many studies describe interruption of these mechanisms, without hemodynamic consequence, in the preservation of function in type 1 diabetes. Possible interactions between the renin-angiotensin-aldosterone system and physiologic glycemic control (through pulsatile insulin release) suggest opportunities for further clinical investigation.
Ammonia absorption by the medullary thick ascending limb of Henle's loop (MTALH) is thought to be a critical step in renal ammonia handling and excretion in urine, in which it is the main acid component. Basolateral Na+/H+ exchangers have been proposed to play a role in ammonia efflux out of MTALH cells, which express 2 exchanger isoforms: Na+/H+ exchanger 1 (NHE1) and NHE4. Here, we investigated the role of NHE4 in urinary acid excretion and found that NHE4-/- mice exhibited compensated hyperchloremic metabolic acidosis, together with inappropriate urinary net acid excretion. When challenged with a 7-day HCl load, NHE4-/- mice were unable to increase their urinary ammonium and net acid excretion and displayed reduced ammonium medulla content compared with wild-type littermates. Both pharmacologic inhibition and genetic disruption of NHE4 caused a marked decrease in ammonia absorption by the MTALH. Finally, dietary induction of metabolic acidosis increased NHE4 mRNA expression in mouse MTALH cells and enhanced renal NHE4 activity in rats, as measured by in vitro microperfusion of MTALH. We therefore conclude that ammonia absorption by the MTALH requires the presence of NHE4 and that lack of NHE4 reduces the ability of MTALH epithelial cells to create the cortico-papillary gradient of NH3/NH4+ needed to excrete an acid load, contributing to systemic metabolic acidosis.
Angiotensin II (Ang II) acutely stimulates thick ascending limb (TAL) NO via an unknown mechanism. In endothelial cells, activation
of Ang II type 2 receptor (AT2) stimulates NO. Akt1 activates NOS3 by direct phosphorylation. We hypothesized that Ang II stimulates TAL NO production via
AT2-mediated Akt1 activation, which phosphorylates NOS3 at serine 1177. We measured NO production by fluorescence microscopy.
In isolated TALs, Ang II (100 nm) increased NO production by 1.1 ± 0.2 fluorescence units/min (p < 0.01). Ang II increased cGMP accumulation by 4.9 ± 1.3 fmol/μg (p < 0.01). Upon adding the AT2 antagonist PD123319 (1 μm), Ang II failed to stimulate NO (0.1 ± 0.1 fluorescence units/min; p < 0.001 versus Ang II); adding the AT1 antagonist losartan (1 μm) resulted in Ang II stimulating NO by 0.9 ± 0.1 fluorescence units/min. Akt inhibitor (5 μm) blocked Ang II-stimulated NO (−0.1 ± 0.2 fluorescence units/min versus inhibitor alone). Phospho-Akt1 increased by 72% after 5 min (p < 0.006), returning to basal after 10 min. Phospho-Akt2 did not change after 5 min but increased by 115 and 163% after 10
and 15 min (p < 0.02). Phospho-Akt3 did not change. An AT2 agonist increased pAkt1 by 78% (p < 0.02), PI3K inhibition blocked this effect. In TALs transduced with dominant negative Akt1, Ang II failed to stimulate
NO (0.1 ± 0.2 fluorescence units/min versus 1.2 ± 0.2 for controls; p < 0.001). Ang II increased phospho-NOS3 at serine 1177 by 130% (p < 0.01) and 150% after 5 and 10 min (p < 0.02). Ang II increased phosphoNOS3 at serine 633 by 50% after 5 min (p < 0.01). Akt inhibition prevented NOS3 phosphorylation. We concluded that Ang II enhances TAL NO production via activation
of AT2 and Akt1-dependent phosphorylation of NOS3 at serines 1177 and 633.
Besides the importance of the renin-angiotensin system (RAS) in the circulation and other organs, the local RAS in the kidney has attracted a great attention in research in last decades. The renal RAS plays an important role in the body fluid homeostasis and long-term cardiovascular regulation. All major components and key enzymes for the establishment of a local RAS as well as two important angiotensin II (Ang II) receptor subtypes, AT1 and AT2 receptors, have been confirmed in the kidney. In additional to renal contribution to the systemic RAS, the intrarenal RAS plays a critical role in the regulation of renal function as well as in the development of kidney disease. Notably, kidney AT1 receptors locating at different cells and compartments inside the kidney are important for normal renal physiological functions and abnormal pathophysiological processes. This mini-review focuses on: 1) the local renal RAS and its receptors, particularly the AT1 receptor and its mechanisms in physiological and pathophysiological processes; and 2) the chemistry of the selective AT1 receptor blocker, losartan, and the potential mechanisms for its actions in the renal RAS-mediated disease.
Renal medullary superoxide (O2-) increases in angiotensin (Ang) II-dependent hypertension. O2- increases thick ascending limb Na transport, but the effect of Ang II-dependent hypertension on the thick ascending limb is unknown. We hypothesized that Ang II-dependent hypertension increases thick ascending limb NaCl transport because of enhanced O2- production and increased protein kinase C (PKC) α activity. We measured the effect of Ang II-dependent hypertension on furosemide-sensitive oxygen consumption (a measure of Na transport), O 2- production, and PKCα translocation (a measure of PKCα activity) in thick ascending limb suspensions. Ang II-dependent hypertension increased furosemide-sensitive oxygen consumption (26.2±1.0% versus 36.6±1.2% of total oxygen consumption; P<0.01). O 2- was also increased (1.1±0.2 versus 3.2±0.5 nmol of O2-/min per milligram of protein; P<0.03) in thick ascending limbs. Unilateral renal infusion of Tempol decreased O2- (2.4±0.4 versus 1.2±0.2 nmol of O2-/min per milligram of protein; P<0.04) and furosemide-sensitive oxygen consumption (32.8±1.3% versus 24.0±2.1% of total oxygen consumption; P<0.01) in hypertensive rats. Tempol did not affect O2- or furosemide-sensitive oxygen consumption in normotensive controls and did not alter systolic blood pressure. Ang II-dependent hypertension increased PKCα translocation (5.7±0.3 versus 13.8±1.4 AU per milligram of protein; P<0.01). Unilateral renal infusion of Tempol reduced PKCα translocation (5.0±0.9 versus 10.4±2.6 AU per milligram of protein; P<0.04) in hypertensive rats. Unilateral renal infusion of the PKCα inhibitor Gö6976 reduced furosemide-sensitive oxygen consumption (37.4±1.5% versus 25.1±1.0% of total oxygen consumption; P<0.01) in hypertensive rats. We conclude that Ang II-dependent hypertension enhances thick ascending limb Na transport-related oxygen consumption by increasing O2- and PKCα activity.
Cells in the renal medulla exist in a hostile milieu characterized by wide variations in extracellular solute concentrations, low oxygen tensions, and abundant reactive oxygen species. This article reviews the strategies adopted by these cells to allow them to survive and fulfill their functions under these extreme conditions.
Previous micropuncture studies have reported nanomolar concentrations of angiotensin II in proximal tubular fluid and have indicated that angiotensin II or a precursor may be secreted into the tubular lumen. Further experiments were performed to determine if proximal tubular fluid angiotensin I concentrations are also greater than plasma and kidney levels and to estimate the degree of intrarenal compartmentalization of the angiotensin peptides. Free-flow proximal tubular fluid samples were collected in micropipets and were pooled for each animal. At the end of each experiment, a blood sample was collected and the micropunctured left kidney was harvested and homogenized in methanol. The angiotensin I concentration in proximal tubular fluid samples averaged 6.1 +/- 1.2 pmol/mL, whereas the angiotensin II concentration averaged 8.1 +/- 1.6 pmol/mL (N = 13). HPLC analysis of a separate sample pooled from collections in five rats indicated that the immunoreactive angiotensin I and angiotensin II primarily represented authentic angiotensin I and II. Plasma concentrations of angiotensin I and angiotensin II averaged 0.39 +/- 0.09 and 0.15 +/- 0.03 pmol/mL, respectively. The kidney contents of angiotensin I and angiotensin II were 1.28 +/- 0.24 and 0.97 +/- 0.17 pmol/g of kidney, respectively. These findings indicate that proximal tubular fluid contains nanomolar concentrations of angiotensin I as well as angiotensin II. These high tubular fluid concentrations, which greatly exceed the plasma and kidney levels, likely reflect net secretion of the angiotensin peptides by proximal tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of angiotensin 1-7 (Ang 1-7) on the proximal tubule has not been well studied. It was hypothesized that Ang 1-7 has a biphasic effect on fluid absorption in the isolated rat proximal straight tubule. Proximal straight tubules were perfused at a rate of 5.81 +/- 0.44 nL/mm per minute and absorbed fluid at 0.98 +/- 0.10 nL/mm per minute. Bicarbonate absorption was 80.1 +/- 11.6 pmol/mm per minute. When 10(-12) M Ang 1-7 was added to the bath, fluid absorption increased to 1.47 +/- 0.10 nL/mm per minute (P < 0.013) and bicarbonate increased to 115.0 +/- 12.8 pmol/mm per minute (P < 0.004). Ang 1-7 had no effect on either the maximum rate of bicarbonate absorption (P > 0.90) or bicarbonate permeability (P > 0.60). Next, 10(-8) M Ang 1-7 was used. During the control period, fluid absorption was 0.90 +/- 0.09 nL/mm per minute. When 10(-8) M Ang 1-7 was added, fluid absorption decreased to 0.62 +/- 0.04 nL/mm per minute (P < 0.05). DuP 753, an AT1 receptor antagonist, blocked both effects induced by Ang 1-7, whereas PD 123319, an AT2 receptor antagonist, did not block the stimulatory effect. From these data, it was concluded that Ang 1-7 binds AT1 receptors and has a biphasic effect on fluid absorption, and at physiologic levels, the heptapeptide induces the stimulation of bicarbonate absorption.
We have demonstrated in previous studies that luminal administration of low doses of angiotensin II (ANG II) stimulate and high doses of ANG II inhibit fluid and HCO3- transport in proximal tubules of rat kidney. However, the role of ANG II on Na+ and HCO3- transport in the distal nephron has not yet been fully elucidated. The superficial early and late distal tubules (DT) of the nephron segments correspond to the distal convoluted tubule and initial collecting tubule. Accordingly, we investigated the effects of ANG II on Na+, HCO3-, and K+ transport in the early and late DT by separate perfusion of these tubule segments in vivo. [3H]inulin, Na+, K+, and total CO2 concentrations were measured in the perfusate and collected fluid, and transport of sodium (JNa), bicarbonate (JHCO3), potassium (JK), and fluid (JV) were analyzed as an index of the hormone effect. Intravenous infusion of the ANG II receptor antagonist [Sar1,Ile8]ANG II (1 microgram.kg-1.min-1) decreased JV, JNa, and JHCO3 in the early DT and decreased Jv and JNa in the late DT. Addition of ANG II (10(-11) M) to the tubular perfusate significantly increased the Jv, JNa, and JHCO3 in the early DT. Similar studies in late DT demonstrated an increase in Jv and JNa, decrease in JK, but no effect on JHCO3. The effects of ANG II on fluid and ion transport were abolished by the luminal application of amiloride (10(-3) M) and of the angiotensin-receptor blocker [Sar1,Ile8]ANG II (10(-6) M). These results suggest that ANG II stimulates Na+/H+ exchange in the early DT (distal convoluted tubule) and amiloride-sensitive Na+ transport (Na+ channels) in the late DT (initial collecting tubule) of cortical nephrons.
Bicarbonate reabsorption was evaluated by stationary microperfusion "in vivo" early distal (ED) and late distal (LD) segments of at kidney. Intratubular pH was recorded by double-barreled of H+ exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of angiotensin II (ANG II) (10(-12) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.930 +/- 0.060 to 2.64 +/- 0.210 nmol.cm-2.s-1 in luminally perfused tubules and from 0.850 +/- 0.040 to 2.03 +/- 0.210 nmol.cm-2.s-1 during capillary perfusion) and LD segments from 0.310 +/- 0.130 to 2.16 +/- 0.151 nmol.cm-2.s-1 during luminal perfusion and from 0.530 +/- 0.031 to 2.16 +/- 0.211 nmol.cm-2.s-1 with capillary perfusion). The addition of the AT1-receptor antagonist losartan (10(-6) M) to luminal perfusion blocked luminal ANG II-mediated stimulation in ED and LD segments. 5-(N,N-hexamethylene)amiloride (10(-4) M) added to luminal perfusion inhibited luminal ANG II-mediated stimulation in ED (by 81%) and LD (by 54%) segments. The addition of bafilomycin A1 (2 x 10(-7) M) to luminal perfusion does not affect luminal ANG II-mediated stimulation in ED segments but reduces it in LD segments (by 33%). During the addition of atrial natriuretic peptide (ANP) (10(-6) M) or ANG II plus ANP in both segments, no significant differences in HCO3- reabsorption were observed. Our results indicate that luminal ANG II acts to stimulate Na+/H+ exchange in ED and LD segments via activation of AT1 receptors, as well as the vacuolar H(+)-adenosinetriphosphatase in LD segments. ANP does not affect HCO3- reabsorption in either ED or LD segments and does not impair the stimulation caused by ANG II.
The effects of angiotensin II (ANG II) on tumor necrosis factor-alpha (TNF) production were determined in freshly isolated tubules from the medullary thick ascending limb (MTAL). ANG II (10(-9) M) increased the accumulation of TNF mRNA associated with enhanced production of TNF by approximately five- to sixfold. ANG II also increased prostaglandin E2 (PGE2) production by the MTAL in a dose-dependent manner and exerted biphasic differential effects on 86Rb uptake, depending on the exposure time of the tubules to the peptide and the doses used. Low-dose ANG II (10(-11) M) increased 86Rb uptake by MTAL tubules after a "short-term" (15 min) challenge, whereas uptake was inhibited after a "long-term" (3 h) incubation period. High-dose ANG II (10(-6) M) inhibited MTAL 86Rb uptake, irrespective of incubation time. Uptake of 86Rb was inhibited by approximately 60% in MTAL tubules that were challenged for 3 h with ANG II. The inhibitory action of ANG II was prevented by eliminating the participation of either TNF with antisera to the cytokine or PGE2 by inhibition of cyclooxygenase with indomethacin. We conclude that ANG II regulates TNF production in the MTAL, an interaction that affects 86Rb uptake via an eicosanoid-dependent mechanism in this nephron segment.
Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na(+)-K+(NH4+)-2Cl- cotransport. ANG II at 10(-16) to 10(-12) M inhibited 30-50% (P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10(-12) M ANG II level cotransport activity; eventually, 10(-6) M ANG II stimulated 34% cotransport activity (P < 0.003). Inhibition by 10(-12) M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10(-12) M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10(-6) M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus < or = 10(-12) M ANG II inhibits via 20-HETE, whereas > or = 5 x 10(-11) M ANG II stimulates via PKC Na(+)-K+(NH4+)-2Cl- cotransport; all ANG II effects involve AT1 receptors and PLC activation.
Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na-Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 +/- 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 +/- 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 +/- 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.
Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of alphaENaC protein without increasing the abundance of the beta and gamma subunits. Thus, alphaENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of gammaENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport.
The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na+ transporters. Rats were on a fixed intake of Na+ and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng x min(-1) x kg(-1) ANG II (sc). Semiquantitative immunoblotting revealed that Na+/H+ exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 +/- 28 (ANG II-50, n = 5), 166 +/- 23 (ANG II-100, n = 7), and 167 +/- 19% (ANG II-200, n = 4) of control levels (n = 6, P < 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (BSC-1) in the ISOM was also increased to 187 +/- 28 (ANG II-50), 162 +/- 23 (ANG II-100), and 166 +/- 19% (ANG II-200) of control levels (P < 0.05), but there were no changes in the abundance of Na(+)-K(+)-ATPase and the electroneutral Na(+)-HCO3 cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng x min(-1) x kg(-1) for 5 days, n = 6) had a higher urinary pH compared with controls. NH4Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na+ and HCO3 reabsorption in response to ANG II.
We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20-HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50-100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.
Inherited renal tubular disorders associated with hypokalemic alkalosis (Bartter-like syndromes) can be subdivided into at least three clinical phenotypes: (i) the hypocalciuric-hypomagnesemic Gitelman variant; (ii) the classic variant; and (iii) the antenatal hypercalciuric variant (also termed hyperprostaglandin E syndrome). Mutations in the Na-Cl cotransporter (NCCT) underlie the pathogenesis of the Gitelman variant and mutations in the Na-K-2Cl cotransporter (NKCC2) have recently been identified in the antenatal hypercalciuric variant. We now describe mutations in the gene encoding the inwardly-rectifying potassium channel, ROMK, in eight kindreds with the antenatal variant of Bartter syndrome. These findings indicate that antenatal Bartter syndrome is genetically heterogeneous and provide new insights into the molecular pathogenesis of Bartter-like syndromes.
1. Two methods of titrating chloride with Ag+ ion using the potentiometric end-point are described.
2. The first method is conventional in that silver nitrate is added from a burette. It deals with volumes down to 0.2µl. and can measure 1µg. of chloride with an error of < ± 1% (standard deviation).
3. According to the second method Ag+ ion is added by passing a current through a silver electrode in series with a condenser. The charge developed on the condenser is a measure of the amount of chloride titrated. This method deals with volumes down to 0.5 x 10-3µl. and can measure 10-4µg. of chloride with an error of < ± 1% (standard deviation).
4. As far as is known these methods are not susceptible to interference from other substances likely to be present in biological fluids.
For purposes of determining the freezing-point of small volumes of aqueous solutions the difficulties of undercooling are avoided by first freezing the sample and then determining the thawing-point. Apparatus and procedure specially designed for simplicity of construction and operation are described. The method works best with volumes of the order of 10-3 to 10-4 mm3 and its accuracy in terms of standard deviation is ±0.003° C for freezing-point depressions of the order of 1 to 2° C.
Micropuncture experiments on the rat kidney were performed to evaluate the effect of angiotensin upon tubular sodium reabsorption.
Half time reabsorption as measured by the split droplet method was constant (9.4±0.4 sec) at glomerular filtration rates ranging from 0.66–1.26 ml/g kidney/min, and remained unchanged when peritubular or tubular angiotensin concentration was elevated, indicating no direct effect of angiotensin upon transporting capacity of the proximal tubule for sodium.
Sodium reabsorption in Henle's loop was studied by means of perfusing single loops with isotonic saline. From the increase in inulin concentration in the perfusat and from the sodium concentration in the early distal segment sodium reabsorption along Henle's loop was calculated. The correlation between perfusion rate and sodium reabsorption was found to be the same in the control experiments and during the perfusion with saline containing angiotensin with concentration of 0.5 and 5.0×10−6 g/100 ml.
These results together with the findings that urine volume and sodium excretion in the anaesthetized rat remain unchanged during the i.v. infusion of angiotensin indicate that angiotensin has no direct effect upon tubular transporting capacity for sodium.
Changes in tubular sodium reabsorption due to angiotensin, as calculated from clearance data, are considered to be the effect of an indirect action of angiotensin upon tubular sodium reabsorption.
This study was designed to improve and validate methods for the accurate and consistent quantitation of angiotensin (ANG) I and II levels in rat kidney and to determine the effects on renal ANG I and II of changes in dietary sodium intake and ANG-converting enzyme (ACE) inhibition. Kidneys from pentobarbital-anesthetized rats were rapidly removed and homogenized in methanol before extraction and purification of ANG peptides by solid-phase extraction and high-performance liquid chromatography (HPLC). Recoveries of 125I-ANG I and II were greater than 80%. Reversed-phase HPLC of the partially purified methanol extract showed that greater than 75% of the ANG I- and greater than 82% of the ANG II-like immunoreactivity coeluted with ANG I and II, respectively. Dietary sodium deprivation (0.003 meq/g) and excess (1.34 meq/g) for 7 days significantly (P less than 0.01) increased and decreased renal ANG I (296 +/- 30 and 82.6 +/- 15.8 vs. 161 +/- 18 fmol/g) and ANG II (216 +/- 16 and 45.6 +/- 11.8 vs. 98 +/- 16 fmol/g) contents, respectively. Plasma ANG I and II levels showed similar changes. ACE activity was significantly upregulated by sodium deprivation in both kidney (44% increase) and plasma (30% increase). In rats fed normal chow, infusion of enalaprilat for 1 h abolished plasma ACE activity but decreased renal ACE activity by only 58%. ACE inhibition increased renal and plasma ANG I levels 2.8- and 12-fold, respectively, and decreased renal and plasma ANG II levels 75-78%.(ABSTRACT TRUNCATED AT 250 WORDS)
Previously, we demonstrated that adenosine (Ado) was released by the medullary thick ascending limb (MTAL) during hypoxia. The present experiments were designed to examine the effects of Ado and adenosine analogues on net chloride (JCl) and bicarbonate (JHCO3) absorption by the isolated, perfused MTAL of the rat. Ado, 10 nM, in the presence or absence of arginine vasopressin (AVP, 10(-10) M) reduced JCl by 50%. The inhibition of Ado was reproduced with the selective A1 agonist, N-6-phenylisopropyladenine (2 nM), and was reversed by 8-cyclopentyl-1,3-dipropylxanthine, an A1-receptor antagonist. Thus the inhibition of JCl is likely mediated through A1 receptors. In contrast, Ado had no effect on (JHCO3) either in the presence or absence of AVP. Ado also had no influence on the effect of AVP to inhibit JHCO3. The lack of effect on JHCO3 suggests that the inhibition of JCl by Ado is unlikely to be mediated through changes in cellular adenosine 3',5'-cyclic monophosphate. These results support the hypothesis that Ado released into the renal medulla during hypoxia may protect the MTAL from ischemic injury by directly inhibiting NaCl absorption and reducing transport-related oxygen consumption.
The effect of angiotensin on HCO3- absorption, fluid absorption, and Na+/K+ ATPase activity in isolated rat proximal straight tubules was investigated. During the control period, tubules absorbed fluid at 0.66 +/- 0.12 nL/mm.min and bicarbonate at 60.2 +/- 10.7 pmol/mm.min. After 10(-10) M angiotensin was added to the bath, tubules absorbed fluid at 0.93 +/- 0.19 nL/mm.min and bicarbonate at 77.4 +/- 15.2 pmol/mm.min, indicating stimulation of both parameters. Time controls showed no significant change in the rate of bicarbonate or fluid absorption. To determine whether this stimulation was due to an increase in the maximum rate of transport, tubules were perfused at greater than or equal to 20 nL/mm.min. During the control period, tubules absorbed bicarbonate at 82.5 +/- 13.0 pmol/mm.min. After 10(-10) M angiotensin was added to the bath, these same tubules absorbed bicarbonate at 75.9 +/- 11.9 pmol/mm.min. Thus, angiotensin did not alter the maximum rate of transport. Angiotensin also had no effect on bicarbonate permeability, which was 1.1 +/- 0.2 x 10(-4) cm/s before treatment and 1.3 +/- 0.3 x 10(-4) cm/s afterward. Finally, the effect of angiotensin on Na+/K+ ATPase activity was measured in paired experiments. Na+/K+ ATPase activity of control tubules was 36 +/- 6 pmol of ADP/mm.min; after angiotensin treatment, it was 47 +/- 6 pmol ADP/mm.min. From these data it was concluded that: (1) angiotensin stimulates bicarbonate absorption in the rat proximal straight tubule; (2) this stimulation is the result of a change in Km rather than of an increase in the maximum rate of transport or permeability; and (3) angiotensin directly stimulates Na+/K+ ATPase activity in the proximal nephron.
Angiotensin II has recently been shown to exert potent control over sodium and water absorption in the proximal convoluted tubule. This transport stimulation is effected by receptors on both the luminal and basolateral membranes of cells located predominantly in the early, S1 proximal tubule. Angiotensin II increases transport primarily by a Gi protein-mediated reduction in intracellular cyclic adenosine monophosphate, which enhances the affinity of the Na(+)-H+ antiporter. Change in early proximal acidification ultimately causes alteration in the amount of sodium chloride leaving the proximal tubule and entering the urine. These direct tubular transport actions by angiotensin II may participate importantly in various physiological actions of the kidney, including the renal response to change in dietary sodium intake and in extracellular volume, as well as in pathophysiological processes such as hypertension.
To examine angiotensin (ANG) concentrations in fluid compartments near known intrarenal ANG receptors, we measured ANG concentrations in glomerular filtrate (GF), star vessel plasma (SVP), and luminal fluid from the early, mid, and late proximal tubule (E, M, and L PT). Samples were collected from euvolemic Munich-Wistar rats by free-flow micropuncture; ANG concentrations were measured by RIA. In one group of rats, concentrations of total immunoreactive ANG (reflecting ANG II and lesser amounts of three fragments) in GF and E, M, and L PT fluid averaged 29-40 nM compared with 32 pM in systemic plasma. In a second group, immunoreactive ANG concentrations in SVP also exceeded systemic levels by a factor of 1,000. In a final group, samples of GF and LPT fluid were purified by HPLC before RIA to measure ANG II and III concentrations specifically: their respective concentrations were 6-8 nM and 14-25 nM. We interpret these results to indicate that substantial amounts of ANG peptides are released into or generated within intrarenal fluid compartments, in which local ANG is likely to effect regulation of renal function independently of systemic ANG.
We studied the effects of dietary NaCl intake on the renal distal tubule by feeding rats high or low NaCl chow or by chronically infusing furosemide. Furosemide-treated animals were offered saline as drinking fluid to replace urinary losses. Effects of naCl intake were evaluated using free-flow micropuncture, in vivo microperfusion, and morphometric techniques. Dietary NaCl restriction did not affect NaCl delivery to the early distal tubule but markedly increased the capacity of the distal convoluted tubule to transport Na and Cl. Chronic furosemide infusion increased NaCl delivery to the early distal tubule and also increased the rates of Na and Cl transport above the rates observed in low NaCl diet rats. When compared with high NaCl intake alone, chronic furosemide infusion with saline ingestion increased the fractional volume of distal convoluted tubule cells by nearly 100%, whereas dietary NaCl restriction had no effect. The results are consistent with the hypotheses that (a) chronic NaCl restriction increases the transport ability of the distal convoluted tubule independent of changes in tubule structure, (b) high rates of ion delivery to the distal nephron cause tubule hypertrophy, and (c) tubule hypertrophy is associated with increases in ion transport capacity. They indicate that the distal tubule adapts functionally and structurally to perturbations in dietary Na and Cl intake.
To evaluate the effects of angiotensin converting enzyme inhibition (SQ 20881, CEI) on superficial nephron function of the non-clipped kidney in Goldblatt hypertensive rats in the absence of alterations in renal arterial pressure, control renal arterial pressure (RAP) was reduced first to the range generally obtained during CEI (124 +/- 4 mm Hg). RAP was maintained during the CEI period by adjustment of a suprarenal aortic clamp. At the reduced RAP, whole kidney and single nephron glomerular filtration rates (GFR) were reduced from the hypertensive levels and were lower than the measurements in normotensive control rats. During CEI, whole kidney GFR and single nephron GFR increased by 55 and 42%, respectively. There were decreases in absolute as well as fractional proximal reabsorption rates. In the intermediate nephron segment, fractional reabsorption was decreased, but absolute fluid reabsorption increased in proportion to the increased delivery rate. Proximal tubule and peritubular capillary hydrostatic pressures increased significantly during CEI also. These results indicate that an increased activity of the renin-angiotensin system occurring in Goldblatt hypertensive rats subjected to aortic constriction exerts effects to lower GFR and increase proximal reabsorption rate. The concomitant superficial nephron and whole kidney GFR responses to CEI when arterial pressure was maintained suggests that the pre-existing levels of angiotensin exerted similar influences on the total nephron population.
Angiotensin II (ANG II) is one of the body's most powerful regulators of Na excretion, operating through extrarenal mechanisms, such as stimulation of aldosterone secretion, as well as intrarenal mechanisms. Considerable evidence suggests that the intrarenal actions of ANG II are quantitatively more important than changes in aldosterone secretion in the normal day-to-day regulation of Na balance and arterial pressure. ANG II at physiological concentrations increases proximal tubular reabsorption, but further studies are needed to determine whether ANG II also has an important effect on more distal tubular segments. ANG II also markedly constricts efferent arterioles, tending to increase Na reabsorption by altering peritubular capillary physical forces and also helping to prevent excessive decreases in glomerular filtration rate. ANG II may also decrease Na excretion and increase urine concentrating ability by reducing renal medullary blood flow. Regulation of Na excretion by ANG II is closely linked with arterial pressure control and volume homeostasis through the renal pressure natriuresis mechanism. Under many physiological conditions, such as changes in Na intake, ANG II greatly multiplies the effectiveness of the pressure natriuresis mechanism to prevent fluctuations in body fluid volume and arterial pressure. In circumstances associated with circulatory depression, such as decreased cardiac function, reductions in blood pressure and increased ANG II formation cause Na retention until arterial pressure is restored to normal. However, in pathophysiological conditions in which ANG II is inappropriately elevated, increased arterial pressure (hypertension) is required for the kidney to "escape" the potent antinatriuretic actions of ANG II and to return Na excretion to normal via the pressure natriuresis mechanism.
The early proximal convoluted tubule (PCT) is the site of 50% of bicarbonate reabsorption in the nephron, but its control by angiotensin II has not been previously studied. In vivo microperfusion was used in both the early and late PCT in Munich-Wistar rats. Systemic angiotensin II administration (20 ng/kg X min) or inhibition of endogenous angiotensin II activity with saralasin (1 microgram/kg X min) caused profound changes in bicarbonate absorption in the early PCT (169 +/- 25 and -187 +/- 15 peq/mm X min, respectively). Because the bicarbonate absorptive capacity of the early PCT under free-flow conditions is 500 peq/mm X min, angiotensin II administration or inhibition affected greater than 60% of proton secretion in this segment. Both agents less markedly affected bicarbonate absorption in the late PCT (+/- 28 peq/mm X min) or chloride absorption (+/- 68-99 peq/mm X min) in both the early and late PCT. Because of its potential for controlling the majority of bicarbonate absorption in the early PCT (hence greater than or equal to 30% of bicarbonate absorption in the entire nephron), angiotensin II may be a powerful physiologic regulator of renal acidification.
Bicarbonate, ammonia, and fluid transport were studied in isolated perfused proximal straight tubules from rats. The mean rate of fluid absorption (0.77 nl X min-1 X mm-1) and the mean rate of total CO2 absorption (42 pmol X min-1 X mm-1) exceeded corresponding rates measured previously in rabbit proximal straight tubules. The limiting total CO2 concentration when the tubules were perfused at slow flow rates was 5 mM, a value similar to those reported previously for rat proximal convoluted tubules and thick ascending limbs. When rat proximal straight tubules were perfused and bathed with solutions containing 1 mM total ammonia at slow perfusion rates, the measured total ammonia concentration in collected fluid rose to a level predicted by the diffusion trapping model of ammonia secretion in the absence of a luminal disequilibrium pH. We conclude the proximal straight tubule of the rat can absorb bicarbonate at a rate that can account for a large portion of the bicarbonate absorption measured in vivo between the late proximal convoluted tubule and the early distal tubule, the rat proximal straight tubule is capable of transepithelial ammonia secretion, most likely by NH3 diffusion down a concentration gradient generated by luminal acidification, and the rat proximal straight tubule apparently does not generate a luminal disequilibrium pH despite the occurrence of proton secretion, implying the presence of endogenous luminal carbonic anhydrase.
Ammonia and bicarbonate transport by the thick ascending limb of rat kidney was studied to determine whether this segment contributes to the regulation of renal ammonia and net acid excretion. Cortical and medullary thick ascending limbs were perfused in vitro at 1.0-1.5 nl X min-1 X mm-1 with HCO3-buffered solutions. There was no significant net fluid transport. With 4 mM ammonia in bath and perfusate, transepithelial voltage averaged 6-9 mV, lumen positive, and did not differ between the two segments. The mean ammonia concentration in collected tubule fluid was 2.8 mM with cortical segments and 2.3 mM with medullary segments, indicating net absorption of ammonia. Furosemide (10(-4) M) in the perfusate eliminated ammonia absorption in medullary thick ascending limbs and converted net absorption to net secretion in cortical thick ascending limbs. Furosemide reduced transepithelial voltage to near zero in every tubule. Cortical and medullary thick ascending limbs also absorbed bicarbonate, indicating that their tubule fluid was acidified relative to the bath. Therefore, absorption of ammonia could not have occurred by nonionic diffusion. The absorption most likely was due to direct transport of NH4+. The possible mechanisms involved are discussed, and it is proposed that absorption of ammonia by thick ascending limbs provides a source for its accumulation in the renal medulla and secretion into the collecting ducts.
To study receptors for angiotensin II, polyclonal rabbit anti-peptide antisera were prepared against the peptide QDDCPKAGRHC corresponding to amino acids 15-24 of the rat AT1A and AT1B receptors. Western analysis of rat tissues showed a major band of approximately 43 kDa. The antisera immunoprecipitated AT1-receptor protein produced in vitro. Immunohistochemical analysis of rat tissues showed intense staining of arterial and arteriolar smooth muscle. Other tissues that contained AT1-receptor protein included hepatocytes, the zona glomerulosa of the adrenal gland, and the smooth muscle of the bronchus, gut, ureter, and epididymis. In the kidney, intense staining was observed in all small arteries and arterioles. Both afferent and efferent arterioles contain approximately equal intensities of immunoreactive AT1 protein. The inner stripe of the outer medulla has a moderate level of receptors within thick ascending limb epithelium. Proximal tubular epithelium also expresses receptor protein. Glomerular immunoreactive AT1 protein is found within mesangial cells and varies in intensity among different rat strains. Lewis and Wistar rats demonstrated moderate glomerular staining, whereas the CD and Sprague-Dawley strains showed lesser levels of reactivity. The fact that glomerular mesangial cells are the primary locus of angiotensin II action within the glomerulus.
1. Little direct information is available on the actions of angiotensin II beyond the proximal tubule. We therefore studied the effect of a mildly vasoconstrictive dose of angiotensin II on tubular handling of water, sodium (Na+) and lithium (Li+) in rats by means of free-flow micropuncture at the late proximal tubule and the early distal tubule.
2. Endogenous angiotensin II was suppressed by pretreament with enalapril. Compared with a control group, angiotensin II increased mean arterial pressure by 15 mmHg. Glomerular filtration rate decreased from 1.32 ± 0.05 to 1.10 ± 0.05 ml/min, Na+ excretion from 0.43 ± 0.09 to 0.13 ± 0.03 μmol/min, fractional delivery of water at the late proximal tubule from 50.1 ± 1.7 to 42.9 ± 3.2%, fractional delivery of Na+ at the late proximal tubule from 46.5 ± 1.3 to 39.1 ± 3.5% and fractional delivery of water at the early distal tubule from 26.4 ± 1.4 to 21.9 ± 1.0% (P < 0.05 for each variable). Fractional delivery of Na+ at the early distal tubule did not change significantly.
3. Similar experiments were performed during partial aortic constriction to exclude the effects of increased perfusion pressure. The data obtained were similar, except that in this group the fractional delivery of Na+ at the early distal tubule decreased from 8.6 ± 0.7 to 6.8 ± 0.9% (P < 0.05).
4. The relation between late proximal tubule Na+ delivery and Na+ reabsorption between late proximal and early distal tubule was not disturbed by angiotensin II. For water, however, this relation tended to shift to a higher reabsorption rate.
5. The decrease in fractional excretion of Li+ followed the decrease in proximal reabsorption as measured directly by micropuncture. Angiotensin II infusion did not appear to affect Li+ reabsorption beyond the proximal tubule. Distal fractional Na+ reabsorption estimated by the Li+ clearance increased significantly during angiotensin II infusion.
6. In conclusion, our data indicate that a Na+-retaining dose of angiotensin II increases Na+ reabsorption in the proximal tubule, an effect correctly indicated by the change in the fractional excretion of Li+, does not influence Na+ reabsorption in the loop of Henle beyond the proximal tubule, but may increase Na+ reabsorption in more distal segments.
The effect of 6 days' s.c. infusions of angiotensin II at increasing doses was determined on the uptake of rat or human low density lipoprotein (LDL) and of human fibrinogen by aorta in normal and spontaneously hypertensive rats. Rat or human LDL or human fibrinogen was injected i.v. 5 days after the start of infusion, and 24 hr later the radioactivity of aortic walls was determined. Body weight was almost constant in control rats and moderately decreased in a dose-dependent way by angiotensin II. Diastolic blood pressure decreased slightly over 6 days in control rats and increased transiently at the lowest dose of angiotensin II and progressively with two higher concentrations. All three angiotensin II concentrations significantly increased the uptake of rat and human LDL and of fibrinogen by aorta. The increase was dose related for rat LDL but not for human LDL or fibrinogen. In spontaneously hypertensive rats of the same age in which blood pressure was higher than in angiotensin II-infused rats, protein uptakes were not increased. The blood content of aortic walls was negligible and not altered by angiotensin II. Therefore, the uptake of atherogenic plasma proteins by rat aorta is increased by angiotensin II, but this effect may be independent of its pressor action.
We have reported that overnight fasting stimulates bicarbonate reabsorption (JtCo2) in rat distal tubules. The present in vivo microperfusion studies evaluated the hypothesis that endogenous angiotensin II (AII) mediates this response. Rat late distal (LD) tubules were perfused at 8 nl/min in vivo with a hypotonic solution containing 28 mM bicarbonate. In overnight-fasted rats, LD JtCO2 was significantly higher than in normally fed rats (50 +/- 4 vs. 16 +/- 6 pmol/min.mm, P < 0.05). When overnight-fasted rats were salt-loaded, JtCO2 fell significantly (38 +/- 3 pmol/min.mm, P < 0.05). Conversely, in fed rats ingesting a zero-salt diet, JtCO2 increased three-fold (45 +/- 5 pmol/min.mm, P < 0.05). Enalaprilat infusion (0.25 micrograms/kg body wt, intravenously), in these zero-salt and overnight-fasted rats, reduced LD JtCO2 values to normal. Further, infusion of losartan (5 mg/kg body wt, intravenously), the specific AII AT1 receptor blocker, reduced JtCO2 in overnight-fasted rats by two-thirds (16 +/- 4 pmol/min.mm, P < 0.05). Finally, we perfused 10(-11) M AII intraluminally with and without 10(-6) M losartan: AII increased JtCO2 to 45 +/- 6 pmol/min.mm, equal to the zero-salt flux. This was completely abrogated by simultaneous losartan perfusion. Therefore, these results suggest that AII is an in vivo stimulator of late distal tubule bicarbonate reabsorption.
There is evidence that angiotensin II is synthesized by the proximal tubule and secreted into the tubular lumen. This study examined the functional significance of endogenously produced angiotensin II on proximal tubule transport in male Sprague-Dawley rats. Addition of 10(-11), 10(-8), and 10(-6) M angiotensin II to the lumen of proximal convoluted tubules perfused in vivo had no effect on the rate of fluid reabsorption. The absence of an effect of exogenous luminal angiotensin II could be due to its endogenous production and luminal secretion. Luminal 10(-8) M Dup 753 (an angiotensin II receptor antagonist) resulted in a 35% decrease in proximal tubule fluid reabsorption when compared to control (Jv = 1.64 +/- 0.12 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). Similarly, luminal 10(-4) M enalaprilat, an angiotensin converting enzyme inhibitor, decreased fluid reabsorption by 40% (Jv = 1.53 +/- 0.23 nl/mm.min vs. 2.55 +/- 0.32 nl/mm.min, P < 0.05). When 10(-11) or 10(-8) M exogenous angiotensin II was added to enalaprilat (10(-4) M) in the luminal perfusate, fluid reabsorption returned to its baseline rate (Jv = 2.78 +/- 0.35 nl/mm.min). Thus, addition of exogenous angiotensin II stimulates proximal tubule transport when endogenous production is inhibited. These experiments show that endogenously produced angiotensin II modulates fluid transport in the proximal tubule independent of systemic angiotensin II.
The intrarenal renin-angiotensin system plays a critical role in the paracrine regulation of renal hemodynamics and tubular transport function. Much of the intrarenal angiotensin II (ANG II) is formed locally as evidenced by intrarenal ANG II contents that are much greater than can be explained from the circulating ANG II concentration. Intrarenal ANG II is formed from systemically delivered ANG I and from intrarenally formed ANG I derived from systemically delivered angiotensinogen as well as locally synthesized angiotensionogen. There is a regional distribution of intrarenal ANG II in that the medullary content per gram of tissue is four to five times higher than the cortical content. In addition, most of the cortical ANG II is compartmentalized in the renal interstitial fluid and in the tubular fluid. Proximal tubule cells contain all the components of the renin-angiotensin system necessary for synthesis and secretion of ANG II. Proximal tubule concentrations of ANG II as well as ANG I and angiotensinogen support the concept that the proximal tubule cells secrete ANG II or precursors of ANG II into the tubular fluid. The intratubular concentrations of ANG II are in the nanomolar range, indicating a substantial capability to influence luminal ANG II receptors on the tubule cell membranes. Thus, much of the ANG II-dependent actions on tubular transport functions could be due to specific effects of locally synthesized ANG II on luminal ANG II receptors. Experimental evidence shows that the intratubular ANG II concentrations are regulated independently of the circulating concentrations, but the specific mechanisms responsible remain to be delineated.
Cleavage of the C-terminal tripeptide of angiotensin I (Ang I) by neutral endopeptidase 24.11 releases angiotensin 1-7 (Ang 1-7). Because Ang I and neutral endopeptidase 24.11 are present in proximal tubular fluid and brush border, respectively, Ang 1-7 could be released into proximal tubular fluid to affect nephron function. Therefore, we studied the effect of intratubular Ang 1-7 (10(-12) to 10(-8) M) on nephron function employing in vivo renal micropuncture in inactin-anesthetized Munich-Wistar-Frömter rats. We observed that: (i) Intratubular application of Ang 1-7 for 3, 15, or 30 min did not affect reabsorption in the microperfused proximal convoluted tubule determined as net fluid reabsorption. (ii) During perfusion of Henle's loop for 15 min with artificial tubular fluid (time control), we observed a decline in fluid, potassium and sodium reabsorption by 20, 18 and 5%, respectively. A similar decline in reabsorption was seen with intratubular application of Ang 1-7 in a concentration of 10(-12) or 10(-10) M. In contrast, intratubular application of Ang 1-7 in a concentration of 10(-8) M increased fluid, potassium and sodium reabsorption in that nephron segment by 11, 9 and 3%, respectively. The latter response was completely abolished by AT1 angiotensin II receptor antagonist losartan (10[-6] M). (iii) Intratubular application of Ang 1-7 did not affect net sodium, potassium, or fluid reabsorption in the distal tubule. (iv) TGF response assessed by measuring proximal tubular stop-flow pressure or single nephron filtration rate during orthograde open-loop perfusion of Henle's loop was not significantly altered by intratubular application of Ang 1-7. These findings show that intratubular application of Ang 1-7 in concentrations which possibly cover the physiological range does not significantly alter (i) tubular reabsorption in proximal convoluted or distal tubule, or (ii) TGF response. Intratubular Ang 1-7 at a concentration of 10(-8) M appears to increase reabsorption in Henle's loop by an AT1 angiotensin II receptor-mediated mechanism, the physiological relevance of which remains to be established.
Changes in ammonium excretion with acid/base perturbations are dependent on changes in medullary ammonium accumulation mediated by active NH4+ absorption by the medullary thick ascending limb. To investigate whether alterations in the abundance of medullary thick ascending limb ion transporters, namely the apical Na+/K+(NH4+)/2Cl- -cotransporter (BSC-1), the apical Na+/H+ -exchanger (NHE3), and the Na+/K+ -ATPase alpha1-subunit, may be responsible in part for altered medullary ammonium accumulation, semiquantitative immunoblotting studies were performed using homogenates from the inner stripe of the rat renal outer medulla. After 7 d of NH4Cl (7.2 mmol/220 g body wt per d) loading (associated with increased medullary ammonium accumulation), neither BSC-1 nor Na+/K+ -ATPase protein expression was altered, but NHE3 protein abundance was significantly increased. On the other hand, both BSC-1 and Na+/K+ -ATPase protein abundance was increased significantly in rats fed NaHCO3 (7.2 mmol/220 g body wt per d) for 7 d. Rats fed a high-NaCl diet (7.7 mEq Na+/220 g body wt per d) for 5 d also showed marked increases in both BSC-1 and Na+/K+ -ATPase expression. The expression level of NHE3 protein did not change with either NaHCO3 or high NaCl intake. None of these three transporters showed a significant difference in abundance between the groups fed equimolar (7.2 mmol/220 g body wt per d for 7 d) NaHCO3 or NaCl. It is concluded that outer medullary BSC-1 and Na+/K+ -ATPase alpha1-subunit protein abundance is increased by chronic Na+ loading but not by acid/base perturbations and that outer medullary NHE3 protein abundance is increased by chronic NH4Cl loading.
The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10(-8) M in the bath) inhibits HCO-3 absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 microM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 microM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M), the major P-450 metabolite in the MTAL, inhibited HCO-3 absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10(-8) M) to the bath had no effect on HCO-3 absorption. The inhibition of HCO-3 absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 microM) or herbimycin A (1 microM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10 microM) or the NO synthase (NOS) substrate L-arginine (1 mM) to the bath stimulated HCO-3 absorption by 35%, suggesting that NO directly regulates MTAL HCO-3 absorption. Addition of 10(-11) to 10(-10) M ANG II to the bath did not affect HCO-3 absorption. We conclude that ANG II inhibits HCO-3 absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of HCO-3 absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.
Previous studies have indicated that angiotensin II (Ang II) concentrations in renal interstitial fluid are much higher than plasma levels. In the present study, we performed experiments to explore renal interstitial fluid concentrations of Ang I and Ang II further and to determine whether these levels are altered by acute arterial infusion of an ACE inhibitor (enalaprilat) or by volume expansion. Microdialysis probes (molecular weight cutoff point: 30 000 Da) were implanted in the renal cortex of anesthetized rats and were perfused at a rate of 2 microL/min. Using relative equilibrium rates, the basal renal interstitial fluid Ang II concentration averaged 3.07+/-0.43 nmol/L, a value much higher than the plasma Ang II concentration of 107+/-8 pmol/L (n=7). Interstitial fluid Ang I concentrations (0.84+/-0.04 nmol/L) were consistently lower than the Ang II concentrations but higher than the plasma Ang I concentrations (112+/-14 pmol/L). Intra-arterial infusion of enalaprilat (7.5 micromol/kg/min, n=5) for 120 minutes resulted in a significant decrease in mean arterial pressure (from 114+/-4 to 68+/-4 mm Hg) along with reductions in plasma and renal ACE activity (by -99% and -52%, respectively). Enalaprilat resulted in a significant increase in plasma Ang I from 133+/-21 to 1167+/-328 pmol/L and a decrease in plasma Ang II from 110+/-12 to 67+/-9 pmol/L. During enalaprilat infusion, interstitial fluid concentration of Ang I was significantly increased from 0.78+/-0.06 to 0.97+/-0.08 nmol/L; however, Ang II concentrations were not altered significantly (3.67+/-0.28 versus 3.67+/-0.25 nmol/L). Acute volume loading with Ringer's solution containing 1% bovine serum albumin at a rate of 150 microL/min for 2 hours (6% to 7% of body weight) lowered plasma concentrations of Ang I from 110+/-23 to 16+/-2 pmol/L and Ang II from 100+/-23 to 36+/-6 pmol/L; however, renal interstitial fluid concentrations of Ang I and Ang II were not altered significantly during volume expansion (Ang I, from 0.77+/-0.05 to 0.69+/-0.03 nmol/L; Ang II, from 3.76+/-0.43 to 3.59+/-0.39 nmol/L, n=5). These data indicate that renal interstitial fluid concentrations of Ang I and Ang II are substantially higher than the corresponding plasma concentrations. Furthermore, the fact that the high interstitial fluid concentrations of Ang II are not responsive to acute ACE inhibition or volume expansion suggests the compartmentalization and independent regulation of renal interstitial fluid Ang II.
Intrarenal angiotensin II (Ang II) is regulated by several complex processes involving formation from both systemically delivered and intrarenally formed substrate, as well as receptor-mediated internalization. There is substantial compartmentalization of intrarenal Ang II, with levels in the renal interstitial fluid and in proximal tubule fluid being much greater than can be explained from the circulating levels. In Ang II--dependent hypertension, elevated intrarenal Ang II levels occur even when intrarenal renin expression and content are suppressed. Studies in Ang II--infused rats have demonstrated that augmentation of intrarenal Ang II is due, in part, to uptake of circulating Ang II via an Ang II type 1 (AT(1)) receptor mechanism and also to sustained endogenous production of Ang II. Some of the internalized Ang II accumulates in the light and heavy endosomes and is therefore potentially available for intracellular actions. The enhanced intrarenal Ang II also exerts a positive feedback action to augment intrarenal levels of angiotensinogen (AGT) mRNA and protein, which contribute further to the increased intrarenal Ang II in hypertensive states. In addition, renal AT(1) receptor protein and mRNA levels are maintained, allowing increased Ang II levels to elicit progressive effects. The increased intrarenal Ang II activity and AGT production are associated with increased urinary AGT excretion rates. The urinary AGT excretion rates show a clear relationship to kidney Ang II content, suggesting that urinary AGT may serve as an index of Ang II--dependent hypertension. Collectively, the data support a powerful role for intrarenal Ang II in the pathogenesis of hypertension.
Endogenous intratubular angiotensin II (Ang II) supports an autocrine tonic stimulation of NaCl absorption in the proximal tubule, and its production may be regulated independently of circulating Ang II. In addition, endogenous Ang II activity may be regulated at the brush border membrane (BBM), by the rate of aminopeptidase A and N (APA and APN) activities and the rate of Ca2+-independent phospholipase A2 (PLA2-dependent endocytosis and recycling of the complex Ang II subtype 1 (AT1) receptor (AT1-R). The aim of the present study was to look for subcellular localization of AT1-R, and APA and APN activities in the medullary thick ascending limb of Henle (mTAL), as well as search for an asymmetric coupling of AT1-R to signal transduction pathways.
Preparations of isolated basolateral membrane (BLMV) and luminal (LMV) membrane vesicles from rat mTAL were used to localize first, AT1-R by 125I-[Sar1, Ile8] Ang II binding studies and immunoblot experiments with a specific AT1-R antibody, and second, APA and APN activities. Microfluorometric monitoring of cytosolic Ca2+ with a Fura-2 probe was performed in mTAL microperfused in vitro, after apical or basolateral application of Ang II.
AT1-R were present in both LMV and BLMV, with a similar Kd (nmol/L range) and Bmax. Accordingly, BLMV and LMV preparations similarly stained specific AT1-R antibody. APA and APN activities were selectively localized in LMV, although to a lesser extent than those measured in BBM. In the in vitro microperfused mTAL, basolateral but not apical Ang II induced a transient increase in cytosolic [Ca2+].
Besides the presence of basolateral AT1-R in mTAL coupled to the classical Ca2+-dependent transduction pathways, AT1-R are present in LMV, not coupled with Ca2+ signaling, and co-localized with APA and APN activities. Thus, apical APA and APN may play an important role in modulating endogenous Ang II activity on NaCl reabsorption in mTAL.
This study was designed to determine the involvement of AT(1) receptors in the uptake of ANG II in the kidney of rats exposed to differing salt intake. Male Wistar-Kyoto rats were treated with a normal-salt (NS; 1% NaCl, n = 7) or a low-salt (LS; 0.025% NaCl, n = 7) diet combined with (LS+Los, n = 7; NS+Los, n = 7) or without losartan (30 mg. kg(-1). day(-1)), an AT(1) receptor antagonist. Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in plasma, renal cortex, and medulla. In LS rats, in both plasma and renal cortex, the increase in RA was associated with an increase in ANG I and ANG II levels compared with NS rats, but intrarenal ANG II levels increased more than ANG I levels. In NS+Los rats, the increase in RA in plasma was followed by a marked increase in plasma ANG I and ANG II levels compared with NS rats whereas in the kidney the increase of renal RA was followed by a decrease of the levels of these peptides. The same pattern was observed in LS+Los rats, but the decrease in renal ANG II levels was much more pronounced in LS+Los rats than in NS+Los rats. Our results suggest that the increase in renal ANG II levels after salt restriction results mainly from an uptake of ANG II, via AT(1) receptors. Such elevated intrarenal ANG II levels could contribute to maintain sodium and fluid balance and arterial blood pressure during salt-deficiency states.