Clinical and Experimental Pharmacology and Physiology (1992) 19,213-222
REGULATION OF PROXIMAL TUBULE FUNCTION BY ANGIOTENSIN Peter J. Harris
Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia (Received 25 October 1991)
SUMMARY 1. Independent of its effects on renal haemodynamics and glomerular filtration, angiotensin I1 (AII) has direct actions on the proximal tubule involving transepithelial Na+, H+, HC03-, and water reabsorption, ammoniagenesis, gluconeogenesis and renal growth. 2. The effects of A11 on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations ( 10-12-10-9 mol/ L) stimulate reabsorption whereas high concentrations ( 10-7-10-6 mol/L) inhibit reabsorption. Similar dose-response relations have been obtained for luminal and peritubular addition of AII. 3. The cellular responses to A11 are mediated via an AT-1 receptor coupled via G-regulatory proteins to several parallel signal transduction pathways. Low doses inhibit the basolateral adenylate cyclase, lower intracellular CAMP and withdraw the inhibitory effect of protein kinase A on the luminal Na/H exchanger. Stimulation of this exchanger may also occur due to AII-receptor activation of phospholipase C to release diacyl glycerol, or by local transduction in the brush-border membrane involving phospholipase Az4. Inhibition of proximal fluid reabsorption is associated with increased intracellular Ca2+ released from intracellular stores, or entering via voltage-sensitive channels in response to the release of inositol-l,4,5,-trisphosphate,or following Ca2+channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. 5 . The stimulatory actions of peritubular A11 on proximal transport are inhibited by physiological concentrations of atrial natriuretic factor (ANF) and by parathyroid hormone (PTH). 6. It is concluded that intrarenal A11 acts to maintain optimal matching of fluid reabsorption and filtered load in response to changes in sodium balance, as well as to promote acidification of the urine during acidosis and perhaps to potentiate tubular growth following renal injury.
Key words: ammoniagenesis, angiotensin 11, glomerulotubular balance, gluconeogenesis, intracellular signalling, Na/H exchanger, proximal tubule, renal acid-base regulation, renal growth, sodium reabsorption.
INTRODUCTION Angiotensin I1 (AII) exerts powerful influences on many functions of the proximal tubule, including salt and water reabsorption, H+ and H C 0 3 transport, ammoniagenesis (Chobanian & Julin 199I), gluconeo-
genesis (Guder 1979) and growth (Norman et al. 1987). The renal haemodynamic responses to A11 have been extensively investigated, and it is well established that A11 modifies solute delivery to the
Correspondence: Peter J. Harris, Department of Physiology, The University of Melbourne, Parkville, Vic. 3052, Australia.
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proximal tubule by altering glomerular filtration rate and inducing changes in peritubular oncotic and hydrostatic pressures with consequent effects on fluid and electrolyte uptake. These aspects of the tubular response to A11 have been reviewed elsewhere (Navar & Rosivall 1984; Mitchell & Navar 1991), and this review will focus on direct actions of A11 on the proximal tubular epithelium. Historically, the notion that A11 might be a physiologically important modulator of proximal fluid reabsorption arose from observations that the ‘loaddependency’ of proximal glomerulo-tubular balance could not be explained by purely intrinsic factors involving tubular and interstitial geometry, pressures and flow dynamics. Bojeson’s (1954) suggestion that a plasma hormone might be involved was examined by Leyssac (1969, who found that intravenous injection of A11 but not noradrenaline prolonged occlusion time in rats, indicating that inhibition of proximal fluid reabsorption was independent of any haemodynamic effects. Development of the perfused, isolated tubule preparation enabled Burg and Orloff (1968) to test more directly whether A11 might affect fluid uptake in the rabbit pars recta and thus explain the discrepancy between net fluid uptake rates estimated from in vivo micropuncture studies and during in vitro tubule perfusion. Unfortunately, no effect of A11 on fluid reabsorption could be found (later shown by Schuster et al. 1984to be due to enzymatic degradation by peptidases in the serum perfusate), and the nature of the hormonal modulator remained unknown. Despite negative or conflicting findings from several laboratories (for review, see Harris & Navar 1985), the Danish group continued to assert the importance of A11 in the proximal tubule, and Steven (1974) confirmed that peritubular infusion of A11 (2 X 10-8 mol/ L) inhibited proximal fluid reabsorption and reduced end-proximal TF/ P inulin ratio. Encouraged by observations that much lower doses of A11 (10-12 mol/ L) stimulated sodium transport in kidney cortex slices (Munday et al. 1971) and other tissues, notably frog skin (McAfee & Locke 1967) and rat colon (Davies et al. 1970), Harris and Young (1977) continued this investigation in anaesthetized rats. Stationary split-drop micropuncture combined with simultaneous capillary perfusion revealed a biphasic, dose-dependent response in which proximal sodium transport was stimulated by peritubular A11 at low concentrations (10-12 mol/L-10-10 mol/L) and inhibited by higher doses (10-6 mol/L). The characteristics of this dose-response relation were confirmed first by Spinelli and Walther (1979) using shrinking split-drop analysis with delivery of A11 by superfusion of the kidney surface, and later in the perfused,
isolated rabbit pars convoluta by Schuster et al. (1984). Subsequent work in many laboratories has been directed toward understanding the cellular mechanism of this action of AII, and to determining its physiological significance.
Cellular mechanisms The main features of current models for the cellular transduction of the action of A11 on proximal tubule electrolyte transport to A11 are summarized in Figs 1 and 2. Depending upon the dose, stimulation (Fig. 1) or inhibition (Fig. 2) of transepithelial sodium and water reabsorption can be elicited by either peritubular (Harris & Young 1977; Harris 1979; Spinelli & Walther 1979; Schuster et al. 1984) or luminal (Harris & Young 1977; Wang & Chan 1990; Yanagawa 1991; Morduchowicz et al. 1991) application of AII. Although the characteristics of the dose-response curves obtained are remarkably similar, there may well be important differences in the intracellular mechanisms involved in responses initiated from basolateral and apical surfaces.
Angiotensin receptors In either case, the chain of events begins with the binding of A11 to high affinity (nanomolar K d ) membrane-bound receptors that have been characterized in enriched membrane fractions according to affinity, density of distribution and cation dependence (Brown & Douglas 1982, 1983). In autoradiographic studies, binding of 12sI{sar’,Ile8] A11 in the renal cortex was potently displaced (K,= 23 nmol/L) by the non-peptide AT1 antagonist DuP 753, whereas AT2 antagonists had little effect (Zhuo 1991). It was concluded that approximately 95% of renal A11 receptors are of the AT1 subtype and that, at most, 5% are AT*. AT1 receptors probably correspond to the ‘subtype Bydescribed by de Gasparo et al. (1 990) and found, also by antagonist displacement studies, to predominate in the kidney. It is necessary to reconcile the conclusion that the majority of renal and proximal tubular A11 receptors are pharmacologically identical, with functional heterogeneity based on relative binding potency of A11 and AIII, response to the reducing agent dithiothreitol, and receptor coupling to different transduction pathways. Applying these criteria, Douglas(1987) concluded that there ace two functional subtypes corresponding to the glomerular mesangial (type A) and tubular epithelial (type B) sites of action. The distinction between glomerular and tubular receptors may represent a further subdivision of A11 receptors (ATIA and ATIB, respectively), but final
Angiotensin and proximal tubule function resolution of this issue will require cloning and expression of renal A11 receptors from each site and subsequent comparison with the known cDNA sequences for adrenal and vascular AT, receptors. The observation that proximal tubular A11 receptors belong predominantly to the ATI subtype implies that proximal reabsorption will be inhibited by the AT, antagonist DuP 753. This hypothesis has been tested and confirmed in recent clearance and micropuncture experiments in anaesthetized rats (Xie et al. 1990; Zhuo et al. 1992), but no comparable studies using AT2 antagonists have been reported. Although proximal epithelial A11 receptors appear to be relatively homogeneous with regard to pharmacology and binding kinetics, there is considerable evidence that they are coupled to a number of parallel signal transduction pathways within the cell corresponding to the variety of physiological responses that can be elicited by this peptide (Douglas et al. 1990).
Low dose stimulation of fluid and electrolyte transport The best described pathway mediates the stimulation of sodium and bicarbonate reabsorption by low (picomolar) concentrations of AII, and involves inhibition of adenylate cyclase with subsequent enhancement of luminal Na/ H exchange providing increased cellular uptake of Na and extrusion of H. Early biochemical studies on cortical tissue (Torres et al. 1978; Woodcock &Johnston 1982) established that cAMP production was inhibited by A11 but at concenK+
Na+ Na+ HC0,Peritubular
Kt
TT\
cAMP
J
DAG.T
I
5
J
PKA
,PKC?
n-
All
Fig. 1. Composite model for stimulation of proximal tubule by angiotensin. Abbreviations: PIP2, phosphatidylinositol4,5,-bisphosphate; PLC, phospholipase C; DAG, diacyl glycerol; PKC, protein kinase C; PLAz, phospholipase A2; G,, inhibitory G-regulatory protein; AC, adenylate cyclase; CAMP, cyclic adenosine monophosphate. represents the angiotensin receptor.
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trations well above those associated with increased electrolyte transport. Douglas et al. (1990) have recently developed a more sensitive assay system using cultured rabbit proximal tubule cells, and report receptor-mediated inhibition of adenylate cyclase by A11 within the picomolar to nanomolar range. Both this action and the AII-induced stimulation of sodium flux across confluent monolayers of similar cells were inhibited by pertussis toxin, indicating the involvement of a Gi regulatory protein coupling receptor and cyclase activity. A link between adenylate cyclase and sodium transport rate has been demonstrated in micropuncture experiments on rats in which luminal N a /H exchange was stimulated in proximal tubules when intracellular cAMP levels were reduced by A11 infusion (Liu & Cogan 1989). In addition, luminal perfusion with dibutyryl cAMP to maintain a high intracellular c AMP level abolished the luminal acidification normally observed with A11 infusion. CAMP-dependent protein kinase (PKA) has been shown to inhibit brush border Na/ H exchange (Kahn ef al. 1985), but it has not yet been demonstrated directly that removal of an inhibitory action of this enzyme occurs during the epithelial transport response to AII. Stronger evidence has been accumulated to support the involvement of the apical N a /H antiporter as the next step in the transduction pathway. Several groups have demonstrated that the stimulatory action of A11 on proton secretion and sodium reabsorption is inhibited by amiloride (Liu & Cogan 1988; Saccomani et al. 1990; Wang & Chan 1990). Liu and Cogan (1988) also noted that this effect was more pronounced in the S1 compared with S 2 segments, and correlated this with the relative abundance of A11 receptors. In the same study, evidence was provided that the major effect of A11 in the S2 segment is to stimulate NaCl reabsorption, and it was suggested that this action is mediated via catecholamine release from sympathetic nerve terminals adjacent to the basement membrane. In the most detailed study to date, Geibel ef al. (1990) loaded perfused, isolated superficial S 1 segments of rabbit tubules with the pH-sensitive dye BCECF and measured intracellular pH (pHi) in response to A11 (10-9 mol/ L) added to the bath solution. A11 caused an initial prolonged alkalinization and enhanced the rate of recovery from an intracellular acid load induced by removal of Na from the bath and luminal perfusates. The latter effect was inhibited by the amiloride analogue ethylisopropylamiloride (EIPA) indicating involvement of the luminal Na/ H exchanger, but the initial alkalinization was not sensitive to EIPA and was presumed to be mediated by some other acidloading or extruding process. A11 was also found to
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stimulate a DIDS-sensitive basolateral Na/ HC03 cotransporter, and it was noted that increased luminal H extrusion, together with stimulated basolateral HC03 efflux, would support a substantial rise in transepithelial HCO3 reabsorption with only a small change in pHi. The question of how Na exit from the proximal tubule cell might be linked to increased luminal entry has also been addressed by Garvin (1991). In proximal straight tubules, A11 (10-10 mol/ L) stimulated fluid absorption and increased K, for HCO3, since there was no change in transepithelial HCO3 permeability of Urn,,. In tubule segments treated with A11 for 30 min, Na/ K ATPase activity was increased by 27%. In this study the cells were made permeable prior to the addition of AII, and were therefore exposed to similar sodium concentrations during incubation, but it remains unclear whether there are direct effects of A11 on the Na/ K ATPase in vivo, or whether stimulation occurs primarily in response to increased Na delivery. Stimulation of basolateral Na/ K exchange must also result in increased K entry, which will tend to raise the intracellular K concentration. Due to the low luminal K permeability of these cells, the resulting increase in transmembrane K gradient is likely to be dissipated across the basolateral membrane. A basolateral K channel has been characterized in this membrane (Goegelein 1990) and appears to be pH-sensitive. Activation of this channel in response to cellular alkalinization following AII-induced stimulation of luminal Na/ H exchange would provide for enhanced K efflux. All available evidence indicates that there is no change in transepithelial potential difference during stimulation of transport by A11 (Harris & Young 1977; Schuster et al. 1984; Garvin 1989). Electroneutrality could be preserved if the net effect of hyperpolarization, due to stimulation of the Na/ K exchanger combined with increased basolateral K conductance, were balanced by the depolarizing influence of increased Na/ HCO3 cotransport. Microelectrode measurements in microdissected rabbit S1 and S2 segments showed no change in the potential difference across the basolateral membrane when A11 (10-10 or 10-7 mol/L) was added to the bath solution (Eitle, Hirst & Harris, unpubl. data). It should be noted that, due to the low electrical resistance of the proximal tubule, transepithelial potential gradients are very small ( cytosolic Ca2+and could thus mediate inhibition of
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Na transport. The source of this Ca2+is uncertain, since although AII-induced inhibition of Jv and JHCO3 was blocked by TMB-8 consistent with release from intracellular stores (Chatsudthipong & Chan 1991), Welsh et al. (1988) perfused cultured proximal cells with Ca2+chelators and found that the rise in cytosolic Ca2+was greatly reduced, indicating substantial influx from the extracellular medium. These investigators have concluded that Ca2+entry occurs through voltage-sensitive channels, since verapamil prevented the increase in intracellular Ca2+in response to high dose A11 (10-6 mol/L; Romero et al. 1991). The discrepancy between these observations regarding the source of Ca2+may be due to differences in species and the experimental system employed (rat in vivo vs rabbit proximal cells in culture), and further work is clearly required. Although most studies report increases in intracellular Ca2+ only with high (inhibitory) concentrations of AII, Jung and Endou (1989) attribute this to depletion of ATP levels. They demonstrated in isolated rat S1 segments that when acetate or pyruvate were provided as TCA cycle substrates, A11 caused a dosedependent biphasic response, with peak rises in cytosolic Ca2' at 10-11 mol/L and 10-7 mol/L, and a trough at 10-9 mol/ L. The coincidence of this pattern with the biphasic response characteristic of Na, fluid and HCO3 reabsorption, is unmistakable, but at present the significance of increased intracellular Ca2+ with low dose A11 remains unclear. A novel proposal for transduction of the inhibitory effect of high concentrations of A11 has been investigated by Douglas et al. (1990). Convinced that the A11 receptor does not couple effectively with phospholipase C in the proximal tubule, this group explored the possibility that high doses of A11 (10-9-10-6 mol/ L) activate phospholipase A2, releasing arachidonate and lysophospholipids from membrane phospholipids. Substantial evidence has been accumulated to support this hypothesis (Douglas et al. 1990) and to show that an epoxide, thought to be 5,6-EET, is produced as a result of metabolism of arachidonate by an NADPH-dependent cytochrome P450 enzyme. Accumulation of this epoxide induced by A11 mol/ L) in cultured rabbit proximal cells was inhibited by ketoconazole, an inhibitor of P450-dependent epoxygenase, while the addition of 5,6-EET to the apical side of monolayers of similar cells mimicked the inhibition of apical to basal Na flux seen with AII. 5,6-EET also increased cytosolic Ca2+in a manner similar to that caused by A11 (10-9-10-6 mol/ L), and the epoxide-induced rise was blocked by verapamil and nifedipine, indicating involvement of voltagedependent Ca2+channels in mediating the influx of
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extracellular Ca2+(Madhun et al. 1991). Inhibition of the basolateral Na/K ATPase by 5,6-EET was also observed (Romero et al. 1991), and it is possible that this effect contributes to the inhibition of Na transport by AII.
Physiological role of A11 regulation of proximal fluid reabsorption If stimulation (or inhibition) of proximal tubule transport is a significant feature of the regulation of this nephron segment under physiological conditions, then removal of A11 should cause corresponding changes in proximal fluid reabsorption and in delivery of filtrate to more distal segments. This prediction has been tested using inhibitors of angiotensinconverting enzyme (Harris et al. 1984; Thomas et al. 1988) and more recently with a non-peptide A11 antagonist, DuP 753 (Xie et al. 1990; Zhuo et al. 1992). According to these studies in anaesthetized rats, approximately 30% of proximal sodium, bicarbonate and water is modulated by A11 and, at least under these conditions, A11 exerts a tonic stimulatory action on the proximal tubule acting to maintain a high level of water and electrolyte reabsorption. It is possible that such a tonic effect of A11 is due in part to activation of the renin-angiotensin system during the anaesthesia and surgery required for micropuncture experiments. However, in an early study in conscious rats, Barraclough et al. (1967) found that intravenous infusions of low doses of A11 caused sodium and water retention only when the animals had previously been salt-loaded, presumably suppressing the endogenous renin and A11 levels. These workers, long before direct effects of A11 on the proximal tubule had been demonstrated, concluded that A11 has a tonic stimulatory action on tubular fluid reabsorption. Although it is clearly not possible to investigate individual tubule function in conscious animals or humans, indirect studies using lithium clearance as an indicator of proximal sodium and water reabsorption have provided valuable information (despite reservations regarding the quantitative relation between lithium clearance and proximal tubule reabsorption). In normal human subjects given a range of dietary sodium intakes, ACE inhibition caused natriuresis accompanied by a decrease in proximal fractional sodium reabsorption (Broyn 1988). Further evidence for a proximal stimulatory action of circulating AII was found when infusion of a low dose of A11 (1 ng/ min/ kg) caused antinatriuresis with reduced fractional lithium clearance (Seidelin et al. 1989). In anaesthetized rats ACE inhibition increased glome-
rular filtration rate accompanied by a lesser increase in proximal fluid reabsorption determined by lithium clearance (Harris et al. 1987b). It was concluded that proximal glomerulo-tubular balance (PGTB), reflecting the fraction of the filtered load of solute and water being reabsorbed, was biased towards increased rejection resulting in enhanced end-proximal flow. This study further demonstrated that the impaired effectiveness of PGTB could be corrected by infusion of either A11 or des-asp' A11 (AIII) indicating that the presence of circulating angiotensin is necessary for optimal matching of proximal reabsorption with delivery of glomerular filtrate. It is difficult to compare the results of studies in which A11 has been infused systemically with the data from in vivo micropuncture, microperfusion or cultured cell experiments where the concentrations of A11 can be controlled directly. Similarly, the concentrations of A11 that exist under various conditions in the interstitial fluid adjacent to the peritubular capillaries are unknown since, at present, it is not possible to obtain sufficiently large samples for assay. The problem is complicated by the presence of immunoreactive products of AII, some of which such as A111 and the heptapeptide 1-7 A11 are biologically active and form a substantial proportion of the total A11 particularly in the rat. Seikaly et al. (1990) recently reported total immunoreactive angiotensin concentrations in the nanomolar range in micropuncture samples of glomerular filtrate, proximal tubular fluid and plasma from star vessels on the kidney surface. Even when these values are corrected for the increase in tubule fluid concentration due to water reabsorption and for the dilution of star vessel plasma during passage through the peritubular capillaries, it is likely that the luminal and basal surfaces of the proximal tubule sells are exposed to A11 and A111 at concentrations approximately 100 times those circulating in plasma. These estimates are consistent with earlier measurements of A11 in renal cortical tissue (Mendelsohn 1979) and with the known affinities of A11 receptors. Angiotensins could pass from the juxtaglomerular cefls to the efferent arteriolar blood via the interstitium, and local production of A11 or A111 from angiotensinogen and renin within the proximal tubule cells (Schunkert et al. 1991) might account for their elevated concentrations in the tubule lumen. These estimates correspond with doses shown to stimulate proximal sodium and HCO3 transport (10-12-10-9 mol/L) and cover the physiological range of variations in circulating and local intrarenal A11 levels. It may be concluded that the proximal tubule is normally stimulated to some extent by A11 and that suppression of the renin-angiotensin system, for example by
Angiotensin and proximal tubule function
sodium loading, will lead to reduced reabsorption and an increase in the delivery of filtrate out of the proximal tubule. Whether these events will subsequently induce urinary loss of salt and water will depend on the capacity of the distal nephron to reabsorb the increased load and the activity of the tubulo-glomerular feedback response, itself modulated by the intrarenal A11 concentration (Mitchell & Navar 1991). Micropuncture, microperfusion and cell culture experiments have all demonstrated the inhibitory action of high concentrations of A11 (10-7 mol/L), but it is not clear in what physiological or pathological situations this effect might operate. Inhibition of proximal salt and water reabsorption by A11 does not appear to be the explanation for the natriuresis observed during systemic infusion of high doses of A11 as this has been shown to be dependent upon a concomitant rise in blood pressure (Olsen et al. 1985). Mitchell and Navar (1987) demonstrated stimulation of proximal fluid uptake when low concentrations of A11 were infused into the peritubular capillaries of anaesthetized rats, but found that higher doses affected the upstream glomerulus, reducing SNGFR and precluding detection of an inhibitory action on transepithelial transport. A luminal action of A11 in the proximal tubule was reported by Harris and Young (1977). Using stationary split-drop micropuncture in rats, they found that stimulation of the steady-state Na concentration gradient occurred when A11 ( mol/L) was added to the luminal fluid, but noted that this concentration was 100 times that required for stimulation from the peritubular side. More recently, continuous microperfusion studies (Wang & Chan 1990) have demonstrated that the dose-response curve for luminal addition of A11 is comparable with those reported for the peritubular action, including both stimulatory and inhibitory components and showing similar sensitivities for each response. The disparity between the doses required for stimulation by luminal A11 delivered by stationary rather than continuous microperfusion is most likely due to reduced enzymic degradation when the peptide is replaced during perfusion. This observation suggests that the concentration of A11 available far luminal receptor binding, and therefore the extent of AII-induced stimulation or inhibition of transport, may depend on the luminal flow that is directly related to the single nephron glomerular filtration rate (GFR). Thus these data may reveal an important physiological role for the brush-border angiotensinase in proximal glomerulo-tubular balance and provide an additional mechanism for the mediation of flow-related fluid reabsorption.
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AII-stimulated transport in hypertension In the spontaneously hypertensive rat (SHR) model of human essential hypertension, the early phase of development is accompanied by inappropriate retention of sodium and water, although neutral sodium balance is achieved in the adult at approximately 12 weeks of age. This pattern of development is associated with changes in the extent of AII-stimulated transport, such that the component of proximal fluid reabsorption in the young SHR (5 weeks) is enhanced when compared with age-matched Wistar-Kyoto (WKY) controls (Thomas et al. 1988). By 12 weeks, AIIstimulated transport is absent in the SHR although unaltered in the corresponding WKY. Further studies involving peritubular capillary perfusion of A11 in young and adult SHR and WKY have shown agedependent alterations in the sensitivity of proximal fluid reabsorption (Thomas et al. 1990). These changes may reflect down-regulation of A11 receptors due to elevation of A11 levels, or may indicate some genetic abnormality in AII-receptor kinetics or signal transduction. In another model of hypertension using the one-clip two-kidney Goldblatt rat, the extent of AIIstimulated proximal fluid transport was assessed by intravenous infusion of CEI and found to be similar (approximately 30%) in normotensive controls and in hypertensive animals 4-6 weeks after clipping (Harris et al. 1984). The maintenance of proximal reabsorption by A11 in the renin-angiotensin dependent phase of these Goldblatt rats contrasts with the absence of AIIstimulated transport in the adult SHR (Thomas et al. 1988) perhaps reflecting the contributions of A11 to sodium homeostasis in each model.
Interactions with other hormones Hormonal factors or neurotransmitters that act on proximal transport will necessarily act to attenuate or reinforce the responses due to angiotensin. Interaction with A11 has been clearly demonstrated for two hormones to date, parathyroid hormone (PTH) and atrial natriuretic factor (ANF). PTH acts via its own specific receptors on proximal tubule cells and exerts an inhibitory effect on bicarbonate and fluid reabsorption that is mediated by intracellular CAMP. Liu and Cogan (1989) have shown that net bicarbonate absorption correlates inversely with tubular fluid CAMP concentration during administration of either A11 or PTH. They concluded that approximately half of total renal acidification is influenced by this cyclic nucleotide. Since stimulation of proximal Na and HCO, reabsorption by A11 involves inhibition of CAMP,while PTH suppresses reabsorption by stimulating CAMP, interaction occurs at the level of the
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common shared pathway for signal transduction. ANF also acts through specific cell membrane receptors but has no action on the proximal tubule when infused into the peritubular capillaries (Harris et al. 1987a) or added to the bath fluid perfusing isolated rat tubules (Garvin 1989). However, at physiological concentrations (10-10 mol/L-10-8 mol/L), peritubular ANF inhibits AII-stimulated fluid reabsorption in vivo (Harris et al. 1987a) and in perfused isolated tubules (Garvin 1989). Since this latter action could be mimicked by the membrane permeant dibutyrylcGMP, it may be inferred that cGMP acts as a second messenger for the ANF response in the proximal tubule, as in other tissues. It is not yet clear how modulation of the intracellular cGMP level might inhibit the stimulatory action of A11 but these data reveal an important antagonism in the proximal tubule between the sodium and water retaining effects of A11 and the natriuretic and diuretic actions of ANF.
Ammoniagenesis and gluconeogenesis The role of A11 as a regulator of proximal tubular acidification is evident from its potent actions on transepithelial HC03 reabsorption. Additional importance has been attached to this role, as A11 also stimulates ammonia production from L-glutamine in suspensions of canine proximal tubule segments (Chobanian & Julin 1991). This action was observed under conditions of acidosis but not alkalosis, and there was an additive effect of A11 and reduced tubule fluid pH resulting in further enhancement of ammonia production. The threshold concentration required for a significant increase in ammoniagenesis was 10-8 mol/L, some three orders of magnitude higher than those associated with stimulation of Na, HC03 and water flux, and closer to the concentrations that inhibit reabsorption. The cellular mechanisms responsible for stimulation of ammoniagenesis are not clear but do not appear to involve cAMP or the luminal Na/ H exchanger. The response was mediated by A11 receptors and could be blocked by removal of extracellular Ca2’ or by inhibitors of the Ca2+-calmodulin dependent pathway. Effects of A11 on proximal tubule metabolism were also reported by Guder (1979), who reported stimulation of ammoniagenesis and gluconeogenesis in rat tubule fragments in response to high concentrations of A11 (10-7 mol/L). These actions did not correlate with changes in intracellular cAMP and were only partially inhibited by removal of CaZ+from the medium. AII-induced stimulation of gluconeogenesis from L-glutamine was confirmed by Chobanian and Julin (1991), who noted that this substrate will support
parallel stimulation of glucose and ammonia production, as the deamination and deamidation of Lglutamine produces ammonia, leaving alpha-ketoglutarate, which may be converted to glucose.
Renal growth Studies designed to assess the effects of A11 on cell growth in primary cultures of proximal tubule cells have found no response in terms of cell numbers or cell size. However, A11 has been shown to potentiate the mitogenic effect of epidermal growth factor (EGF) on cultured proximal cells, and it appears that this effect operates at some stage after the agonist-receptor interaction (Norman ef al. 1987). Although it has been suggested that mobilization of intracellular Ca2+ might be involved in the interaction between A11 and EGF, it is also possible that alkalinization of the cell as a consequence of stimulation of the luminal Na/ H exchanger provides an internal environment favourable to mitogenesis.
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Chatsudthipog, V. 4 Chn, Y-L. (1991) Inhibitory effect of angiotensin I1 on renal tubular transport. American Journal of Physiology, 260 (Renal Fluid & Electrolyte Physiology, 29), F340-346. Chobanian, M. C. & Julin, C. M. (1991) Angiotensin I1 stimulates ammoniagenesis in canine renal proximal tubule segments. American Journal of Physiology, 260 (Renal Fluid & Electrolyte Physiology, 29), F19-26. Davles, N. T., Munday, K. A. & Parsons, B. J. (1 970) The effect of angiotensin on rat intestinal fluid transfer. Journal of Endocrinology, 48,3946. de Gasparo, M., Whitebread, S., Mele, M., Motmi, A. S., Whiteombe, P. J., Ramjoue, H-P. & Kamber, B. (1990)
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Biochemical characterization of two angiotensin 11 receptor subtypes in the rat. Journal of Cardiovascular Pharmacology, 16 (Suppl. 4), S31-35. Dominguez, J. H., Snowdowne, K. W., Freudenrich, C. C., Brown, T. & Borle, A. B. (1987) lntracellular messenger for action of angiotensin I1 on fluid transport in rabbit proximal tubule. American Journal of Physiofogy, 252 (Renal Fluid & Electrolyte Physiology, 21), F423-428. Douglas, J. G. (1987) Angiotensin receptor subtypes of the kidney cortex. American Journal of Physiology, 253 (Renal Fluid & Electrolyte Physiology, 22), F1-7. Douglas, J. G., Romero, M. & Hopfer, U. (1990) Signalling mechanisms coupled to the angiotensin receptor of proximal tubular epithelium. Kidney International, 38 (SUPPI.30), S43-47. Freidman, P. A., Figueiredo, J. F., Maack, T. & Windhager, E. E. (1981) Sodium calcium interactions in the renal proximal convoluted tubule of the rabbit. American Journal of Physiology, 240 (Renal Fluid & Electrolyte Physiology, 9), F558-568. Garvin, J. L. (1989) Inhibition of Jv by ANF in rat proximal straight tubules requires angiotensin. American Journal of Physiology, 257 (Renal Fluid & Electrolyte Physiology, 26), F907-911. Garvin, J. L. (1991) Angiotensin stimulates bicarbonate transport and Na+/K+ ATPase in rat proximal straight tubules. Journal of the American Society of Nephrology, 1, 1146-1 152. Geibel, J., Giebisch, G. & Boron, W. F. (1990) Angiotensin I1 stimulates both Na+-H+ exchange and Na+/HCOj cotransport in the rabbit proximal tubule. Proceedings of the National Academy of Science, USA, 87, 79177920. Goegelein, H. (1990) Ion channels in mammalian proximal renal tubules. Renal Physiology and Biochemistry, 13, 8-25. Guder, W. G. (1979) Stimulation of renal gluconeogenesis by angiotensin 11. Biochimica Biophysica Acta, 584, 507-5 19. Harris, P. J. (1979) Stimulation of proximal tubular sodium reabsorption by ile5-angiotensin I1 in the rat kidney. Pflugers Archiv. European Journal of Physiology, 381, 83-85. Harris, P. J. & Navar, L. G. (1985) Tubular transport responses to angiotensin. American Journal of Physiology, 248 (Renal Fluid & Electrolyte Physiology, 17), F62 1-630. Harris, P. J., Navar, L. G . & Ploth, D . W. (1984) Evidence for angiotensin-stimulated proximal tubular fluid reabsorption in normotensive and hypertensive rats: effect of acute administration of captopril. Clinical Science, 66, 541-544. Harris, P. J., Thomas, D . & Morgan, T. 0. (1987a) Atrial natriuretic peptide inhibits angiotensin-stimulated proximal tubular sodium and water reabsorption. Nature, 326,695-696. Harris, P . J. & Young, J. A. (1977) Dose-dependent stimulation and inhibition of proximal tubular sodium
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reabsorption by angiotensin I1 in the rat kidney. pflugers Archiv. European Journal of Physiology, 367,295-297. Harris, P. J., Zhuo, J. & Skinner, S. L. (1987b) Effects of angiotensins I1 and 111 on glomerulotubular balance in rats. Clinical and Experimental Pharmacology and Physiology, 14,489-502. Jung, K. Y. & Endou, H. (1989) Biphasic increasing effect of angiotensin [I on intracellular free calcium in isolated rat early proximal tubule. Biochemical and Biophysical Research Communications, 165, 1221-1228. Kahn, A. M., Dalson, G . M., Hise, M. K., Bennet, S. C. & Weinmann, E. J. (1985) Parathyroid hormone and dibutyryl CAMP inhibit Na/ H exchange in renal brush border vesicles. American Journal of Physiology, 248 (Renal Fluid & Electrolyte Physiology, 17), F212-218. Leyssac, P. P. (1965) The in vivo effect of angiotensin and noradrenaline on the proximal tubular reabsorption of salt in mammalian kidneys. Acta Physiologica Scandinavica, 64, 167-175. Liu, F-Y. & Cogan, M. G. (1988) Angiotensin I1 stimulation of hydrogen ion secretion in the rat early proximal tubule. Journal of Clinical Investigation, 82, 601-607. Liu, F-Y. & Cogan, M. G. (1989) Angiotensin I I stimulates early proximal bicarbonate absorption in the rat by decreasing cyclic adenosine monophosphate. Journal of Clinical Investigation, 84, 83-91. Liu, F-Y. & Cogan, M. G. (1990) Role of protein kinase C in proximal bicarbonate absorption and angiotensin signalling. American Journal of Physiology, 258 (Renal Fluid & Electrolyte Physiology, 27), F927-933. Mahhun, Z. T., Goldthwait, D. A., McKay, D., Hopfer, U. & Douglas, J. G. (1991) An epoxygenase metabolite of arachidonic acid mediates angiotensin 11-induced rises in cytosolic calcium in rabbit proximal tubule epithelial cells. Journal of Clinical Investigation, 88,456-461. McAfee, R. D. & Locke, W. (1967) Effect of angiotensin amide on sodium isotope flux and short-circuit current of isolated frog skin. Endocrinology, 81, 1301-1305. Mendelsohn, F. A. 0. (1979) Evidence for the local occurrence of angiotensin I1 in the rat kidney and its modulation by dietary sodium intake and converting enzyme blockade. Clinical Science, 57, 173-179. Mitchell, K. D . & Navar, L. G. (1987) Superficial nephron responses to peritubular capillary infusion of angiotensin I and 11. American Journal of Physiology, 252 (Renal Fluid & Electrolyte Physiology, 21), F8 18-824. Mitchell, K. D. & Navar, L. G. (1991) Influence of intrarenally generated angiotensin I1 on renal haemodynamics and tubular reabsorption. Renal Physiology and Biochemistry, 14, 155-163. Morduchowicz, G. A., Sheikh-Hamad, D., Dwyer, B. E., Stern, N., Jo, 0.D. & Yanagawa, N. (1991) Angiotensin I1 directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system. Journal of Membrane Biology, 122,43-53. Munday, K. A., Parsons, B. J. & Poat, J. A. (1971) The effect of angiotensin on cation transport by rat kidney cortex slices. Journal of Physiology, 215, 269-282. Navar, L. G. & Rosivall, L. (1984) Contribution of the
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renin-angiotensin system to the control of intrarenal haemodynamics. Kidney International, 25, 857-868. Norman, J., Badie-Dezfooly, B., Nord, E. P., Kurtz, I., Schlosser, J., Chaudhari, A. & Fine, J. G. (1987) EGFinduced mitogenesis in proximal tubular cells: Potentiation by angiotensin 11. American Journal of Physiology, 253 (Renal Fluid & Electrolyte Physiology, 22), F299309. Olsen, M. E., Hall, J. E., Montani, J-P., Guyton, A. C., Langford, H. G. & Cornell, J. E. (1985) Mechanisms of angiotensin I1 natriuresis and antinatriuresis. American Journal of Physiology, 249 (Renal Fluid & Electrolyte Physiology, 18), F299-307. Romero, M. F., Hopfer, U., Madhun, Z. T., Zhou, W. & Douglas, J. G. (1991) Angiotensin I1 actions in the rabbit proximal tubule. Renal Physiology and Biochemistry, 14, 199-207. Saccomani, G., Mitchell, K. D. & Navar, L. G. (1990) Angiotensin I1 stimulation of Na+-H+exchange in proximal tubule cells. American Journal of Physiology, 258 (Renal Fluid & Electrolyte Physiology, 27), F1188-1195. Schunkert, H., Ingelfinger, J. R. & Dzau, V. J. (1991) Evolving concepts of the intrarenal renin-angiotensin system in health and disease: Contributions of molecular biology. Renal Physiology and Biochemistry, 14, 146-154. Schuster, V. L., Kokko, J. P. & Jacobson, H. R. (1984) Angiotensin I1 directly stimulates sodium transport in rabbit proximal convoluted tubules. Journal of Clinical Investigation, 13, 507-515. Seidelin, P. H., McMurray, J. J. & Struthers, A. D. (1989) Mechanisms of the antinatriuretic action of physiological doses of angiotensin I1 in man. Clinical Science, 76, 653-658. Seikaly, M. G., Arant, B. S. Jr & Seney, F. D. Jr (1990) Endogenous angiotensin concentrations in specificintrarenal fluid compartments of the rat. Journal of Clinical Investigation, 86, 1352- 1357. Sehr, M. C., Young, M., Meezan, E. & Pillion, D. J. (1990) Angiotensin I1 and bradykinin stimulate phosphoinositide breakdown in intact rat glomeruli but not in proximal tubules: Glomerular response modulated by phorbol ester. Biochemical Biophysical Research Communications, 166, 373-379. Spinelli, F. & Walther, A. (1979) Modulation by prostaglandins of angiotensin 11-induced stimulation of sodium transport in the proximal tubule of rat. Colloquium INSERM, 85,273-278. Steven, K. (1974) Effect of peritubular infusion of angiotensin 11 on rat proximal nephron function. Kidney International, 6, 73-80. Taub, M. & Saier, M. H. Jr (1979) Regulation of nNa+ transport by calcium in an established kidney epithelial cell line. Journal of Biological Chemistry, 254, 1144011444. Taylor, A. & Whindhager, E. E. (1979) Possible role of
cytosolic calcium and Na-Ca exchange in regulation of transepithelial sodium transport. American Journal of Physiology, 236 (Renal Fluid & Electrolyte Physiology, 5), F505-512. Thomas, D., Hams, P. J. & Morgan, T. 0. (1988) Agerelated changes in angiotensin 11-stimulated proximal tubule fluid reabsorption in the spontaneously hypertensive rat. Journal of Hypertension, 6 (Suppl. 4), S449-45 1. Thomas, D., Hams, P. J. & Morgan, T. 0. (1980) Altered responsiveness of proximal tubule fluid reabsorption in spontaneously hypertensive rats. Journal of Hypertension, 8,407-410. Torres, V. E., Northrup, T. E., Edwards, R. M.,Shah, S. V. & Dousa, T. P. (1978) Modulation of cyclic nucleotides in isolated rat glomeruli. Journal of Clinical Investigation, 62, 1334-1343. Wang, T. & Chan, Y-L. (1990) Mechanism of angiotensin I1 action on proximal tubular transport. Journal of Pharmacology and Experimental Therapeutics,252,689-695. Wang, T. & Chan, Y-L. (1991) The role of phosphoinositide turnover in mediating the biphasic effect of angiotensin I1 on renal tubular transport. Journal of Pharmacology and merapeutics, 256,309-317. Welsh, C., Dubyak, G. & Douglas, J. G. (1988) Relationship between phospholipase C and prostaglandin E2 and cyclic adenosine monophosphate production in rabbit tubular epithelial cells. Journal of Clinical Investigation, 81,710-719. Wirthensohn, G., Lefrank, S. & Guder, W. G. (1984) Phospholipid metabolism in rat kidney cortical tubules. 11. Effects of hormones on 33P incorporation. Biochimica et Biophysica Acta, 795,50 1-510. Wirthensohn, G. & Guder, W. G. (1985) Stimulation of phospholipid turnover by angiotensin I1 and phenylephrine in proximal tubules microdissected from mouse nephron. Pjlugers Archiv. European Journal of Physiology, 404,94-96. Woodcock, E. A. & Johnston, C. I. (1982) Inhibition of adenylate cyclase by angiotensin I1 in rat renal cortex. Endocrinology, 111,1687-1691. Xie, M-H., Liu, F-Y., Wong, P. C., Timmermans, P. B. M. W. M. & Cogan, M. G. (1990) Proximal nephron and renal effects of DuP 753, a nonpeptide angiotensin I1 antagonist. Kidney International, 38,473-479. Yu~gawa,M (1991) Angiotensin 11 and proximal tubule sodium tramport. Ranal Phlr~wlogyund Biochemistry, 14,208-215. Zhuo, J., Song, K., Harris, P. J. & Mendelsohn, F. A. 0.In vitro autoradiography reveals predominantly AT-1 angiotensin I1 receptor subtype in rat kidney. Renal Physiology and Biochemistry (in press). Zhuo, J., Thomas, D., Harris, P. J. & Skinner, S. L. (1992) The role of endogenous angiotensin I1 in the regulation of renal haemodynamics and proximal reabsorption. Journal of Physiology (in press).
REGULATION OF PROXIMAL TUBULE FUNCTION BY ANGIOTENSIN Peter J. Harris
Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia (Received 25 October 1991)
SUMMARY 1. Independent of its effects on renal haemodynamics and glomerular filtration, angiotensin I1 (AII) has direct actions on the proximal tubule involving transepithelial Na+, H+, HC03-, and water reabsorption, ammoniagenesis, gluconeogenesis and renal growth. 2. The effects of A11 on water and electrolyte transport are biphasic and dose-dependent, such that low concentrations ( 10-12-10-9 mol/ L) stimulate reabsorption whereas high concentrations ( 10-7-10-6 mol/L) inhibit reabsorption. Similar dose-response relations have been obtained for luminal and peritubular addition of AII. 3. The cellular responses to A11 are mediated via an AT-1 receptor coupled via G-regulatory proteins to several parallel signal transduction pathways. Low doses inhibit the basolateral adenylate cyclase, lower intracellular CAMP and withdraw the inhibitory effect of protein kinase A on the luminal Na/H exchanger. Stimulation of this exchanger may also occur due to AII-receptor activation of phospholipase C to release diacyl glycerol, or by local transduction in the brush-border membrane involving phospholipase Az4. Inhibition of proximal fluid reabsorption is associated with increased intracellular Ca2+ released from intracellular stores, or entering via voltage-sensitive channels in response to the release of inositol-l,4,5,-trisphosphate,or following Ca2+channel opening induced by the arachidonic acid metabolite 5,6,-epoxy-eicosatrienoic acid. 5 . The stimulatory actions of peritubular A11 on proximal transport are inhibited by physiological concentrations of atrial natriuretic factor (ANF) and by parathyroid hormone (PTH). 6. It is concluded that intrarenal A11 acts to maintain optimal matching of fluid reabsorption and filtered load in response to changes in sodium balance, as well as to promote acidification of the urine during acidosis and perhaps to potentiate tubular growth following renal injury.
Key words: ammoniagenesis, angiotensin 11, glomerulotubular balance, gluconeogenesis, intracellular signalling, Na/H exchanger, proximal tubule, renal acid-base regulation, renal growth, sodium reabsorption.
INTRODUCTION Angiotensin I1 (AII) exerts powerful influences on many functions of the proximal tubule, including salt and water reabsorption, H+ and H C 0 3 transport, ammoniagenesis (Chobanian & Julin 199I), gluconeo-
genesis (Guder 1979) and growth (Norman et al. 1987). The renal haemodynamic responses to A11 have been extensively investigated, and it is well established that A11 modifies solute delivery to the
Correspondence: Peter J. Harris, Department of Physiology, The University of Melbourne, Parkville, Vic. 3052, Australia.
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proximal tubule by altering glomerular filtration rate and inducing changes in peritubular oncotic and hydrostatic pressures with consequent effects on fluid and electrolyte uptake. These aspects of the tubular response to A11 have been reviewed elsewhere (Navar & Rosivall 1984; Mitchell & Navar 1991), and this review will focus on direct actions of A11 on the proximal tubular epithelium. Historically, the notion that A11 might be a physiologically important modulator of proximal fluid reabsorption arose from observations that the ‘loaddependency’ of proximal glomerulo-tubular balance could not be explained by purely intrinsic factors involving tubular and interstitial geometry, pressures and flow dynamics. Bojeson’s (1954) suggestion that a plasma hormone might be involved was examined by Leyssac (1969, who found that intravenous injection of A11 but not noradrenaline prolonged occlusion time in rats, indicating that inhibition of proximal fluid reabsorption was independent of any haemodynamic effects. Development of the perfused, isolated tubule preparation enabled Burg and Orloff (1968) to test more directly whether A11 might affect fluid uptake in the rabbit pars recta and thus explain the discrepancy between net fluid uptake rates estimated from in vivo micropuncture studies and during in vitro tubule perfusion. Unfortunately, no effect of A11 on fluid reabsorption could be found (later shown by Schuster et al. 1984to be due to enzymatic degradation by peptidases in the serum perfusate), and the nature of the hormonal modulator remained unknown. Despite negative or conflicting findings from several laboratories (for review, see Harris & Navar 1985), the Danish group continued to assert the importance of A11 in the proximal tubule, and Steven (1974) confirmed that peritubular infusion of A11 (2 X 10-8 mol/ L) inhibited proximal fluid reabsorption and reduced end-proximal TF/ P inulin ratio. Encouraged by observations that much lower doses of A11 (10-12 mol/ L) stimulated sodium transport in kidney cortex slices (Munday et al. 1971) and other tissues, notably frog skin (McAfee & Locke 1967) and rat colon (Davies et al. 1970), Harris and Young (1977) continued this investigation in anaesthetized rats. Stationary split-drop micropuncture combined with simultaneous capillary perfusion revealed a biphasic, dose-dependent response in which proximal sodium transport was stimulated by peritubular A11 at low concentrations (10-12 mol/L-10-10 mol/L) and inhibited by higher doses (10-6 mol/L). The characteristics of this dose-response relation were confirmed first by Spinelli and Walther (1979) using shrinking split-drop analysis with delivery of A11 by superfusion of the kidney surface, and later in the perfused,
isolated rabbit pars convoluta by Schuster et al. (1984). Subsequent work in many laboratories has been directed toward understanding the cellular mechanism of this action of AII, and to determining its physiological significance.
Cellular mechanisms The main features of current models for the cellular transduction of the action of A11 on proximal tubule electrolyte transport to A11 are summarized in Figs 1 and 2. Depending upon the dose, stimulation (Fig. 1) or inhibition (Fig. 2) of transepithelial sodium and water reabsorption can be elicited by either peritubular (Harris & Young 1977; Harris 1979; Spinelli & Walther 1979; Schuster et al. 1984) or luminal (Harris & Young 1977; Wang & Chan 1990; Yanagawa 1991; Morduchowicz et al. 1991) application of AII. Although the characteristics of the dose-response curves obtained are remarkably similar, there may well be important differences in the intracellular mechanisms involved in responses initiated from basolateral and apical surfaces.
Angiotensin receptors In either case, the chain of events begins with the binding of A11 to high affinity (nanomolar K d ) membrane-bound receptors that have been characterized in enriched membrane fractions according to affinity, density of distribution and cation dependence (Brown & Douglas 1982, 1983). In autoradiographic studies, binding of 12sI{sar’,Ile8] A11 in the renal cortex was potently displaced (K,= 23 nmol/L) by the non-peptide AT1 antagonist DuP 753, whereas AT2 antagonists had little effect (Zhuo 1991). It was concluded that approximately 95% of renal A11 receptors are of the AT1 subtype and that, at most, 5% are AT*. AT1 receptors probably correspond to the ‘subtype Bydescribed by de Gasparo et al. (1 990) and found, also by antagonist displacement studies, to predominate in the kidney. It is necessary to reconcile the conclusion that the majority of renal and proximal tubular A11 receptors are pharmacologically identical, with functional heterogeneity based on relative binding potency of A11 and AIII, response to the reducing agent dithiothreitol, and receptor coupling to different transduction pathways. Applying these criteria, Douglas(1987) concluded that there ace two functional subtypes corresponding to the glomerular mesangial (type A) and tubular epithelial (type B) sites of action. The distinction between glomerular and tubular receptors may represent a further subdivision of A11 receptors (ATIA and ATIB, respectively), but final
Angiotensin and proximal tubule function resolution of this issue will require cloning and expression of renal A11 receptors from each site and subsequent comparison with the known cDNA sequences for adrenal and vascular AT, receptors. The observation that proximal tubular A11 receptors belong predominantly to the ATI subtype implies that proximal reabsorption will be inhibited by the AT, antagonist DuP 753. This hypothesis has been tested and confirmed in recent clearance and micropuncture experiments in anaesthetized rats (Xie et al. 1990; Zhuo et al. 1992), but no comparable studies using AT2 antagonists have been reported. Although proximal epithelial A11 receptors appear to be relatively homogeneous with regard to pharmacology and binding kinetics, there is considerable evidence that they are coupled to a number of parallel signal transduction pathways within the cell corresponding to the variety of physiological responses that can be elicited by this peptide (Douglas et al. 1990).
Low dose stimulation of fluid and electrolyte transport The best described pathway mediates the stimulation of sodium and bicarbonate reabsorption by low (picomolar) concentrations of AII, and involves inhibition of adenylate cyclase with subsequent enhancement of luminal Na/ H exchange providing increased cellular uptake of Na and extrusion of H. Early biochemical studies on cortical tissue (Torres et al. 1978; Woodcock &Johnston 1982) established that cAMP production was inhibited by A11 but at concenK+
Na+ Na+ HC0,Peritubular
Kt
TT\
cAMP
J
DAG.T
I
5
J
PKA
,PKC?
n-
All
Fig. 1. Composite model for stimulation of proximal tubule by angiotensin. Abbreviations: PIP2, phosphatidylinositol4,5,-bisphosphate; PLC, phospholipase C; DAG, diacyl glycerol; PKC, protein kinase C; PLAz, phospholipase A2; G,, inhibitory G-regulatory protein; AC, adenylate cyclase; CAMP, cyclic adenosine monophosphate. represents the angiotensin receptor.
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trations well above those associated with increased electrolyte transport. Douglas et al. (1990) have recently developed a more sensitive assay system using cultured rabbit proximal tubule cells, and report receptor-mediated inhibition of adenylate cyclase by A11 within the picomolar to nanomolar range. Both this action and the AII-induced stimulation of sodium flux across confluent monolayers of similar cells were inhibited by pertussis toxin, indicating the involvement of a Gi regulatory protein coupling receptor and cyclase activity. A link between adenylate cyclase and sodium transport rate has been demonstrated in micropuncture experiments on rats in which luminal N a /H exchange was stimulated in proximal tubules when intracellular cAMP levels were reduced by A11 infusion (Liu & Cogan 1989). In addition, luminal perfusion with dibutyryl cAMP to maintain a high intracellular c AMP level abolished the luminal acidification normally observed with A11 infusion. CAMP-dependent protein kinase (PKA) has been shown to inhibit brush border Na/ H exchange (Kahn ef al. 1985), but it has not yet been demonstrated directly that removal of an inhibitory action of this enzyme occurs during the epithelial transport response to AII. Stronger evidence has been accumulated to support the involvement of the apical N a /H antiporter as the next step in the transduction pathway. Several groups have demonstrated that the stimulatory action of A11 on proton secretion and sodium reabsorption is inhibited by amiloride (Liu & Cogan 1988; Saccomani et al. 1990; Wang & Chan 1990). Liu and Cogan (1988) also noted that this effect was more pronounced in the S1 compared with S 2 segments, and correlated this with the relative abundance of A11 receptors. In the same study, evidence was provided that the major effect of A11 in the S2 segment is to stimulate NaCl reabsorption, and it was suggested that this action is mediated via catecholamine release from sympathetic nerve terminals adjacent to the basement membrane. In the most detailed study to date, Geibel ef al. (1990) loaded perfused, isolated superficial S 1 segments of rabbit tubules with the pH-sensitive dye BCECF and measured intracellular pH (pHi) in response to A11 (10-9 mol/ L) added to the bath solution. A11 caused an initial prolonged alkalinization and enhanced the rate of recovery from an intracellular acid load induced by removal of Na from the bath and luminal perfusates. The latter effect was inhibited by the amiloride analogue ethylisopropylamiloride (EIPA) indicating involvement of the luminal Na/ H exchanger, but the initial alkalinization was not sensitive to EIPA and was presumed to be mediated by some other acidloading or extruding process. A11 was also found to
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stimulate a DIDS-sensitive basolateral Na/ HC03 cotransporter, and it was noted that increased luminal H extrusion, together with stimulated basolateral HC03 efflux, would support a substantial rise in transepithelial HCO3 reabsorption with only a small change in pHi. The question of how Na exit from the proximal tubule cell might be linked to increased luminal entry has also been addressed by Garvin (1991). In proximal straight tubules, A11 (10-10 mol/ L) stimulated fluid absorption and increased K, for HCO3, since there was no change in transepithelial HCO3 permeability of Urn,,. In tubule segments treated with A11 for 30 min, Na/ K ATPase activity was increased by 27%. In this study the cells were made permeable prior to the addition of AII, and were therefore exposed to similar sodium concentrations during incubation, but it remains unclear whether there are direct effects of A11 on the Na/ K ATPase in vivo, or whether stimulation occurs primarily in response to increased Na delivery. Stimulation of basolateral Na/ K exchange must also result in increased K entry, which will tend to raise the intracellular K concentration. Due to the low luminal K permeability of these cells, the resulting increase in transmembrane K gradient is likely to be dissipated across the basolateral membrane. A basolateral K channel has been characterized in this membrane (Goegelein 1990) and appears to be pH-sensitive. Activation of this channel in response to cellular alkalinization following AII-induced stimulation of luminal Na/ H exchange would provide for enhanced K efflux. All available evidence indicates that there is no change in transepithelial potential difference during stimulation of transport by A11 (Harris & Young 1977; Schuster et al. 1984; Garvin 1989). Electroneutrality could be preserved if the net effect of hyperpolarization, due to stimulation of the Na/ K exchanger combined with increased basolateral K conductance, were balanced by the depolarizing influence of increased Na/ HCO3 cotransport. Microelectrode measurements in microdissected rabbit S1 and S2 segments showed no change in the potential difference across the basolateral membrane when A11 (10-10 or 10-7 mol/L) was added to the bath solution (Eitle, Hirst & Harris, unpubl. data). It should be noted that, due to the low electrical resistance of the proximal tubule, transepithelial potential gradients are very small ( cytosolic Ca2+and could thus mediate inhibition of
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Na transport. The source of this Ca2+is uncertain, since although AII-induced inhibition of Jv and JHCO3 was blocked by TMB-8 consistent with release from intracellular stores (Chatsudthipong & Chan 1991), Welsh et al. (1988) perfused cultured proximal cells with Ca2+chelators and found that the rise in cytosolic Ca2+was greatly reduced, indicating substantial influx from the extracellular medium. These investigators have concluded that Ca2+entry occurs through voltage-sensitive channels, since verapamil prevented the increase in intracellular Ca2+in response to high dose A11 (10-6 mol/L; Romero et al. 1991). The discrepancy between these observations regarding the source of Ca2+may be due to differences in species and the experimental system employed (rat in vivo vs rabbit proximal cells in culture), and further work is clearly required. Although most studies report increases in intracellular Ca2+ only with high (inhibitory) concentrations of AII, Jung and Endou (1989) attribute this to depletion of ATP levels. They demonstrated in isolated rat S1 segments that when acetate or pyruvate were provided as TCA cycle substrates, A11 caused a dosedependent biphasic response, with peak rises in cytosolic Ca2' at 10-11 mol/L and 10-7 mol/L, and a trough at 10-9 mol/ L. The coincidence of this pattern with the biphasic response characteristic of Na, fluid and HCO3 reabsorption, is unmistakable, but at present the significance of increased intracellular Ca2+ with low dose A11 remains unclear. A novel proposal for transduction of the inhibitory effect of high concentrations of A11 has been investigated by Douglas et al. (1990). Convinced that the A11 receptor does not couple effectively with phospholipase C in the proximal tubule, this group explored the possibility that high doses of A11 (10-9-10-6 mol/ L) activate phospholipase A2, releasing arachidonate and lysophospholipids from membrane phospholipids. Substantial evidence has been accumulated to support this hypothesis (Douglas et al. 1990) and to show that an epoxide, thought to be 5,6-EET, is produced as a result of metabolism of arachidonate by an NADPH-dependent cytochrome P450 enzyme. Accumulation of this epoxide induced by A11 mol/ L) in cultured rabbit proximal cells was inhibited by ketoconazole, an inhibitor of P450-dependent epoxygenase, while the addition of 5,6-EET to the apical side of monolayers of similar cells mimicked the inhibition of apical to basal Na flux seen with AII. 5,6-EET also increased cytosolic Ca2+in a manner similar to that caused by A11 (10-9-10-6 mol/ L), and the epoxide-induced rise was blocked by verapamil and nifedipine, indicating involvement of voltagedependent Ca2+channels in mediating the influx of
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extracellular Ca2+(Madhun et al. 1991). Inhibition of the basolateral Na/K ATPase by 5,6-EET was also observed (Romero et al. 1991), and it is possible that this effect contributes to the inhibition of Na transport by AII.
Physiological role of A11 regulation of proximal fluid reabsorption If stimulation (or inhibition) of proximal tubule transport is a significant feature of the regulation of this nephron segment under physiological conditions, then removal of A11 should cause corresponding changes in proximal fluid reabsorption and in delivery of filtrate to more distal segments. This prediction has been tested using inhibitors of angiotensinconverting enzyme (Harris et al. 1984; Thomas et al. 1988) and more recently with a non-peptide A11 antagonist, DuP 753 (Xie et al. 1990; Zhuo et al. 1992). According to these studies in anaesthetized rats, approximately 30% of proximal sodium, bicarbonate and water is modulated by A11 and, at least under these conditions, A11 exerts a tonic stimulatory action on the proximal tubule acting to maintain a high level of water and electrolyte reabsorption. It is possible that such a tonic effect of A11 is due in part to activation of the renin-angiotensin system during the anaesthesia and surgery required for micropuncture experiments. However, in an early study in conscious rats, Barraclough et al. (1967) found that intravenous infusions of low doses of A11 caused sodium and water retention only when the animals had previously been salt-loaded, presumably suppressing the endogenous renin and A11 levels. These workers, long before direct effects of A11 on the proximal tubule had been demonstrated, concluded that A11 has a tonic stimulatory action on tubular fluid reabsorption. Although it is clearly not possible to investigate individual tubule function in conscious animals or humans, indirect studies using lithium clearance as an indicator of proximal sodium and water reabsorption have provided valuable information (despite reservations regarding the quantitative relation between lithium clearance and proximal tubule reabsorption). In normal human subjects given a range of dietary sodium intakes, ACE inhibition caused natriuresis accompanied by a decrease in proximal fractional sodium reabsorption (Broyn 1988). Further evidence for a proximal stimulatory action of circulating AII was found when infusion of a low dose of A11 (1 ng/ min/ kg) caused antinatriuresis with reduced fractional lithium clearance (Seidelin et al. 1989). In anaesthetized rats ACE inhibition increased glome-
rular filtration rate accompanied by a lesser increase in proximal fluid reabsorption determined by lithium clearance (Harris et al. 1987b). It was concluded that proximal glomerulo-tubular balance (PGTB), reflecting the fraction of the filtered load of solute and water being reabsorbed, was biased towards increased rejection resulting in enhanced end-proximal flow. This study further demonstrated that the impaired effectiveness of PGTB could be corrected by infusion of either A11 or des-asp' A11 (AIII) indicating that the presence of circulating angiotensin is necessary for optimal matching of proximal reabsorption with delivery of glomerular filtrate. It is difficult to compare the results of studies in which A11 has been infused systemically with the data from in vivo micropuncture, microperfusion or cultured cell experiments where the concentrations of A11 can be controlled directly. Similarly, the concentrations of A11 that exist under various conditions in the interstitial fluid adjacent to the peritubular capillaries are unknown since, at present, it is not possible to obtain sufficiently large samples for assay. The problem is complicated by the presence of immunoreactive products of AII, some of which such as A111 and the heptapeptide 1-7 A11 are biologically active and form a substantial proportion of the total A11 particularly in the rat. Seikaly et al. (1990) recently reported total immunoreactive angiotensin concentrations in the nanomolar range in micropuncture samples of glomerular filtrate, proximal tubular fluid and plasma from star vessels on the kidney surface. Even when these values are corrected for the increase in tubule fluid concentration due to water reabsorption and for the dilution of star vessel plasma during passage through the peritubular capillaries, it is likely that the luminal and basal surfaces of the proximal tubule sells are exposed to A11 and A111 at concentrations approximately 100 times those circulating in plasma. These estimates are consistent with earlier measurements of A11 in renal cortical tissue (Mendelsohn 1979) and with the known affinities of A11 receptors. Angiotensins could pass from the juxtaglomerular cefls to the efferent arteriolar blood via the interstitium, and local production of A11 or A111 from angiotensinogen and renin within the proximal tubule cells (Schunkert et al. 1991) might account for their elevated concentrations in the tubule lumen. These estimates correspond with doses shown to stimulate proximal sodium and HCO3 transport (10-12-10-9 mol/L) and cover the physiological range of variations in circulating and local intrarenal A11 levels. It may be concluded that the proximal tubule is normally stimulated to some extent by A11 and that suppression of the renin-angiotensin system, for example by
Angiotensin and proximal tubule function
sodium loading, will lead to reduced reabsorption and an increase in the delivery of filtrate out of the proximal tubule. Whether these events will subsequently induce urinary loss of salt and water will depend on the capacity of the distal nephron to reabsorb the increased load and the activity of the tubulo-glomerular feedback response, itself modulated by the intrarenal A11 concentration (Mitchell & Navar 1991). Micropuncture, microperfusion and cell culture experiments have all demonstrated the inhibitory action of high concentrations of A11 (10-7 mol/L), but it is not clear in what physiological or pathological situations this effect might operate. Inhibition of proximal salt and water reabsorption by A11 does not appear to be the explanation for the natriuresis observed during systemic infusion of high doses of A11 as this has been shown to be dependent upon a concomitant rise in blood pressure (Olsen et al. 1985). Mitchell and Navar (1987) demonstrated stimulation of proximal fluid uptake when low concentrations of A11 were infused into the peritubular capillaries of anaesthetized rats, but found that higher doses affected the upstream glomerulus, reducing SNGFR and precluding detection of an inhibitory action on transepithelial transport. A luminal action of A11 in the proximal tubule was reported by Harris and Young (1977). Using stationary split-drop micropuncture in rats, they found that stimulation of the steady-state Na concentration gradient occurred when A11 ( mol/L) was added to the luminal fluid, but noted that this concentration was 100 times that required for stimulation from the peritubular side. More recently, continuous microperfusion studies (Wang & Chan 1990) have demonstrated that the dose-response curve for luminal addition of A11 is comparable with those reported for the peritubular action, including both stimulatory and inhibitory components and showing similar sensitivities for each response. The disparity between the doses required for stimulation by luminal A11 delivered by stationary rather than continuous microperfusion is most likely due to reduced enzymic degradation when the peptide is replaced during perfusion. This observation suggests that the concentration of A11 available far luminal receptor binding, and therefore the extent of AII-induced stimulation or inhibition of transport, may depend on the luminal flow that is directly related to the single nephron glomerular filtration rate (GFR). Thus these data may reveal an important physiological role for the brush-border angiotensinase in proximal glomerulo-tubular balance and provide an additional mechanism for the mediation of flow-related fluid reabsorption.
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AII-stimulated transport in hypertension In the spontaneously hypertensive rat (SHR) model of human essential hypertension, the early phase of development is accompanied by inappropriate retention of sodium and water, although neutral sodium balance is achieved in the adult at approximately 12 weeks of age. This pattern of development is associated with changes in the extent of AII-stimulated transport, such that the component of proximal fluid reabsorption in the young SHR (5 weeks) is enhanced when compared with age-matched Wistar-Kyoto (WKY) controls (Thomas et al. 1988). By 12 weeks, AIIstimulated transport is absent in the SHR although unaltered in the corresponding WKY. Further studies involving peritubular capillary perfusion of A11 in young and adult SHR and WKY have shown agedependent alterations in the sensitivity of proximal fluid reabsorption (Thomas et al. 1990). These changes may reflect down-regulation of A11 receptors due to elevation of A11 levels, or may indicate some genetic abnormality in AII-receptor kinetics or signal transduction. In another model of hypertension using the one-clip two-kidney Goldblatt rat, the extent of AIIstimulated proximal fluid transport was assessed by intravenous infusion of CEI and found to be similar (approximately 30%) in normotensive controls and in hypertensive animals 4-6 weeks after clipping (Harris et al. 1984). The maintenance of proximal reabsorption by A11 in the renin-angiotensin dependent phase of these Goldblatt rats contrasts with the absence of AIIstimulated transport in the adult SHR (Thomas et al. 1988) perhaps reflecting the contributions of A11 to sodium homeostasis in each model.
Interactions with other hormones Hormonal factors or neurotransmitters that act on proximal transport will necessarily act to attenuate or reinforce the responses due to angiotensin. Interaction with A11 has been clearly demonstrated for two hormones to date, parathyroid hormone (PTH) and atrial natriuretic factor (ANF). PTH acts via its own specific receptors on proximal tubule cells and exerts an inhibitory effect on bicarbonate and fluid reabsorption that is mediated by intracellular CAMP. Liu and Cogan (1989) have shown that net bicarbonate absorption correlates inversely with tubular fluid CAMP concentration during administration of either A11 or PTH. They concluded that approximately half of total renal acidification is influenced by this cyclic nucleotide. Since stimulation of proximal Na and HCO, reabsorption by A11 involves inhibition of CAMP,while PTH suppresses reabsorption by stimulating CAMP, interaction occurs at the level of the
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common shared pathway for signal transduction. ANF also acts through specific cell membrane receptors but has no action on the proximal tubule when infused into the peritubular capillaries (Harris et al. 1987a) or added to the bath fluid perfusing isolated rat tubules (Garvin 1989). However, at physiological concentrations (10-10 mol/L-10-8 mol/L), peritubular ANF inhibits AII-stimulated fluid reabsorption in vivo (Harris et al. 1987a) and in perfused isolated tubules (Garvin 1989). Since this latter action could be mimicked by the membrane permeant dibutyrylcGMP, it may be inferred that cGMP acts as a second messenger for the ANF response in the proximal tubule, as in other tissues. It is not yet clear how modulation of the intracellular cGMP level might inhibit the stimulatory action of A11 but these data reveal an important antagonism in the proximal tubule between the sodium and water retaining effects of A11 and the natriuretic and diuretic actions of ANF.
Ammoniagenesis and gluconeogenesis The role of A11 as a regulator of proximal tubular acidification is evident from its potent actions on transepithelial HC03 reabsorption. Additional importance has been attached to this role, as A11 also stimulates ammonia production from L-glutamine in suspensions of canine proximal tubule segments (Chobanian & Julin 1991). This action was observed under conditions of acidosis but not alkalosis, and there was an additive effect of A11 and reduced tubule fluid pH resulting in further enhancement of ammonia production. The threshold concentration required for a significant increase in ammoniagenesis was 10-8 mol/L, some three orders of magnitude higher than those associated with stimulation of Na, HC03 and water flux, and closer to the concentrations that inhibit reabsorption. The cellular mechanisms responsible for stimulation of ammoniagenesis are not clear but do not appear to involve cAMP or the luminal Na/ H exchanger. The response was mediated by A11 receptors and could be blocked by removal of extracellular Ca2’ or by inhibitors of the Ca2+-calmodulin dependent pathway. Effects of A11 on proximal tubule metabolism were also reported by Guder (1979), who reported stimulation of ammoniagenesis and gluconeogenesis in rat tubule fragments in response to high concentrations of A11 (10-7 mol/L). These actions did not correlate with changes in intracellular cAMP and were only partially inhibited by removal of CaZ+from the medium. AII-induced stimulation of gluconeogenesis from L-glutamine was confirmed by Chobanian and Julin (1991), who noted that this substrate will support
parallel stimulation of glucose and ammonia production, as the deamination and deamidation of Lglutamine produces ammonia, leaving alpha-ketoglutarate, which may be converted to glucose.
Renal growth Studies designed to assess the effects of A11 on cell growth in primary cultures of proximal tubule cells have found no response in terms of cell numbers or cell size. However, A11 has been shown to potentiate the mitogenic effect of epidermal growth factor (EGF) on cultured proximal cells, and it appears that this effect operates at some stage after the agonist-receptor interaction (Norman ef al. 1987). Although it has been suggested that mobilization of intracellular Ca2+ might be involved in the interaction between A11 and EGF, it is also possible that alkalinization of the cell as a consequence of stimulation of the luminal Na/ H exchanger provides an internal environment favourable to mitogenesis.
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