Angiotensin regulates endothelin-B receptor in rat inner medullary collecting duct

Our recent studies showed that endothelin (ET)(B) receptors are downregulated in congestive heart failure. These changes in ET(B) receptor density can be prevented by angiotensin-converting enzyme inhibitors, suggesting a possible role for angiotensin. Using isolated inner medullary collecting ducts (IMCD), we examined the possibility that angiotensin-induced downregulation of ET(B) receptors is accompanied by a decrease in ET(B) receptor mRNA. Binding studies showed that overnight incubation with angiotensin II induced a downregulatiion of ET(A) and ET(B) receptors' density in IMCD by 39% and 29%, respectively. This downregulation in ET receptor density was abolished when IMCD was coincubated with angiotensin II and its receptor antagonist saralasin. Furthermore, when the cells were exposed to phorbol myristate acetate (PMA), it resulted in a reduction in ET(A) and ET(B) receptor binding sites by 41% and 34%, respectively, suggesting the involvement of protein kinase C (PKC). In isolated IMCD, ET-1 induced an increase in cyclic guanosine monophosphate (cGMP) accumulation (705 + 63 to 1,015 + 88 fmol/microg protein/5min, P <.01), and the ET-1-induced accumulation was attenuated in the presence of angiotensin II (641 + 45 to 809 + 46 fmol/microg protein/5min, P <.01). Using competitive polymerase chain reaction (PCR) method, we also observed downregulation of ET(A) and ET(B) receptors mRNA in IMCD treated with angiotensin II (ET(A), 1.09+0.11 v 0.77 + 0.07 amol/microg of total RNA, P <.01; ET(B), 14.80 + 1.95 v 8.65 + 0.67 amol/microg of total RNA, P <.01). The addition of a PKC inhibitor abolished the downregulation of ET(A) and ET(B) receptor mRNA induced by angiotensin II (ET(A), 1.25 + 0.07 v 1.19 + 0.06 amol/microg of total RNA, not significant [NS]; ET(B), 14.36 + 0.83 to 13.68 + 0.64 amol/microg of total RNA, NS). These results suggest that angiotensin II-induced downregulation of ET(A) and ET(B) receptors mRNA is mediated by a mechanism involving PKC.     

Sgk1 mediates osmotic induction of NPR-A gene in rat inner medullary collecting duct cells

We have shown previously that increased extracellular osmolality stimulates expression and promoter activity of the type A natriuretic peptide receptor (NPR-A) gene in rat inner medullary collecting duct (IMCD) cells through a mechanism that involves activation of p38 mitogen-activated protein kinase (MAPK). The serum and glucocorticoid inducible kinase (Sgk) is thought to participate in the regulation of sodium handling in distal tubular segments. We sought to determine whether this kinase might be involved in the osmotic stimulation of NPR-A gene promoter activity. Exposure of cultured IMCD cells to an additional 75 mmol/L NaCl in culture media (final osmolality 475 mosm/kg) resulted in an approximately 4-fold increase in Sgk1 protein levels after 7 hours. The Sgk1 induction was almost completely inhibited by the p38 MAPK inhibitor SB203580, indicating that NaCl activates Sgk1 through the p38 MAPK pathway. Transient transfection of a mouse Sgk1 expression vector along with a -1590 NPR-A luciferase reporter resulted in an approximately 3-fold increment in reporter activity, which was significantly reduced by cotransfection with a kinase-dead Sgk1 mutant. The NaCl-dependent induction was partially blocked (approximately 40% inhibition) by cotransfection of the kinase-dead Sgk1 mutant. Neither Sgk1 nor the kinase-dead mutant had any effect on endothelial nitric oxide synthase (eNOS) promoter activity, and the Sgk1 mutant and 8-bromo-cyclic guanosine monophosphate were, to some degree, additive in reducing osmotically stimulated NPR-A promoter activity. Collectively, these data imply that Sgk1 operates over an eNOS-independent, p38 MAPK-dependent pathway in mediating osmotic induction of the NPR-A gene promoter.     

Angiotensin II increases H+-ATPase B1 subunit expression in medullary collecting ducts

Metabolic alkalosis is a common feature of hypokalemic hypertensive syndromes associated with angiotensin II excess. The alkalosis-generating effect of angiotensin II is usually ascribed to its stimulatory effect on aldosterone secretion, a hormone that upregulates collecting duct hydrogen ion secretion. We studied the effect of angiotensin II infusions on the expression of B1 and a4 protein, subunits of the renal H+-ATPase in adrenalectomized rats. Adrenalectomized rats were given either angiotensin II or vehicle for 7 days via osmotic mini-pumps. H+-ATPase B1 protein expression was evaluated by Western blot analysis in isolated medulla and cortex plasma membrane preparations from one kidney, whereas the contralateral kidney was used for immunostaining. By Western blotting, the relative abundance of B1 protein was 2-fold higher in renal medulla membranes from rats with intact adrenal glands (sham surgery) than from adrenalectomized rats (219+/-47%, n=12; P<0.05). In contrast to renal medulla, adrenalectomy did not significantly alter the relative abundance of B1 protein in renal cortex. Angiotensin II also did not significantly alter the relative levels of B1 protein in the cortex, but it increased it significantly in renal medullary membranes (231+/-56%, n=8; P<0.005). Moreover, enhanced H+-ATPase B1 subunit protein immunoreactivity was found in medullary collecting duct segments of rats infused with angiotensin II. In contrast to B1, expression of a4, another subunit of the H+-ATPase was not altered by adrenalectomy or angiotensin II. We conclude that adrenalectomy decreases whereas angiotensin II increases H+-ATPase B1 subunit expression in medullary, but not in cortical collecting ducts. By increasing the relative abundance of the B1 subunit of H+-ATPase in the collecting duct, angiotensin II excess may lead to increased hydrogen ion secretion and thus metabolic alkalosis-a common feature of hypertensive syndromes associated with angiotensin II overactivity.     

Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat.

AQP-CD is a vasopressin-regulated water channel expressed exclusively in the renal collecting duct. We have previously shown that AQP-CD is present in the apical plasma membrane and subapical vesicles of collecting duct cells, consistent with membrane-shuttling mechanisms that have been proposed to explain the short-term action of [Arg8] vasopressin (AVP) to regulate apical water permeability. We propose here that AVP may also have long-term actions on the collecting duct to regulate the expression of the AQP-CD water channel. We used immunoblotting, immunohistochemistry, and in vitro perfusion of renal tubules to investigate water channel regulation in collecting ducts of diabetes insipidus (Brattleboro) rats treated with a 5-day infusion of AVP or vehicle. Immunoblotting and immunohistochemistry demonstrated that collecting ducts of vehicle-infused Brattleboro rats had markedly reduced expression of AQP-CD relative to normal rats. In response to AVP infusion there was a nearly 3-fold increase in AQP-CD expression as detected by immunoblotting. Immunocytochemistry demonstrated that the increased expression was predominantly in the apical plasma membrane and subapical vesicles of collecting duct cells. Inner medullary collecting ducts of AVP-infused Brattleboro rats displayed a 3-fold increase in osmotic water permeability relative to vehicle-infused controls, in parallel with the change in AQP-CD expression. Based on these findings, we conclude that (i) long-term infusion of AVP, acting either directly or indirectly, regulates expression of the AQP-CD water channel and (ii) AQP-CD is the predominant AVP-regulated water channel.     

Angiotensin II stimulates renin in inner medullary collecting duct cells via protein kinase C and independent of epithelial sodium channel and mineralocorticoid receptor activity

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

Postnatal ontogenesis of vasopressin receptors in the rat collecting duct.

The ontogenesis of vasopressin receptors in the rat collecting duct was studied by measuring the binding of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-O-methyltyrosine,4-threonine,8-ornithine,9-125I-tyrosylamide+ ++]-vasotocin (125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH(9)2]-OVT) to isolated cortical collecting ducts (CCD), outer medullary collecting ducts (OMCD) and inner medullary collecting ducts (IMCD) microdissected from collagenase-treated kidneys of 2- to 34-day-old rats and adult animals. The stereospecificity for recognition of a series of seven vasopressin structural analogues by CCD and OMCD receptors reveals that the labeled binding sites identified in 11- to 16-day-old and adult rats are homologous respectively and contain a major population of V2 type and a minor population of V1a type of vasopressin receptors. At all postnatal stages examined, the receptor density (expressed as 10(-18) mol radioligand bound per square millimeter tubular outer surface area) decreases gradually from the CCD to the IMCD. For the three segments, the numbers of receptors detected remained constant during the first 2 weeks after birth and increased sharply after 20 days to reach the corresponding adult levels during the fifth week.     

Angiotensin I conversion to angiotensin II stimulates cortical collecting duct sodium transport

Angiotensin (Ang) II directly stimulates epithelial sodium channel activity in the rabbit cortical collecting duct. Because Ang I and converting enzyme analogues might be present in the distal nephron, this raises the possibility of intraluminal generation of Ang II. Conversion of Ang I to Ang II was monitored by Ang II-dependent changes in intracellular sodium concentration as a reflection of sodium transport across the apical membrane. This involved imaging-based fluorescence microscopy with sodium-binding benzofuran isophthalate in isolated, perfused, cortical collecting-duct segments from rabbit kidney. Principal and intercalated cells were differentiated by rhodamine-conjugated peanut lectin. Control principal cell intracellular sodium concentration, during perfusion with 25 mmol/L NaCl and zero sodium in the bath plus monensin (10(-5) mol/L) averaged 5.8+/-0.14 mmol/L (n=156). The increase in intracellular sodium concentration, when luminal NaCl was increased from 25 to 150 mmol/L, was elevated by 3.5-fold in the presence of intraluminal Ang I (10(-6) mol/L). Also, the effects of Ang I on sodium transport were not significantly different from the effects of Ang II (10(-9) mol/L). Ang I was used in micromolar concentrations to ensure that there was sufficient substrate available for conversion to Ang II. Inhibition of the angiotensin-converting enzyme with captopril reduced the stimulatory effect of Ang I. These results suggest that intraluminal conversion of Ang I to Ang II can occur in the cortical collecting duct, resulting in enhanced apical sodium entry.     

Rapid stimulation of cyclic AMP production by aldosterone in rat inner medullary collecting ducts

Aldosterone stimulates sodium transport in the inner medullary collecting duct (IMCD) via the classic genomic pathway, but it is not known whether it also acts via a rapid, non-conventional pathway in this part of the nephron. The IMCD regulates the final sodium content of urine and expresses vasopressin receptors coupled to adenylate cyclase. The recently reported rapid, non-genomic actions of aldosterone have been associated mainly with an increase in intracellular Ca(2+); however, it has also been shown to stimulate camp generation. Thus the aim of this study was to determine whether aldosterone stimulates rapid generation of cAMP in isolated IMCD segments. IMCD segments were microdissected from Sprague-Dawley rat kidneys and incubated at 37 degrees C for 4 min with aldosterone (10(-12) to 10(-6) M), vasopressin (10(-12) to 10(-6) M), or a combination of hormones in the presence of a phosphodiesterase inhibitor. cAMP was measured by radioimmunoassay. While corticosterone and dexamethasone were ineffective, aldosterone stimulated a dose-dependent increase in cAMP within 4 min (P<0.05). This action of aldosterone was not inhibited by the MR antagonist spironolactone. Co-incubation of aldosterone with vasopressin resulted in a further increase in cAMP generation above that induced by the neurohypophysial hormone alone. Aldosterone-mediated cAMP generation was not inhibited by a vasopressin V(1) or V(2) receptor antagonist. These data support a novel and rapid, non-genomic effect of aldosterone in IMCD. Aldosterone does not apparently interact with the vasopressin receptor to stimulate cAMP generation.     

Effects of urinary ouabain-like factors and vanadium diascorbate on calcium mobilization in porcine inner medullary collecting duct cells: Comparison with the effects of ouabain and vasopressin

It is speculated that ouabain-like factors (OLF) play a role in the pathogenesis of volume-dependent hypertension. In previous studies we isolated a more polar OLF-1 and a more apolar OLF-2 from the urine of healthy subjects after 5 days on a high sodium intake (>400 mmol/day) by gel chromatography (Sephadex G-25 and G-10) and reverse-phase HPLC. We subsequently identified the chemical structure of OLF-2 as vanadium (V^IV ) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. Because the inner medullary collecting duct (IMCD) plays a crucial role in the long-term regulation of body fluid volume, in the present study we investigated the effects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comparison to ouabain and vasopressin (AVP) on calcium mobilization, ie, on free calcium concentration [Ca²⁺]_i, in cultured porcine IMCD cells. [Ca²⁺]_i was determined by the fura-2 method in IMCD cells isolated by hypotonic treatment and density gradient centrifugation from fresh porcine kidneys. Assuming an approximate molecular weight (MW) of 400 for OLF-1 and OLF-2, OLF-1 (10⁻⁴ mol/L) produced a slow increase in [Ca²⁺]_i from 39 ± 10 to 169 ± 21 nmol/L (n = 7 ) after 4 min. Similarly, OLF-2 (10⁻⁴ mol/L) resulted in an increase in [Ca²⁺]_i from 74 ± 20 to 216 ± 52 nmol/L (n = 7) after 4 min. Vanadium diascorbate (MW 403) dose-dependently increased [Ca²⁺]_i . At a concentration of 10⁻⁶ mol/L it increased [Ca²⁺]_i from 46 ± 5 to 149 ± 9 nmol/L (n = 5) after 4 min. A similar slow increase in [Ca²⁺]_i was found with ouabain (10⁻⁶ mol/L), which increased [Ca²⁺]_i from 61 ± 22 to 180 ± 29 nmol/L (n = 5) after 4 min in contrast to AVP (10⁻⁷ mol/L), which rapidly increased [Ca²⁺]_i from 48 ± 10 to 299 ± 32 nmol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorbate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca²⁺]_i as expected from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.     

Physiological effects of vasopressin and atrial natriuretic peptide in the collecting duct

Vasopressin plays a primary role in the concentration of urine to maintain body fluid homeostasis. The collecting duct as well as thick ascending limb is a major target site of vasopressin. The antidiuretic action of vasopressin is mediated by the V2 receptor in the basolateral membrane of principal cells in the collecting ducts. The binding of vasopressin to V2 receptors causes an activation of adenylate cyclase and a synthesis of cAMP. Vasopressin regulates water and ion transport through V2 receptor-mediated ion channels and transporters. In contrast, the V1a receptor mainly in the luminal membrane of distal nephron regulates basolateral V2 receptor-mediated action with regard to water and ion transport through the activation of G(q/11) and phosphoinositide turnover. Guanylate cyclase forms three types of ANP receptors, although NPR-A and B (GC-A and B) are biologically active and related to the synthesis of cGMP. Urodilatin, synthesized by the kidney, causes natriuresis by binding to GC-A in the collecting ducts. ANP causes diuresis and natriuresis, at least in part by inhibiting the V2 receptor-mediated action of AVP in the collecting ducts. The site of interaction of ANP and AVP is post cAMP synthesis, at least in the collecting ducts. The roles of AVP and ANP under pathophysiological conditions have been reported.     

Prompt inhibition of arginine vasopressin-induced cellular adenosine 3',5'-monophosphate production by extracellular sodium depletion in rat renal inner medullary collecting duct cells in culture

The present study was undertaken to determine whether the absence of extracellular Na+ affects cellular action of arginine vasopressin (AVP) in rat renal inner medullary collecting duct cells in culture. AVP increased cellular cAMP production in a dose-dependent manner. Na+ depletion promptly diminished the cellular cAMP response to AVP (1 nM AVP; 405.9 +/- 26.1 vs. 189.8 +/- 12.1 fmol/micrograms protein, P less than 0.01). The dose-response relation shifted to the right. The inhibition of the ability of AVP to produce cAMP was observed with an extracellular Na+ concentration less than 60 mM. Similar results were obtained with 2 x 10(-8) M forskolin, a diterpene activator of adenylate cyclase. Such inhibition was easily released, since only 10-min reexposure of the Na(+)-depleted cells to the control medium totally recovered the cAMP response to AVP. Extracellular Na+ depletion promptly decreased the cellular Na+ concentration from 15.8 +/- 1.0 to 5.4 +/- 0.6 mM (P less than 0.01), measured using the fluorescence dye sodium-binding benzofuran isophthalate. If the Na(+)-depleted cells were again incubated with the control medium, intracellular Na+ rapidly recovered to the precontrol level. Such a change was closely related to the change in cellular pH, which decreased from 7.19 +/- 0.02 to 6.97 +/- 0.02, measured using the fluorescence dye 2',7'-bis-(2-carboxymethyl)-5 (and -6)carboxyfluorescein,acetamethylester. However, Na+ depletion did not affect the cellular free calcium concentration or cellular protein and ATP contents. These results indicate that Na+ depletion promptly attenuated the ability of AVP to produce cAMP mediated through either the decrease in intracellular Na+ or cellular pH in renal inner medullary collecting duct cells.     

Stimulation of rat renal medullary Na+/K(+)-ATPase by arginine vasopressin is mediated by the V2 receptor

In previous studies, we have demonstrated that 1-10 fmol arginine vasopressin (AVP)/l maximally stimulates the activity of the enzyme Na+/K(+)-ATPase in the rat renal medullary thick ascending limb (MTAL) of Henle's loop after 4 or 10 min of stimulation when measured using a cytochemical bioassay. We have tested the hypothesis that this stimulation is mediated by the V2 receptor in the MTAL. A cytochemical bioassay was used to investigate the effect of specific V1 and V2/V1 antagonists and a synthetic V2 agonist [1-deamino,8-D-arginine]-vasopressin (dDAVP), on the activity of Na+/K(+)-ATPase. There was no effect of the V1 antagonist (1 fmol-1 mumol/l) in inhibiting the activity of Na+/K(+)-ATPase stimulated by 1 fmol AVP/l. In contrast, 100 pmol of the V2/V1 antagonist/l significantly (P less than 0.001) inhibited the stimulation of Na+/K(+)-ATPase activity by 1 fmol AVP/l from 55.5 +/- 4.3 (S.E.M.) to 31.9 +/- 1.6 mean integrated extinction (MIE) after 4 min of stimulation and from 67.0 +/- 3.2 to 36.9 +/- 0.7 MIE after 10 min of stimulation. Similarly, the stimulation of Na+/K(+)-ATPase by 10 fmol dDAVP/l was inhibited by the V2/V1 antagonist from 55.1 +/- 1.0 to 26.1 +/- 0.5 MIE after 4 min of stimulation. We conclude that the stimulation of Na+/K(+)-ATPase by AVP is mediated by the V2 receptor in the rat renal MTAL.