The collecting duct, dopamine and vasopressin-dependent hypertension.

AVP not only increases osmotic water permeability (Pf) in the rat cortical collecting duct (CCD), but also acts synergistically with aldosterone to augment sodium reabsorption (JNa). These effects are inhibited by catecholamines via alpha2 adrenergic receptors, and by dopamine. We review here studies designed to determine the mechanism and receptor involved in dopamine action. The inhibitory effect of dopamine on Na+ and water transport was found to be reversible, and was not produced by agonists specific to D1A and D1B receptors. D2-type (D2, D3 or D4) receptors and activation of the GTP-binding protein Gi were implicated by the observation that dopamine had no inhibitory effect when JNa and Pf were stimulated by a cyclic AMP analogue plus isobutylmethylxanthine. The only dopaminergic antagonist that reversed the inhibitory effect of dopamine was clozapine, which is relatively D4-specific. We also found that dopamine or D1-specific agonists by themselves had no effect on cAMP production. However, dopamine inhibited the high rate of AVP-dependent cAMP production, and this effect of dopamine was reversed by clozapine but not other antagonists or by inhibitors of protein kinase C. The D4 receptor was observed in western blots of renal cortical proteins, and it was localized to the collecting duct by RT-PCR and immuno-histochemistry using a D4-specific antibody. These results show that at least a portion of the natriuretic effect of dopamine can be attributed to inhibition of AVP-dependent Na+ reabsorption by the CCD, and they introduce another signalling system as a candidate in the aetiology of low-renin, salt-dependent hypertension.

Interaction of modeling and experimental approaches to understanding renal salt and water balance.

Understanding of renal function has profited greatly from quantitative and modeling approaches throughout this century. One of the most salient examples is the concentration and dilution of the urine--a fundamental characteristic of the mammalian kidney. Only in the last three decades have the necessary components of this and other renal mechanisms been confirmed at the molecular level, but there have also been surprises. For example, the high water permeability of the proximal tubule has been shown to be imparted by the water channel, aquaporin AQP1; however, genetic knockout of this water channel is at least partially compensated by intrarenal feedback mechanisms. Also, the critical role played by the fine regulation of Na+ reabsorption in the collecting duct for the maintenance of normal blood pressure presents challenges to our understanding of the integrated interaction among systems. As a first step in placing the kidney in the physiome paradigm, we need to integrate our currently restricted mathematical models, to develop accessible databases of critical parameter values together with indices of their degrees of reliability and variability, and to describe regulatory mechanisms and their interactions from the molecular to the interorgan level. However, even greater will be the challenge to anticipate, and hopefully predict, the subtle ways in which the organism typically adapts to aberrations in these normal functional parameters, but sometimes does not.

The concerns of patients and spouses after the diagnosis of colon cancer: a qualitative analysis.

PURPOSE: The purposes of this study were to describe the concerns of patients and their spouses after a diagnosis of colon cancer and to identify ways in which health care professionals could assist both patients and their spouses to cope more effectively with this illness and its treatment. DESIGN: Descriptive, cross-sectional study. SETTING AND SUBJECTS: Thirty patients with colon cancer and their spouses completed interviews in their homes. Most patients (83%) had undergone partial colon resection, and 77% had no evidence of cancer in adjacent lymph nodes. Twelve of the patients (40%) had a colostomy at the time the data were collected. METHODS: Patients and spouses participated in semistructured interviews that elicited information concerning their reactions to a diagnosis of colon cancer, to a colostomy (if present), to changes in lifestyle, to their satisfaction with information they received, and to ways that health care professionals could assist them with the illness and treatments. Content analysis was used to analyze the interview data and to group data into inductively derived categories. Interrater reliability was obtained by having 2 researchers independently code the data. RESULTS: Spouses tended to regard a colon cancer diagnosis more negatively than did patients. Both patients and spouses reported that fear of cancer's recurrence was their greatest concern. Most also reported lifestyle changes (80%) as a result of the illness; but the patients reported more changes in their functional ability, whereas spouses reported more changes in their roles and relationships. Half of the patients and most of the spouses (75%) reported a favorable reaction to the colostomy. Approximately half of the couples expressed satisfaction with the information they received; they reported a need for more information about treatments and management of side effects. When asked how professionals could help them, most couples stated that they wanted more information about the expected course of recovery. CONCLUSION: Spouses should be included in health assessment and teaching because they regard the illness more negatively than do patients. Both patients and spouses desire information that will help them to understand the typical course of recovery, assist them to plan for lifestyle changes, and enable them to manage the side effects of treatment.

Dopamine D4 receptor isoform mRNA and protein are expressed in the rat cortical collecting duct.

We reported previously [Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F391-F400, 1996] that dopamine inhibits vasopressin (AVP)-dependent water permeability and Na+ transport in the rat cortical collecting duct (CCD) apparently through a D4 dopamine receptor. The present experiments used RT-PCR of total RNA extracted from microdissected rat CCD to determine whether the D4 and D1A dopamine receptor isoforms are expressed. Specific primers were used to amplify three regions of the D4 cDNA. All three gave products with 98-100% nucleotide identity to the known rat D4 sequence; however, there was an extra 6-bp insert at the 3' end of the second transmembrane region that was identical to the human and mouse sequences but which had not been documented in the rat sequence. D4 receptor protein was also localized exclusively to the CCD and medullary collecting ducts by immunohistochemistry. Two regions of the D1A dopamine receptor message were also amplified by RT-PCR of RNA from rat CCD and were verified by sequencing and immunohistochemistry. We conclude that both D4 and D1A dopamine receptors are expressed in the rat CCD, but the physiological effects are attributable to a D4 receptor.

Dopamine inhibits vasopressin-dependent cAMP production in the rat cortical collecting duct.

Dopamine inhibits Na+ and water reabsorption in the rat cortical collecting duct (CCD) in the presence of arginine vasopressin (AVP). This inhibition appears to involve the D4 dopamine receptor isoform, which inhibits cAMP production; however, the D1A receptor, which stimulates cAMP production, is also expressed in the CCD. To discriminate between these opposing effects, we measured cAMP production in intact CCD segments. The basal rate of cAMP production ranged from 6.5 to 10 fmol/mm of tubule length over a 7-min incubation period, and it was unaffected by either dopamine or the D1A-specific agonist fenoldopam. AVP increased cAMP production to the range of 85-153 fmol . mm-1 . 7 min-1. Whereas neither 0.1 nor 1.0 microM fenoldopam affected AVP-dependent cAMP production, dopamine reduced it in a dose-dependent manner, achieving a maximum inhibition of 50% at 10 microM. This effect was reversed by the D4 receptor antagonist clozapine but not by pimozide or spiperone (antagonists of D2 and D3 receptors) or by calphostin C or chelerythrine (inhibitors of protein kinase C). We conclude that dopamine inhibits transepithelial Na+ transport and osmotic water permeability in the presence of AVP by inhibition of cAMP production, which is mediated by the D4 receptor isoform linked via the inhibitory G protein Gi.

Expression of multiple alpha-adrenoceptor isoforms in rat CCD.

In the rat cortical collecting duct (CCD), epinephrine inhibits vasopressin (AVP)-dependent water permeability and Na+ reabsorption. Although inhibition is reversed by the alpha2-adrenoceptor (AR) antagonist yohimbine, suggesting the epinephrine effect is primarily mediated by an alpha2-AR [C. T. Hawk, L. H. Kudo, A. J. Rouch, and J. A. Schafer. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F449-F460, 1993], there are also suggestions of an effect at an additional receptor, perhaps an alpha1-AR. For the present experiments, we used RT-PCR of total RNA extracted from 1 to 5 mm of microdissected CCDs from rat kidney to identify the alpha-AR isoforms expressed. Specific primers for the alpha2-ARs amplifying from the 6th transmembrane (TM) to the 3'-untranslated regions, revealed the presence of alpha2A and alpha2B. Western blot analysis also indicated the presence of alpha2B-AR at the protein level. Degenerate alpha1-AR primers that amplify from conserved regions of TM-1 to TM-5, as well as specific primers that amplify either the same region (alpha1B), the carboxy terminus (alpha1A), or within the third cytoplasmic loop (alpha1D), indicated the presence of all three alpha1-ARs. Measurement of transepithelial voltage in isolated perfused renal tubules indicated a small inhibitory effect mediated by alpha1-ARs. Although the functional effects of epinephrine on AVP-dependent transport processes appear to be mediated predominantly by an alpha2-AR, a small contribution to the overall alpha-AR effect may be due to simultaneous activation of an alpha1-AR.

Low Na+ diet inhibits Na+ and water transport response to vasopressin in rat cortical collecting duct.

BACKGROUND: We previously demonstrated that vasopressin (AVP) produces a sustained increase in Na+ reabsorption by the isolated perfused cortical collecting duct (CCD) from rats on a normal diet, and that this effect is synergistic with that of pharmacological doses of deoxycorticosterone (DOC) or physiological levels of aldosterone. The present experiments examined the effect of AVP under the more physiological circumstances when plasma aldosterone was elevated by prior volume depletion. METHODS: Rats were volume depleted by a single dose of furosemide followed by a low-salt diet (0.3% NaCl) for four to nine days. Some of these rats were also implanted with a pellet containing 2.5 mg DOC. Rats in a third group were not injected with furosemide but were implanted with the DOC pellet and maintained on a standard (approximately 1% NaCl) diet. CCD were perfused and the lumen-to-bath Na+ flux (JNA), transepithelial voltage (VT), and osmotic water permeability (Pf) were measured in the presence and absence of 200 pm AVP. RESULTS: Although Na+ depletion by a single injection of furosemide and the low salt diet elevated plasma aldosterone and Vt, JNA remained low and there was a decreased response to AVP in comparison with DOC-treated rats on a standard diet. In CCD from rats on the low salt-diet with DOC, JNa was less than observed in CCD from DOC-treated rats on a standard diet. AVP-dependent Pf in CCD from rats on the low salt-diet, with or without DOC treatment, was also markedly lower. CONCLUSIONS: We interpret the results to demonstrate that maximal rates of Na+ reabsorption in the CCD depend not only on the synergistic stimulatory effects of aldosterone and AVP, but also require normal to high rates of salt delivery in vivo for the effects of the hormones on Na+ transport to be maximized in vitro.

Microtubule disruption inhibits AVT-stimulated Cl- secretion but not Na+ reabsorption in A6 cells.

The effects of microtubule disruption on arginine vasotocin (AVT)-stimulated Na+ and Cl- transport were studied in A6 cells by measuring short-circuit currents (Isc) across cell layers grown in tissue culture on permeable supports. Microtubule disruption inhibited an AVT-stimulated secretory Cl- current but did not prevent activation of amiloride-sensitive Na+ transport. This AVT-stimulated secretory Cl- current was significantly inhibited by glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). Reverse transcription of RNA isolated from A6 cells followed by polymerase chain reaction (PCR) using primers designed to amplify a portion of the R-domain of CFTR cloned from Xenopus laevis skin and immunocytochemistry demonstrated the presence of CFTR in A6 cells and an apparent recruitment of cytoplasmic CFTR to the apical cell surface after AVT stimulation. In contrast, indirect immunofluorescent labeling of Na+ channels using a polyclonal antibody raised against a biochemically isolated Na+ channel complex from bovine renal medulla labeled the apical plasma membrane but failed to demonstrate intracellular labeling of Na+ channels (except in subconfluent cells) or recruitment of Na+ channels to the apical membrane region after AVT stimulation.

A simplified method for isolation of large numbers of defined nephron segments.

We describe a simplified method for the isolation of large numbers of nephron segments from rat and rabbit kidneys. In contrast to most previous protocols, the kidneys are not perfused. After removal from the animal, the kidney is sliced and torn in pieces that are subsequently digested in culture medium containing 0.5 mg/ml of collagenase at 37 degrees C. If the preparation is agitated only very gently and infrequently, then the tissue gradually falls apart into a suspension containing long nephron fragments, often consisting of multiple connected segments. These are easily sorted into homogeneous segment populations that can be used for enzyme assays, protein extraction for immunoblotting, and RNA extraction for reverse transcription-polymerase chain reaction, all of which have been done successfully in our laboratory. For comparison, we have also examined cortical collecting tubule segments and cells prepared by the more rigorous protocol described previously (E. Schlatter, U. Fröbe, and R. Greger. Pflügers Arch. 421: 381-387, 1992). Even after the isolation of single cells in a Ca2+-free medium, the cells maintain their normal architecture and a distinct separation of apical and basolateral membranes.

Effects of sodium nitroprusside in the rat cortical collecting duct are independent of the NO pathway.

Recently we described K+ channels in the basolateral membrane of principal cells of rat cortical collecting duct (CCD) which are regulated by a cGMP-dependent protein kinase (Pflugers Arch 429:338-344, 1995). We examined the effects of the NO-liberator sodium nitroprusside (SNP) on single channel activity and membrane voltage (Vm) in principal cells of rat CCD, and on transepithelial voltage, lumen-to-bath Na+ fluxes, and osmotic water permeability in isolated perfused rat CCD tubules. While in patch clamp experiments SNP (10 microM) hyperpolarized principal cells from -54 +/- 10 mV to -71 +/- 5 mV (N = 5) and increased the activity of the described K+ channels from 0.05 +/- 0.03 to 0.45 +/- 0.14 (N = 5) in cell-attached and from 0.04 +/- 0.02 to 0.25 +/- 0.05 (N = 4) in excised patch clamp experiments, it had no effect on basal or AVP-dependent transepithelial voltage, Na+ fluxes, or the osmotic water permeability. In addition, neither 50 microM SIN-1, another liberator of NO, nor 1 mM L-NAME, an inhibitor of the NO-synthase, changed Vm significantly. Furthermore, in cGMP-assays SNP failed to increase intracellular cGMP in CCD segments. Thus, we conclude that in the rat CCD transport is not regulated via the NO-pathway and that SNP acts as an cGMP independent activator of K+ channels in the basolateral membrane of these cells.

AVP and aldosterone at physiological concentrations have synergistic effects on Na+ transport in rat CCD.

These studies examined whether the previously reported synergism between pharmacological doses of deoxycorticosterone (DOC) and arginine vasopressin (AVP) also occur at physiological concentrations of aldosterone and AVP. We examined the dose-response of salt transport, as measured by the lumen-to-bath 22Na+ flux (J1 --> b) and transepithelial voltage (VT), and of osmotic water permeability (Pf), to AVP in isolated perfused cortical collecting ducts (CCDs) from three groups of rats: (1) implanted with a 1 mg d-aldosterone pellet, which produced a moderately elevated, but physiologically relevant, plasma aldosterone concentration of 18.4 +/- 2.6 ng/dl; (2) implanted with a 2.5 mg DOC pellet (a high pharmacological dose); and (3) untreated rats, Pf reached the same maximal value in all three groups, and the concentration of AVP producing one-half the maximal Pf response (K0.5) was not significantly different among the three groups, ranging from 5 to 10 pM. There was a significantly greater increase in J1 --> b and hyperpolarization of VT with increasing AVP in both groups of treated rats than in the untreated group. The maximum values of J1 --> b and VT achieved at high AVP concentrations were not significantly different in CCDs from the aldosterone-treated and DOC-treated groups, but they were significantly higher than in the CCDs from untreated rats. Although maximal VT values achieved with DOC and aldosterone treatment were the same, the AVP K0.5 for VT were significantly lower in the DOC-treated than in the aldosterone-treated group. Although not statistically significant, the same trend was observed for J1 --> b. We conclude that AVP and aldosterone synergistically stimulate Na+ reabsorption at physiological concentrations of both hormones; however, VT (and probably Na+ reabsorption, which is generally proportional to VT) reaches maximum values at lower AVP concentrations when pharmacological doses of DOC are employed.

cGMP-activating peptides do not regulate electrogenic electrolyte transport in principal cells of rat CCD.

K+ channels in the basolateral membrane of rat cortical collecting duct (CCD) are regulated by a cGMP-dependent protein kinase (J. Hirsch and E. Schlatter. Pfluegers Arch. 429: 338-344, 1995). Conflicting data exist on the effects of cGMP-activating agonists on Na+ transport in these cells. Thus we tested members of the family of peptides that increase intracellular cGMP [cardiodilatin/atrial natriuretic peptide (CDD/ANP), brain natriuretic peptide, C-type natriuretic peptide, urodilatin, guanylin, and uroguanylin], as well as bradykinin +/- CDD/ANP on membrane voltages (Vm) of principal cells of isolated rat CCD using the slow whole cell patch-clamp technique (E. Schlatter, U. Fröbe, and R. Greger. Pfluegers Arch. 421: 381-387, 1992). None of the agonists tested changed Vm significantly. There was also no effect of dibutyryl guanosine 3',5'-cyclic monophosphate (DBcGMP) on AVP-dependent lumen-to-bath Na+ flux, transepithelial voltage, or osmotic water permeability in isolated perfused rat CCD. Finally, CDD/ANP increased intracellular cGMP only in glomeruli but not in CCD. Thus the findings provide no evidence for control of electrogenic electrolyte transport by these natriuretic peptides in principal cells of rat CCD, and the agonist that physiologically regulates the cGMP-dependent K+ channels remains to be identified.

Dopamine inhibits AVP-dependent Na+ transport and water permeability in rat CCD via a D4-like receptor.

We studied the receptor responsible for dopamine action in isolated perfused cortical collecting ducts (CCD) from rats treated with deoxy-corticosterone. (Critical experiments were repeated in CCD from untreated rats with the same results.) At doses > or = 1 microM, dopamine inhibited arginine vasopressin (AVP)-dependent Na+ and water transport (measured by the unidirectional lumen-to-bath 22Na+ flux and the transepithelial voltage) and osmotic water permeability (Pf). The effects of dopamine were not reversed by the dopamine-1 (D1) antagonist SCH-23390, and no inhibition was produced by the D1 agonists fenoldopam or SKF-81247. When Na+ transport and Pf were stimulated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine, dopamine did not inhibit, suggesting a "D2-type" receptor. However, the D2 agonist quinpirole had no effect on the AVP-dependent transepithelial voltage (VT), and the D2 and D3 antagonists domperidone and pimozide did not reverse dopamine inhibition of VT. The only agent tested that reversed the effects of dopamine was the D4-specific antagonist clozapine. We conclude that dopamine inhibition of salt and water transport in the CCD is mediated by a D4-like receptor.

Differential expression of PKC isoforms in fresh and cultured rabbit CCD.

Growth of rabbit cortical collecting duct (CCD) cells in primary culture results in a phenotype in which there is a stable stimulation of Na+ transport by arginine vasopressin. Our objective was to determine whether this altered phenotype was associated with altered expression of protein kinase C (PKC) isoforms. Western blot analysis of extracted proteins and reverse transcription-polymerase chain reaction analysis of extracted RNA showed expression of PKC-alpha, -epsilon, and -zeta in microdissected CCD segments and in fresh and cultured immunodissected CCD cells. These techniques also suggested that the rabbit CCD expresses PKC-eta- and -theta-like isoforms, which have not been identified in this species. Growth of CCD cells in primary culture produced no apparent change in the level of expression of PKC-alpha, -epsilon, or -zeta; however, the theta-like isoform was strongly expressed in fresh CCDs but only weakly expressed in cultured CCDs. The putative eta-isoform was more heavily expressed in cultured than in fresh CCD cells. The consequences of altered expression of these two isoforms in fresh and cultured rabbit CCD remain unknown, but the possibility exists that they are involved in the altered arginine vasopressin response of cultured cells.