Cultured Inner Medullary Collecting Duct Cells Research Articles

Unexpected localization of AQP3 and AQP4 induced by migration of primary cultured IMCD cells

Aquaporin-2–4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2–4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2–3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Vasopressin-Independent Regulation of Aquaporin-2 by Tamoxifen in Kidney Collecting Ducts.

Arginine vasopressin (AVP) mediates water reabsorption in the kidney collecting ducts through regulation of aquaporin-2 (AQP2). Also, estrogen has been known to regulate AQP2. Consistently, we previously demonstrated that tamoxifen (TAM), a selective estrogen receptor modulator, attenuates the downregulation of AQP2 in lithium-induced nephrogenic diabetes insipidus (NDI). In this study, we investigated the AVP-independent regulation of AQP2 by TAM and the therapeutic effect of TAM on the dysregulation of AQP2 and impaired urinary concentration in a unilateral ureteral obstruction (UUO) model. Primary cultured inner medullary collecting duct (IMCD) cells from kidneys of male Sprague-Dawley rats were treated with TAM. Rats subjected to 7 days of UUO were treated with TAM by oral gavage. Changes of intracellular trafficking and expression of AQP2 were evaluated by quantitative PCR, Western blotting, and immunohistochemistry. TAM induced AQP2 protein expression and intracellular trafficking in primary cultured IMCD cells, which were independent of the vasopressin V2 receptor (V2R) and cAMP activation, the critical pathways involved in AVP-stimulated regulation of AQP2. TAM attenuated the downregulation of AQP2 in TGF-β treated IMCD cells and IMCD suspensions prepared from UUO rats. TAM administration in vivo attenuated the downregulation of AQP2, associated with an improvement of urinary concentration in UUO rats. In addition, TAM increased CaMKII expression, suggesting that calmodulin signaling pathway is likely to be involved in the TAM-mediated AQP2 regulation. In conclusion, TAM is involved in AQP2 regulation in a vasopressin-independent manner and improves urinary concentration by attenuating the downregulation of AQP2 and maintaining intracellular trafficking in UUO.

Bile Acid Membrane Receptor TGR5 Regulates Renal AQP2 and Improves Lithiuminduced NDI

The bile acid‐activated receptors, which include the membrane G protein‐coupled receptor (TGR5) and nuclear farnesoid X receptor (FXR), are shown to play roles in kidney diseases. The purpose of current study was to investigate the role of TGR5 receptors in renal water handling and the underlying molecular mechanisms. The activation of TGR5 by lithocholic acid (LCA, a TGR5 endogenous ligand) induced aquaporin‐2 (AQP2) protein expression and intracellular trafficking in rat primary cultured inner medullary collecting duct (IMCD) cells via cAMP‐PKA signaling pathway. A synthetic specific TGR5 agonist INT‐777 has the same effects as LCA in IMCD cells. In mice with lithium‐induced nephrogenic diabetes insipidus (NDI), the activation of TGR5 by LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly up‐regulated AQP2 expression in the inner medulla, whereas chenodeoxycholic acid (CDCA, a FXR endogenous ligand) had no effects. In primary cultured IMCD cells prepared from lithium‐treated rats, INT‐777 or INT‐767 (a FXR and TGR5 dual agonist), but not INT‐747 (a FXR agonist) increased AQP2 protein expression. Compared with wildtype mice, tgr5−/− mice exhibited a mild urine concentration defect after dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium‐treated tgr5−/− mice, LCA treatment failed to prevent reduction of AQP2 expression. In conclusion, TGR5 activation increases renal AQP2 expression via cAMP‐PKA signaling pathway and improves impaired urinary concentration in lithium‐induced NDI. TGR5 is thus involved in regulation of water metabolism in the kidney.Support or Funding InformationThis work was supported by the Natural Science Foundation of China (Grants 81370822, 81570635, and 81670646), by the 111 Project, China (No. B13037), and by the Natural Science Foundation of Guangdong Province, China (Grants 2014A020212623, 2014A030313168, and 2016A020215034).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Depletion of vacuolar protein sorting-associated protein 35 is associated with increased lysosomal degradation of aquaporin-2.

The carboxyl terminus of aquaporin-2 (AQP2c) undergoes posttranslational modifications, including phosphorylation and ubiquitination, in the process of regulating aquaporin-2 (AQP2) translocation and protein abundance. We aimed to identify novel proteins interacting with AQP2c. Recombinant AQP2c protein was made in Escherichia coli BL21 (DE3) cells by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct (IMCD) tubule suspensions interacted with rat AQP2c bound to Ni2+-resin were subjected to LC-MS/MS proteomic analysis. Potential interacting proteins were identified, including vacuolar protein sorting-associated protein 35 (Vps35). Coimmunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were partly colocalized at the intracellular vesicles in collecting duct cells. The role of Vps35 in AQP2 regulation induced by 1-deamino-8D-arginine vasopressin (dDAVP) was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under siRNA-mediated Vps35 knockdown. dDAVP-induced AQP2 upregulation was less prominent in the cells with Vps35 knockdown. Moreover, AQP2 protein abundance was decreased to a greater extent during the withdrawal period after dDAVP stimulation under Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) but not by MG132 (a proteasome inhibitor). Immunocytochemistry demonstrated that internalized AQP2 was more associated with lysosomal-associated membrane protein 1 (LAMP-1) in primary cultured IMCD cells under a Vps35 knockdown situation. Taken together, our results show that Vps35 interacts with AQP2c, and depletion of Vps35 is likely to be associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2 protein.

Cyclophosphamide-induced vasopressin-independent activation of aquaporin-2 in the rat kidney.

Because cyclophosphamide-induced hyponatremia was reported to occur without changes in plasma vasopressin in a patient with central diabetes insipidus, we hypothesized that cyclophosphamide or its active metabolite, 4-hydroperoxycyclophosphamide (4-HC), may directly dysregulate the expression of water channels or sodium transporters in the kidney. To investigate whether intrarenal mechanisms for urinary concentration are activated in vivo and in vitro by treatment with cyclophosphamide and 4-HC, respectively, we used water-loaded male Sprague-Dawley rats, primary cultured inner medullary collecting duct (IMCD) cells, and IMCD suspensions prepared from male Sprague-Dawley rats. In cyclophosphamide-treated rats, significant increases in renal expression of aquaporin-2 (AQP2) and Na-K-2Cl cotransporter type 2 (NKCC2) were shown by immunoblot analysis and immunohistochemistry. Apical translocation of AQP2 was also demonstrated by quantitative immunocytochemistry. In both rat kidney and primary cultured IMCD cells, significant increases in AQP2 and vasopressin receptor type 2 (V2R) mRNA expression were demonstrated by real-time quantitative PCR analysis. Confocal laser-scanning microscopy revealed that apical translocation of AQP2 was remarkably increased when primary cultured IMCD cells were treated with 4-HC in the absence of vasopressin stimulation. Moreover, AQP2 upregulation and cAMP accumulation in response to 4-HC were significantly reduced by tolvaptan cotreatment in primary cultured IMCD cells and IMCD suspensions, respectively. We demonstrated that, in the rat kidney, cyclophosphamide may activate V2R and induce upregulation of AQP2 in the absence of vasopressin stimulation, suggesting the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis (NSIAD).

Renal medullary cyclooxygenase-2 and (pro)renin receptor expression during angiotensin II-dependent hypertension

The (pro)renin receptor [(P)RR] upregulates cyclooxygenase-2 (COX-2) in inner medullary collecting duct (IMCD) cells through ERK1/2. Intrarenal COX-2 and (P)RR are upregulated during chronic ANG II infusion. However, the duration of COX-2 and (P)RR upregulation has not been determined. We hypothesized that during the early phase of ANG II-dependent hypertension, membrane-bound (P)RR and COX-2 are augmented in the renal medulla, serving to buffer the hypertensinogenic and vasoconstricting effects of ANG II. In Sprague-Dawley rats infused with ANG II (0.4 μg·min(-1)·kg(-1)), systolic blood pressure (BP) increased by day 7 (162 ± 5 vs. 114 ± 10 mmHg) and continued to increase by day 14 (198 ± 15 vs. 115 ± 13 mmHg). Membrane-bound (P)RR was augmented at day 3 coincident with phospho-ERK1/2 levels, COX-2 expression, and PGE2 in the renal medulla. In contrast, membrane-bound (P)RR was reduced and COX-2 protein levels were not different from controls by day 14. In cultured IMCD cells, ANG II increased secretion of the soluble (P)RR. In anesthetized rats, COX-2 inhibition decreased the glomerular filtration rate (GFR) and renal blood flow (RBF) during the early phase of ANG II infusion without altering BP. However, at 14 days of ANG II infusions, COX-2 inhibition decreased mean arterial BP (MABP), RBF, and GFR. Thus, during the early phase of ANG II-dependent hypertension, the increased (P)RR and COX-2 expression in the renal medulla may contribute to attenuate the vasoconstrictor effects of ANG II on renal hemodynamics. In contrast, at 14 days the reductions in RBF and GFR caused by COX-2 inhibition paralleled the reduced MABP, suggesting that vasoconstrictor COX-2 metabolites contribute to ANG II hypertension.

The effects of altered extracellular pH on dDAVP‐induced phosphorylation (serine 256) and intracellular trafficking of AQP2 (1137.1)

The effects of altered extracellular pH (pHe) on dDAVP‐induced phosphorylation (S256, p‐AQP2) and trafficking of AQP2 were examined in the rat kidney inner medullary collecting duct (IMCD) cells. Freshly prepared IMCD suspension was exposed to buffer with pH 6.4, 7.4, or 8.4 for 1 h. dDAVP (10‐10 M, 3 min)‐induced AQP2 phosphorylation was more prominent when tubule suspension was exposed to pH 7.4 or 8.4, compared to pH 6.4. When primary cultured IMCD cells were exposed to pHe 6.4, 7.4 or 8.4 for 1 h, intracellular pH was changed to 6.1, 7.2 and 8.1, respectively. IMCD cells cultured in transwell chamber were exposed to transepithelial pH gradient for 1 h (pH 6.4, 7.4 or 8.4 at the apical side vs. pH 7.4 at the basolateral side). Laser scanning confocal microscopy and cell surface biotinylation assay revealed that exposure to lower pH (pH 6.4, 1 h) significantly reduced dDAVP (10‐9M, 15 min, basolateral)‐induced AQP2 trafficking and surface expression of p‐AQP2. Importantly FRET analysis revealed that dDAVP (10‐10 M)‐induced increase of PKA activity was attenuated when cells were exposed to pHe 6.4, whereas forskolin (10‐7 M)‐induced PKA activation was not affected. Taken together, decreased pHe is likely to exert an inhibitory effect on dDAVP‐induced AQP2 phosphorylation and intracellular trafficking in IMCD cells, presumably via an inhibition of G‐protein‐cAMP‐PKA action.

Glucocorticoids Improve Renal Responsiveness to Atrial Natriuretic Peptide by Up-Regulating Natriuretic Peptide Receptor-A Expression in the Renal Inner Medullary Collecting Duct in Decompensated Heart Failure

In heart failure, the renal responsiveness to exogenous and endogenous atrial natriuretic peptide (ANP) is blunted. The mechanisms of renal hyporesponsiveness to ANP are complex, but one potential mechanism is decreased expression of natriuretic peptide receptor-A (NPR-A) in inner medullary collecting duct (IMCD) cells. Newly emerging evidence shows that glucocorticoids could produce potent diuresis and natriuresis in patients with heart failure, but the precise mechanism is unclear. In the present study, we found dexamethasone (Dex) dramatically increased the expression of NPR-A in IMCD cells in vitro. The NPR-A overexpression induced by Dex presented in a time- and dose-dependent manner, which emerged after 12 h and peaked after 48 h. The cultured IMCD cells were then stimulated with exogenous rat ANP. Consistent with the findings with NPR-A expression, Dex greatly increased cGMP (the second messenger for the ANP) generation in IMCD cells, which presented in a time- and dose-dependent manner as well. In rats with decompensated heart failure, Dex dramatically increased NPR-A expression in inner renal medulla, which was accompanied by a remarkable increase in renal cGMP generation, urine flow rate, and renal sodium excretion. It is noteworthy that Dex dramatically lowered plasma ANP, cGMP levels, and left ventricular end diastolic pressure. These favorable effects induced by Dex were glucocorticoid receptor (GR)-mediated and abolished by the GR antagonist 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU486). Collectively, glucocorticoids could improve renal responsiveness to ANP by up-regulating NPR-A expression in the IMCD and induce a potent diuretic action in rats with decompensated heart failure.

Loss of Bicc1 impairs tubulomorphogenesis of cultured IMCD cells by disrupting E-cadherin-based cell-cell adhesion

The Bicaudal-C ( Bic-C) gene was originally discovered in Drosophila melanogaster. The gene product Bic-C is thought to serve as an RNA-binding molecule targeting diverse proteins at the post-transcriptional level. Recent research has shown this gene to be conserved in many species, from Caenorhabditis elegans to humans. Disruption of this protein can disturb the normal migration direction of the anterior follicle cell of Drosophila oocytes, while mutation of a mouse Bicc1 (a mouse homologue of Bic-C) results in phenotypes mimicking human hereditary polycystic kidney disease (PKD). However, the cellular function of Bicc1 gene products in mammalian systems remains largely unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which Bicc1 was silenced by short hairpin RNA inhibition (shRNA). We show that inhibition of Bicc1 disrupted normal tubulomorphogenesis and induced cystogenesis of IMCD cells grown in three dimensional cultures. To determine what factors contributed to the defect, we systematically examined biological changes of Bicc1-silenced IMCD cells. We found that the cells had significant defects in E-cadherin-based cell-cell adhesion, along with abnormalities in actin cytoskeleton organization, cell-extracellular matrix interactions, cell proliferation, and apoptosis. These findings suggest that lack of Bicc1 leads to disruption of normal cell-cell junctions, which in turn impedes establishment of epithelial polarity. These cellular defects may initiate abnormal tubulomorphogenesis and cystogenesis of IMCD cells grown in vitro. The observation of aberrant cellular behaviors in Bicc1-silenced IMCD cells reveal functions for Bicc1 in renal epithelial cells and provides insight into a potential pathogenic mechanism of polycystic kidney disease.

Increased AQP2 targeting in primary cultured IMCD cells in response to angiotensin II through AT1receptor

Vasopressin and angiotensin II (ANG II) play a major role in renal water and Na(+) reabsorption. We previously demonstrated that ANG II AT(1) receptor blockade decreases dDAVP-induced water reabsorption and AQP2 levels in rats, suggesting cross talk between these two peptide hormones (Am J Physiol Renal Physiol 288: F673-F684, 2005). To directly address this issue, primary cultured inner medullary collecting duct (IMCD) cells from male Sprague-Dawley rats were treated for 15 min with 1) vehicle, 2) ANG II, 3) ANG II + the AT(1) receptor blocker candesartan, 4) dDAVP, 5) ANG II + dDAVP, or 6) ANG II + dDAVP + candesartan. Immunofluorescence microscopy revealed that 10(-8) M ANG II or 10(-11) M dDAVP (protocol 1) was associated with increased AQP2 labeling of the plasma membrane and decreased cytoplasmic labeling, respectively. cAMP levels increased significantly in response to 10(-8) M ANG II and were potentiated by cotreatment with 10(-11) M dDAVP. Consistent with this finding, immunoblotting revealed that this cotreatment significantly increased expression of phosphorylated AQP2. ANG II-induced AQP2 targeting was blocked by 10(-5) M candesartan. In protocol 2, treatment with a lower concentration of dDAVP (10(-12) M) or ANG II (10(-9) M) did not change subcellular AQP2 distribution, whereas 10(-12) M dDAVP + 10(-9) M ANG II enhanced AQP2 targeting. This effect was inhibited by cotreatment with 10(-5) M candesartan. ANG II-induced cAMP accumulation and AQP2 targeting were inhibited by inhibition of PKC activity. In conclusion, ANG II plays a role in the regulation of AQP2 targeting to the plasma membrane in IMCD cells through AT(1) receptor activation and potentiates the effect of dDAVP on AQP2 plasma membrane targeting.

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.

Syntaxin Isoform Specificity in the Regulation of Renal H+-ATPase Exocytosis

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.

Regulation of cAMP production in initial and terminal inner medullary collecting ducts

The inner medullary collecting duct (IMCD) is composed of at least two functionally and morphologically distinct segments, the initial (IMCDi) and the terminal (IMCDt) portions. However, most studies of receptor signaling have been performed on cells obtained from the entire inner medulla. The purpose of this study was to determine whether the patterns of receptor-activated cAMP accumulation were different between these segments. We measured cAMP accumulation stimulated by vasopressin and isoproterenol, and the effect of epinephrine in freshly dissected IMCDi and IMCDt segments cultured and IMCDi and IMCDt cells in primary culture. The maximum response to vasopressin was twofold higher in fresh IMCDt verus IMCDi (P < 0.05), however, it increased in cultured IMCDi by 40% verus fresh cells with no change in the response in fresh verus cultured IMCDt. The maximum response to isoproterenol was small in fresh cells but increased by five- and sixfold, respectively, in cultured IMCDi and IMCDt cells. alpha 2-Adrenoceptor stimulation almost completely inhibited both vasopressin and isoproterenol-stimulated cAMP accumulations in fresh IMCDi and IMCDt cells, but only partially inhibited either accumulation by 34 to 49% in cultured cells. (1) IMCDi and IMCDt cells are both subject to vasopressin and alpha 2- and beta-adrenergic regulation of adenylyl cyclase activity; (2) the relative influence of beta-adrenergic, alpha 2-adrenergic and V2 receptors to affect cAMP accumulation is altered in primary culture versus freshly dissected IMCD segments, suggesting that caution must be exercised in the extrapolation of data from cultured IMCD cells to in vivo models.

Characterization of anion exchangers in an inner medullary collecting duct cell line.

Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells, rat renal medulla, and freshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney homogenate and therefore probably represents AE1. The mobility of the 180-kD band is similar to that for rat stomach and kidney AE2 and therefore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that both AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these cells was found to be virtually identical to that reported for kidney AE1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, AE1 and AE2, and both of these exchangers probably mediate the electroneutral Cl--dependent HCO3-transport observed in this cell line.

Hormonal regulation of inner medullary collecting duct sodium transport

The inner medullary collecting duct (IMCD) is the final arbiter of renal Na+ excretion, and Na+ transport in this segment is controlled by a wide variety of hormones and renal autacoids. This review examines the mechanisms of IMCD Na+ transport and its regulation using results obtained from micropuncture and microcatheterization studies in the intact animal, as well as data from isolated perfused tubules, freshly prepared cell suspensions, and cultured IMCD cells. Where appropriate, results from closely related tissues such as the cortical collecting duct and model urinary epithelia are examined. Na+ reabsorption in this segment occurs predominantly via apical amiloride-sensitive Na+ channels and basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase). Although there is some evidence for the activities of other transporters such as Na(+)-K(+)-2Cl- and Na-Cl cotransporters and Na+/H+ exchanger, their role in Na+ homeostasis remains undefined. Mineralocorticoids augment the activities of both apical Na+ channels and basolateral Na(+)-K(+)-ATPase by a variety of complex mechanisms. Prostaglandin E2 inhibits Na(+)-K(+)-ATPase and appears to mediate the actions of several peptide hormones, including endothelin, interleukin-1, and atrial natriuretic peptide [ANP-(31-67)]. Several peptides in the ANP family [ANP-(99-126), urodilatin, and brain natriuretic peptide] bind to guanylate cyclase-linked receptors, leading to inhibition of apical Na+ channel function. These mechanisms of regulation of IMCD Na+ transport likely play important roles in total body Na+ balance in health and disease.

Prostaglandin E2 production in rat IMCD cells. II. Possible role for locally formed dopamine

Dopamine has been proposed as an intrarenal natriuretic hormone. We reported previously that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to stimulation of prostaglandin E2 (PGE2) production. In this study we examined whether locally formed dopamine could stimulate PGE2 production in cultured IMCD cells. L-Dopa stimulated PGE2 production dose dependently in cultured IMCD cells (concentration for half-maximal stimulation, 54.3 microM; maximal stimulation, 212.7% of basal), with the maximal stimulation similar to that obtained with dopamine. This effect was blocked by aromatic L-amino acid decarboxylase (AADC) inhibitors and DA2-receptor antagonists. IMCD cells also had measurable AADC activity and produced dopamine from exogenously added L-dopa. AADC inhibitors and DA2 antagonists also lowered basal PGE2 levels, suggesting that dopamine was being formed constitutively in culture. These results suggest that cultured IMCD cells have the capacity to take up and convert L-dopa to dopamine, which then stimulates PGE2 production via DA2K receptors. These results further suggest that locally formed dopamine could act as an autocrine/paracrine hormone in the kidney inner medulla to regulate PGE2 synthesis and water and electrolyte excretion.

Prostaglandin E2 production in rat IMCD cells. I. Stimulation by dopamine

Previous studies from our laboratory have determined that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to prostaglandin E2 (PGE2) production. In the present study, we have further characterized the dopamine-stimulated PGE2 response. Dopamine stimulated PGE2 production in cultured IMCD cells dose dependently (concentration for half-maximal stimulation, 11.1 microM; maximal stimulation, 235.1% of basal), an effect that was blocked by the DA2 antagonists domperidone and (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)-methyl] benzamine. Inhibition of intracellular calcium release with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (100 microM) blocked the dopamine response, whereas voltage-dependent calcium-channel blockers had no effect. Inhibition of phospholipase A2 (PLA2) activity with quinacrine (100 microM) completely blocked the dopamine-stimulated PGE2 production, whereas inhibition of polyphosphoinositol hydrolysis with neomycin (100 microM) or inhibition of protein kinase C with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 microM) did not. Pertussis toxin (PT) treatment completely blocked the dopamine-stimulated PGE2 production but not the arachidonic acid-stimulated PGE2 production. These results suggest that dopamine, acting through the DA2K receptor, may be an important regulator of PGE2 production in IMCD cells. Furthermore, our results are most consistent with either a direct interaction of the DA2K receptor with PLA2 through a PT-sensitive G protein or an indirect interaction with PLA2 through mobilization of intracellular calcium.

Endothelin synthesis by rabbit renal tubule cells

Endothelins regulate nephron sodium and water transport, prostaglandin E2 (PGE2) synthesis, and phospholipid metabolism. Recent studies suggest that renal tubule cells synthesize endothelins. To determine which nephron sites have such potential, endothelin production by cells derived from different nephron segments was examined. Immunoreactive endothelin 1 (ET-1) and endothelin 3 (ET-3) were measured in supernatants of cultured rabbit proximal tubule (PT), medullary thick ascending limb (MTAL), cortical collecting tubule (CCT), and inner medullary collecting duct (IMCD) cells. All cell types released immunoreactive ET-1 and ET-3. However, the amounts of endothelin produced differed as follows: IMCD greater than MTAL greater than CCT much greater than PT for ET-1 and IMCD greater than MTAL = PT = CCT for ET-3; in all cases ET-1 much greater than ET-3. To confirm de novo ET-3 synthesis, IMCD cells were labeled with [35S]cysteine, and the supernatant was immunoprecipitated with anti-ET-3 antibody. Sample and standard ET-3 eluted at identical positions on high-performance liquid chromatographs, confirming de novo synthesis of ET-3 by cultured IMCD cells. These data raise the possibility of an important functional role for nephron-derived endothelin and, in particular, endothelin produced by tubule cells in the medulla.

Inner medullary collecting duct Na(+)-H+ exchanger.

Cells from the inner medullary collecting duct (IMCD) exhibit Na(+)-H+ exchange. The present studies were performed to address certain important characteristics of this process in cultured IMCD cells. First, Na(+)-H+ exchange was found to be present both at 37 degrees C and at 25 degrees C, in contrast to Na(+)-independent H+ extrusion, which was only observed in some cultures and only at 37 degrees C. Second, with the use of image analysis techniques, virtually all cells in IMCD cultures were demonstrated to possess Na(+)-H+ exchange, whether or not the cells exhibited Na(+)-independent intracellular pH recovery from acid loads. Also, Na(+)-H+ exchange was found to be expressed on the basolateral aspect of these cells, but not on the apical membrane. These properties of IMCD Na(+)-H+ exchange are consistent with a function to regulate intracellular pH rather than mediate transepithelial acid-base transport. Na(+)-H+ exchange in IMCD cells was also compared with that in cultured renal proximal tubule cells. Despite physiologically distinct roles in vivo, Na(+)-H+ exchange in these two cell types in culture was found to be similar with respect to the Km for Na+ and the Ki for 5-(N-ethyl-N-isopropyl)amiloride. These data are consistent with functionally similar (if not identical) processes mediating Na(+)-H+ exchange in these two cell types, but with opposite polarity.