Renal Cortical Collecting Duct Cells Research Articles

Lithium sensitizes P2Y2-dependent intracellular Ca2+ signaling in the collecting duct cells

INTRODUCTION. Lithium salts are used as mood stabilizers to treat bipolar disorder and depression. A common renal side effect of lithium therapy is nephrogenic diabetes insipidus (NDI), manifesting in polyuria. Lithium decreases the numbers of aquaporin 2 (AQP2) water channels in collecting duct cells reducing water reabsorption in response to vasopressin (AVP). Stimulation of purinergic P2Y2 receptors (P2Y2R) can impair AVP signaling and deletion of P2Y2R in mice slows down the progression of lithium-induced NDI. Interestingly, stimulation of P2Y2R triggers Ca2+ release from the endoplasmic reticulum (ER) in collecting duct cells. The interplay between P2Y2R and intracellular Ca2+ ([Ca2+]i) signaling in lithium-induced NDI has not been tested. We hypothesize that lithium facilitates P2Y2-mediated Ca2+ signaling in collecting duct cells, attenuating AVP-dependent water transport and disrupting [Ca2+]i balance. METHODS. This project combines water permeability tests in cultured cell monolayers, immunoblotting and ratiometric Fura-2 Ca2+-imaging to study the effects of lithium on P2Y2-dependent [Ca2+]i mobilization in a culture of mouse renal cortical collecting duct cells, mpkCCDcl4. RESULTS. Upon stimulation with 1 nM AVP, mpkCCDcl4 cells incubated in the medium containing 10 mM LiCl for 4 days exhibited a 2-fold lower abundance of membrane resident AQP2, phosphorylated at serine 269, when compared to control cells. Transepithelial water transport was also significantly slower in LiCl-treated than in control cells, further validating the detrimental effects of Li+ in our cell model. Saturating (10-100 uM) concentrations of a P2Y2R-selective ligand, uridine-5'-triphosphate (UTP), induced comparable [Ca2+]i responses in in control and LiCl-treated cells. When we assessed the P2Y2-mediated [Ca2+]i responses to a range of UTP concentrations (0.1-100 uM), the dose response curve was shifted to the left in LiCl-treated cells when compared to controls. The half-maximal effective UTP concentration, EC50, was 0.4±0.03 uM and 2.0±0.07 uM in Li-treated and control cells, respectively, indicating higher sensitivity to UTP in LiCl-treated cells. Next, we evaluated how P2Y2-dependent depletion of the ER Ca2+ stores, affects store-operated calcium entry (SOCE) in mpkCCDcl4 cells. The magnitude of SOCE-reporting Ca2+ signal was significantly (~25%) higher in LiCl-treated cells than in controls. CONCLUSIONS. Lithium facilitates P2Y2-mediated [Ca2+]i mobilization, reduces AVP-dependent transepithelial water transport and AQP2 trafficking in collecting duct cells. Sensitization of P2Y2-dependent Ca2+ signaling causes excessive depletion of the ER stores and requires higher activity of SOCE to compensate for Ca2+ loss and restore the balance. Our findings reveal previously unrecognized signaling determinants underlying the pathophysiological effects of lithium, pertaining to Ca2+ homeostasis in collecting duct cells. This work was supported by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) R01DK125464. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increase in Salt Concentration in the Culture Media Enhances Protein Expression of Tumor Necrosis Factor‐Alpha Receptor Type 1 in Cultured Renal Cortical Collecting Duct Cells But Not in Proximal Tubular Cells

Tumor necrosis factor‐alpha (TNFα) exerts its biological responses via interaction with its’ two cell surface receptors, type 1 (TNFR1) and type 2 (TNFR2) which are differentially expressed and regulated in the kidney. Previously, we have demonstrated that circulating TNFα exerts natriuresis via its action on TNFR1 but not on TNFR2 and such TNFR1 activation exerts inhibitory action on distal tubular sodium transport, particularly tubular ENaC activity. As TNFα level is elevated during dietary intake of high salt (HS), we hypothesized that increases in salt concentration in renal tissue enhances TNFα ‐TNFR1 activity in the renal tubules to induce natriuretic response to HS intake. To test this hypothesis, we examined the protein expression of TNFR1 and TNFR2 in cultured proximal (human kidney [HK‐2] cell line) and distal (murine cortical collecting duct [M‐1] cell line) tubular cells at different concentrations of NaCl in the culture media. Both these HK‐2 and M‐1 cells were incubated for 24 hours in control media with normal NaCl concentration (120 mM) and at media containing higher NaCl (140 mM and 160 mM) concentrations. TNF receptors protein expressions were analyzed by the Western Blot technique using appropriate antibodies for TNFR1 (rabbit anti‐TNFR1; Abcam cat # 19139) and TNFR2 (rabbit anti‐TNFR2; Abcam cat # 109322), which were normalized by expression of β‐actin or GAPDH proteins using appropriate antibodies. The blot image was quantified and measured as band intensity density with Image J software. The quantitative immunoblot analysis in HK‐2 cells showed that there were no significant changes in the band intensity unit (IU) in TNFR1 (120 mM, 70.1±12.8 IU; 140 mM, 73.7±9.9 IU; 160 mM, 57.2±9.8 IU; n=6 in each condition) or TNFR2 protein (120 mM, 83.9 ±6.3 IU; 140 mM, 66.8±14.1 IU; 160 mM, 77.8±2.3 IU; n=6 in each condition) levels. On the other hand, TNFR1 protein expression was significantly higher in M‐1 cells incubated in 160 mM NaCl conc. (135.6 ±7.8 IU, n=7; P< 0.005) than that in 120 mM (109.3±2.8 IU, n=7) and in 140 mM (91.1±10.8 IU, n=7) NaCl conc. However, TNFR2 protein expression in M‐1 cells did not significantly alter with the changes in NaCl conc. in the media (120 mM, 137±20.1 IU; 140 mM, 128±7.2 IU; 160 mM, 143±7.2 IU; n=7 in each condition). These data indicate that TNFR1 activity is upregulated in renal collecting duct cells (CCD) in response increases in tissue NaCl concentration in the kidney. Such upregulation of TNFR1 activity in the CCD induced by high salt concentration, exerts inhibitory action on distal sodium transport and plays a role in enhancing urinary sodium excretion during dietary HS intake.

Functional role of histamine receptors in the renal cortical collecting duct cells.

Histamine is an important immunomodulator, as well as a regulator of allergic inflammation, gastric acid secretion, and neurotransmission. Although substantial histamine level has been reported in the kidney, renal pathological and physiological effects of this compound have not been clearly defined. The goal of this study was to provide insight into the role of histamine-related pathways in the kidney, with emphasis on the collecting duct (CD), a distal part of the nephron important for the regulation of blood pressure. We report that all four histamine receptors (HRs) as well as enzymes responsible for histamine metabolism and synthesis are expressed in cultured mouse mpkCCDcl4 cells, and histamine evokes a dose-dependent transient increase in intracellular Ca2+ in these cells. Furthermore, we observed a dose-dependent increase in cAMP in the CD cells in response to histamine. Short-circuit current studies aimed at measuring Na+ reabsorption via ENaC (epithelial Na+ channel) demonstrated inhibition of ENaC-mediated currents by histamine after a 4-h incubation, and single-channel patch-clamp analysis revealed similar ENaC open probability before and after acute histamine application. The long-term (4 h) effect on ENaC was corroborated in immunocytochemistry and qPCR, which showed a decrease in protein and gene expression for αENaC upon histamine treatment. In summary, our data highlight the functional importance of HRs in the CD cells and suggest potential implications of histamine in inflammation-related renal conditions. Further research is required to discern the molecular pathways downstream of HRs and assess the role of specific receptors in renal pathophysiology.

Protein kinase D2 regulates epithelial sodium channel activity and aldosterone non-genomic responses in renal cortical collecting duct cells

Protein kinase D2 (PKD2) is a serine/threonine protein kinase which plays an important role in vesicle fission at the trans-Golgi network (TGN) to coordinate subcellular trafficking with gene expression. We found that in the rat kidney, PKD2 is specifically expressed in collecting duct principal cells predominantly at the apical membrane and with lower basal expression in cytosolic compartments. When rats were maintained on a Na+ depleted diet (<0.87 mmol Na+/kg) to increase plasma aldosterone levels, PKD2 became internalized to a cytoplasmic compartment. Treatment of murine M1 cortical collecting duct (M1-CCD) cells with aldosterone (10 nM) promoted PKD2 co-localization with the trans-Golgi network within 30 min. PKD2 underwent autophosphorylation at Ser876 within 10 min of aldosterone treatment and remained phosphorylated (active) for at least 24 h. A stable PKD2 shRNA knock-down (PKD2 KD) M1-CCD cell line was developed to study the role of PKD2 in epithelial Na+ channel (ENaC) trafficking and transepithelial Na+ transport (SCC) in epithelial monolayers grown in Ussing chambers. The PKD2 KD cells developed transepithelial resistance with kinetics equivalent to wild-type cells, however the transepithelial voltage and Na+ current were significantly elevated in PKD2 knock-down CCD epithelia. The higher basal SCC was due to increased ENaC activity. Aldosterone treatment for 24 h resulted in a decline in ENaC activity in the PKD2 KD cells as opposed to the increase observed in the wild-type cells. The paradoxical inhibition of SCC by aldosterone in PKD2 KD epithelium was attributed to a reduction in ENaC current and lower membrane abundance of ENaC, demonstrating that PKD2 plays a critical tonic role in ENaC trafficking and channel subunit stability. The rapid activation of PKD2 by aldosterone is synergistic with the transcriptional activity of MR and contributes to increased ENaC activity.

Drowning in Data: Early Responses of Renal Cortical Collecting Duct Cells to Lithium

It is now more than fifty years since Burg made a signal advance in the study of renal physiology by reporting a method of isolating and perfusing tubules from selected regions of the kidney. Prior to this, knowledge of renal tubular function had been derived either from micropuncture experiments, limited to the superficial regions of the kidney, from kidney slices, which were subject to injury and represented an aggregate of tubular function, or from renal cells in tissue culture

SUMOylation Landscape of Renal Cortical Collecting Duct Cells.

Protein post-translational modification by the smallubiquitin-like modifier (SUMO) is a mechanism that allows a diverse response of cells to stress. Five SUMO family members, SUMO1-5, are expressed in mammals. We hypothesized that because kidney epithelial cells are often subject to stresses arising from various physiological conditions, multiple proteins in the kidney will be SUMOylated. Here, we profiled SUMO1- and SUMO2-modified proteins in a polarized epithelial cell model of the renal cortical collecting duct (mpkCCD14 cells). Modified forms of SUMO1 or SUMO2, with a histidine tag and a Thr to Lys mutation preceding the carboxyl-terminal di-gly motif, were expressed in mpkCCD14 cells, allowing SUMO-conjugated proteins to be purified and identified. Protein mass spectrometry identified 1428 SUMO1 and 1957 SUMO2 sites, corresponding to 741 SUMO1 and 971 SUMO2 proteins. Gene ontology indicated that the function of the majority of SUMOylated proteins in mpkCCD14 cells was related to gene transcription. After treatment of the mpkCCD14 cells for 24 h with aldosterone, the levels of SUMOylation at a specific site on the proton and oligopeptide/antibiotic cotransporter protein Pept2 were greatly increased. In conclusion, the SUMOylation landscape of mpkCCD14 cells suggests that protein modification by SUMOylation is a mechanism within renal epithelial cells to modulate gene transcription under various physiological conditions.

Abstract P2039: Nitric Oxide Inhibition Enhances the Protein Expression of TNF-α Receptor Type 1 in Cultured M-1 Cells With Characteristics of Renal Collecting Duct Epithelium

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37 0 C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.

Activation of the Epithelial Sodium Channel (ENaC) by Aldosterone in mCCDcl1 Mouse Renal Cortical Collecting Duct Cells is Dependent on SGK1 and Can be Prevented by Inhibiting Endogenous Proteases

The epithelial sodium channel (ENaC) is a heteromeric channel composed of three subunits (α, β, γ) critically important for renal sodium homeostasis and the long term control of arterial blood pressure. In the cortical collecting duct aldosterone is the main hormonal regulator of ENaC activity, and SGK1 (serum and glucocorticoid‐regulated kinase 1) is thought to play a pivotal role in mediating ENaC stimulation by aldosterone. Proteolytic cleavage by the Golgi‐associated convertase furin at three putative furin sites (two in α‐ and one in γENaC) is thought to be important for ENaC maturation in the biosynthetic pathway before the channel reaches the plasma membrane. The final step in proteolytic ENaC activation probably takes place at the plasma membrane where γENaC is cleaved by membrane‐bound proteases and/or extracellular proteases in a region distal to the furin cleavage site. In this study we investigated whether ENaC activation by aldosterone is dependent on SGK1 and requires proteolytic channel activation. A highly differentiated murine principal cell line (mCCDcl1) derived from microdissected cortical collecting duct was used, and cells were grown on permeable supports. ENaC‐mediated sodium transport was assessed by recording the amiloride‐sensitive equivalent short circuit current (ISC‐Ami) in Ussing chambers for up to 6 h. Exposure of the mCCDcl1 cells to 3 nM aldosterone for 2.5 h stimulated ISC‐Ami about 2.5‐fold. Subsequent application of chymotrypsin (24 or 240 μg/ml) had no additional stimulatory effect. The stimulatory response to aldosterone was completely abolished by the SGK1 inhibitor GSK650394 (10 μM). In untreated control cells apical application of chymotrypsin also showed no stimulatory effect on ISC‐Ami. Therefore, we hypothesized that in mCCDcl1 cells ENaC is fully activated by endogenous proteases in the presence and absence of aldosterone. To inhibit these endogenous proteases, we used an intracellularly acting convertase inhibitor (furin inhibitor‐1) and extracellularly acting serine protease inhibitors (aprotinin or nafamostat). A combination of aprotinin (30 μg/ml)/nafamostat (1 μM) and furin inhibitor‐1 (40 μM) essentially abolished baseline ISC‐Ami. Subsequent apical application of chymotrypsin stimulated ISC‐Ami back to baseline levels. Importantly, pre‐treatment of the cells with furin inhibitor‐1 in combination with aprotinin/nafamostat essentially prevented ENaC stimulation by aldosterone. Subsequent chymotrypsin rapidly and completely rescued the stimulatory effect of aldosterone in mCCDcl1 cells treated with protease inhibitors. We conclude that stimulation of ENaC activity by aldosterone is not only dependent on SGK1 but also requires proteolytic channel activation. It is tempting to speculate that SGK1 may contribute to the regulation of endogenous proteases necessary for channel activation. Endogenous proteases relevant for proteolytic ENaC activation under physiological and pathopyhsiological conditions remain to be elucidated.Support or Funding InformationThis work was supported by The Alexander von Humboldt Foundation (MKM) and Bayerische Forschungsstiftung (CK).

PS 18-16 Renal sodium handling in relation to plasma advanced glycation end products concentration in untreated subjects

Objective: previous experimental study showed that advanced glycation end products (AGEs) might enhance epithelial sodium channel in the renal cortical collecting duct cells. In the present study, we investigated renal sodium handling, as assessed by endogenous lithium clearance indexes, in relation to plasma AGEs concentration in subjects who did not take antihypertensive medication. Design and Method: The study subjects were previously never treated with or stopped antihypertensive medication for at least 2 weeks. We calculated fractional excretion of lithium and fractional distal reabsorption rate of sodium, as markers of proximal and distal sodium handling, respectively. Plasma AGEs concentration was measured by the enzyme-linked immunosorbent assay method and logarithmically transformed for statistical analysis. Results: The 1006 study participants (mean age 51.3 years) included 499 (49.6%) men and 40 (4.0%) diabetic patients. After adjustment for sex, age, body mass index, mean arterial pressure, pulse rate, current smoking and alcohol intake, plasma fasting glucose, serum triglycerides, serum total-to-high-density lipoprotein cholesterol ratio, plasma AGEs concentration (geometric mean [95% confidence interval] 13.4 [12.8 to 14.0] mg/mL) was significantly (P = 0.0007) associated with fractional lithium clearance (mean [±standard deviation] 21.7% ± 12.1%). For each 10-time increase in plasma AGEs concentration, fractional lithium clearance was 4.3% (absolute percentage change) lower. In tertile 3 of sodium urinary excretion, higher plasma AGEs concentration was also associated with lower fractional distal reabsorption rate of sodium (93.3% in tertile 3 vs. 95.1% in tertile 1 of plasma AGEs concentration, P = 0.002). Conclusions: Plasma AGEs concentration was positively associated with sodium renal reabsorption in the proximal tubule, as assessed by fractional lithium clearance. However, on the condition of high sodium intake, it was negatively associated with sodium reabsorption in the distal tubule, probably as a compensation of increased sodium intake and increased sodium reabsorption in the proximal tubule.

Mitochondria form an apical calcium signaling domain in renal cortical collecting duct cells

In several epithelia, Mitochondria sequester intracellular Ca2+ to create Ca2+ signaling domains. We asked whether mitochondria could form barriers to Ca2+ diffusion in renal cortical collecting duct (CCD) cells. We began by staining each of two cell lines (from Xenopus and mouse) with mitotracker red. Mitochondria in both cell lines were localized in bands with a prominent band near the apical surface and a weaker band at the basal pole. To determine the proximity of the apical band to the membrane, we fluorescently tagged PIP2 to label the apical membrane and found that the apical mitochondrial band was localized just beneath the membrane. Many apical membrane proteins are regulated by endoplasmic reticular Ca2+ release. If mitochondria do sequester calcium to create an apical membrane Ca2+ signaling domain, the ER must be able to cross the mitochondrial barrier at certain points so that release of Ca2+ from the ER can reach its desired targets. Cells co‐stained with mitotracker red and ERtracker blue showed that ER crossed the mitochondrial barrier in many places. Next we sought to determine whether mitochondrial bands can prevent movement of intracellular Ca2+ from one pole to another. Live cells stained with the mitochondrial Ca2+ dye Rhodamine 2‐AM showed Ca2+ uptake in the apical but not basal band after treatment with the Ca2+ ionophore ionomycin. Cells transfected with a membrane‐tethered intracellular Ca2+ sensor showed increased Ca2+ at the apical but not basal pole following apical ionomycin. These data suggest that mitochondrial bands in the CCD create an apical signaling domain for Ca2+ regulation of membrane proteins. This work was supported by NIH R37‐DK037963 and T32‐ DK07656 and AHA 13POST16820072.

Estrogen increases ENaC activity via PKCδ signaling in renal cortical collecting duct cells.

The most active estrogen, 17β‐estradiol (E2), has previously been shown to stimulate a female sex‐specific antisecretory response in the intestine. This effect is thought to contribute to the increase in whole body extracellular fluid (ECF) volume which occurs in high estrogen states, such as in the implantation window during estrous cycle. The increased ECF volume may be short‐circuited by a renal compensation unless estrogen exerts a proabsorptive effect in the nephron. Thus, the effect of E2 on ENaC in kidney cortical collecting duct (CCD) cells is of interest to understand estrogen regulation of ECF volume. Previous studies showed a rapid stimulatory effect of estrogen on ENaC in bronchial epithelium. In this study we examined if such a rapid effect on Na+ absorption could occur in the kidney. Experiments were carried out on murine M1‐CCD cell cultures. E2 (25 nmol/L) treatment caused a rapid‐onset (<15 min) and sustained increase in the amiloride‐sensitive Na+ current (INa) in CCD monolayers mounted in Ussing chambers (control, 1.9 ± 0.2 μA/cm2; E2, 4.7 ± 0.3 μA/cm2; n = 43, P < 0.001), without affecting the ouabain‐sensitive Na+/K+ pump current. The INa response to E2 was inhibited by PKCδ activity antagonism with rottlerin (5 μmol/L), inhibition of matrix metalloproteinases activity with GM6001 (1 μmol/L), inhibition of EGFR activity with AG1478 (10 μmol/L), inhibition of PLC activity with U‐73122 (10 μmol/L), and inhibition of estrogen receptors with the general ER antagonist ICI‐182780 (100 nmol/L). The estrogen activation of INa could be mimicked by the ERα agonist PPT (1 nmol/L). The nuclear excluded estrogen dendrimer conjugate (EDC) induced similar stimulatory effects on INa comparable to free E2. The end target for E2 stimulation of PKCδ was shown to be an increased abundance of the γ‐ENaC subunit in the apical plasma membrane of CCD cells. We have demonstrated a novel rapid “nongenomic” function of estrogen to stimulate ENaC via ERα‐EGFR transactivation in kidney CCD cells. We propose that the salt‐retaining effect of estrogen in the kidney together with its antisecretory action in the intestine are the molecular mechanisms causing the expanded ECF volume in high‐estrogen states.

Human Urinary Exosomes as Innate Immune Effectors

Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

Sphingosine‐1‐phosphate Modulates Aldosterone‐induced Epithelial Sodium Channel Subunit Trafficking in Renal Cortical Collecting Duct Cells

We reported previously that sphingosine‐1‐phosphate (S1P) inhibited amiloride‐sensitive epithelial Na+ channels (ENaC) in the renal medulla. The present study aimed to investigate the underlying molecular mechanisms using cultured M1‐cortical collecting duct cell line. Transepithelial voltage and resistance were measured with a volt ohm meter. Equivalent current (Ieq) was calculated as the ratio of voltage to resistance. Results showed that S1P significantly inhibited aldosterone (ALDO)‐induced Ieq increase. In the presence of S1P1 receptor antagonist W146, however, the inhibitory effect of S1P was blocked (Control, 1.79 ± 0.11; ALDO, 3.57 ± 0.32; ALDO + S1P, 2.42 ± 0.05; ALDO + S1P + W146, 3.22 ± 0.30). In contrast, S1P2 or S1P3 antagonists had no affect. Furthermore, protein kinase A inhibitor 8‐ bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) attenuated ALDO‐induced Ieq increase, but it had no additive inhibitory effect with S1P (Control, 1.82 ± 0.10; ALDO, 3.69 ± 0.30; ALDO + 8‐Br‐cAMP, 2.41 ± 0.39; ALDO + 8‐Br‐cAMP + S1P, 2.35 ± 0.34). In addition, S1P blocked ALDO‐induced cAMP production. Finally, S1P prevented ALDO‐induced α‐ENaC subunit translocation to apical membrane. It is suggested that S1P reduces epithelial Na+ transport via S1P1 receptor‐mediated inhibition of cAMP production and ENaC translocation. (NIH grants HL89563, HL106042 and DK54927).

Evidence for the existence of calcium signaling domains in a renal cortical collecting duct cell line

The epithelial sodium channel of the kidney (ENaC) is inhibited by activation of the purinergic P2Y2 receptor in the apical membrane but stimulated by basolateral activation of the P2X4 receptor. We hypothesized that regional regulation of [Ca2+]i is responsible for the polarized effects of ATP on ENaC function. To test this, we analyzed [Ca2+]i responses downstream of purinergic agonists in renal A6 cells. Apically, the P2Y2 agonist UTP caused a rapid, transient increase (15–60 sec) in [Ca2+]i observed by loading cells with fluo4‐AM. The P2X4 receptor agonist 2‐meSATP applied basolaterally significantly increased the open probability of ENaC, an effect which was abolished by pretreatment with the Ca2+chelator EDTA (2 mM). In other epithelial cell types, mitochondria form barriers that compartmentalize Ca2+. To test whether such a barrier exists in renal A6 cells, we loaded cells with MitoTracker Red to visualize mitochondria. In Z‐axis sections, we observed strong bands of mitochondria both apically and basolaterally. Our results suggest that the differing effects of apical vs basolateral ATP on ENaC occur through activation of different purinergic receptors, both of which cause increases in [Ca2+]i but that the Ca2+ is confined to two separate compartments, producing differing effects. This work was supported by NIH grants R37‐DK037963 and T32‐DK07656.

Role of Per1 and the mineralocorticoid receptor in the coordinate regulation of αENaC in renal cortical collecting duct cells

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.

Aquaporin 2-increased renal cell proliferation is associated with cell volume regulation

We have previously demonstrated that in renal cortical collecting duct cells (RCCD(1)) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT-RCCD(1) (not expressing aquaporins) and AQP2-RCCD(1) (transfected with AQP2). Our results showed that when most RCCD(1) cells are in the G(1)-phase (unsynchronized), the blockage of barium-sensitive K(+) channels implicated in rapid RVD inhibits cell proliferation only in AQP2-RCCD(1) cells. Though cells in the S-phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down-regulation in the rapid RVD response only in AQP2-RCCD(1) cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2-besides increasing water permeability-would play some other role. These observations together with evidence implying a cell-sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G(1), volume tends to increase but it may be efficiently regulated by an AQP2-dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down-regulated when volume needs to be increased in order to proceed into the S-phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2.

Functional interaction between AQP2 and TRPV4 in renal cells

We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.

Mice Lacking the Circadian Clock Protein Per1 are Hypotensive

Increasing evidence suggests that the circadian clock regulates renal function and blood pressure, but the molecular mechanisms responsible are poorly understood. The circadian clock protein Per1 regulates expression of the epithelial sodium channel (ENaC) α subunit, suggesting that the clock may affect renal Na reabsorption, and hence, blood pressure. Direct evidence in support of this hypothesis was obtained by the novel finding that Per1 knockout (KO) mice are hypotensive. Unrestrained mice were maintained on normal light-dark and dietary conditions. Per1 KO mice exhibited a 24 hr mean arterial pressure 18 mm Hg less than control mice. The Per1 KO mice had significantly lower systolic and diastolic pressures, but no differences in heart rate or activity were observed. Consistent with positive regulation of Na reabsorption by the circadian clock, identification of novel Per1 targets in renal cortical collecting duct cells demonstrated that Per1 coordinately regulated genes whose products are known to modulate ENaC or Na, K ATPase activity. Together these data suggest an essential role for the circadian clock in the maintenance of blood pressure. Supported by NIDDK, AHA.

Protein kinase D1 modulates aldosterone-induced ENaC activity in a renal cortical collecting duct cell line

Aldosterone treatment of M1-CCD cells stimulated an increase in epithelial Na + channel (ENaC) α-subunit expression that was mainly localized to the apical membrane. PKD1-suppressed cells constitutively expressed ENaCα at low abundance, with no increase after aldosterone treatment. In the PKD1-suppressed cells, ENaCα was mainly localized proximal to the basolateral surface of the epithelium both before and after aldosterone treatment. Apical membrane insertion of ENaCβ in response to aldosterone treatment was also sensitive to PKD1 suppression as was the aldosterone-induced rise in the amiloride-sensitive, trans-epithelial current ( I TE ). The interaction of the mineralocorticoid receptor (MR) with specific elements in the promoters of aldosterone responsive genes is stabilized by ligand interaction and phosphorylation. PKD1 suppression inhibited aldosterone-induced SGK-1 expression. The nuclear localization of MR was also blocked by PKD1 suppression and MEK antagonism implicating both these kinases in MR nuclear stabilization. PKD1 thus modulates aldosterone-induced ENaC activity through the modulation of sub-cellular trafficking and the stabilization of MR nuclear localization.

Protein kinase D1 modulates aldosterone‐induced ENaC trafficking and regulation in M1 renal cortical collecting duct cells

Aldosterone stimulates the phosphorylation and activation of Protein Kinase D1 in M1 cells through the transactivation of EGFR (1). PKD1 mediates rapid aldosterone‐induced subcellular trafficking of ENaC (2). ENaCα is under transcriptional control of the mineralocorticoid receptor while ENaCβ and ENaCγ are constitutively expressed in the distal nephron. Using siRNA‐mediated stable knockdown of PKD1, transepithelial current measurements (ITE) and confocal microscopy, we examined the role of PKD1 in the regulation of ENaC trafficking and activity in M1 cells. Aldosterone (10nM) increased amiloride‐sensitive ITE from 1 ± 0.21 μA/cm2 to 7 ± 0.86 μA/cm2 (n= 8, p &lt; 0.005) in wild type M1 cells within 24h. This effect was reduced by 71.5% in the PKD1 suppressed cells. ENaCα expression was increased in wild type cells treated with aldosterone for 24h and this response was absent in PKD1 knockdown cells. Aldosterone stimulated the apical membrane insertion of ENaCβ in wild type cells, whereas no trafficking effect was observed in the PKD1 suppressed cells. PKD1 plays a central role in the aldosterone‐mediated regulation of ENaC through both transcriptional control and subcellular trafficking. Funded by Wellcome Trust, Higher Education Authority of Ireland and Science Foundation Ireland.