Abstract P229: Site-1 Protease-derived Soluble (pro) Renin Receptor Mediates Aldosterone-induced Enac Activation In The Collecting Duct

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Fei Wang ◽  
Renfei Luo ◽  
KEXIN PENG ◽  
Peng Wu ◽  
Xiyang Liu ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Vehicle Group ◽  
Α Subunit ◽  
Collecting Duct Cells

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na + channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), generated by site-1 protease (S1P), a newly identified PRR cleavage protease, in ENaC regulation, and further tested its relevance to Aldo signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM for 24 h induced a significant increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique ( I eq : 7.5 ± 0.7 μA/cm 2 in sPRR group vs. 3.5 ± 0.5 μA/cm 2 in vehicle group, n = 6, p < 0.01) . In primary cultured rat IMCD cells, the same sPRR-His treatment induced a 1.7 fold increase in protein expression of the α-subunit but not β- or γ-subunit of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The upregulation of α-ENaC transcription depended on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Using chamber determination of short-circuit current showed that Aldo-induced ENaC activity was almost completely abolished by PF-429242 (PF), a S1P inhibitor, and the response was restored by supplement of sPRR-His ( I eq : 7.2 ± 0.7 μA/cm 2 in Aldo group vs. 5.0 ± 0.3 μA/cm 2 in Aldo/PF group vs. 6.8 ± 0.3 μA/cm 2 in Aldo/PF/sPRR-His group, n = 5, p < 0.05). Medium sPRR was elevated by Aldo and inhibited by PF. Male C57BL/6 mice were pretreated with PF (30 mg/kg/day) or vehicle via minipump, followed by 3 days of aldosterone (0.2 mg/kg/day via a second minipump). Amiloride-sensitive Na+ current in freshly isolated CCD as measured by using patch clamp lower in Aldo + PF group than in Aldo group. Together, these results support an essential role of S1P-derived sPRR in mediating Aldo-induced ENaC activation.

scholarly journals Soluble (pro)renin receptor regulation of ENaC involved in aldosterone signaling in cultured collecting duct cells

2020 ◽  
Vol 318 (3) ◽  
pp. F817-F825 ◽  
Author(s):  
Fei Wang ◽  
Renfei Luo ◽  
Kexin Peng ◽  
Xiyang Liu ◽  
Chuanming Xu ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Α Subunit ◽  
Duct Cells ◽  
Nadph Oxidase 1 ◽  
Collecting Duct Cells ◽  
Two Phases

We have previously shown that activation of (pro)renin receptor (PRR) induces epithelial Na+ channel (ENaC) activity in cultured collecting duct cells. Here, we examined the role of soluble PRR (sPRR), the cleavage product of PRR in ENaC regulation, and further tested its relevance to aldosterone signaling. In cultured mpkCCD cells, administration of recombinant histidine-tagged sPRR (sPRR-His) at 10 nM within minutes induced a significant and transient increase in the amiloride-sensitive short-circuit current as assessed using the Ussing chamber technique. The acute ENaC activation was blocked by the NADPH oxidase 1/4 inhibitor GKT137892 and siRNA against Nox4 but not the β-catenin inhibitor ICG-001. In primary rat inner medullary collecting duct cells, administration of sPRR-His at 10 nM for 24 h induced protein expression of the α-subunit but not β- or γ-subunits of ENaC, in parallel with upregulation of mRNA expression as well as promoter activity of the α-subunit. The transcriptional activation of α-ENaC was dependent on β-catenin signaling. Consistent results obtained by epithelial volt ohmmeter measurement of equivalent current and Ussing chamber determination of short-circuit current showed that aldosterone-induced transepithelial Na+ transport was inhibited by the PRR decoy inhibitor PRO20 and PF-429242, an inhibitor of sPRR-generating enzyme site-1 protease, and the response was restored by the addition of sPRR-His. Medium sPRR was elevated by aldosterone and inhibited by PF-429242. Taken together, these results demonstrate that sPRR induces two phases of ENaC activation via distinct mechanisms and functions as a mediator of the natriferic action of aldosterone.

scholarly journals Coupling of epithelial Na+ and Cl− channels by direct and indirect activation by serine proteases

2012 ◽  
Vol 303 (9) ◽  
pp. C936-C946 ◽  
Author(s):  
Veronika Gondzik ◽  
Wolf Michael Weber ◽  
Mouhamed S. Awayda
Keyword(s):  
Serine Proteases ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Epithelial Cell Line ◽  
Extracellular Proteases ◽  
Direct Role ◽  
Renal Epithelial Cell ◽  
Transport Pathways

The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na+ channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na+ transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na+ homeostasis. To explore this process, we examined the coordinated effect of this protease on Na+ and Cl− transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current ( Isc) was used to measure transport of these ions. The Isc exhibited an ENaC-dependent Na+ component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl− component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated IENaC but was without effects on ICFTR. Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na+ and Cl− transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na+ and Cl− transport in these epithelia.

scholarly journals Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II–Induced Hypertension in Mice

Hypertension ◽  
2020 ◽  
Author(s):  
Ye Feng ◽  
Kexin Peng ◽  
Renfei Luo ◽  
Fei Wang ◽  
Tianxin Yang
Keyword(s):  
Blood Pressure ◽  
Angiotensin Ii ◽  
Collecting Duct ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Duct Cell ◽  
Ang Ii ◽  
Cortical Collecting Duct ◽  
Induced Hypertension

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II–induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II–induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II–induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1337-F1350 ◽  
Author(s):  
Darren P. Wallace ◽  
Marcy Christensen ◽  
Gail Reif ◽  
Franck Belibi ◽  
Brantley Thrasher ◽  
...  
Keyword(s):  
Fluid Transport ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Transport Studies ◽  
Collecting Ducts ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCDi) and terminal (hIMCDt) inner medullary regions of human kidneys. AVP, PGE2, and forskolin increased cAMP in both hIMCDi and hIMCDt cells. The effects of AVP and PGE2 were greatest in hIMCDi; however, forskolin increased cAMP to the same extent in hIMCDi and hIMCDt. Basal short-circuit current ( I SC) of hIMCDi monolayers was 1.4 ± 0.5 μA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE2, and forskolin increased I SC; the current was reduced by blocking PKA, apical Cl− channels, basolateral NKCC1 (a Na+-K+-2Cl−cotransporter), and basolateral Cl−/HCO[Formula: see text]exchangers. In fluid transport studies, hIMCDi monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCDt monolayers, basal current was not different from zero and cAMP had no effect on I SC. We conclude that AVP and PGE2stimulate cAMP-dependent Cl− secretion by hIMCDi cells, but not hIMCDt cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

NH2-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

2001 ◽  
Vol 280 (3) ◽  
pp. C451-C458 ◽  
Author(s):  
James R. Broughman ◽  
Kathy E. Mitchell ◽  
Roger L. Sedlacek ◽  
Takeo Iwamoto ◽  
John M. Tomich ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Glycine Receptor ◽  
Short Circuit Current ◽  
Disulfonic Acid ◽  
Α Subunit ◽  
Anion Secretion ◽  
Anion Conductance ◽  
Selective Channel

A synthetic, channel-forming peptide, derived from the α-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH2 terminus (N-K4-M2GlyR). In Ussing chamber experiments, apical N-K4-M2GlyR (250 μM) increased transepithelial short-circuit current ( I sc) by 7.7 ± 1.7 and 10.6 ± 0.9 μA/cm2 in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672–C1679, 1997). N-K4-M2GlyR caused a concentration-dependent increase in I sc ( k [1/2] = 190 μM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K4-M2GlyR-mediated increases in I sc were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl− conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4′-dinitrostilben-2,2′-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K4-M2GlyR-mediated I sc. Whole cell membrane patch voltage-clamp studies revealed an N-K4-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K4-M2GlyR forms a 50-pS anion conductance with a k [1/2] for Cl−of 290 meq. These results indicate that N-K4-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.

Characteristics of papillary collecting duct cells in primary culture

1988 ◽  
Vol 255 (6) ◽  
pp. F1160-F1169 ◽  
Author(s):  
R. F. Husted ◽  
M. Hayashi ◽  
J. B. Stokes
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Calf Serum ◽  
Morphological Characteristics ◽  
Short Circuit Current ◽  
Papillary Collecting Duct ◽  
Collecting Duct Cells ◽  
Na Uptake

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

scholarly journals Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

2012 ◽  
Vol 302 (7) ◽  
pp. F801-F808 ◽  
Author(s):  
Takamitsu Saigusa ◽  
Ryan Reichert ◽  
Jennifer Guare ◽  
Brian J. Siroky ◽  
Monika Gooz ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Renal Cysts ◽  
Short Circuit Current ◽  
Polycystic Kidney ◽  
Collecting Duct Cells ◽  
And Function ◽  
Apical Membranes ◽  
Apical Localization

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (−), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/ Ift88 gene). Interestingly, only cilia (−) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (−) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (−) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current ( Isc) in cilia (−) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of Isc in cilia (−) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors

2015 ◽  
Vol 308 (5) ◽  
pp. F450-F458 ◽  
Author(s):  
Morag K. Mansley ◽  
Winfried Neuhuber ◽  
Christoph Korbmacher ◽  
Marko Bertog
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Nerve Fibers ◽  
Good Evidence ◽  
Fine Tuning ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na+ absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na+ excretion takes place. Here, the appropriate regulation of transepithelial Na+ transport, mediated by the amiloride-sensitive epithelial Na+ channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na+ transport, we performed short-circuit current ( Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na + absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

scholarly journals Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells.

1994 ◽  
Vol 4 (11) ◽  
pp. 1855-1860
Author(s):  
B A Stoos ◽  
O A Carretero ◽  
J L Garvin
Keyword(s):  
Nitric Oxide ◽  
Maximum Rate ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Cortical Collecting Duct ◽  
Duct Cells ◽  
Collecting Duct Cells

Previously, it has been shown that the addition of bradykinin (Bk) to M-1 cortical collecting duct cells in the presence of endothelial cells decreased short-circuit current (Isc), a measure of net active transport. This effect is presumably due to the release of endothelium-derived nitric oxide (EDNO), because the decrease in Isc could be blocked with Nw-nitro-L-arginine. To show that the inhibition of Isc was due to EDNO rather than prostaglandins, the ability of a cyclooxygenase inhibitor to block the inhibition was examined. When Bk was added to cocultures in the presence of meclofenamate (10(-5) M), Isc decreased from 62 +/- 12 to 44.5 +/- 7 muA/cm2, not significantly different from that in the absence of meclofenamate. To determine if the effect was due to an alteration of sodium absorption, Bk (10(-9) M) was added to cocultures, resulting in a decrease in Na flux from 28 +/- 3.1 to 20 +/- 2.2 nEq/min (P < 0.05), with Isc decreasing from 25 +/- 2.4 to 20 +/- 3.6 nEq/min (P < 0.05). To examine if the inhibition was due to blockade at the apical membrane sodium channel or the basolateral Na+/K+ ATPase, the cation-selective ionophore nystatin was used. Nystatin reversed the effect of EDNO on Isc. The effects of EDNO on Na+/K+ ATPase were also measured directly. Under maximum rate conditions, the Na+/K+ ATPase activity of control and Bk-treated cocultures was 5.2 +/- 0.3 and 6.8 +/- 1.0 nmol/min per square centimeter, respectively (not significantly different).(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor inhibits amiloride-sensitive sodium absorption in renal collecting duct cells

2003 ◽  
Vol 284 (1) ◽  
pp. F57-F64 ◽  
Author(s):  
Jie-Pan Shen ◽  
Calvin U. Cotton
Keyword(s):  
Growth Factor ◽  
Chronic Exposure ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mrna Levels ◽  
Erk Pathway ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Renal Collecting Duct

The effects of the ERK pathway on electrogenic transepithelial Na+ absorption by renal collecting duct cells were determined. Approximately 90% of the unstimulated short-circuit current (15 ± 1 μA/cm2, n = 10) across conditionally immortalized murine collecting duct epithelial cells (mCT1) is amiloride sensitive and is likely mediated by apical epithelial Na+ channels. Chronic exposure (24 h) of the epithelial monolayers to either EGF (50 ng/ml) or transforming growth factor-α (TGF-α; 20 ng/ml) reduced amiloride-sensitive short-circuit current by >60%. The inhibitory effect of EGF on Na+ absorption was not due to inhibition of basolateral Na+-K+-ATPase, because the pump current elicited by permeabilization of apical membrane with nystatin was not reduced by EGF. Chronic exposure of the mCT1 cells to EGF (20 ng/ml, 24 h) elicited a 70–85% decrease in epithelial Na+ channel subunit mRNA levels. Exposure of mCT1 cells to either EGF (20 ng/ml) or PMA (150 nM) induced rapid phosphorylation of p42/p44 (ERK1/2) and pretreatment of the monolayers with PD-98059 (an ERK kinase inhibitor; 30 μM) prevented phosphorylation of p42/p44. Similarly, pretreatment of mCT1 monolayers with PD-98059 prevented the EGF- and PMA-induced inhibition of amiloride-sensitive Na+ absorption. The results of these studies demonstrate that amiloride-sensitive Na+ absorption by renal collecting duct cells is regulated by the ERK pathway. This pathway may play a role in alterations in ion transport that occur in polycystic kidney disease.

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