scholarly journals Molecular evidence for a role for K+-Cl− cotransporters in the kidney

2013 ◽  
Vol 305 (10) ◽  
pp. F1402-F1411 ◽  
Author(s):  
Zesergio Melo ◽  
Silvia Cruz-Rangel ◽  
Rocio Bautista ◽  
Norma Vázquez ◽  
María Castañeda-Bueno ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Protein Expression ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Intercalated Cells ◽  
Salt Diet ◽  
High K ◽  
Low Salt ◽  
Mrna And Protein Expression ◽  
Tubule Cells

K+-Cl− cotransporter (KCC) isoforms 3 (KCC3) and 4 (KCC4) are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low-salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low- or high-K+ diets. Both KCC3 mRNA and protein expression were increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not with a low-salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low-sodium diet in the whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low-salt diet was also observed in WNK4 knockout mice, suggesting that upregulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low- or high-K+ diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the thick ascending loop of Henle's loop and acid secretion of the collecting duct.

Renal expression of the ammonia transporters, Rhbg and Rhcg, in response to chronic metabolic acidosis

2006 ◽  
Vol 290 (2) ◽  
pp. F397-F408 ◽  
Author(s):  
Ramanathan M. Seshadri ◽  
Janet D. Klein ◽  
Shelley Kozlowski ◽  
Jeff M. Sands ◽  
Young-Hee Kim ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Protein Expression ◽  
Mrna Expression ◽  
Collecting Duct ◽  
Broad Band ◽  
Ammonia Excretion ◽  
Intercalated Cells ◽  
Outer Medulla ◽  
Chronic Metabolic Acidosis ◽  
Medullary Collecting Duct

Chronic metabolic acidosis induces dramatic increases in net acid excretion that are predominantly due to increases in urinary ammonia excretion. The current study examines whether this increase is associated with changes in the expression of the renal ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). Chronic metabolic acidosis was induced in Sprague-Dawley rats by HCl ingestion for 1 wk; control animals were pair-fed. After 1 wk, metabolic acidosis had developed, and urinary ammonia excretion increased significantly. Rhcg protein expression was increased in both the outer medulla and the base of the inner medulla. Intercalated cells in the outer medullary collecting duct (OMCD) and in the inner medullary collecting duct (IMCD) in acid-loaded animals protruded into the tubule lumen and had a sharp, discrete band of apical Rhcg immunoreactivity, compared with a flatter cell profile and a broad band of apical immunolabel in control kidneys. In addition, basolateral Rhcg immunoreactivity was observed in both control and acidotic kidneys. Cortical Rhcg protein expression and immunoreactivity were not detectably altered. Rhcg mRNA expression was not significantly altered in the cortex, outer medulla, or inner medulla by chronic metabolic acidosis. Rhbg protein and mRNA expression were unchanged in the cortex, outer and inner medulla, and no changes in Rhbg immunolabel were evident in these regions. We conclude that chronic metabolic acidosis increases Rhcg protein expression in intercalated cells in the OMCD and in the IMCD, where it is likely to mediate an important role in the increased urinary ammonia excretion.

scholarly journals Regulation of (pro)renin receptor expression in mIMCD via the GSK-3β-NFAT5-SIRT-1 signaling pathway

2014 ◽  
Vol 307 (5) ◽  
pp. F593-F600 ◽  
Author(s):  
Syed Quadri ◽  
Helmy M. Siragy
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Collecting Duct ◽  
Receptor Expression ◽  
Low Salt ◽  
Mrna And Protein Expression ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Gsk 3Β ◽  
Si Rna

The localization and regulation of (pro)renin receptor (PRR) expression in kidney collecting duct cells are not well established. We hypothesized that low salt (LS) contributes to the regulation of PRR expression in these cells via the GSK-3β-NFAT5-sirtuin1 (SIRT-1) signaling pathway. Mouse inner medullary collecting duct (mIMCD) cells were treated with NaCl at 130 (normal salt; NS), 63 (LS), or 209 mM (high salt; HS) alone or in combination with NFAT5 scrambled small interfering (si) RNA, NFAT5 siRNA, or the SIRT-1 inhibitor EX-527. Compared with NS, LS increased the mRNA and protein expression of PRR by 71% and 69% ( P < 0.05), and reduced phosphorylation of GSK-3β by 62% ( P < 0.01), mRNA and protein expressions of NFAT5 by 65% and 45% ( P < 0.05), and SIRT-1 by 44% and 50% ( P < 0.01), respectively. LS also enhanced p65 NF-κB by 102% ( P < 0.01). Treatment with HS significantly reduced the mRNA and protein expression of PRR by 32% and 23% ( P < 0.05), and increased the mRNA and protein expression of NFAT5 by 39% and 45% ( P < 0.05) and SIRT-1 by 51% and 56% ( P < 0.05), respectively. HS+NFAT5 siRNA reduced the mRNA and protein expression of NFAT5 by 51% and 35% ( P < 0.01) and increased the mRNA and protein expression of PRR by 148% and 70% ( P < 0.01), respectively. HS+EX-527 significantly increased the mRNA and protein expression of PRR by 96% and 58% ( P < 0.05), respectively. We conclude that expression of PRR in mIMCD cells is regulated by the GSK-3β-NFAT5- SIRT-1 signaling pathway.

scholarly journals Intercalated cell-specific Rh B glycoprotein deletion diminishes renal ammonia excretion response to hypokalemia

2013 ◽  
Vol 304 (4) ◽  
pp. F422-F431 ◽  
Author(s):  
Jesse M. Bishop ◽  
Hyun-Wook Lee ◽  
Mary E. Handlogten ◽  
Ki-Hwan Han ◽  
Jill W. Verlander ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Ammonia Concentration ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Intercalated Cell ◽  
Transporter Family ◽  
Total Ammonia ◽  
Normal Increase

The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K+-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia.

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

1998 ◽  
Vol 9 (5) ◽  
pp. 746-754
Author(s):  
G Obrador ◽  
H Yuan ◽  
T M Shih ◽  
Y H Wang ◽  
M A Shia ◽  
...  
Keyword(s):  
Cell Line ◽  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Rat Stomach ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

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.

scholarly journals SAT-006 A New Concept for the Endocrinology of Pre-Eclampsia: The Role of Spiral Steroids

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Fred I Chasalow ◽  
Ron Bochner
Keyword(s):  
Type A ◽  
Structural Features ◽  
Type B ◽  
Salt Diet ◽  
Breast Cyst ◽  
High K ◽  
Epithelial Na Channels ◽  
Low Salt

Abstract Background: In 1987, Graves observed that during the 3rd trimester, some patients with pre-eclampsia had high levels of unknown materials that could be detected with assays for digoxin (DLM). In 2018, we characterized a new candidate for the DLM, Ionotropin. It is a phosphocholine (PC) ester of a novel steroid with 23 carbon atoms. As Ionotropin shares structural features (a) with spironolactone (both have spiral lactones in the E-ring) and (b) with digoxin (E-ring lactone and 3α-5β configuration), we have proposed that Ionotropin may function as a potassium (K+) sparing diuretic. This suggestion is supported by the observations that [1] patients who cannot make Ionotropin (7-dehydrosterol reductase deficiency) are K+ wasting and [2] breast cyst fluids with high K+ levels also have high Ionotropin levels. Hypothesis: During the 3rd trimester, fetal requirements for K+ reach a maximum, fetal blood pressure increases and aldosterone signaling is blocked. This blockage leads to fetal sodium (Na+) wasting and is essential for formation of amniotic fluid. These events are consistent with a normal role for an unknown endogenous K+ sparing hormone and would be the basis for a modest elevation of maternal DLM during the 3rd trimester. Our hypothesis is that if any of the functions were inadequate, then the fetal-placental unit would synthesize excess PC-spiral steroids; the woman would exhibit symptoms of K+ sparing hormone excess (hypertension and proteinuria) and would be diagnosed with pre-eclampsia. Experimental Results: We have just reported a pilot study associating elevated PC esters of spiral steroids in women with pre-eclampsia. In brief, 12 of 19 women had elevated levels of at least one of the PC steroids (Z-score &gt; 2) when compared to the levels in 20 pregnant women matched for gestational age and fetal sex. There are two basic mechanisms for this dichotomy: (a) there may be episodic secretion with of a DLM with a short half-life or (b) there may be two different underlying biochemical causes. In prior studies, there has been no indication of episodic secretion of DLM similar to that observed with glucocorticoids, Ionotropin or other PC spiral steroids. Discussion: There are two basic types of K+ sparing diuretics. Type A: Spironolactone functions by regulating the NaK-ATPase. Type B: Triamterene functions by blocking synthesis of epithelial Na+ channels. Thus, Type A would have high levels of spiral steroids and Type B would have low levels of spiral steroids. Type A patients would be expected to have higher risk of long-term consequences when compared to the Type B patients. Conclusion: The recognition of the division of pre-eclampsia into two separate diseases might be the key observation for developing Type-specific diagnosis and therapy. For example, a Type A patient might benefit from a low salt diet but that diet would not be expected to benefit a patient with Type B disease.

scholarly journals ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load

2019 ◽  
Vol 317 (2) ◽  
pp. F435-F443
Author(s):  
Lydie Cheval ◽  
Naziha Bakouh ◽  
Christine Walter ◽  
Dignê Tembely ◽  
Luciana Morla ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Urinary Excretion ◽  
Secretory Pathway ◽  
Collecting Duct ◽  
Type A ◽  
Intercalated Cells ◽  
Membrane Expression ◽  
Acid Load ◽  
Anp Receptors ◽  
Stimulation Of

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl− cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

Proximal tubular dopamine production regulates basolateral Na-K-ATPase

1992 ◽  
Vol 262 (4) ◽  
pp. F566-F571 ◽  
Author(s):  
A. D. Baines ◽  
P. Ho ◽  
R. Drangova
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Sch 23390 ◽  
Basolateral Membrane ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Western Blots ◽  
Salt Diet ◽  
Low Salt ◽  
Proximal Tubular

Regulation of proximal tubular Na-K-adenosine-triphosphatase (ATPase), brush-border membrane Na(+)-H+ antiporter and Na(+)-Pi symporter activity by endogenously produced dopamine was examined in Wistar rats. Na-K-ATPase was measured in basolateral membrane (BLM) fractions permeabilized with alamethicin or sodium dodecyl sulfate (SDS). Carbidopa (5 mg/kg) injected 18 h before removal of kidneys increased maximal activity (Vmax) noncompetitively in cortical BLM but not in other membrane fractions or outer medullary BLM (-2 +/- 4%). Chronic renal denervation did not alter the response. Carbidopa stimulated Na-K-ATPase in cortical BLM from rats eating a normal salt diet with and without 1% saline to drink (+18 +/- 4% and +22 +/- 4%, respectively; P greater than 0.001). Carbidopa did not increase Vmax of BLM Na-K-ATPase from rats eating a low-salt diet (+1.5 +/- 4%); however, when the low-salt diet was supplemented with 1 mM dihydroxyphenylalanine (dopa) to drink for 1 day carbidopa, increased Vmax by 18 +/- 3% (P = 0.018). Carbidopa did not alter the Michaelis constant (Km) for Na or K or inhibitory constant (Ki) for ouabain. Injection of the DA1 antagonist Sch 23390 (2 mg/kg) also increased Na-K-ATPase (18 +/- 4%; P = 0.014). Western blots using a monoclonal alpha-subunit antibody revealed a 22 +/- 8% increase following carbidopa treatment (P = 0.033; n = 19 pairs). Carbidopa had no effect on Na(+)-H+ antiporter activity (22Na uptake) or on Na(+)-32Pi cotransport in brush-border membrane vesicles. These results indicate that dopamine produced in proximal tubules tonically reduces Na-K-ATPase Vmax by decreasing the number of alpha-subunits associated with the BLM.

scholarly journals Cortical distal nephron Cl− transport in volume homeostasis and blood pressure regulation

2013 ◽  
Vol 305 (4) ◽  
pp. F427-F438 ◽  
Author(s):  
Susan M. Wall ◽  
Alan M. Weinstein
Keyword(s):  
Blood Pressure ◽  
Pressor Response ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Extracellular Volume ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base Balance ◽  
Relative Contribution ◽  
And Function

Renal intercalated cells mediate the secretion or absorption of Cl− and OH−/H+ equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume, and blood pressure. Cl− absorption is either electrogenic and amiloride-sensitive or electroneutral and thiazide-sensitive. However, which Cl− transporter(s) are targeted by these diuretics is debated. While epithelial Na+ channel (ENaC) does not transport Cl−, it modulates Cl− transport probably by generating a lumen-negative voltage, which drives Cl− flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl− secretion via a type A intercalated cells. During ENaC blockade, Cl− is taken up across the basolateral membrane through the Na+-K+−2Cl− cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl− channel or an electrogenic exchanger). The mechanism of this apical Cl− secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl− absorption mediated by a Na+-dependent Cl−/HCO3− exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl− absorption varies between studies and probably depends on the treatment model employed. Cl− absorption increases markedly with angiotensin and aldosterone administration, largely by upregulating the Na+-independent Cl−/HCO3− exchanger pendrin. In the absence of pendrin [ Slc26a4 (−/−) or pendrin null mice], aldosterone-stimulated Cl− absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3−. This review summarizes mechanisms of Cl− transport in CNT and CCD and how these transporters contribute to the regulation of extracellular volume and blood pressure.

Endogenous renal dopamine production regulates phosphate excretion

1994 ◽  
Vol 266 (6) ◽  
pp. F858-F867 ◽  
Author(s):  
A. Debska-Slizien ◽  
P. Ho ◽  
R. Drangova ◽  
A. D. Baines
Keyword(s):  
Brush Border Membrane ◽  
Membrane Vesicle ◽  
Basolateral Membrane ◽  
High Salt Diet ◽  
Salt Diet ◽  
Endogenous Dopamine ◽  
Low Salt ◽  
32P Uptake ◽  
22Na Uptake ◽  
Renal Dopamine

We examined the effect of endogenous dopamine production on Pi and citrate excretion by Wistar rats. Carbidopa (20-40 mumol/kg ip) decreased dopamine, Pi, and citrate excretion within 20 min (86%, 47%, and 38%, respectively); Pi reabsorption increased 11 +/- 4% (P = 0.03). The decreases were sustained for at least 18 h. 3-Hydroxybenzylhydrazine (45 mumol/kg ip) reduced Pi excretion 24%. Benserazide (40 mumol/kg ip and 0.1 mumol/min iv) reduced dopamine excretion (94%) and blocked the effect of carbidopa on Pi and citrate excretion. In isolated perfused kidneys benserazide, carbidopa, and 3-hydroxybenzylhydrazine all decreased Pi excretion. Dopamine (1 mumol/l) added to cortical minceates reduced brush-border membrane vesicle (BBMV) 32P uptake by 8% (P < 0.02) and amiloride-inhibitable 22Na uptake by 19%. Carbidopa added to minceates increased 32P uptake by 12%. Carbidopa pretreatment increased (75%) amiloride-sensitive 22Na uptake into BBMV of rats fed a high-salt diet. Uptake was not increased into BBMV from rats fed a low-salt diet. Carbidopa increased (17%) basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) gradually over 4 h. Na(+)-K(+)-ATPase did not increase in rats fed a low-phosphorous diet, but did increase when dopa was added to the diet. Thus endogenous dopamine appears to directly control Na(+)-Pi and Na+/H+ transport and secondarily alter basolateral membrane Na(+)-K(+)-ATPase.

Structural-functional relationships along the distal nephron

1986 ◽  
Vol 250 (1) ◽  
pp. F1-F15 ◽  
Author(s):  
K. M. Madsen ◽  
C. C. Tisher
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Structural Heterogeneity ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Functional Relationships

The distal tubule, which includes the thick ascending limb (TAL), the macula densa, and the distal convoluted tubule (DCT), and the collecting duct are structurally heterogeneous, thus reflecting the functional heterogeneity that is also present. As the TAL ascends from medulla to cortex, the surface area of the apical plasma membrane increases while that of the basolateral membrane decreases. The structure of the DCT resembles that of the medullary TAL. An excellent correlation exists between structure, Na-K-ATPase activity, and NaCl reabsorptive capacity in the distal tubule. The collecting duct is subdivided into the initial collecting tubule (ICT), and cortical (CCD), outer medullary (OMCD), and inner medullary (IMCD) collecting ducts. Between the distal tubule and the collecting duct is a transition region termed the connecting segment or connecting tubule (CNT). Considerable structural heterogeneity exists along the collecting duct within the two major cell populations, the intercalated cells and the principal cells. In the CNT, the ICT, and the CCD, potassium loading and mineralocorticoids stimulate Na-K-ATPase activity and cause proliferation of the basolateral membrane of CNT cells and principal cells, thus identifying the cells responsible for mineralocorticoid-stimulated potassium secretion in these regions. Finally, at least two morphologically distinct populations of intercalated cells exist, types A and B. In the rat, type A predominates in the CNT and the OMCD and is believed to be responsible for H+ secretion, at least in the OMCD. Type B predominates in the CCD, where it may be involved in bicarbonate secretion.

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