Bradykinin increases cytosolic free [Ca2+] in proximal tubule cells

1988 ◽  
Vol 255 (3) ◽  
pp. F486-F493 ◽  
Author(s):  
A. Aboolian ◽  
E. P. Nord
Keyword(s):  
Proximal Tubule ◽  
Dependent Manner ◽  
Ang Ii ◽  
Transduction Pathway ◽  
Free Medium ◽  
Proximal Tubule Cells ◽  
Heterologous Desensitization ◽  
Tubule Cells ◽  
Dose Dependent

The role of bradykinin (BK) as a calcium-mobilizing agonist in cells of renal proximal tubule origin was examined. Experiments were performed on confluent cultures of rabbit proximal tubule cells in primary culture and changes in cytosolic free Ca2+ concentration, [Cai2+], were monitored by use of the Ca2+-sensitive fluorescent probe fura-2. Under steady-state conditions, [Cai2+] was 210 +/- 7 nM in a Ca2+-replete medium vs. 135 +/- 5 nM in a medium devoid of Ca2+. Acute challenge with BK resulted in a transient increment in [Cai2+], which peaked at 150% the resting value within 10 s and was independent of medium [Ca2+]. The K0.5 for the process was 2.5 X 10(-10) M. The BK receptor displayed properties of the beta 2-variety. In a Ca2+-free medium 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) blocked the BK-elicited Ca2+ transient in a time- and dose-dependent manner. In contrast to TMB-8, the Ca2+-channel inhibitor, verapamil, was without effect. Prior exposure of cells to ionomycin completely obviated the BK-induced Ca2+ transient. Cells challenged with BK were nonresponsive to subsequent challenge by a second Ca2+-mobilizing agonist, angiotensin II (ANG II). In summary, these data suggest that BK is an extremely sensitive activator of the phosphoinositol transduction pathway in rabbit proximal tubule cells. Furthermore, the heterologous desensitization between BK and ANG II, in terms of elevating [Cai2+], suggests that these two agonists release Ca2+ from a common intracellular store.

Regulation of Na(+)-K(+)-2Cl- cotransport activity in rabbit proximal tubule primary culture

1994 ◽  
Vol 267 (1) ◽  
pp. F63-F69
Author(s):  
N. J. Raat ◽  
A. Hartog ◽  
C. H. van Os ◽  
R. J. Bindels
Keyword(s):  
Proximal Tubule ◽  
Primary Culture ◽  
Pulse Method ◽  
Rabbit Kidney ◽  
Free Medium ◽  
Hypertonic Medium ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Uptake Studies

The presence of Na(+)-K(+)-2Cl- cotransport in rabbit kidney proximal tubule cells in primary culture was demonstrated by bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake studies. After addition of 10 microM bumetanide, 86RB+ uptake was inhibited from 11.1 +/- 0.8 to 1.1 +/- 0.1 nmol.mg protein-1.min-1 under isotonic (300 mosM) conditions. Na(+)-K(+)-2Cl- cotransport activities ranged from 2.2 +/- 0.3 to 13.2 +/- 1.0 nmol.mg protein-1.min-1 depending on the osmolarity of the medium (150-500 mosM). Decreasing extracellular pH to 6.5 inhibited, whereas increasing to 8.5 stimulated, transport at all osmolarities. Decreasing intracellular pH (pHi) by the NH4Cl pulse method showed similar results, suggesting a possible regulatory role of pHi on cotransport activity. Ca(2+)-free medium increased cotransport activity 35 and 20% at iso- and hypertonicity, respectively. At 300 mosM, ionomycin (5 microM) inhibited cotransport by 25%. The combination of forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM) resulted in inhibition of cotransport activity by 38% at hypertonic conditions. Calyculin (1 microM) increased cotransport activity 134 and 128% at 150 and 300 mosM, respectively. In hypertonic medium calyculin did not influence cotransport activity. Okadaic acid (1 microM) had no effect on cotransport activity at all three osmolarities. NaF (10 mM) increased cotransport at all osmolarities tested.

scholarly journals Mechanisms of AT1a receptor-mediated uptake of angiotensin II by proximal tubule cells: a novel role of the multiligand endocytic receptor megalin

2014 ◽  
Vol 307 (2) ◽  
pp. F222-F233 ◽  
Author(s):  
Xiao C. Li ◽  
Jia L. Zhuo
Keyword(s):  
Proximal Tubule ◽  
Map Kinases ◽  
Signaling Proteins ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Downstream Signaling ◽  
Tubule Cells ◽  
At1a Receptor ◽  
Si Rna

The present study tested the hypothesis that the multiligand endocytic receptor megalin is partially involved in the uptake of ANG II and downstream signaling responses in mouse proximal tubule cells (mPCT) by interacting with AT1a receptors. mPCT cells of wild-type (WT) and AT1a receptor-deficient (AT1a-KO) mice were treated with vehicle, the AT1 receptor blocker losartan (10 μM), or a selective megalin small interfering (si) RNA for 48 h. The uptake of fluorescein (FITC)-labeled ANG II (10 nM, 37°C) and downstream signaling responses were analyzed by fluorescence imaging and Western blotting. AT1a receptors and megalin were abundantly expressed in mPCT cells, whereas AT1a receptors were absent in AT1a-KO mPCT cells ( P < 0.01). In WT mPCT cells, FITC-ANG II uptake was visualized at 30 min in the cytoplasm and in the nuclei 1 h after exposure. Losartan alone completely blocked the uptake of FITC-ANG II, whereas megalin siRNA inhibited only 30% of the response ( P < 0.01). The remaining FITC-ANG II uptake in the presence of megalin siRNA was completely abolished by losartan. ANG II induced threefold increases in phosphorylated MAP kinases ERK1/2 and a onefold increase in phosphorylated sodium and hydrogen exchanger 3 (NHE3) proteins, which were also blocked by losartan and megalin-siRNA. By contrast, losartan and megalin siRNA had no effects on these signaling proteins in AT1a-KO mPCT cells. We conclude that the uptake of ANG II and downstream MAP kinases ERK1/2 and NHE3 signaling responses in mPCT cells are mediated primarily by AT1a receptors. However, megalin may also play a partial role in these responses to ANG II.

Signaling cascade of ANG II-induced inhibition of α-MG uptake in renal proximal tubule cells

2004 ◽  
Vol 286 (4) ◽  
pp. F634-F642 ◽  
Author(s):  
Ho Jae Han ◽  
Soo Hyun Park ◽  
Yun Jung Lee
Keyword(s):  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Receptor Blocker ◽  
Dependent Manner ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Tk Inhibitors ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Mapk Inhibitor

ANG II and Na+-glucose cotransporter have been reported to be associated with the onset of diverse renal diseases. However, the effect of ANG II on Na+-glucose cotransporter activity was not elucidated. The effects of ANG II on α-methyl-d-[14C]glucopyranoside (α-MG) uptake and its related signal pathways were examined in the primary cultured rabbit renal proximal tubule cells (PTCs). ANG II (>2 h; >10-9 M) inhibited α-MG uptake in a time- and concentration-dependent manner and decreased the protein level of Na+-glucose cotransporters, the expression of which was abrogated by both actinomycin D and cycloheximide exposure. ANG II-induced inhibition of α-MG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD-123319, an ANG II type 2 receptor blocker. ANG II-induced inhibition of α-MG uptake was blocked by genistein, herbimycin A [tyrosine kinase (TK) inhibitors], mepacrine, and AACOCF3 (phospholipase A2 inhibitors), suggesting the role of TK phosphorylation and arachidonic acid (AA). Indeed, ANG II increased AA release, which was blocked by losartan or TK inhibitors. The effects of ANG II on AA release and α-MG uptake also were abolished by staurosporine and bisindolylmaleimide I (protein kinase C inhibitors) or PD-98059 (p44/42 MAPK inhibitor), but not SB-203580 (p38 MAPK inhibitor), respectively. Indeed, ANG II increased p44/42 MAPK activity. ANG II-induced activation of p44/42 MAPK was blocked by staurosporine. In conclusion, ANG II inhibited α-MG uptake via PKC-MAPK-cPLA2 signal cascade through the AT1 receptor in the PTCs.

scholarly journals Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

2010 ◽  
Vol 299 (6) ◽  
pp. F1496-F1506 ◽  
Author(s):  
Alan C. Pao ◽  
Aditi Bhargava ◽  
Francesca Di Sole ◽  
Raymond Quigley ◽  
Xinli Shao ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Proximal Tubule ◽  
Cell Surface Expression ◽  
Thick Ascending Limb ◽  
Proximal Tubule Cells ◽  
Mammalian Kidney ◽  
Tubule Cells ◽  
Exchange Activity

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na+) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na+/H+ exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na+/H+ exchange activity by Na+-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na+/H+ exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+ exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na+ transport through NHE3 in the proximal tubule.

scholarly journals Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner

2015 ◽  
Vol 308 (11) ◽  
pp. F1268-F1275 ◽  
Author(s):  
Yixin Su ◽  
Jianli Bi ◽  
Victor M. Pulgar ◽  
Jorge Figueroa ◽  
Mark Chappell ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Fetal Programming ◽  
Specific Effect ◽  
Ang Ii ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells ◽  
Female Sheep ◽  
Male Sheep ◽  
In Cells

We have shown a sex-specific effect of fetal programming on Na+ excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na+ uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na+ uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na+/H+ exchanger 3. Basal Na+ uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na+ uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na+ uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na+ uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells.

Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis

1994 ◽  
Vol 266 (3) ◽  
pp. C669-C675 ◽  
Author(s):  
J. R. Schelling ◽  
S. L. Linas
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Receptor Internalization ◽  
Ang Ii ◽  
Na Transport ◽  
Cellular Mechanisms ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Receptor Mediated Endocytosis ◽  
Tubule Cells

Angiotensin II (ANG II) receptors are present on apical and basolateral surfaces of proximal tubule cells. To determine the cellular mechanisms of proximal tubule ANG II receptor-mediated Na transport, apical-to-basolateral 22Na flux was measured in cultured proximal tubule cells. Apical ANG II caused increases in 22Na flux (maximum response: 100 nM, 30 min). Basolateral ANG II resulted in 22Na flux that was 23-56% greater than 22Na flux observed with equimolar apical ANG II. Apical ANG II-induced 22Na flux was prevented by preincubation with amiloride, ouabain, and the AT1 receptor antagonist losartan. Because apical ANG II signaling was previously shown to be endocytosis dependent, we questioned whether endocytosis was required for ANG II-stimulated proximal tubule Na transport as well. Apical (but not basolateral) ANG II-dependent 22Na flux was inhibited by phenylarsine oxide, an agent which prevents ANG II receptor internalization. In conclusion, apical and basolateral ANG II caused proximal tubule Na transport. Apical ANG II-dependent Na flux was mediated by AT1 receptors, transcellular transport pathways, and receptor-mediated endocytosis.

Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells

1998 ◽  
Vol 274 (5) ◽  
pp. F897-F905 ◽  
Author(s):  
Thomas J. Thekkumkara ◽  
Rochelle Cookson ◽  
Stuart L. Linas
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Epithelial Tissue ◽  
Proximal Tubule Cell ◽  
Ang Ii ◽  
Binding Studies ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
And Function ◽  
In Cells

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1A are present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood, because specific binding of ANG II to intact PTEC has not been found and because a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells, which stably express AT1A( K d = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of nontransfected OK cells revealed no evidence of AT1A mRNA (reverse transcriptase-polymerase chain reaction analysis) or protein (125I-labeled ANG II binding studies) expression. In cells stably expressing AT1A, ANG II binding was saturable, reversible, and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions that allowed selective access to AP or BL surfaces. AT1A were expressed on both AP ( K d = 8.7 nM, Bmax = 3.33 pmol/mg protein) and BL ( K d = 10.1 nM, Bmax = 5.50 pmol/mg protein) surfaces. Both AP and BL AT1Areceptors underwent agonist-dependent endocytosis (AP receptor: t 1/2 = 7.9 min, Ymax = 78.5%; BL receptor: t 1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, ANG II caused time- and concentration-dependent increases in transepithelial22Na transport (2-fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line that expresses AT1A on both AP and BL surfaces, undergoes agonist-dependent receptor endocytosis, and is functional, as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC.

Role of MAPK pathways in light chain-induced cytokine production in human proximal tubule cells

2003 ◽  
Vol 284 (6) ◽  
pp. F1245-F1254 ◽  
Author(s):  
Sule Sengul ◽  
Craig Zwizinski ◽  
Vecihi Batuman
Keyword(s):  
Proximal Tubule ◽  
Light Chain ◽  
Cytokine Production ◽  
Tubulointerstitial Injury ◽  
Proximal Tubule Cells ◽  
Mapk Pathways ◽  
Cytokine Responses ◽  
Tubule Cells ◽  
Human Proximal Tubule

We previously demonstrated that light chain (LC) endocytosis by human proximal tubule cells (PTCs) leads to production of cytokines through activation of NF-κB. Here, we examined the role of MAPK pathways in these responses using four species of myeloma LCs (κ1, κ2, κ3, and λ1) previously shown to induce cytokine production by PTCs. Among these, κ1-LC, which yielded the strongest cytokine responses, was selected for detailed studies. Activation of MAPKs was probed by Western blot analysis for the active kinases, ERK 1/2, JNK 1/2, and p38 in κ1-LC-exposed human PTCs. To evaluate the functional role of MAPKs in LC-induced cytokine responses, we tested the effects of U-0126, an ERK inhibitor; SP-600125, an inhibitor of JNK; SB-203580, a p38 inhibitor; and curcumin, a JNK-AP-1 inhibitor, all added to media before 4-h exposure to 1.5 mg/ml κ1-LC. IL-6 and monocyte chemotactic protein-1 (MCP-1) were determined by ELISA. Both LC and human serum albumin (HSA) activated ERK, although the HSA effect was weaker. κ1-LC stimulated all three MAPKs, although phosphorylation of ERK was more pronounced and sustained than others. Inhibitors of ERK, JNK, and p38 reduced LC-induced IL-6 and MCP-1 production. These findings suggest that activation of MAPKs plays a role in LC-induced cytokine responses in PTCs. Activation of MAPKs may be involved in cytokine responses induced by other proteins as well as LCs and may be pivotal in the pathophysiology of tubulointerstitial injury in proteinuric diseases.

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