Metabolic inhibition-induced transient Ca2+ increase depends on mitochondria in a human proximal renal cell line

2007 ◽  
Vol 293 (2) ◽  
pp. F533-F540 ◽  
Author(s):  
Adrian Caplanusi ◽  
Andrew J. Fuller ◽  
Romer A. Gonzalez-Villalobos ◽  
Timothy G. Hammond ◽  
L. G. Navar
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Fold Increase ◽  
Recovery Phase ◽  
Metabolic Inhibition ◽  
Human Origin ◽  
Renal Epithelial Cells ◽  
Proximal Tubular ◽  
Renal Cell Line ◽  
Signaling Process

During ischemia or hypoxia an increase in intracellular cytosolic Ca2+ induces deleterious events but is also implicated in signaling processes triggered in such conditions. In MDCK cells (distal tubular origin), it was shown that mitochondria confer protection during metabolic inhibition (MI), by buffering the Ca2+ overload via mitochondrial Na+-Ca2+ exchanger (NCX). To further assess this process in cells of human origin, human cortical renal epithelial cells (proximal tubular origin) were subjected to MI and changes in cytosolic Ca2+ ([Ca2+]i), Na+, and ATP concentrations were monitored. MI was accomplished with both antimycin A and 2-deoxyglucose and induced a 3.5-fold increase in [Ca2+]i, reaching 136.5 ± 15.8 nM in the first 3.45 min. Subsequently [Ca2+]i dropped and stabilized to 62.7 ± 7.3 nM by 30 min. The first phase of the transient increase was La3+ sensitive, not influenced by diltiazem, and abolished when mitochondria were deenergized with the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The subsequent recovery phase was impaired in a Na+-free medium and weakened when the mitochondrial NCX was blocked with 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Thus Ca2+ entry is likely mediated by store-operated Ca2+ channels and depends on energized mitochondria, whereas [Ca2+]i recovery relied partially on the activity of mitochondrial NCX. These results indicate a possible mitochondrial-mediated signaling process triggered by MI, support the hypothesis that mitochondrial NCX has an important role in the Ca2+ clearance, and overall suggest that mitochondria play a preponderant role in the regulation of responses to MI in human renal epithelial cells.

Abstract P415: ACE N-domain Regulates High-glucose Mediated Interleukin-1 beta Production by Renal Epithelial Cells Independently From Angiotensin II Generation

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Jorge F Giani ◽  
Ellen A Bernstein ◽  
Masahiro Eriguchi ◽  
Romer A Gonzalez-Villalobos ◽  
Kenneth E Bernstein
Keyword(s):  
Diabetic Nephropathy ◽  
Angiotensin Ii ◽  
Epithelial Cells ◽  
High Glucose ◽  
Interleukin 1 ◽  
Kidney Injury ◽  
Fold Increase ◽  
Renal Epithelial Cells ◽  
Proximal Tubular ◽  
Sense And Respond

Research studies demonstrated that interleukin (IL)-1β contributes to the development of diabetic nephropathy and hypertension. However, the origin and regulation of IL-1β synthesis during diabetic kidney injury are still unknown. Here, we hypothesize that renal epithelial cells produce IL-1β in response to a high glucose stress and that angiotensin converting enzyme (ACE) plays a key role in this process. To study this, we isolated proximal tubular (PT) epithelial cells from wild-type (WT) and mice lacking either the ACE N-domain (NKO) or the C-domain (CKO) catalytic activity. These cells were exposed to normal (5 mM) or high (30 mM) glucose for 24 hours. IL-1β produced by PT cells were assessed by ELISA and RT-PCR. High glucose induced WT PT cells to release significant amounts of IL-1β (from 5±1 to 70±6 pg/ml, p<0.001; n=6). When WT PT cells were exposed to a high glucose media in the presence of an ACE inhibitor (lisinopril, 10 mM), IL-1β levels were significantly reduced (from 70±6 to 38±6 pg/ml, p<0.01). In contrast, AT1 receptor blockade by losartan did not change the amount of IL-1β produced by WT PT cells. To determine which ACE domain is associated with IL-1β production, NKO and CKO PT cells were exposed to high glucose. Strikingly, NKO PT cells released lower amounts of IL-1β when exposed to high glucose compared to WT (NKO: 15±7 vs. WT: 79±9 pg/ml, p<0.01, n=4). No differences were observed between WT and CKO PT cells. Since the ACE N-domain degrades the anti-inflammatory tetrapeptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), we tested whether the lower IL-1β production in NKO PT cells was due to an accumulation of AcSDKP. For this, we pre-treated NKO PT cells with a prolyl endopeptidase inhibitor (S17092, 50μM) to prevent the production of AcSDKP. Notably, this treatment increased the IL-1β response to high glucose in NKO PT cells (2.1±0.3-fold increase, p<0.01, n=4). Our data indicate that: 1) PT cells can sense and respond to high glucose by secreting IL-1β and 2) the absence of the ACE N-domain blunts the production of IL-1β through a mechanism that involves AcSDKP accumulation. In conclusion, ACE might contribute to the inflammatory response that underlays diabetic nephropathy independently from angiotensin II generation.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

2006 ◽  
Vol 290 (2) ◽  
pp. C492-C498 ◽  
Author(s):  
Diego Alvarez de la Rosa ◽  
Ignacio Gimenez ◽  
Biff Forbush ◽  
Cecilia M. Canessa
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Direct Consequence ◽  
Fold Increase ◽  
Inducible Promoter ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells ◽  
Important Regulator ◽  
Renal Epithelial Cell Line

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na+ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na+ transport. Previous studies have shown that SGK1 increases Na+ transport and epithelial Na+ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na+-K+-ATPase activity, the transporter responsible for basolateral Na+ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na+-K+-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1 TS425D) increased the transport activity of Na+-K+-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na+-K+-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na+ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1 TS425D, induced an ∼2.5-fold increase in total protein and plasma membrane Na+-K+-ATPase α1-subunit abundance. We conclude that aldosterone increases the abundance of Na+-K+-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli.

scholarly journals Polycystin-1 and Gα12 regulate the cleavage of E-cadherin in kidney epithelial cells

2015 ◽  
Vol 47 (2) ◽  
pp. 24-32 ◽  
Author(s):  
Jen X. Xu ◽  
Tzong-Shi Lu ◽  
Suyan Li ◽  
Yong Wu ◽  
Lai Ding ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signaling Pathway ◽  
Cell Polarity ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Renal Epithelial Cells ◽  
Extracellular Milieu ◽  
Kidney Epithelial Cells ◽  
Autosomal Dominant Polycystic Kidney ◽  
E Cadherin

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused β-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023 X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/β-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD.

scholarly journals Total flavones of Desmodium styracifolium antagonize calcium oxalate monohydrate-triggered IL-2Rβ expression in renal epithelial cells

2021 ◽  
Vol 18 (5) ◽  
pp. 1077-1081
Author(s):  
Haijie Xie ◽  
Rui Yang ◽  
Yong Xu ◽  
Chunyu Liu
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Calcium Oxalate ◽  
Human Kidney ◽  
Calcium Oxalate Monohydrate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Renal Epithelial Cells ◽  
Desmodium Styracifolium ◽  
Proximal Tubular

Purpose: To explore the effect of total flavone of Desmodium styracifolium (TFDS) on calcium oxalate monohydrate (COM)-triggered IL-2Rβ expression in human kidney proximal tubular epithelial cells. Methods: Human kidney proximal tubular epithelial cell line HK-2 was treated with COM, TFDS or both. The expression of IL-2Rβ was evaluated by quantitative polymerase chain reaction (qPCR) or flow cytometry. The responsiveness of HK-2 cells to IL-2 was determined by enzyme-linked immunosorbent assay (ELISA), qPCR and western blot. The signaling mechanism underlying the effect of TFDS was studied using western blot and qPCR. The clinical relevance of IL-2Rβ to renal inflammation was investigated by re-analyzing a Gene Expression Omnibus (GEO) dataset. Results: Total flavones of Desmodium styracifolium (TFDS) antagonize COM-triggered IL-2Rβ expression in HK-2 cells, thus reducing the responsiveness of HK-2 cells to IL-2 stimulation. Mechanistically, TFDS dampens IL-2Rβ expression by preventing the activation of STAT3. The level of IL-2Rβ is positively correlated with the inflammatory status of the kidney. Conclusions: The total flavones of Desmodium styracifolium (TFDS) prevent the upregulation of IL2Rβ in renal epithelial cells upon COM stimulation in a STAT3-dependent manner.

scholarly journals Apical Proteins Stimulate Complement Synthesis by Cultured Human Proximal Tubular Epithelial Cells

1999 ◽  
Vol 10 (1) ◽  
pp. 69-76 ◽  
Author(s):  
SYDNEY TANG ◽  
NEIL S. SHEERIN ◽  
WUDING ZHOU ◽  
ZARIN BROWN ◽  
STEVEN H. SACKS
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Epithelial Cells ◽  
Serum Proteins ◽  
Fold Increase ◽  
Complement C3 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tubular Epithelial Cells ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular

Abstract. There is increasing evidence to suggest that the renal tubular epithelium is important in the pathogenesis of progressive renal failure resulting from persistent proteinuria. The role of complement in the progression of chronic renal failure is not well defined. The purpose of this study was to characterize the production of complement by human proximal tubular epithelial cells exposed to serum proteins at the apical surface. Complement C3 gene expression was analyzed by reverse transcription and PCR. C3 protein biosynthesis was confirmed by metabolic labeling followed by immunoprecipitation and quantified by enzyme-linked immunosorbent assay. In the quiescent state, proximal tubular epithelial cells grown on permeable membrane supports secreted C3 predominantly into the apical medium. The addition of 5 mg/ml serum proteins led to an 8.9-fold increase in basolateral C3 secretion and a 2.1-fold increase in apical C3 secretion, altering the ratio of basolateral: apical C3 secretion from 0.44 ± 0.16 to 1.87 ± 0.52. C3 mRNA expression was also upregulated in a time- and dose-dependent manner. Serum fractionation demonstrated that the stimulant responsible for these effects was in the molecular weight range 30 to 100 kD. The observed phenomenon was not reproduced when purified human albumin alone was used as the stimulant. These findings could provide a possible mechanism for the link between proteinuria and interstitial fibrosis. This may have potential implications for strategies directed against complement in retarding the progression of chronic renal failure.

Macrophage interactions with collecting duct epithelial cells are capable of driving tubulointerstitial inflammation and fibrosis in immunoglobulin A nephropathy

2020 ◽  
Vol 35 (11) ◽  
pp. 1865-1877
Author(s):  
Izabella Z A Pawluczyk ◽  
Maria S F Soares ◽  
William A Barratt ◽  
Jeremy R Brown ◽  
Jasraj S Bhachu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Collecting Duct ◽  
Immunoglobulin A ◽  
Human Monocyte ◽  
Fold Increase ◽  
Tubulointerstitial Fibrosis ◽  
Fibrotic Process ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Factor Α

Abstract Background Tubulointerstitial fibrosis is a powerful predictor of future progression inimmunoglobulin A (IgA) nephropathy (IgAN). Proximal tubular epithelial cells (PTECs), in concert with infiltrating macrophages, are regarded as the agents provocateurs for driving this fibrotic process. However, evidence is now emerging for a contributory role of the distal nephron. The aim of this study was to examine the potential influence of macrophages on collecting duct epithelial cells (CDECs) and their combined role in the progression of IgAN. Methods CDECs were cultured with macrophage-conditioned media (MCM) generated from human monocyte cell lines U937 and THP-1 stimulated with or without 100 μg/mL galactose-deficient IgA1. CDECs were analysed for evidence of inflammation and fibrosis. Results Staining of IgAN biopsies for CD68+ macrophages revealed the presence of macrophages juxtaposed to collecting ducts and within their lumina. CDEC exposed to MCM from IgA1-stimulated THP-1 cells (THP-1-IgA-MCM) exhibited markedly increased expression of neutrophil-associated gelatinase (NGAL) and proinflammatory cytokinesinterleukin (IL)-1β, tumour necrosis factor-α, IL-6 and IL-8 compared with MCM from non-IgA-stimulated THP-1 cells (THP-1-MCM). U937-IgA-MCM increased fibronectin levels and reduced E-cadherinmRNA expression. THP-1-IgA-MCM-derived exosomes induced similar increases in NGAL and cytokine expression while in cross-over experiments exosomes extracted from IL-1β-exposed CDEC induced IL-1β and IL-6 mRNA expression in both sets of macrophages. MiRnome analysis revealed that microRNA (miR)-146a, -155 and -200b exhibited a &gt;2-fold increase in expression in CDEC treated with THP-1-IgA-MCM compared with THP-1-MCM. Enforced miR-146a suppression further enhanced NGAL expression, while ectopic miR-146a over-expression downregulated it. NGAL mRNA and miR-146a were upregulated in the biopsies of patients with progressive IgAN compared with non-progressive IgAN. Conclusions Taken together, these data suggest that CDEC–macrophage interactions potentially contribute to the tubulointerstitial fibrosis characteristic of progressive IgAN.

scholarly journals Angiotensin II stimulates fibronectin protein synthesis via a Gβγ/arachidonic acid-dependent pathway

2014 ◽  
Vol 307 (3) ◽  
pp. F287-F302 ◽  
Author(s):  
Larry D. Alexander ◽  
Yaxian Ding ◽  
Suganthi Alagarsamy ◽  
Xiaolan Cui
Keyword(s):  
Arachidonic Acid ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Immunoblot Analysis ◽  
Sh2 Domain ◽  
Eicosatetraenoic Acid ◽  
Ang Ii ◽  
Renal Epithelial Cells ◽  
Fibronectin Expression ◽  
Proximal Tubular

In rabbit proximal tubular cells, ANG II type 2-receptor (AT2)-induced arachidonic acid release is PLA2 coupled and dependent of G protein βγ (Gβγ) subunits. Moreover, ANG II activates ERK1/2 and transactivates EGFR via a c-Src-dependent mechanism. Arachidonic acid has been shown to mimic this effect, at least in part, by an undetermined mechanism. In this study, we determined the effects of ANG II on fibronectin expression in cultured rabbit proximal tubule cells and elucidated the signaling pathways associated with such expression. We found that ANG II and transfection of Gβγ subunits directly increased fibronectin protein expression, and this increase was inhibited by overexpression of β-adrenergic receptor kinase (βARK)-ct or DN-Src. Moreover, ANG II-induced fibronectin protein expression was significantly abrogated by the AT2 receptor antagonist PD123319. In addition, inhibition of cystolic PLA2 diminished ANG II-induced fibronectin expression. Endogenous arachidonic acid mimicked ANG II-induced fibronectin expression. We also found that overexpression of Gβγ subunits induced c-Src, ERK1/2, and EGFR tyrosine phosphorylation, which can be inhibited by overexpression of βARK-ct or DN-Src. Gβγ also induced c-Src SH2 domain association with the EGFR. Supporting these findings, in rabbit proximal tubular epithelium, immunoblot analysis indicated that βγ expression was significant. Interestingly, arachidonic acid- and eicosatetraenoic acid-induced responses were preserved in the presence of βARK-ct. This is the first report demonstrating the regulation of EGFR, ERK1/2, c-Src, and fibronectin by Gβγ subunits in renal epithelial cells. Moreover, this work demonstrates a role for Gβγ heterotrimeric proteins in ANG II, but not arachidonic acid, signaling in renal epithelial cells.

scholarly journals A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ce Zhang ◽  
Yue Chen ◽  
Shijin Sun ◽  
Yikai Zhang ◽  
Lina Wang ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Function ◽  
Mdck Cells ◽  
Ldl Receptor ◽  
Membrane Targeting ◽  
Protein Distribution ◽  
Recognition Mechanism ◽  
Renal Epithelial Cells

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin–Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

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