High glucose induces the activity and expression of Na+/H+exchange in glomerular mesangial cells

2000 ◽  
Vol 278 (1) ◽  
pp. F91-F96 ◽  
Author(s):  
Michael B. Ganz ◽  
Karen Hawkins ◽  
Robert F. Reilly
Keyword(s):  
Steady State ◽  
High Glucose ◽  
Prolonged Exposure ◽  
Mesangial Cells ◽  
Fold Increase ◽  
Cell Behavior ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Exchange Contribution ◽  
Acid Extrusion

.—Changes in activity or expression of transporters may account for alterations in cell behavior in diabetes. We sought to ascertain if mesangial cells (MC) grown in different glucose concentrations exhibit changes in activity and expression of acid-extruding transporters, the Na+/H+and Na+-dependent Cl−/[Formula: see text]exchanger. pHi was determined by the use of the fluorescent pH-sensitive dye BCECF. In MCs grown in 5 mM glucose (control), the Na+/H+exchanger was responsible for 31.8 ± 5.1% of steady-state pHi, whereas Na+-dependent Cl−/[Formula: see text]contributed 62.9 ± 4.0% ( n = 11). In MCs grown in high glucose for 2 wk, Na+/H+exchange contribution to acid-extrusion increased as follows: 42.3 ± 4.6% [ n = 8, 10 mM, not significant (NS)], 51.1 ± 5.1% ( n = 8, 20 mM, P < 0.01), and 64.8 ± 5.5% ( n = 7, 30 mM, P < 0.001). The Na+-dependent Cl−/[Formula: see text]exchanger contributed less [47.0 ± 4.6, 38.6 ± 5.8, and 21.1 ± 3.8%, for 10, 20, and 30 mM glucose, respectively ( n > 7)]. We sought to ascertain if the magnitude of the acute stimulated response to ANG II by the Na+/H+and Na+-dependent Cl−/[Formula: see text]exchanger is changed. Na+/H+exchanger (1.89-fold increase in 30 vs. 5 mM, P < 0.002), but not Na+-dependent Cl−/[Formula: see text]exchange (0.17-fold, NS), exhibited an enhanced response to ANG II (1 μM). Na+/H+exchange (NHE1) expression was significantly different (1.72-fold) after prolonged exposure to high glucose. These results suggest that the Na+/H+exchanger, but not Na+-dependent Cl−/[Formula: see text]exchanger, may play an early role in the response to hyperglycemia in the diabetic state.

Downregulation of TRPC6 protein expression by high glucose, a possible mechanism for the impaired Ca2+ signaling in glomerular mesangial cells in diabetes

2007 ◽  
Vol 293 (4) ◽  
pp. F1381-F1390 ◽  
Author(s):  
Sarabeth Graham ◽  
Min Ding ◽  
Sherry Sours-Brothers ◽  
Thomas Yorio ◽  
Jian-Xing Ma ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Protein Expression ◽  
High Glucose ◽  
Mesangial Cells ◽  
Membrane Currents ◽  
Maximum Effect ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Fura 2 ◽  
Fluorescence Measurements

The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca2+ signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess angiotensin II (ANG II)-stimulated membrane currents and Ca2+ responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC1 or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day 7 and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC1, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as 1 day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca2+ influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca2+ entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca2+ signaling of MCs seen in diabetes.

Role of the JAK/STAT signaling pathway in diabetic nephropathy

2006 ◽  
Vol 290 (4) ◽  
pp. F762-F768 ◽  
Author(s):  
Mario B. Marrero ◽  
Amy K. Banes-Berceli ◽  
David M. Stern ◽  
Douglas C. Eaton
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Intracellular Signaling ◽  
Mesangial Cells ◽  
Janus Kinase ◽  
Cellular Growth ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Stat Signaling ◽  
Vasoactive Peptide

Excessive cellular growth is a major contributor to pathological changes associated with diabetic nephropathy. In particular, high glucose-induced growth of glomerular mesangial cells is a characteristic feature of diabetes-induced renal complications. Glomerular mesangial cells respond to traditional growth factors, although in diabetes this occurs in the context of an environment enriched in both circulating vasoactive mediators and high glucose. For example, the vasoactive peptide ANG II has been implicated in the pathogenesis of diabetic renal disease, and recent findings suggest that high glucose and ANG II activate intracellular signaling processes, including the polyol pathway and generation of reactive oxygen species. These pathways activate the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling cascades in glomerular mesangial cells. Activation of the JAK/STAT signaling cascade can stimulate excessive proliferation and growth of glomerular mesangial cells, contributing to diabetic nephropathy. This review focuses on some of the key elements in the diabetic microenvironment, especially high glucose and the accumulation of advanced glycoxidation end products and considers their impact on ANG II and other vasoactive peptide-mediated signaling events in vitro and in vivo.

Effects of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells

1988 ◽  
Vol 254 (6) ◽  
pp. F787-F794 ◽  
Author(s):  
M. B. Ganz ◽  
G. Boyarsky ◽  
W. F. Boron ◽  
R. B. Sterzel
Keyword(s):  
Angiotensin Ii ◽  
Intracellular Ph ◽  
Mesangial Cells ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Initial Value ◽  
Ph Change ◽  
Acid Load ◽  
Ethanesulfonic Acid ◽  
Acid Extrusion

We investigated changes in intracellular pH (pHi) of cultured rat glomerular mesangial cells (MCs) exposed to angiotensin II (ANG II) and arginine vasopressin (AVP). pHi of quiescent MCs, passage 2–5, and grown on glass cover slips, was assessed by spectrofluorometry using the pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The steady-state pHi of MCs in a pH 7.4, HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.10 +/- 0.02 (n = 68) and in a pH 7.4, HCO3-containing solution, was 7.23 +/- 0.03 (n = 47) (P less than 0.01). The pHi recovery following an NH+4-induced acid load was inhibited by removal of Na+ from the bath or by addition of the amiloride analogue, ethyl isopropyl amiloride (EIPA). These effects were observed in MCs bathed in HEPES- or in HCO3-buffered solutions, consistent with the action of a Na+-H+ exchanger. When cells were bathed in HEPES, a 10-min exposure to ANG II or AVP (10(-10) to 10(-6) M) caused early and transient acidification of MCs (maximal pH change was -0.10), followed by gradual alkalinization (maximal pHi change +0.15 above the initial value). The increase of pHi was dependent on the presence of Na+ in the bath and was inhibited by EIPA. In the presence of HCO3, ANG II or AVP induced merely a small gradual acidification of MCs (pHi change -0.05). These findings demonstrate that MCs utilize a Na+-H+ exchanger for acid extrusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Mesangial cell-reduced Ca2+ signaling in high glucose is due to inactivation of phospholipase C-β3 by protein kinase C

2005 ◽  
Vol 289 (5) ◽  
pp. F1078-F1087 ◽  
Author(s):  
Helena Frecker ◽  
Snezana Munk ◽  
Hong Wang ◽  
Catharine Whiteside
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
High Glucose ◽  
Mesangial Cells ◽  
Fold Increase ◽  
Glomerular Mesangial Cells ◽  
Kinase C ◽  
Normal Glucose ◽  
Intracellular Ca

In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca2+ signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca2+ was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca2+ response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca2+ responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca2+ vs. 1.8 mM Ca2+. Depletion of intracellular Ca2+ stores with thapsigargin eliminated ET-1-stimulated Ca2+ responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca2+ response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with Gö6976 in HG normalized the Ca2+ response to ET-1. Because ET-1 and PDGF stimulate Ca2+ signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-β3. Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-β3 Ser1105 phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-β3 phosphorylation, without change in total PLC-β3, which was reversed with 24-h PMA or Gö6976. We conclude that reduced Ca2+ signaling in HG cannot be explained by reduced Ca2+ stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-β3.

Extracellular glucose reduces the responsiveness of mesangial cell ion channels to angiotensin II

1995 ◽  
Vol 269 (3) ◽  
pp. F389-F397 ◽  
Author(s):  
E. E. Seal ◽  
D. C. Eaton ◽  
L. M. Gomez ◽  
H. Ma ◽  
B. N. Ling
Keyword(s):  
Angiotensin Ii ◽  
Ion Channels ◽  
High Glucose ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Ion Homeostasis ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Extracellular Glucose ◽  
Normal Glucose

Abnormal cellular ion homeostasis is a well-recognized component of diabetic glomerular disease. In cultured rat glomerular mesangial cells, we have previously shown that insulin regulates Ca(2+)-dependent activation of 4-pS Cl- channels and 27-pS nonselective cation channels (NSCC) by angiotensin II (ANG II). To assess whether extracellular glucose also affects mesangial ion channels, we applied patch-clamp techniques to cells incubated in constant insulin (100 mU/ml) and either "normal" (5 mM) or "high" (30 mM) glucose for 1 wk. In normal glucose, 100 nM ANG II increased Cl- and NSCC activity by > 16-fold and > 60-fold, respectivley. Direct release of intracellular Ca2+ ([Ca2+]i) stores (0.25 microM thapsigargin) mimicked ANG II-induced channel stimulation. In high glucose, Cl- and NSCC stimulation by ANG II was attenuated (< 7-fold), whereas channel activation by thapsigargin was unaffected. Protein kinase C (PKC) inhibition (30-min exposure to 0.5 microM calphostin) or downregulation (24-h exposure to 0.1 microM 4 beta-phorbol 12-myristate 13-acetate), but not aldose reductase inhibition (0.5 mM sorbinil), restored channel responsiveness to ANG II despite high glucose. Channel responsiveness was also restored if mesangial cells were coincubated in both high glucose and 500 microM myo-inositol. Acute exposure to a synthetic diacylglycerol (100 microM 1-oleoyl-2-acetyl glycerol) reestablished channel unresponsiveness to ANG II. We conclude the following in rat mesangial cell cultures: 1) Activation of Ca(2+)-dependent Cl- and NSCCs by ANG II is reduced by high extracellular glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-

1988 ◽  
Vol 255 (6) ◽  
pp. C844-C856 ◽  
Author(s):  
G. Boyarsky ◽  
M. B. Ganz ◽  
R. B. Sterzel ◽  
W. F. Boron
Keyword(s):  
Steady State ◽  
Loading Rate ◽  
Mesangial Cells ◽  
Ph Regulation ◽  
Disulfonic Acid ◽  
Glomerular Mesangial Cells ◽  
Fluorescence Excitation ◽  
Dependent Transport ◽  
Acid Extrusion ◽  
Acid Loading

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

scholarly journals Jak2-Independent Activation of Stat3 by Intracellular Angiotensin II in Human Mesangial Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Rekha Singh
Keyword(s):  
Transcription Factor ◽  
High Glucose ◽  
Mesangial Cells ◽  
Binding Activity ◽  
Matrix Proteins ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Human Mesangial Cells ◽  
Stat3 Transcription Factor ◽  
Stat3 Phosphorylation

Ang II is shown to mediate the stimulatory effect of high glucose on TGF-b1 and extracellular matrix proteins in glomerular mesangial cells. Also inhibition of Ang II formation in cell media (extracellular) and lysates (intracellular) blocks high-glucose effects on TGF-b1 and matrix more effectively compared to inhibition of extracellular Ang II alone. To investigate whether intracellular Ang II can stimulate TGF-b1 and matrix independent of extracellular Ang II, cultured human mesangial cells were transfected with Ang II to increase intracellular Ang II levels and its effects on TGF-b1 and matrix proteins were determined. Prior to transfection, cells were treated with candesartan to block extracellular Ang II-induced responses via cell membrane AT1 receptors. Transfection of cells with Ang II resulted in increased levels of intracellular Ang II which was accompanied by increased production of TGF-b1, collagen IV, fibronectin, and cell proliferation as well. On further examination, intracellular Ang II was found to activate Stat3 transcription factor including increased Stat3 protein expression, tyrosine 705 phosphorylation, and DNA-binding activity. Treatment with AG-490, an inhibitor of Jak2, did not block intracellular Ang II-induced Stat3 phosphorylation at tyrosine 705 residue indicating a Jak2-independent mechanism used by intracellular Ang II for Stat3 phosphorylation. In contrast, extracellular Ang II-induced tyrosine 705 phosphorylation of Stat3 was inhibited by AG-490 confirming the presence of a Jak2-dependent pathway. These findings suggest that intracellular Ang II increases TGF-b1 and matrix in human mesangial cells and also activates Stat3 transcription factor without involvement of the extracellular Ang II signaling pathway.

High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells

2004 ◽  
Vol 286 (6) ◽  
pp. F1039-F1045 ◽  
Author(s):  
D. B. Vidotti ◽  
D. E. Casarini ◽  
P. C. Cristovam ◽  
C. A. Leite ◽  
N. Schor ◽  
...  
Keyword(s):  
High Glucose ◽  
Cathepsin B ◽  
Mesangial Cells ◽  
Renin Angiotensin System ◽  
Fold Increase ◽  
Renin Activity ◽  
Mrna Levels ◽  
Ang Ii ◽  
Renin Substrate ◽  
Angiotensin System

Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.

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