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.

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.

scholarly journals Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Michelle T. Barati ◽  
James C. Gould ◽  
Sarah A. Salyer ◽  
Susan Isaacs ◽  
Daniel W. Wilkey ◽  
...  
Keyword(s):  
Protein Expression ◽  
Posttranslational Modifications ◽  
High Glucose ◽  
Mesangial Cells ◽  
Expression Patterns ◽  
Growth Conditions ◽  
Glomerular Mesangial Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Glucose Levels ◽  
Flight Mass Spectrometry

The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG⁎) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

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.

scholarly journals Impairment of hepatic nuclear factor-4α binding to the Stim1 promoter contributes to high glucose-induced upregulation of STIM1 expression in glomerular mesangial cells

2015 ◽  
Vol 308 (10) ◽  
pp. F1135-F1145 ◽  
Author(s):  
Yanxia Wang ◽  
Sarika Chaudhari ◽  
Yuezhong Ren ◽  
Rong Ma
Keyword(s):  
Protein Expression ◽  
High Glucose ◽  
Mesangial Cells ◽  
Therapeutic Option ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Glomerular Mesangial Cells ◽  
Expression Levels ◽  
293 Cells ◽  
Interfering Rna

The present study was carried out to investigate if hepatic nuclear factor (HNF)4α contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4α expression in MCs. Knockdown of HNF4α using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4α reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4α protein in MCs but significantly attenuated HNF4α binding activity to the Stim1 promoter. Moreover, knockdown of HNF4α significantly augmented store-operated Ca2+ entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4α significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4α negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4α binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4α repression. Since the STIM1-gated store-operated Ca2+ entry pathway in MCs has an antifibrotic effect, inhibition of HNF4α in MCs might be a potential therapeutic option for diabetic kidney disease.

Dihydropyridine-sensitive Ca2+ channels in human glomerular mesangial cells

2000 ◽  
Vol 278 (1) ◽  
pp. F97-F103 ◽  
Author(s):  
David A. Hall ◽  
Pamela K. Carmines ◽  
Steven C. Sansom
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Mesangial Cells ◽  
Extracellular Fluid ◽  
Bay K 8644 ◽  
Sustained Response ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Open Probability ◽  
Pipette Solution

In mesangial cells (MC), the response of intracellular Ca2+ concentration ([Ca2+]i) to a contractile agonist is biphasic with a large, transient increase in [Ca2+]i followed by a smaller but sustained elevation as Ca2+ flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca2+ channels in the plasmalemmal membrane. However, a role for voltage-gated Ca2+ channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca2+ entry pathway responsible for the sustained response of [Ca2+]i in human MC. We found that ANG II at 10 nM, 100 nM, and 1 μM increased [Ca2+]i to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 μM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca2+. None of the peak responses (due to release of intracellular stores of Ca2+) were affected by removal of external Ca2+ or addition of diltiazem. Upon elevating the extracellular [K+] from 5 mM to 75 mM, [Ca2+]i reached a sustained level of 48% greater than baseline. This effect of high K+ was attenuated by either Ca2+ removal or addition of diltiazem. In the presence of 75 or 140 mM K+, the dihydropyridine agonist BAY K 8644 (1 μM and 10 μM) initiated sustained [Ca2+]i responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca2+ in the external solution, BAY K 8644 did not significantly affect [Ca2+]i. In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl2 and 10 μM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between −30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca2+ channels responsible for the voltage-regulated entry of Ca2+ into the cell during an agonist-induced contraction.

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)

Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells

1986 ◽  
Vol 251 (1) ◽  
pp. F94-F102 ◽  
Author(s):  
J. V. Bonventre ◽  
K. L. Skorecki ◽  
J. I. Kreisberg ◽  
J. Y. Cheung
Keyword(s):  
Calcium Concentration ◽  
Mesangial Cells ◽  
Free Calcium ◽  
Base Line ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Cytosolic Free Calcium ◽  
Fura 2 ◽  
Free Calcium Concentration ◽  
Cytosolic Free Calcium Concentration

Cytosolic free calcium concentration ([Ca2+]f) was determined in cultured rat glomerular mesangial cells under basal conditions and after exposure to arginine vasopressin (AVP) or angiotensin II (ANG II). [Ca2+]f was determined using quin 2 or fura-2, two intracellular fluorescent probes. [Ca2+]f was measured to be 102 +/- 3 nM (n = 154) using quin 2 and 82 +/- 4 (n = 34) using fura-2. AVP and ANG II increased [Ca2+]f. Maximal levels of [Ca2+]f were achieved in less than 10 s after addition of the hormone. This peak value was followed by a rapid fall toward the base line. With fura-2 as the intracellular Ca2+ indicator, [Ca2+]f increased from 74 +/- 7 to a peak of 578 +/- 39 nM (n = 17) with 100 nM AVP. At 115 s after addition of AVP, [Ca2+]f was 125 +/- 9 nM. Similar peak levels of [Ca2+]f were observed using quin 2. The increase in [Ca2+]f was due in large part to release of Ca2+ from intracellular stores, since reduction in extracellular free [Ca2+] with EGTA did not prevent the hormone-induced increase in [Ca2+]f, although it did result in a decreased peak level and a more rapid return to base line. The AVP-induced increase in [Ca2+]f was blocked by the V1 receptor antagonist (CH2)5Tyr(Me)VDAVP. Neither isoproterenol, which increased adenylate cyclase activity, nor dibutyryl cAMP had any affect on [Ca2+]f directly or on the AVP-induced increase in [Ca2+]f. In this report we present the first direct measurements of [Ca2+]f and hormone-induced changes in [Ca2+]f in glomerular mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The Mechanism of miR-192 in Regulating High Glucose-Induced MCP-1 Expression in Rat Glomerular Mesangial Cells

2019 ◽  
Vol 19 (7) ◽  
pp. 1055-1063
Author(s):  
Fenqin Chen ◽  
Guozhu Wei ◽  
Yang Zhou ◽  
Xiaoyu Ma ◽  
Qiuyue Wang
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Cell Lines ◽  
Western Blotting ◽  
High Glucose ◽  
Messenger Rna ◽  
Mesangial Cells ◽  
Inflammatory Factor ◽  
Glomerular Mesangial Cells ◽  
Regulatory Loop

Background: Although the pathogenetic mechanism of Diabetic Kidney Disease (DKD) has not been elucidated, an inflammatory mechanism may be a potential contributor. Monocyte chemotactic protein-1 (MCP-1) is suggested to be implicated in the development of DKD by playing a role in the infiltration of monocyte/macrophage. The aim of this study was to investigate the expression of MCP-1 under high glucose conditions, as well as the effects of microRNA-192 (miR-192) under these conditions, and to study the regulatory mechanism of MCP-1 in DKD. <p></p> Methods: Rat glomerular mesangial cells were cultured in high glucose or isotonic mannitol. The messenger RNA(mRNA) expression of miR-192, miR-200b, miR-200c, E-box-binding homeobox 1 (Zeb1), and MCP-1 was then detected by real-time PCR, and the protein expression of Zeb1 and MCP- 1 was assessed by western blotting. The rat mesangial cells were transfected with an miR-192 inhibitor, NC inhibitor , and transfected with siRNA Zeb1, siNC. The cells were then cultured in high glucose to detect the mRNA expression of miR-192, miR-200b, miR-200c, Zeb1, and MCP-1 using realtime PCR, and Zeb1 and MCP-1 protein expression were determined by western blotting. <p></p> Results: MiR-192, miR-200b, miR-200c, and MCP-1 were overexpressed, whereas Zeb1 was downregulated when cultured in high glucose (P < 0.05). After transfection with an miR-192 inhibitor, the expression of miR-192, miR-200b, miR-200c, and MCP-1 was downregulated, whereas Zeb1 was increased, and these differences were statistically significant (P < 0.05). The observed changes in the expression in the NC inhibitor transfection group were similar to that of non-transfected cell lines. Silencing the expression of Zeb1 resulted in a significant increase in the expression of miR-192, miR- 200b, miR-200c, and MCP-1 (P < 0.05). The observed changes in the SiNC transfection group were similar to those of non-transfected cell lines. <p></p> Conclusions: MiR-192 expression was upregulated to increase the expression of inflammatory factor MCP-1 by inhibiting the expression of Zeb1, which was mediated by breaking the regulatory loop of Zeb1 and miR-200b/c in rat mesangial cells cultured in high glucose.

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.

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