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Transforming growth Factor Beta 1 drives a switch in connexin mediated cell-to-cell communication in tubular cells of the diabetic kidney

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Hills, Claire E., Price, G. W., Wall, Mark J., Kauffman, T. J., Tang, S. C., Yiu, W. H. and Squires, P. E. (2018) Transforming growth Factor Beta 1 drives a switch in connexin mediated cell-to-cell communication in tubular cells of the diabetic kidney. Cellular Physiology and Biochemistry, 45 (6). pp. 2369-2388. doi:10.1159/000488185

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Official URL: https://doi.org/10.1159/000488185

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Abstract

Background/Aims:

Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose- evoked changes in the beta1 isoform of transforming growth factor (TGFβ1), on connexion expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron.

Methods:

Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFβ1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFβ1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP- biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-β1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100μM) or TGF-β1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression.

Results:

Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 ( P< 0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFβ1 (10ng/ml) for 7days ( P<0.001, n=3). High glucose significantly increased TGFβ1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFβ1 and to 0.42±0.2nS after 7days TGFβ1 incubation (P <0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFβ1 produced a carbenoxolone (200μM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06μM in control to 1.99±0.47μM after 48hr incubation with TGFβ1 (10ng/ml; P<0.05, n=3). TGF-β1 (2- 10ng/ml) and ATPγs (1-100μM) increased expression of IL-6 (P< 0.001 n=3) and fibronectin (P< 0.01 n=3). The effect of TGF-β1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3).

Conclusion:

These data suggest that chronic exposure to glucose-evoked TGFβ1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy.

Item Type: Journal Article
Subjects: R Medicine > RC Internal medicine
Divisions: Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH): Diabetic nephropathies, Diabetes -- Physiological aspects, Connexins
Journal or Publication Title: Cellular Physiology and Biochemistry
Publisher: Karger
ISSN: 1015-8987
Official Date: April 2018
Dates:
DateEvent
April 2018Published
16 March 2018Available
9 January 2018Accepted
Volume: 45
Number: 6
Page Range: pp. 2369-2388
DOI: 10.1159/000488185
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
11/0004215Diabetes UKhttp://dx.doi.org/10.13039/501100000361
16/0005427 Diabetes UKhttp://dx.doi.org/10.13039/501100000361
16/0005509Diabetes UKhttp://dx.doi.org/10.13039/501100000361
11/0004215European Foundation for the Study of Diabeteshttp://dx.doi.org/10.13039/501100001648
16/0005427 European Foundation for the Study of Diabeteshttp://dx.doi.org/10.13039/501100001648
16/0005509European Foundation for the Study of Diabeteshttp://dx.doi.org/10.13039/501100001648
UNSPECIFIEDPhysiological Societyhttp://dx.doi.org/10.13039/501100000674
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