PMID- 29587265 OWN - NLM STAT- MEDLINE DCOM- 20180619 LR - 20190212 IS - 1421-9778 (Electronic) IS - 1015-8987 (Linking) VI - 45 IP - 6 DP - 2018 TI - Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney. PG - 2369-2388 LID - 10.1159/000488185 [doi] AB - 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 (TGFbeta1), on connexin 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) +/- TGFbeta1 (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 TGFbeta1 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-beta1 on connexin mediated cell communication was assessed by incubating cells with ATPgammaS (1-100microM) or TGF-beta1 +/- 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 TGFbeta1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFbeta1 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 TGFbeta1 and to 0.42+/-0.2nS after 7days TGFbeta1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFbeta1 produced a carbenoxolone (200microM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29+/-0.06muM in control to 1.99+/-0.47muM after 48hr incubation with TGFbeta1 (10ng/ml; P<0.05, n=3). TGF-beta1 (2-10ng/ml) and ATPgammas (1-100microM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-beta1 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 TGFbeta1 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. CI - (c) 2018 The Author(s). Published by S. Karger AG, Basel. FAU - Hills, Claire AU - Hills C AD - Joseph Banks Laboratories, Green Lane, University of Lincoln, Lincoln, United Kingdom. FAU - Price, Gareth William AU - Price GW AD - Joseph Banks Laboratories, Green Lane, University of Lincoln, Lincoln, United Kingdom. FAU - Wall, Mark John AU - Wall MJ AD - School of Life Sciences, Gibbet Hill, University of Warwick, Warwick, United Kingdom. FAU - Kaufmann, Timothy John AU - Kaufmann TJ AD - School of Life Sciences, Gibbet Hill, University of Warwick, Warwick, United Kingdom. FAU - Chi-Wai Tang, Sidney AU - Chi-Wai Tang S AD - Division of Nephrology, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China. FAU - Yiu, Wai Han AU - Yiu WH AD - Division of Nephrology, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China. FAU - Squires, Paul Edward AU - Squires PE AD - Joseph Banks Laboratories, Green Lane, University of Lincoln, Lincoln, United Kingdom. LA - eng PT - Journal Article DEP - 20180326 PL - Germany TA - Cell Physiol Biochem JT - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology JID - 9113221 RN - 0 (Connexin 43) RN - 0 (TGFB1 protein, human) RN - 0 (Transforming Growth Factor beta1) RN - 127120-53-0 (Connexin 26) RN - IY9XDZ35W2 (Glucose) SB - IM MH - *Cell Communication MH - Cell Line MH - Cells, Cultured MH - Connexin 26/*analysis/metabolism MH - Connexin 43/*analysis/metabolism MH - Diabetic Nephropathies/metabolism/*pathology MH - Glucose/metabolism MH - Humans MH - Kidney Tubules, Proximal/cytology/metabolism/*pathology MH - Transforming Growth Factor beta1/*analysis/metabolism OTO - NOTNLM OT - ATP OT - Bio-sensing OT - Connexin OT - Diabetic nephropathy OT - Fibrosis OT - Gap-junctions OT - Hemi-channels OT - Inflammation OT - TGFbeta OT - Tubular epithelia EDAT- 2018/03/28 06:00 MHDA- 2018/06/21 06:00 CRDT- 2018/03/28 06:00 PHST- 2017/09/04 00:00 [received] PHST- 2018/01/09 00:00 [accepted] PHST- 2018/03/28 06:00 [pubmed] PHST- 2018/06/21 06:00 [medline] PHST- 2018/03/28 06:00 [entrez] AID - 000488185 [pii] AID - 10.1159/000488185 [doi] PST - ppublish SO - Cell Physiol Biochem. 2018;45(6):2369-2388. doi: 10.1159/000488185. Epub 2018 Mar 26.