Cell-cell communication and diabetes. Professor Paul Squires University of Lincoln

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1 Cell-cell communication and diabetes Professor Paul Squires University of Lincoln

2 Dr Claire Hills University of Lincoln

3 Dr Claire Hills University of Lincoln Joseph Banks Laboratories Lincoln Connexin mediated cell communication in the diabetic kidney"

Diabetic Nephropathy, Gap Junctions and Cell Communication Diabetic

that require dialysis or transplantation in the UK each year.

Tubulointerstitial fibrosis (TIF) is key underlying pathology Glucose-evoked

Pivotal in initiation of tubulointerstitial fibrosis is loss of E-cadherin

4 Diabetic Nephropathy, Gap Junctions and Cell Communication Diabetic Nephropathy (DN) accounts for a significant proportion of the 64, patients that require dialysis or transplantation in the UK each year. Characterised by a number of structural and functional disturbances. Tubulointerstitial fibrosis (TIF) is key underlying pathology Glucose-evoked changes in TGF-b1 are thought instrumental in instigating these changes Pivotal in initiation of tubulointerstitial fibrosis is loss of E-cadherin mediated cell adhesion. Adhesion is not only important for adherens junction but also for formation of gap junctions

5 Why are gap junctions so important? Gap-junctional intercellular communication Cell 1 Cell 2 Gap-junctions facilitate the direct transmission of metabolic and electrical signals between cells, enabling them to entrain cellular activity and synchronise tissue function. The junctions are formed when connexins (CX) cluster into a hexameric structure (connexon) enabling formation of a trans-membrane pore between the cytosol of adjacent cells. P2-receptor Hemi-channel mediated ATP release Cell 1 Cell 2 In the absence of binding partners on neighbouring cells, uncoupled connexons form hemi-channels. These channels permit the local release of small molecules to the extracellular space (e.g. ATP). Regulation of connexin synthesis and channel activity is critical to cellular function and a number of diseases have been attributed to these important proteins. In the proximal nephron, alteration in connexin-mediated cell-to-cell communication may represent an early sign of tubulointerstital fibrosis and injury. [ATP] e Diabetologia (215) 58:

6 Overarching Hypothesis: Early changes in cell-to-cell communication, hemi-channel activity and cellcoupling are associated with renal fibrosis and loss of function in diabetic nephropathy Specific aim 1: To identify a role for glucose evoked changes in pro-fibrotic cytokine TGF-β1 in mediating altered connexin-mediated cell-to-cell communication.

7 Methodology Renal biopsies from patients with biopsy-proven diabetic nephropathy were selected for this study (disease group, n=2), and from the normal portion of nephrectomized specimen removed from patients with renal carcinoma (control group, n=12). Human proximal tubule epithelial cells (hptecs) and immortalised human kidney (HK2) cells were used throughout this study Western blot analysis was used to determine expression of key candidate proteins AFM force spectroscopy was used to measure cell tethering Gap-junctional intercellular communication was examined using paired whole cell patch-clamp recordings to measure junctional conductance +/- TGF-β1 (1ng/mL). Hemi-channel opening/activity was assessed by carboxyfluorescein uptake in cells cultured +/- TGF-β1 (1ng/mL). ATP biosensing measured real time release of ATP from cells +/- TGF-β1 (1ng/mL) hptecs were incubated with either TGF-b1 (2-1ng/mL) +/- Apyrase (5U/mL) or ATPgS (1-1mM) +/- P2Y receptor blocker Suramin (1mM)

8 Is connexin expression altered in the diabetic kidney? CX26 Expression CX43 Expression CX43 Expression CX26 Expression Connexin 26 DN tissue HK2 cells HK2 cells Primary hptecs Primary hptecs CX26 26kDa CX26 26kDa Control tissue Connexin 43 DN tissue TGF-b1 (ng/ml) HK2 cells CX43 43kDa TGF-b1 (ng/ml) Primary hptecs CX43 43kDa Control tissue TGF-b1 (ng/ml) TGF-b1 (ng/ml)

What are the implications of this altered

biopsy material from people with diabetic

9 What are the implications of this altered Cx expression on gap junction intercellular communication? Cx26 and Cx43 expression exhibit decreased expression in HK2 and hptec cells at 48hrs Cx26 and Cx43 expression exhibit increased expression in HK2 and hptec cells at 7days Cx26 and Cx43 expression increased expression in biopsy material from people with diabetic nephropathy A B * * Control 2days 7days Why do cells exhibit an increase in expression yet a loss of Gap Junction mediated communication?

10 % ECAD expression as compared to control In diabetic nephropathy, a loss of E-cadherin mediated cell adhesion represents a pivotal step in initiation of tubular injury ECAD 12kDa ECAD 12kDa a-tubulin a-tubulin 5mM glucosetransitional 1ng/mL TGFb1 Phenotype 25mM glucose mM glucose 1ng/mL TGFb1 25mM glucose 5 5mM 25mM 25mM + TGF-b1 neut ab Control nditioned Media (25mM) Mannitol (25mM) Siamantouras E, Hills CE et al Nanomedicine 216 Hills CE et al, Diabetologia 215

5 1 the glue that binds -1 them together?? Control TGF-β1 1. 1-9 1. 1-14.75 7.5 1-1.75 7.5 1-15.5 5. 1-1.5 5. 1-15.25 2.

11 Force (nn) Maximum unbinding Unbinding Force force (nn) Work of adhesion (fjoule) Work Of Adhesion (fjoule) Maximum Unbinding Force TGF-β1 reduces E-cadherin mediated cell-cell adhesion What happens when -1 cells lose the glue that binds -1 them together?? Control TGF-β Displacement (μm) Control TGF-β1 (1ng/mL) Control TGF-β1 TGF-b1 1ng/ml (1ng/mL) Hills CE et al Nanomedicine 216 / Hills CE et al, Diabetologia 212

Hemi-channel mediated ATP release Cell 1 Cell 2 Cell 1 Cell 2

12 Intercellular adhesion is a pre-requisite for gap junction mediated communication Gap-junctional intercellular communication Hemi-channel mediated ATP release Cell 1 Cell 2 Cell 1 Cell 2 P2-receptor [ATP] e Can we measure hemichannel activity via release of ATP?

% Change in intensity (as compared to control) Hills

of TGF-β1 on hemi-channel mediated dye uptake We can

carboxyfluorescein Removing extracellular calcium

carboxyfluorescein uptake 7day Control Control + CBX

13 % Change in intensity (as compared to control) Hills CE et al 218 Cell Phys Biochem Chronic (7day) effect of TGF-β1 on hemi-channel mediated dye uptake We can look at hemichannel activity by loading cells with carboxyfluorescein Removing extracellular calcium opens hemi-channels in HK2 cells and increases carboxyfluorescein uptake 7day Control Control + CBX 7day TGF-β1 TGF-β1 + CBX Control Control + CBX TGF-b1 TGF-b1 + CBX

Effect of TGF-β1 on hemichannel mediated ATP release at 48hrs and 7days in HK2 cells ma 1mA ATP Release (µm) 5 4

14 Effect of TGF-β1 on hemichannel mediated ATP release at 48hrs and 7days in HK2 cells ma 1mA ATP Release (µm) * Control Ca 2+ -FREE 1μM 1 1secs Control TGF-β1 Control TGF-β1 Ca 2+ -FREE 48 hours 7 days TGF-β1 (1ng/mL) 1μM

15 What are phenotypic implications of altered Cx expression/function activity for the cell? TGF-b1 induced expression of Cx s is paralleled by increased hemi-channel mediated ATP release Elevated levels of nucleotides have been linked to increased expression of inflammatory/fibrotic markers in multiple disease states Expression and secretion of markers central to inflammation and fibrosis were examined hptecs were incubated with TGF-b1 (2-1ng/mL) for 48h These proteins include E-cadherin Claudin -2 Zona-occludin Collagen I Fibronectin Interleukin 6 Does ATP work downstream of TGF-b1 in mediating this response?

29kDa Fibronectin 22kDa Collagen I kda 4 3 2 1 * 3 2 1 * 5 4 3 2 1 1 1 1 ATPγS (µm) 1 1 1 ATPγS (µm) 1

16 ECAD Expression Does ATP mediate changes in expression linked to tubular injury? IL-6 Expression Fibronectin Expression ZO-1 Expression Collagen I Expression Claudin-2 Expression IL-6 29kDa Fibronectin 22kDa Collagen I kda * * ATPγS (µm) ATPγS (µm) ATPγS (µm) ECAD 135kDa Zo-1 22kDa Claudin-2 25kDa ATPγS (µm) ATPγS (µm) ATPγS (µm)

17 Does TGF-b1 mediate its phentoypic effects via ATP? IL-6 Expression Fibronectin Expression Col1 Expression 5mM glucose 5mM glucose + 5U/mL Apyrase 1ng/mL TGF-β1 TGF-b1 (1ng/mL) + 5U/mL Apyrase IL-6 29kDa Fibronectin 22kDa Col1 3kDa 4 * * C TGF-β1 C TGF-β1 C TGF-β1 C TGF-β1 C TGF-β1 C TGF-β1 + Apyrase + Apyrase + Apyrase

18 ECAD Expression Claudin-2 Expression ZO-1 Expression Does TGF-b1 mediate changes in expression of tight junction and adherences junction proteins its via ATP? ECAD 135kDa Claudin-2 25kDa ZO-1 22kDa C TGF-β1 C TGF-β1 C TGF-β1 C TGF-β1 C TGF-β1 C TGF-β1 + Apyrase + Apyrase + Apyrase

19 Does TGF-b1 regulate protein expression through activation of downstream purinergic signalling? 5mM glucose 5mM glucose + Suramin (1mM) 1ng/mL TGF-β1 TGF-b1 (1ng/mL) +Suramin (1mM) IL-6 29kDa Fibronectin 22kDa Col1 3kDa C C+S TGF-β1 TGF-β1 +S C C+S TGF-β1 TGF-β1 +S C C+S TGF-β1 TGF-β1 +S

20 ECAD Expression Claudin-2 Expression ZO-1 Expression Does TGF-b1 regulate protein expression through activation of downstream purinergic signalling? ECAD 135kDa Claudin-2 25kDa ZO-1 22kDa 15 * Cont. Cont + S TGF-b1 TGF-b1 + S Cont. Cont + S TGF-b1 TGF-b1 + S Cont. Cont + S TGF-b1 TGF-b1 + S

21 ATP mediates impaired adhesion and decreased transepithelial resistance Transepithelial Resistance (Ω.cm2) Cell to cell adhesion Transepithelial resistance 1. Approach 2. Contact 3. Separation Bradley M. Denker et al JASN 211;22: Control TGF-β1 ATPγS

22 Summary so far.. Cx26 and Cx43 expression exhibit elevated levels of expression in biopsy material from patients with DN and in glucose/tgf-b treated hptec This increased expression is paralleled by a reduced GJIC with increased hemi-channel mediated ATP release Elevated levels of ATP have been linked to fibrosis in multiple tissue types, yet we know nothing of a role for ATP in mediating inflammation and fibrosis in the diabetic kidney. TGF-b1 evoked changes in [ATP] e drive increased expression of markers of tubular injury through activation of downstream purinergic signalling Can we block glucose-evoked changes in hemichannel mediated ATP release and improve function?

23 Currently.. Utilising peptides which block hemichannel activity/alter GJIC to determine both in vivo and in vitro if connexins represent a viable therapeutic target for treatment of fibrosis in nephropathy (collaboration with industrial colleagues) In addition. We will use a Cx43 -/- (tubule specific) knockout mouse model to determine if removal of Cx43 negates inflammation and fibrosis ahead of studying the mechanisms which appear to be affected (Collaboration with Christos Chadjichristos INSERM). Acknowledged as an endogenous danger signal, ATP can trigger a series of downstream inflammatory pathways involving the NOD-like receptor and Interleukin 1b and Interleukin 6 New data suggests that increased ATP can stimulate the NLRP3 inflammasome in human primary proximal tubule cells Mugishoo et al 218, Biochem Biophys Acta

Siamantouras (EFSD, PDRA) Mr Joe Potter (Diabetes UK, PhD

24 The Team University of Lincoln Dr Claire Hills Prof. Paul E. Squires Dr Gareth Price (Diabetes UK, PDRA) Dr Eleftherios Siamantouras (EFSD, PDRA) Mr Joe Potter (Diabetes UK, PhD Student) Dr Katrina Firth (Back to Science Research Fellow)

25 The collaborators Professor Sydney Tang Dr Isaac Liu Dr Mark Wall Dr Jian Yao Prof Nigel Brunskill Dr Christos Chadjichristos Dr Patricia Martin

26 Acknowledgements: EFSD/Janssen Kidney Programme for the study of the role of the kidney in diabetes EFSD/Boehringer Ingelheim Research Programme in Microvascular Complications of Diabetes Diabetes UK Project Grants (12/4546 & 16/5427) Diabetes Research and Wellness Foundation

Dr Mahmood S Choudhery, PhD, Postdoc (USA) Assistant Professor Tissue Engineering and Regenerative Medicine King Edward Medical University/Mayo

Integration of Cells into Tissues Dr Mahmood S Choudhery, PhD, Postdoc (USA) Assistant Professor Tissue Engineering and Regenerative Medicine King Edward Medical University/Mayo Hospital Lahore 1. How