Alexander Staruschenko, PhD

Transcription

1 Role of the epithelial Na + channel (ENaC) in salt-sensitive hypertension and mechanisms of its regulation by EGF Alexander Staruschenko, PhD Department of Physiology Институт эволюционной физиологии и биохимии им. И. М. Сеченова РАН 26 декабря 212 г.

2 PCT Bowman s capsule CNT DCT CCD Glomerulus PST TAL OMCD tdlh talh IMCD Principal cell Intercalated cell Staruschenko (212) Comp. Physiol. 2(2):

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5 The two-membrane model of epithelial transport in principal cells H 2 O AQP2 Na + ROMK K + URINE ENaC 3Na + H 2 O Na + /K + ATPase 2K + AQP3,4 BLOOD

6 Hormonal factors ENaC regulation Balance of ions Stretch Proteolytic cleavage Small GTPase-regulated processes Phosphorylation ROS production intracellular signaling Arachidonic acid metabolites PIP pathways Gene expression Cytoskeletal rearrangements Ubiquitylation ENaC Increased/decreased expression of the channels Changes in the open probability Altered trafficking

7 Kidney Physiological significance of ENaC Regulation of the body fluid volume Lungs sweat and salivary ducts Keeps the alveoli dry Clears the lungs of fluid at birth Retrieves salt from the sweat ENaC-associated diseases Liddle s syndrome hypertension hypokalemia metabolic alcalosis Pseudohypoaldosteronism type I salt wasting hyperkalaemia unresponsiveness to mineralocorticoids Cystic fibrosis, polycystic kidney disease etc

8 Role of ENaC in the development of salt-sensitive hypertension in the Dahl Salt-Sensitive (SS) rats EGF and its related growth factors regulate ENaC Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins EGF might regulates ENaC via Rho GDP-dissociation inhibitor RhoGDIα EGF modulates ENaC-mediated Na + transport in the ASDN and participate in the development of salt-sensitive hypertension

9 Dahl SS rats Blood pressure salt-sensitivity Chronic kidney injury Reduced renal function Low renin hypertension SS.13 BN consomic rats developed at MCW exhibit only 2% allelic differences compared to the SS strain, but saltinduced hypertension and renal injury are greatly attenuated.

10 Mean arterial pressure (mmhg) Amiloride inhibits the blood pressure in the SS rats A.4% 8% high salt diet Vehicle or Amiloride (6mg.kg.day) infusion Measure blood pressure & collect urine daily Day 1 Start Day 3 Begin High salt Day 6 Begin Drug or Vehicle Day 17 End experiment Plasma/histology B 2 8% NaCl.4% NaCl Arterial pressure Vehicle C % NaCl Urine Na + /K + ratio 8% NaCl Amiloride Vehicle 15 Amiloride Na + /K Drug or vehicle Drug or vehicle

11 Mean arterial pressure (mmhg) Benzamil (given through drinking water 15 mg/l) attenuates the development of hypertension in SS rats.4% 4% diet Control Benzamil Benzamil/vehicle days

12 Relative abundance High salt diet increases ENaC expression SS rats (cortex) LS HS α-enac β-enac γ-enac GAPDH LS HS *** *** α-enac β-enac γ-enac

13 Im_scaled (pa) Im_scaled (pa) Time (s) Time (s) High salt diet increases ENaC activity LS HS 1 pa 2 s c o 1 o 2 c o 1 o 2 o 3 NP o ** n=8 n=7 LS HS

14 MAP (mmhg) Im_scaled (pa) Im_scaled (pa) Im_scaled (pa) Time (s) 34 NPo High salt diet increases MAP and ENaC activity in SS rats fed a high salt diet compare to SS.13 BN 1.5 SS.13 BN (LS) SS.13 BN (HS) c o 1 c * SS (HS) o pA 1s c o 1 o 2.5. n=4 n=9 n=1 SS.13 BN LS SS-13 BN HS SS HS Time (s) Time (s) * 1 SS SS-13 BN

15 Relative abundance High salt diet does not change ENaC expression in SS.13 BN rats LS HS α-enac β-enac γ-enac β-actin LS HS α-enac β-enac γ-enac

16 β-enac signal intensity (% of max) High salt diet increases ENaC abundance in the CCD of SS rats compare to SS.13 BN SS rats SS.13 BN * n=27 n=24 SS SS.13 BN

17 Servocontrolled Uncontrolled β-enac signal intensity ENaC expression is upregulated in chronic servo-controlled rats Control Servo-controlled (left) Uncontrolled (right) 1 * µm 6 5 n=1 n=1

18 Conclusion: Dahl salt-sensitive (SS) rats develop severe hypertension on high-salt diet and ENaC contributes to the development of hypertension in the SS rat strain.

19 Role of ENaC in the development of salt-sensitive hypertension in the Dahl Salt-Sensitive (SS) rats EGF and its related growth factors regulate ENaC Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins EGF might regulates ENaC via Rho GDP-dissociation inhibitor RhoGDIα EGF modulates ENaC-mediated Na + transport in the ASDN and participate in the development of salt-sensitive hypertension

20 ErbB receptors and their ligands From: J Clin Invest. 27; 117:251 8

21 Relative current Biphasic effect of EGF on sodium transport in mpkccd c14 cells Control EGF (1 ng/ml) Amil Time, hours Levchenko et al. (21) J Cell Physiol. 223(1): Liu et al. (29) AJP Renal 296(6): F

22 Relative current Relative current EGF regulates Na + transport in a concentration-dependent manner Amil 1.8 EC 5 = ng/ml Control.1 ng/ml.1 ng/ml.5 ng/ml 1 ng/ml 1 ng/ml 1 ng/ml Time, hours EGF, ng/ml

23 Relative current Relative current Relative current Effect of growth factors in the EGF-family on Na + reabsorption across mpkccd c14 monolayer TGF-α Amil Control.5 ng/ml 1 ng/ml 1 ng/ml 1 ng/ml Time, hours Amil Amil Control HB-EGF (5 ng/ml).4 Control Amphiregulin (1 ng/ml) Time, hours Time, hours

24 Expression profiles of ErbB receptor family proteins in mpkccd c14 cells analyzed by Western Blot analysis A ErbB ErbB actin B ErbB actin

25 ErbB2 expression in rat kidney tissue control ErbB2 ErbB2 5 µm 4X 4X 1X Zheleznova et al. (211) BBA - Mol Basis Disease 48:

26 Relative current Relative current Relative current Effect of ErbB2 inhibitors (tyrphostin AG825 and ErbB2 Inhibitor II) on EGF-induced changes of amiloride-sensitive Na + absorption A B C Control EGF EGF+AG Time, hours hrs 48 hrs * * hrs *

27 Conclusions: Our data are consistent with the idea that EGF and its related growth factors (TGF-α, HB-EGF and amphiregulin) have a biphasic effect on sodium absorption in the mammalian kidney as represented by mpkccd c14 cells. In addition, we show that the EGF effect is mediated via the ErbB2 receptor, most probably via formation of ErbB2/EGFR heterodimers.

28 Role of ENaC in the development of salt-sensitive hypertension in the Dahl Salt-Sensitive (SS) rats EGF and its related growth factors regulate ENaC Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins EGF might regulates ENaC via Rho GDP-dissociation inhibitor RhoGDIα EGF modulates ENaC-mediated Na + transport in the ASDN and participate in the development of salt-sensitive hypertension

29 IN 1 (mv) Time (ms) Sw eep:14 Visible:18 of 18 Current density (pa/pf) Rac1 but not Cdc42 enhances ENaC activity overexpressed in CHO cells 75 *** menac 6 *** 1nA 1 ms mv 6 mv -1 mv 4 mv 15 Whole-cell (CHO cells) Pavlov et al. 21 J Am Soc Nephrol. 21(5):

30 _Ipatch (pa) _Ipatch (pa) Vitality, % _Ipatch (pa) 3.5 Rac1 is essential for ENaC activity Cell-attached 3.5 A B C c -.52 o c o c o I NSC I (before NSC23766) II (after NSC23766) 1 Time (min) II 15 2 NP o pa 3 min/3 s. * control + NSC control + NSC Time (s) Karpushev et al. (211) Hypertension 57:

31 % Rac1 protein expression Creation of M-1 stable cell line with lentivirus-mediated knock down of Rac1 M-1 β-actin Rac *

32 I sc (μa/cm 2 ) _Ipatch (pa) _Ipatch (pa) Time (s) Time (s) 4 8 shrna-mediated silencing of Rac1 inhibit transepithelial Na + currents and ENaC activity in M-1 cells * c o 1 o 2 o 3 c o.5 pa 1 s control Rac1 shrna P o NS N *

33 Expression profiles of N-WASP/WAVEs in rat kidney tissue and in epithelial cells A B 75 WAVE1 control 5 μm 5 WAVE-2 N-WASP 75 WAVE-3 WAVE1 N-WASP Takenawa and Suetsugu Nat Rev Mol Cell Biol 8, mpkccd c14 M-1 WAVE2

34 Current density (pa/pf) Current density (pa/pf) Rac1 enhances ENaC activity via WAVE1 or WAVE2 A 8 6 * * * B 8 6 * * *

35 Conclusions: Co-expression of Rac1 with ENaC markedly increased channel activity, whereas co-expression of Cdc42 failed to change ENaC activity. Inhibition of Rac1-GEF interaction resulted in a decrease of ENaC activity in native principal cells. shrna-mediated silencing of Rac1 inhibits transepithelial current in M-1 cells via decrease in the number of channels at the plasma membrane. Rac1 and WAVE1/2 are parts of the same signaling pathway with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.

36 Role of ENaC in the development of salt-sensitive hypertension in the Dahl Salt-Sensitive (SS) rats EGF and its related growth factors regulate ENaC Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins EGF might regulates ENaC via Rho GDP-dissociation inhibitor RhoGDIα EGF modulates ENaC-mediated Na + transport in the ASDN and participate in the development of salt-sensitive hypertension

37 Small GTPases cycle between a GDP- and GTP-bound state Nethe M, Hordijk P L J Cell Sci 21;123:

38 EGF upregulates RhoGDIα in mpkccd c14 cells mpkccd c14 actin RhoGDIα Control +EGF (1 ng/ml) 24 hrs

39 ENaC + Rho GDIα +Rac1 +Rac1 DN +Rac1 +Rho GDIα Current Density (pa/pf) RhoGDIα downregulates ENaC activity via Rac1 5 * * * # 1

40 % RhoGDIα protein expression Knockdown of RhoGDIα in M-1 cells upregulates ENaC expression A β-actin RhoGDIα C α-enac β-actin B 1 β-enac β-actin γ-enac 25 control * n=4 n=4 RhoGDIα shrna 5 37 β-actin

41 Conclusions: Chronic treatment with EGF increases RhoGDIα expression RhoGDIα downregulates ENaC activity via Rac1

42 Role of ENaC in the development of salt-sensitive hypertension in the Dahl Salt-Sensitive (SS) rats EGF and its related growth factors regulate ENaC Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins EGF might regulates ENaC via Rho GDP-dissociation inhibitor RhoGDIα EGF modulates ENaC-mediated Na + transport in the ASDN and participate in the development of salt-sensitive hypertension

43 EGF, ng/mg Level of EGF measured by ELISA in the cortex of SS rats fed a normal and high salt diets 6 4 * 2.4% NaCl (2 weeks) 4% NaCl (2 weeks)

44 MAP (mmhg) MAP (mmhg) Chronic intravenous infusion of EGF prevents development of hypertension in SS rats fed a HS diet A B 5 Day 1 Start C Day 1 Start 1% 4% high salt diet I/V vehicle or EGF (2μg/h) infusion Blood pressure measurements via arterial catheter Day 3 Begin High salt.4% Day 6 Begin Drug or Vehicle (intravenous infusion) 8% high salt diet I/V vehicle or EGF (2μg/h) infusion Blood pressure measurements via arterial catheter Day 3 Begin High salt and Drug or Vehicle (intravenous infusion) Day 13 (End) IHC, Biochemistry Day 9 (End) Electrophysiology D control * +EGF Control EGF * * *

45 Im_scaled (pa) Ipatch (pa) Time (s) Chronic intravenous infusion of EGF decreases ENaC activity in SS rats fed a high salt diet 3 c o o c o pa 2 s SS rat (high salt) EGF infusion 5 1 Time (s) NP o control * +EGF

46 Glomerular injury score Protein casts, % EGF attenuated renal glomerular and tubular damage A B C x 6 3 mm D 3 control * +EGF 4. 2x * 3.2 SS rat (8%; 2 weeks) 1 µm SS rat (8%; 2 weeks; + i/v EGF) 3. control +EGF

47 Conclusions: EGF concentration is reduced in the SS rats fed a high diet, which we propose would enhance ENaC activity, sodium retention and hypertension. Elevation of EGF is associated with diminished sodium reabsorption, an attenuation of hypertension, and a reduction of renal damage in Dahl SS rats fed high salt. EGF deficiency in the renal cortex contributes to salt-sensitive hypertension in Dahl SS rats via upregulation of ENaC activity Thus, elucidation of the interaction between EGF, small GTPases and ENaC will advance our understanding of the basic mechanisms of sodium regulation in salt-dependent forms of hypertension and might provide novel targets and strategies for their treatment.

48 SD rats Na + WAVE1/2 Rac1 RhoGDIα SS rats high salt Na + WAVE1/2 Rac1 RhoGDIα EGFR ErbB2 EGFR ErbB2 EGF, TGF-α, HB-EGF, amphiregulin EGF, TGF-α, HB-EGF, amphiregulin Na + retention Blood volume Cardiac output Arterial pressure

49 Thanks to: Tengis Pavlov, PhD Daria Ilatovskaya, PhD Vlad Levchenko, BS Allen W. Cowley, Jr., PhD Julian Lombard, PhD Andrey Sorokin, PhD David L. Mattson, PhD Nadezhda Zheleznova, PhD (MCW) Paul M. O Connor, PhD (GHSCU) Patricia D. Wilson, PhD (UCL) Research was supported by the American Heart Association, American Diabetes Association and NIH Heart, Lung and Blood Institute Отдельное спасибо фонду Династия Alexey Karpushev, PhD