Matthew Sparks, MD, explores the vascular-tubular crosstalk in the renin-angiotensin system that may play a key role in the regulation of pressure natriuresis. A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT1) receptors in the kidney, stimulating Na+ reabsorption. AT1 receptors in tubular epithelia control expression and stimulation of Na+ transporters and channels. Recently, Dr. Sparks’ team found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT1 receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na+ excretion in SMKO mice, they considered whether alterations in Na+ transporter expression might also play a role. Using proteomic analysis, they found that levels of Na+-K+-2Cl- cotransporter isoform 2 (NKCC2) and Na+/H+ exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, they found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser71), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na+ channel. However, the increases in α- and γ-epithelial Na+ channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na+ excretion observed after cell-specific deletion of AT1 receptors from smooth muscle cells is associated with altered Na+ transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na+ transporters and contributing to the regulation of pressure natriuresis.

1. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Vascular-Tubular Crosstalk in
the Renin-Angiotensin
System
Matthew Sparks, MD
Nephrology
Duke University
Associate Professor

2. Duke Nephrology
2
Matthew A. Sparks, MD
Associate Professor
Director, Nephrology Fellowship
Director of Medical Student Research, Department of Medicine
Duke University School of Medicine
@Nephro_Sparks
Vascular-Tubular Crosstalk in the
Renin-Angiotensin System

3. 3
Conflict of Interest
None

4. 4
Outline
•Background of the renin-angiotensin system
•What is known about vascular actions from experimental
systems (mouse models)
•Discuss new findings of vascular-tubular crosstalk

5. 5

6. 6
1971 Biochemistry

7. 7
1974 NEJM
Captopril

8. 8
Brenner BM et al. N Engl J Med 2001;345:861-869.
Effects of Losartan on Renal and Cardiovascular Outcomes in Patients
with Type 2 Diabetes and Nephropathy RENAAL

9. 9

10. 10

11. 11
Renin-Angiotensin System (RAS) Blockade
•RAS activation plays a critical role in the pathogenesis of
cardiovascular and kidney diseases.
•RAS blockade reduces morbidity and mortality from
cardiovascular and renal diseases.
•Prevention of cardiovascular events by RAS blockade is
partly independent of blood pressure.

13. 13

14. 14
Ito et al 1995, PNAS

15. 15
Ito et al 1995, PNAS
AT1A receptors and BP

16. 16
Almost complete absence of systemic vasoconstrictor response to Ang II in AT1A KO mice
AT1A receptors and BP
Ito et al 1995, PNAS

17. 17
Crowley et al 2005, JCI

18. 18
Crowley et al 2005, JCI
Key role for the kidney and systemic tissues in BP control
Both the kidney and systemic AT1A receptor pools contribute to baseline BP

19. 19
Crowley et al 2006, PNAS
Dominate role of the KIDNEY in Hypertension
The kidney AT1A receptor pool plays crucial role in BP response to chronic Ang II

20. 20
1.How do vascular angiotensin receptors
control blood pressure?
2.What is the contribution of kidney
versus systemic vascular angiotensin
receptors?

21. 21
 We generated mice lacking the AT1A receptor in all smooth muscle cells
(SMKOs) using Cre/LoxP technology
 Use a mouse line in which Cre recombinase
 is knocked-in to the Sm22 gene locus (KISm22)
 These mice were crossed with a second mouse line bearing a conditional
Agtra1a allele
1Zhang J et al. Arterioscler Thromb Vasc Biol. 2006 Mar;26(3):e23-4.
2Wirth A et al. Nature Medicine. 2008 Jan;14(1):64-8
Methods

22. 22

23. 23
KIsm22 Cre+ mTmG
mTmG
Aorta
Kidney
Sparks et al J Am Soc Nephrol. 2015

24. 24
A B
B
A
C
Sparks et al Am J Physiol Renal Physiol. 2021

25. 25
Sparks et al J Am Soc Nephrol. 2015
Reduced AT1A receptor mRNA levels in vascular tissue of SMKOs

26. 26
Blood pressure with altered dietary salt intake
Sparks et al J Am Soc Nephrol. 2015

27. 27
A B
Blood pressure responses to chronic Ang II infusion
Sparks et al J Am Soc Nephrol. 2015

28. 28
Wolf et al AJP Renal Phys. 2018
Female mice have similar blood pressure response to chronic Ang II

29. 29

30. 30
Systemic vasoconstrictor responses in SMKOs and Controls
Sparks et al J Am Soc Nephrol. 2015

31. 31
• What is the mechanism underlying the
persistent vasoconstriction in SMKOs?
• AT1B receptors
• Stimulation of sympathetic nervous system
Preserved systemic vasoconstrictor responses in SMKOs

32. 32
• Control 1B+/+ (SMMHC ERT2Cre+ AT1A
flox/flox AT1B
+/+ No Tamoxifen)
• Control 1B-/- (SMMHC ERT2 Cre+ AT1A
flox/flox AT1B
-/- No Tamoxifen)
• I-SMKO 1B+/+ (SMMHC ERT2Cre+ AT1A
flox/flox AT1B
+/+ Tamoxifen)
• I-SMKO 1B-/- (SMMHC ERT2Cre+ AT1A
flox/flox AT1B
-/- Tamoxifen)
Experimental Groups

33. 33
Acute vasocontrictor response to Ang II after phentolamine
Sparks et al J Am Soc Nephrol. 2015

34. 34
Increased urinary norepinephrine in SMKOs
Sparks et al J Am Soc Nephrol. 2015

35. 35
WARNING

36. 36

37. 37
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Angiotensin II
0.001 10ug/kg
Minutes

38. 38
A
B
Increased Baseline RBF & Attenuated Renal Vasoconstrictor Responses to Ang II in SMKO Mice
Sparks et al J Am Soc Nephrol. 2015

39. 39
Sodium Balance Study
3 days baseline
• Mice are fed normal sodium gel diet (10g/day)
• Urine collection daily for Na determination
5 days Ang II
Ang II (1000ng/kg/min)

40. 40
IN
OUT
Urinary sodium excretion after Ang II is enhanced in SMKO mice
Sparks et al J Am Soc Nephrol. 2015

41. 41
Summary-1
• Deletion of AT1A receptors from VSMCs led to reduced BP and exaggerated salt
sensitivity.
• Hypertensive response to Ang II was attenuated in SMKOs and was associated
with enhanced sodium excretion.
• Despite elimination of AT1A receptors from VSMCs, there was substantial
preservation of Ang II-dependent vasoconstriction in the systemic, but not in the
renal circulation.

42. 42
Do alterations in vascular function affect
tubular function?

43. 43
Hypothesis- compensation would occur
enhanced sodium channel abundance and
function

44. 44
Alicia McDonough, PhD

45. 45
NHE3 T
NHE3-P
Control
Control
+ AngII SMKO
SMKO
+ AngII
1.00 ± 0.11 0.59 ± 0.06*
NaPi2
Myosin VI
NKCC
total
NKCC-P
NCC total
NCCpS71
SPAK
total
FL ̶
SPAK2 ̶
KS-SPAK
0.78 ± 0.09
0.60 ± 0.08* 0.80 ± 0.13
1.26 ± 0.13
1.05 ± 0.09
1.00 ± 0.07 1.14 ± 0.15
1.00 ± 0.03
0.98 ± 0.09
0.99 ± 0.07
1.00 ± 0.08
0.99 ± 0.07
1.00 ± 0.11
1.00 ± 0.05
1.01 ± 0.03
1.00 ± 0.05
0.76 ± 0.09
0.92 ± 0.05
0.69 ± 0.06*
0.69 ± 0.07
1.62 ± 0.15*
1.45 ± 0.14*
1.51 ± 0.16*
1.67 ± 0.16*
1.01 ± 0.06
2.06 ± 0.30* 1.01 ± 0.08 2.17 ± 0.28*
0.84 ± 0.04
0.90 ± 0.05
0.81 ± 0.07
0.88 ± 0.03
0.81 ± 0.02
0.71 ± 0.03
1.19 ± 0.05*
1.07 ± 0.03
0.87 ± 0.03
0.76 ± 0.03*
0.89 ± 0.29
0.76 ± 0.07
0.92 ± 0.08*
1.08 ± 0.06
SPAKpS373 ̶
OSR1pS373 ̶
ENaC
alpha - FL
ENaC
alpha - Cl
Renin
0.99 ± 0.05
0.96 ± 0.11
0.96 ± 0.14
1.00 ± 0.02
0.96 ± 0.11
0.86 ± 0.11
1.00 ± 0.08
0.87 ± 0.09
1.11 ± 0.08
1.13 ± 0.08
1.09 ± 0.05
1.06 ± 0.05
1.17 ± 0.07
2.58 ± 0.17*
1.10 ± 0.06
1.58 ± 0.17*
1.00 ± 0.04
0.98 ± 0.13
0.49 ± 0.03*
0.91 ± 0.17
0.66 ± 0.04*
1.31 ± 0.23
1.29 ± 0.17
1.67 ± 0.31
Cortex
Sparks et al Am J Physiol Renal Physiol. 2021

46. 46
mNHE3
total
mNHE3-P
mNKCC
total
mNKCC-P
mENaC
alpha - FL
SPAK
total
SPAKpS373 ̶
OSR1pS373 ̶
0.99 ± 0.07
0.98 ± 0.04
1.01 ± 0.05
0.97 ± 0.22
0.99 ± 0.03
0.60 ± 0.07
0.94 ± 0.11
FL ̶
SPAK2 ̶
KS-SPAK
0.88 ± 0.13
0.59 ± 0.02 0.56 ± 0.03
0.85 ± 0.05
0.99 ± 0.07
0.68 ± 0.09
0.95 ± 0.12
0.95 ± 0.14
0.85 ± 0.07
0.79 ± 0.08
0.63 ± 0.05
0.63 ± 0.08
0.71 ± 0.05
0.68 ± 0.06
mENaC
alpha - Cl
0.64 ± 0.02*
0.75 ± 0.11
0.71 ± 0.04*
0.76 ± 0.08*
1.38 ± 0.08*
0.97 ± 0.07
2.22 ± 0.31*
1.05 ± 0.05
0.95 ± 0.10
1.00 ± 0.07
0.97 ± 0.10
1.00 ± 0.08
1.01 ± 0.06
0.56 ± 0.05*
0.86 ± 0.09
0.79 ± 0.06
0.73 ± 0.05
0.88 ± 0.07
0.90 ± 0.10
0.94 ± 0.04
0.88 ± 0.04
0.83 ± 0.02*
0.47 ± 0.01
Medulla
Sparks et al Am J Physiol Renal Physiol. 2021

47. 47
Sparks et al Am J Physiol Renal Physiol. 2021

48. 48
Sparks et al Am J Physiol Renal Physiol. 2021

49. 49
0 .0
0 .5
1 .0
1 .5
R
e
la
tiv
e
d
iffe
r
e
n
c
e
W T -u n if
S M K O (n o rm a liz e d to W T )
W T
S M K O
N K C C to ta l
co rte x m edulla
c o rte x m e d u lla
*
*
0 .0
0 .5
1 .0
1 .5
m edulla
R
e
la
tiv
e
d
iffe
r
e
n
c
e
W T -u n if
S M K O (n o rm a liz e d to W T )
W T
S M K O
N H E 3 to ta l
*

50. 50
Reduced natriuretic response to furosemide in SMKOs
#P<0.0001
**P<0.002
***P<0.0001
Control SMKO Control SMKO
0
10
20
30
40
50
60
Vehicle Furosemide
**
***
#
Na
mol/hour
Furosemide 25mg/kg IP (500ul NS) Collect urine 4 hours later
Sparks et al Am J Physiol Renal Physiol. 2021

51. 51
Our original hypothesis is that we would see enhanced sodium
channel abundance and function
We saw the opposite.
Thus, changes in tubular function seem to be primary disturbance
in mediating sodium excretion and blood pressure effect

52. 52
Tubular function during Ang II-induced Hypertension
Day 5

53. 53
Sparks et al Am J Physiol Renal Physiol. 2021

54. 54
Sparks et al Am J Physiol Renal Physiol. 2021

55. 55
Sparks et al Am J Physiol Renal Physiol. 2021

56. 56
Sparks et al Am J Physiol Renal Physiol. 2021

57. 57
Sparks et al Am J Physiol Renal Physiol. 2021

58. 58
A
C
o
n
t
r
o
l
-
u
n
i
f
C
o
n
t
r
o
l
-
A
n
g
I
I
S
M
K
O
-
u
n
i
f
S
M
K
O
-
A
n
g
I
I
0.0
0.5
1.0
1.5
Relative
difference
*
Medullary NHE3
**
***
B
C
o
n
t
r
o
l
-
u
n
i
f
C
o
n
t
r
o
l
-
A
n
g
I
I
S
M
K
O
-
u
n
i
f
S
M
K
O
-
A
n
g
I
I
0.0
0.5
1.0
1.5
Relative
difference
Cortical NHE3
*
Sparks et al Am J Physiol Renal Physiol. 2021

59. 59
0
1
2
3
4
R
e
la
tiv
e
d
iffe
r
e
n
c
e
W T -u n if
W T + A n g II
W T (c o rte x )
S M K O (c o rte x )
c le a v e d  E N a C
S M K O -u n if
S M K O + A n g II
u n in fu s e d A n g II
(n o r m a liz e d to W T -u n if)
c o rte x
* *
* *
0
1
2
3
4
R
e
la
tiv
e
d
iffe
r
e
n
c
e
W T -u n if
W T + A n g II
S M K O -u n if
S M K O + A n g II
(n o r m a liz e d to W T -u n if)
m e d u lla
W T (m e d u lla )
S M K O (m e d u lla )
c le a v e d  E N a C
u n in fu s e d A n g II
* * *
* *
0 .0
0 .5
1 .0
1 .5
2 .0
R
e
la
tiv
e
d
iffe
r
e
n
c
e
W T -u n if
W T + A n g II
S M K O -u n if
S M K O + A n g II
(n o r m a liz e d to W T -u n if)
m e d u lla
W T (m e d u lla )
S M K O (m e d u lla
c le a v e d  E N a C
u n in fu s e d A n g II
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
R
e
la
tiv
e
d
iffe
r
e
n
c
e
W T -u n if
W T + A n g II
W T (c o rte x )
S M K O (c o rte x )
c le a v e d  E N a C
S M K O -u n if
S M K O + A n g II
u n in fu s e d A n g II
(n o r m a liz e d to W T -u n if)
c o rte x
* * *
* *
⍺ENaC γENaC
Sparks et al Am J Physiol Renal Physiol. 2021

60. 60
Vehicle Amiloride
0
5
10
15
20
25
Urine
Na
+
μmol/hour
*
***
**
Ang II
Vehicle Amiloride
0.0
0.1
0.2
0.3
0.4
Urine
Volume
ml/hour
Control
SMKO
Ang II
✱
A B
Sparks et al Am J Physiol Renal Physiol. 2021
Reduced response to amiloride in SMKO

61. 61
No change in urinary aldosterone
U n in fu s e d D a y 5 o f A n g II
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
U
r
in
e
A
ld
o
s
te
r
o
n
e
p
g
/2
4
h
r
s
#
*
C o n tro l
S M K O
Sparks et al Am J Physiol Renal Physiol. 2021

62. 62
No change in glomerular filtration rate
Sparks et al Am J Physiol Renal Physiol. 2021
Control SMKO
0
10
20
30
40
50
GFR
L/min/g
BW

63. 63
Summary of Sodium Transporter and/or Channel Abundance

64. 64
Ang II Summary of Sodium Transporter and/or Channel Abundance

65. 65
Summary-2
• Mice lacking vascular AT1A receptors fail to generate proximal tubule
pressure natiuresis response
• Lack of vascular AT1A receptors leads to reduced NKCC2 abundance along
the loop of Henle at baseline
• Improved natriuresis during AngII infusion in mice that lack vascular AT1A
receptors is associated
• with a reduction in NKCC2.
• diminished ENaC activation and reduced natriuresis to amliloride

66. 66
• Our data suggests that vascular-epithelial cross-talk modulates renal Na+ handling and
thereby contributes to control of BP at baseline and during hypertension.
• Vascular-epithelial cross-talk provides important insight into the physiologic mechanism of
the pressure-natriuresis response underlying hypertension pathogenesis.
Conclusion

67. 67
Thanks
Thomas Coffman Neha Mehta (Ishan/Meera)
Susan Gurley Samira Farouk
Steve Crowley Kenar Jhaveri
Myles Wolf
Bob Spurney
Mary Foster
Fitra Rianto Duke Post Doc
Emre Dilmen- Radboud University- Nijmedgen Netherlands
Ritika Revoori Duke Undergrad
Ed Diaz UNC Med Student
Hooman Azad Northwestern Med Student
Aaron Kupin High School Teacher
Rishav Adhikari Johns Hopkins Med Student
Erin Wolf UTSW Resident
Alison Hollis UNC Med Student
Thien Hoang UT Galveston Med Student
Lucas Bouknight UNC Med Student
National Scientist Development Grant
Funding Support
Mandel Foundation
VA Career Development Award
Bob Griffiths
Cindy Chen
Kat Bendt
Dennis Abraham
Sudar Rajagopal
Alicia McDonough
Duke Department of Medicine Chairs Award

68. Duke Nephrology
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Potential Mediators- Nitric Oxide

69. Duke Nephrology
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70. Duke Nephrology
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C o n tro l S M K O
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
P ro s ta g la n d in 2 4 H o u r E x c re tio n (p g )
P
G
E
M
(
p
g
/2
4
h
r
)
C o n tro l S M K O
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
P ro s ta c y c lin (6 -K e to P ro s ta g la n d in F 1 a lp h a ) 2 4 H o u r E x c re tio n (p g )
P
r
o
s
t
a
c
y
lc
in
(
p
g
/2
4
h
r
)
C o n tro l S M K O
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
T h ro m b o x a n e 2 4 H o u r E x c re tio n (p g )
T
h
r
o
m
b
o
x
a
n
e
(
p
g
/2
4
h
r
)
20-Hydroxyeicosatetraenoic acid (20-HETE) and prostaglandin E2 (PGE2) inhibit NaCl reabsorption by the TAL

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RNA Sequencing of baseline kidney tissue

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ENaC cleavage
Prostasin
transmembrane protease serine 4 (TMPRSS4)
Matriptase
Kallikrein
Plasmin
elastase

74. Duke Nephrology
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Frindt et al AJP Renal Physiology 2015
Acute effect of aldosterone on ENaC cleavage

75. Q&A Session
Please submit questions through the
'Ask a Question' box. While all questions
cannot be addressed live, stay tuned for
the Q&A report following our event.
Matthew Sparks, MD
Nephrology
Duke University
Associate Professor

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